Readers’ Tribune

Readers Tribune
ENDOTOXIN TESTING ih PHARMACEUTICALS INDUSTRY
A N EXAMPLE FOR A N E>( -ERNAL QUALITY CONTROL SCHEME
Jan Willem van der Laan(’)

INTRODUCTION Each participant was asked to use their own “in-house”

LAL-Test and their own routinely used control standard
Since the adoption of the LAL-te as a method of endotoxin. The dilution steps in the gel-clot tests were
control for bacterial endotoxins by tf USP and EP, this left to the responsibility of the participant in order to
test is now being used in the Qualit Control laborato- imitate the routine procedure as far as possible. Partici-
ries of the pharmaceutical industry i Europe. pants were asked to report their results on the forms
Both the qualitative test, the LAL-ge dot, as well as the provided.
quantitative tests, the chromogenic a d the turbidimetric
tests, are used more or less rout iely in endotoxin
testing of raw materials, “in-proces: control” and end- RESULTS
product testing.
Thirty-nine participants sent in 53 reports which could
The aim of this external Quality Cl itrol scheme is to be evaluated.
verify if there is agreement in the re ilts so that quality
assurance of endotoxin testing in t e Quality Control Only a few laboratories perform all three tests routinely.
laboratories of the pharmaceutical i iustry and in hos- In the table below, the gel-clot and the chromogenic
pita1 pharmacies is given. The aim v .s to make clear to methods are shown to be the most frequently used
each participant the extent to wh h the results ob- methods among the participants. No distinction could
tained are in agreement with thos of other partici- be made between end-point and kinetic methods.
pants.
Two reports could not be evaluated because they had
The present paper describes the re ilts of a pilot trial
not been completed.
and includes the data for 39 Euror an laboratories.
A statistical description of the results obtained from all
MATERIALS AND METHODS participants has been performed according to a method
developed by Jan Henriksson in the department of
Two vials of autoclaved water conti iinated with envi- Statistics and Medical Data Management of Kabi Stock-
ronmental endotoxins (QC-water)ar I two vials of 2 ng holm, and agreed upon by Dr. Rose E. Gaines Das of
endotoxin (E. coli 0111:B4), ! ipplied by Kabi NIBSC.
Diagnostica AB, were distributed y the RIVM (the
National Institute of Public Health nd Environmental Results obtained utilising the various assays are as
Protection). Each vial was assayed I duplicate. follows:

Vial n Mean Median Min

EST METHOL) EU/ml
1 28 0.71 0.48 0.07
water 2 28 0.75 0.42 0.05 3.13 6.6-417
endotoxin 3 28 25.26 24.00 10.00 68.85 40-273
endotoxin 4 28 26.04 27.50 10.00 63.45 38-244
Turbidimetric water 1 5 0.80 0.62 0.52 1.49 65-186
water 2 5 0.70 0.64 0.49 1.24 70-177
endotoxin 3 5 24.43 23.81 12.75 35.00 52-143
endotoxin 4 5 25.61 24.49 12.90 42.00 50-164
Chromogenic water 1 20 0.47 0.45 0.09 0.89 19-189
water 2 20 0.51 0.49 0.10 0.92 20-180
endotoxin 3 20 24.19 23.50 9.98 49.90 41-206
endotoxin 4 20 23.75 23.65 10.30 46.70 44-197

t
(1) Laboratory for Medicines and Medica Devices, National Institute for Public Health and Environmental Protection, P.O. Box 1,
3720 BA Bilthoven, The Netherlands.

O PHARMEUROPA Vol. 6, NQ 1, Mwch 1994 3

Readers’ Tribune

DISCUSSION PLANS FOR FUTURE ACTION

It is remarkable that despite the inherent differences
between the three methods used for quantification of
the LAL-assay, the mean values are similar, especially
for the 2 ng endotoxin vials. The semi-quantitative
nature of the gel-clot method may be reflected by the
greater differencebetween the minimum and maximum
values.
In Fig. 1 the distribution of all the individual data are
shown. Fig. 1A shows the data for vials 1 and 2,and
Fig. 1B shows the results for vials 3 and 4. It is clear
that the bulk of the results is in the order of magnitude
of the mean. Only a few outliers appeared to be present.
The duplicate values for vials 1and 2 are close together,
as are the duplicate values for vials 3 and 4, indicating
that the inner-test reproducibility appears to be accept-
able.
The median values of the vials 1 and 2 are well above
the limit of 0.25EU per ml for Water for Injection. This
indicates that, assuming the sample of water was of-
fered as a sample for preparation of a product for
parenteral use, the majority of the participants would
have rejected this batch. However, a few participants
would still have passed the sample.
A full collaborative study has been set up which will last
two years which comprises about 100 participants.
Two sets of samples have been distributed and one
more will be distributed in 1994 before the results will
then be evaluated.
Each institute will test the sample with its own routine
assay according to Ph. Eur. (V.2.1.9) or USP approved
and validated method.
In the course of the study the laboratories will be aware
whether their results are in the order of magnitude of
the mean or are outliers. In the latter case the labora-
tories are asked to evaluate their procedures critically to
obtain a better result in the future.
Each participant will be encouraged to apply a quanti-
tative chromogenic or turbidimetric test in addition to
the limit gel-clot test.
ACKNOWLEDGEMENT
S. Sandin (Kordia BV Netherlands), C. Kortman (Kabi
Pharmacia Sweden) and R. Gaines (NIBSC UK) are
thanked for their contribution in the design and evalu-
ation of the study.

FIGURE 1
DISTRIBUTION OF ALL INDIVIDUAL DATA IN THE PILOT TRIAL.

The data for all three types of tests were taken together. So the number of data is higher than the
number of participating laboratories. Each vial was assayed twice.

A B

i -1
I

t
1
10 16 21 es W M 4 0 444öMo55 . 10 16 Pl a6 ao 40 4 4 4 6 0 0 .

Laboirtory No,
0 W1:l A W.I1:2 0 WJZl V WalP:P

A. Vial 1 and 2:
Autoclaved water contaminated B. Vial 3 and 4:
with enoironmental endotoxins Endotoxin of E. coli O1 11:B4.

4 O PHARMEUROPA Vol. 6, N” 1, March 1994

 Styl¡/Tam ponae
~

Draft m
forcom
IMPORTANT NOTICE I
This section contains proposals new and revised monographs and other texts, intended for inclusion
in the European submitted for public comment. You can comment through the
appropriate address listed on the back cover page of the present issue. Only
will be considered before the final version is prepared. Readers
Pharmacopoeia Commission should send their comments
to facilitate the work of the Secretariats of the National
mention in any correspondence, the reference number

It is stressed that these proposals ave not been adopted by the European Pharmacopoeia Commission
and must not be regarded as modifications of limits should
be supported by analytical of batches. Proposed changes of
methodology shall be results of a comparative trial of the method published
in Pharmeuropa for

In the case of proposals for n, text to be deleted is crossed out and replacements or additions
are displayed on a grey

In certain cases, draft monograp for appropriate checking, the use of a reference material
which is not yet commercially in exceptional circumstances, we will t r y to make the necessary
substance available; they are by (*); please enquire to the Technical Secretariat.

PA/PH/Exp. 12/T (93) 20 ANP PA/PH/Exp. 12/T (93) 21 ANP

l .

STYLI TAMPONAE

Sticks Tampons

Sticks are solid preparations Tampons are single-dose preparations intended to be

cation. They are rod-shaped inserted into body cavities for a limited period of
consisting of a simple or time. They consist of a suitable material impregnated
one or more active with one or more active ingredients.
dispersed.
Several categories of tampons may be distinguished,
Several categories of sticks may be distinguished, such as:
such as:
- rectal tampons;
- dental sticks;
- vaginal tampons;
- nasal sticks;
- topical sticks; - dental tampons;
- urethral sticks. - nasal tampons.

O PHARMEUROPA Vol. 6, N" 1, Maich 1994 5

 Alcohol Polyvinylicus
PA/PH/Exp. 16/T (91) 17 ANP
NOTES ON THE MONOGRAPH
ALCOHOL POLYVINYLICUS
The intention is that this text shall be applied to
all grades of polyvinyl alcohol for meàico-
pharmaceutical use. These uses include: protective
colloids f o r water dispersions, suspension
stabilizers for syrups, and components of lini-
ments, pastes and jellies. Polyuinyl alcohol is also
used in solutions for injection, as an agglutinant
d- f in opthalmic preparations to
increase the u iscosi ty.
The generalities, characters and tests in the
monograph concern the materials which are most
frequently in use.
ALCOHOL POLYVINYLICUS
Polyvinyl Alcohol
Polyvinyl alcohol is obtained by polymerisation of
vinyl acetate, followed by partial or almost complete
alkaline catalysed hydrolysis.
Polyvinyl alcohol complies with the formula:
-( -CH-CH2- ) -( CH-CHz-)-
I m lO
OH
L O
I of phenolphthalein to 50 ml of solution S and titrate
CH3
Polyvinyl alcohol polymers comply with following
indices:
n 2
O 5 - 50,35
m where V = ml of 0.05 N potassium hydroxide.
The molecular weight lies between 20 O00 and
50 000.
CHARACTERS
White powder or translucent granules. Partially
hydrolysed polymers dissolve more readily in water
than fully hydrolysed polymers. All polymers are
soluble in N-methylpyrrolidone, slightly soluble in
ethanol and methanol, practically insoluble in
acetone, chlorinated hydrocarbons, ether and toluene.
IDENTIFICATION
A. Examine by infrared absorption spectro-
photometry (V.6.18). The spectrum obtained
shows absorption maxima corresponding to
polyvinyl alcohol with m = 100:n = O (fully
hydrolysed) a t 3340 cm-l, 2930 cm-l,
2910 cm-' and to polyvinyl alcohol with m >
88:n < 12 (partially hydrolysed) at 3340 cm-l,
6 O PHARMEUROPA Vol. 6, N" 1 , March 1994
2930cm-l, 2910 cm-l, 1735 cm-l and
1245 cm-l. The spectrum obtained is identical to
the spectrum obtained with the material selected
for the type sample.
B. To 2 ml of solution S (see Tests) add 0.4 ml of
3 per cent M b o r i c acid solution R and 0.1 ml
of 0.01N iodine solution. A greenish-blue colour
is produced.
TESTS
Solution S Suspend 10.0 g in 200 ml of cold water
with continuous stirring. Heat the mixture with
stirring to 90 ' C for 30 min. Cool to room temper-
ature and continue stirring to obtain a homogenous
solution. Filter the solution through a tared sintered
glass filter (No. 16). Wash the residue with 10 ml of
hot water (80 C) and pour the filtrates into a 250 ml
o
graduated flask. Dilute to 250 ml with water.
Appearance of solution Solution S is clear (V.6.1)
and not more intensely coloured than reference so-
lution G (Method II, V.6.2).
pH (V.6.3.1)The pH of solution S is 4.5 to 7.0.
Viscosity (V.6.7.1) Determine the viscosity
immediately after preparation of solution S at 20 " C
f 0.1 ' C by using a capillary viscometer(l). The
viscosity is not less than 85 per cent and not more
than 115 per cent of the average value specified for
the respective polymer.
Acid value (V.3.4.1)Not more than 3.0. Add 1 ml
with potassium hydroxide solution 0.05N until the
pink colour persists for 15 s.
2805 V
IA = -
Ester value (V.3.4.2) 90 to 280.
Heavy metals (V.3.2.8)1.0 g of the polyvinyl alcohol
polymer complies with limit test D (20 ppm). Prepare
the standard using lead standard solution
(10 ppm Pb) R.
Water-insoluble substances Not more than 10 mg
(0.1 per cent). Wash the tared filter used in the
preparation of solution S with two 25 ml portions of
water and dry the residue at 100 OC to 105 OC to
constant weight.
Loss on drying (V.6.22.) Not more than 5 per cent
determined on 1.000 g by drying at 100 OC to
105 OC for 3 h.
Sulphated ash (V.3.2.14) Not more than 1 per
cent, determined on 1.0s.
(1)international Standard: Ubbelohde viscometer No. 1
 Methylprednosoloni Hydrogenosuccinas

CHARACTERS
PA/PHmp.
A white or nearly white, hygroscopic powder,
. 4
practically insoluble in water, slightly soluble in
acetone and in ethanol, practically insoluble in ether.
It dissolves in dilute solutions of alkali hydroxides.

The substance is described in US IDENTIFICATION

editions) from whe
rotation and loss o identification tests A and €3 may be omitted if
style of the mo tdentification tests C, and D a r e carried out. Zden-
cort icos te ro id mon og rap h s in tification tests C, and D may be omitted if iden-
Pharmacopoeia. tification tests A and B are carried out.
The identification A. Examine by infrared absorption spectro-
photometry (V.6.18). The absorption maxima
in the spectrum obtained with the substance to
be examined correspond in position and relative
intensity to those in the spectrum obtained with
methylprednisolone hydrogen succinate CRS.
corticos tero ids.
B. Examine by thin layer chromatography (V.6.20.2),
using as the coating substance a suitable silica
gel with a fluorescent indicator having an opti-
mal intensity at 254 nm.
Test solution Dissolve 10 mg of the substance to
be examined in a mixture of 1 volume of
methanol R and 9 volumes of methylene chloride
R and dilute to 10 ml with the same solvent.
Reference solution (a) Dissolve 10 mg of
methylprednisolone hydrogen succinate CRS in
a mixture of 1 volume of methanol R and 9 vo-
lumes of methylene chloride R and dilute to
10 ml with the same sovent.
Reference solution (b) Dissolve 10 mg of
methylprednisolonehydrogen succinate CRS and
10 mg of hydrocortisone hydrogen succinate
CRS in a mixture of 1volume of methanol R and
9 volumes of methylene chloride R and dilute to
10 ml with the same solvent.
Methylpredn isoIone hydrogen succinate
Apply separately to the plate 10 pl of each
solution. Develop over a path of 15 cm using a
HIC-O-C
~ t
Il
CH2-CH2-COOH
mixture of 0.1 volume of formic acid R, 1volume
of ethanol R and 15 volumes of methylene
chloride R. Allow the plate to dry in air and
examine in ultraviolet light at 254 nm. The prin-
cipal spot in the chromatogram obtained with
the test solution is similar in position and size to
the principal spot in the chromatogram obtained
with reference solution (a). The test is not valid
unless the chromatogram obtained with reference
CHj I solution (b)shows two spots which may however
not be completely separated. Spray the plate
C26H340û

Methylprednisolone hydrogen succi

l Mr 474.5
with alcoholic solution of sulfuric acid R.Heat at
120 "C for 10 min or until the spots appear.
Allow to cool. Examine in daylight and in ultra-
less than 97.0 per cent and violet light at 365 nm. The principal spot in the
chromatogram obtained with the test solution is
similar in position, colour in daylight, fluores-
cence in ultraviolet light at 365 nm and size to
reference to the dried substance. the principal spot in the chromatogram obtained

O PHARMEUROPA Vol. 6, NQ1, Match 1994 7

Methylprednosoloni Hydrogenosuccinas
with reference solution (a). The test is not valid
unless the chromatogram obtained with reference
solution (b) shows two spots which may however
not be completely separated.
C. Examine by thin layer chromatography (V.6.20.2),
using as the coating substance a suitable silica
gel with a fluorescent indicator having an opti-
mal intensity at 254 nm.
Test solution (a) Dissolve 2 5 mg of the subs-
tance to be examined in methanol R with gentle
heating and dilute to 5 ml with the same solvent.
This solution is also used to prepare test solution
(b). Dilute 2 ml of the solution to 10 ml with
methylene chloride R.
Test solution (b) Transfer 2 ml of the solution
obtained during preparation of test solution (a)to
a 15 ml glass tube with a ground-glassstopper or
a polytetrafluoroethylene cap. Add 10 ml of
0.02M methanolic sodium hydroxide solution
and immediately pass a stream of nitrogen R
briskly through the solution for 5 min. Stopper
the tube. Heat in a water-bath at 45 OC, protected
from light, for 30 min. Allow to cool.
Reference solution (a) Dissolve 2 5 mg of
methylprednisolone hydrogen succinate CRS in
methanol R with gentle heating and dilute to
5 ml with the same solvent. This solution is also
used to prepare reference solution (b). Dilute
2 ml of the solution to 10 ml with methylene
chloride R.
Reference solution (b) Transfer 2 ml of the
solution obtained during preparation of reference
solution (a) to a 15 ml glass tube with a ground-
glass stopper or a polytetrafluoroethylene cap.
Add 10 ml of 0.02M methanolic sodium hydr-
oxide solution and immediately pass a stream of
nitrogen R briskly through the solution for 5 min.
Stopper the tube. Heat in a water-bath at 45 OC,
protected from light, for 30 min. Allow to cool.
Apply separately to the plate 5p1 of each solu-
tion. Prepare the mobile phase by adding a mix-
ture of 1.2 volumes of water and 8 volumes of
methanol R to a mixture of 15volumes of ether R
and 77 volumes of methylene chloride R. Develop
over a path of 15 cm. Allow the plate to dry in
air and examine in ultraviolet light at 254 nm.
The principal spot in each of the chromatograms
obtained with the test solutions is similar in
position and size to the principal spot in the
chromatogram obtained with the corresponding
reference solution. Spray the plate with alcoholic
solution of sulfuric acid R. Heat at 120 OC for
10 min or until the spots appear. Allow to cool.
Examine in daylight and in ultraviolet light at
365 nm. The principal spot in the chromatograms
obtained with the test solutions is similar in pos-
ition, colour in daylight, fluorescence in ultravio-
let light at 365 nm and size to the principal spot
in the chromatogram obtained with the corres-
ponding reference solution. The principal spot in
8 O PHARMEUROPA Vol. 6, N" 1 , March 1994
each of the chromatograms obtained with test
solution (b) and reference solution (b) has an R
value distinctly higher than that of the principai
spots in each of the chromatograms obtained
with test solution (a) and reference solution (a).
D. Add about 2 mg to 2 ml of sulfuric acid and
shake to dissolve. Within 5 min a reddish-brown
colour develops. When examined in ultraviolet
light at 365 nm an orange-yellow fluorescence is
seen. Add the solution to 10 ml of water and
mix. The colour fades and there is a yellowish-
green fluorescence in ultraviolet light at 365 nm.
TESTS
Appearance of solution. Dissolve 0.100 g in 5 ml
of sodium hydrogen carbonate solution R. The subs-
tance dissolves completely and the clarity of the
solution is the same as that of the sodium hydrogen
carbonate solution R.
Specific optical rotation (V.6.6).Dissolve 0.250 g
in dioxane R and dilute to 25.0 ml with the same
solvent. The specific optical rotation is +87" to + 9 5 O ,
calculated with reference to the dried substance.
Related substances. Examine by liquid chroma-
tography (V.6.20.4).
Test solution Dissolve 50 mg of the substance to be
examined in the mobile phase and dilute to 25.0 ml
with the same solvent.
Reference solution(a) Dissolve 2.5 mg of methyl-
prednisolone CRS and 2.5 mg of methylprednisolone
17-hydrogen succinate CRS in the mobile phase and
dilute to 50.0 ml with the same solvent.
Reference solution (b) Dilute 1.0 ml of the test
solution to 100.0 ml with the mobile phase.
The chromatographic procedure may be carried out
using:
- a stainless steel column 0.25 m long and 4.0 mm
in internal diameter packed with octadecylsilyl
silica gel for chromatography R (5 pm),
- as mobile phase at a flow rate of 1ml per minute
a mixture of 33 volumes acetonitrile R and 67
volumes of 3 per cent u/u glacial acetic acid R,
- as detector a spectrophotometer set at 254 nm.
Equilibrate the column with the mobile phase at a
flow rate of 1 ml per minute for about 30 minutes.
Adjust the sensitivity so that the height of the prin-
cipal peak in the chromatogram obtained with 20 pl
of reference solution (b) is at least 50 per cent of the
full scale of the recorder.
Inject 20 yl of reference solution (a). When the
chromatograms are recorded in the conditions
described above the retention times are: methyl-
prednisolone, about 1O .2 min; methylprednisolone
17-hydrogen succinate, about 1 1 . 6 min and
methylprednisolone 21-hydrogen succinate, about
 irrigation Solutions
~

22 min. The test is n Irrigation solutions may contain one or more active
between the peaks ingredients, electrolytes or osmotically active subs-
prednisolone arid methylpred tances in varying concentrations and are usually
succinate is not less than 3.0. If adjusted to isotonicity.
concentration of acetonitrile in
Irrigation solutions are supplied in single-dose con-
Inject separately 20 p1 of the tainers which differ from containers of large volume
of reference solution (b). parenteral solutions in the way they are packaged
tography for twice the retention (e.g. screw cap container). The containers and
peak. In the chromatogra closures comply with the requirements for containers
solution: the area of any peak for preparations for parenteral use (VI.2.1.and VI.2.2)
cipal peak is not greater than but are readily distinguishable from containers for
the principal peak in th preparations intended for parenteral administration.
with reference solution (b) (0.5 The containers are tamper-proof and sealed so as to
the areas of all peaks ap exclude micro-organisms.
is not greater than the a
the chromatogram obtained w
(b)(l.O per cent). Disre TESTS
solvent and any peak
times the area of the principal Appearance of the solutions Examined in suitable
ogram obtained wi conditions of visibility, irrigation solutions are
(0.05 per cent). practically clear and practically free from particles.

Loss on drying (V.6. pH (V.6.3.1) 5.0 to 7.4, unless otherwise justified

cent, determined on 1 and authorised.
at 100 OC to 105 OC.
Sterility (V.2.1.1) The solution complies with the
Sulfated ash (V.3.2.14). Not mo test for sterility.
cent, determined on 1.0 g.
Bacterial endotoxins (V.2.1.1)Not more than 0.5
I.U. of endotoxin per millilitre.
ASSAY I
Solutions for which a validated test for bacterial
Dissolve 50.0 mg in alcohol R and endotoxins cannot be carried out comply with the
following requirement:
(V.9.19)at the maximum Pyrogens (V.2.1.4) Inject per kilogramme of the
rabbit's mass, 10 ml of the solution, unless otherwise
justified and authorised.

STORAGE I STORAGE

e
Store in an airtight container, prot cted from light. Store at a temperature not below 4 O C and not above
30 O C .

monograph include:
LABELLING
methylprednisolone,
The label on the container states:
- the name and concentration of the active
ingredient(s),
- the name and concentration of any added subs-
PA/PH/Exp. 12/T (93) 6 ANP
- t I.
tance,
- the pH,
- the calculated osmolality, expressed in milli-
osmoles per litre,
- where appropriate, that the solution is apyrogenic,
- that the solution is not to be used for injections,
- that any unused portion of solution is to be
discarded,
- the storage conditions.
I

O PHARMEUROPA Vol. 6, Ne 1, Makch 1994 9

Pentamidini Diisetionas
PA/PH/Exp. 1OCP (92) 24 ANP
PENTAMIDINI DIISETIONAS
Comments on the monograph
identifica tion
It appears that the IR test alone cannot be used to
differentiate hexamidine from pentamidine. I t is
therefore supplement by another test.
Test
pH: the measurement of the buffering capacity
indicates that the test for pH should be replaced
by a test for acidity-alkalinity. However, in this
case, the test for p H has been kept as the sub
stance tends to precipitate in acidic and in alkaline
conditions.
Heavy metals: test C was preferred as the acidic
buffer used in test A causes precipitation of the
subs tance.
Assay
Contrary to current practice, a colour indicator
was preferred as potentiometric determination of
the end point has been found to be less
reproducible (standard deoiation is 0.57 for
potentiometry as against 0.20 for the colour
indica tor).
PENTAMIDINI DIISETIONAS
Pentamidine Diisethionate
M, 592.7
Pentamidine diisethionate contains not less than 98.5
per cent and not more than the equivalent of 101.5
per cent of 4,4’-[pentane l15-diylbis(oxy)]dibenza-
midine di(2-hydroxyethanesulphonate), calculated
with reference to the dried substance.
CHARACTERS
A white or almost white powder or crystals,
hygroscopic, freely soluble in water, sparingly soluble
in alcohol, practically insoluble in ether and in
methylene chloride.
10
IDENTIFICATION
Identification test B may be omitted if identifica-
tion tests A, C, 0, € and F are carried out. Iden-
tification tests A, D and € may be omitted if
identification tests B, C and F are carried out.
A. Dissolve 20.0 mg in alcohol R and dilute to
100.0 ml with the same solvent. Dilute 5.0 ml of
this solution to 100.0 ml with alcohol R.
Examined between 230 nm and 340 nm (V.6.19),
the solution shows an absorption maximum at
265 nm. The specific absorbance at the maxi-
mum, calculated with reference to the dried subs-
tance, is 520 to 560.
B. Examine by infrared absorption spectro-
photometry (V.6.18). The absorption maxima in
the spectrum obtained with the substance to be
examined correspond in position and relative
intensity to those in the spectrum obtained with
pentamidine diisethionate CRS. Examine the
substances as discs.
C. Dissolve 40.0 mg in 5 ml of water and add
dropwise with shaking 1 ml of a 1 per cent
m/V solution of sodium chloride R. Allow to
stand for 5 min. The mixture remains clear.
D. Dissolve 0.5 g in 5 ml of water with heating to
80 OC. Add 10 ml of 1 N sodium hydroxide. Cool
in ice and filter. To 2 ml of the solution add
0.2 ml of nitric acid R and then 0.2 ml of a
40 per cent m/V solution of ammonium and
cerium nitrate R in dilute nitric acid R. An orange
red colour is produced. A blank test prepared at
the same time in the same manner is yellow.
E. Dissolve 30 mg and 30 mg of ninhydrin R in
5 ml of water. Add 1 ml of a 2 per cent m/V
solution of sodium borate R. An abundant white
precipitate forms slowly.
E Treat 0.150 g by the oxygen-flask method
(V.3.5.3). Use 10 ml of dilute hydroxide peroxide
solution R to absorb the combustion products.
The solution gives reaction (a) of sulphates
(V.3.1.1).
TESTS
Appearance of solution Dissolve 1.0 g in water
and dilute to 10 ml with the same solvent. The
solution is not more opalescent than reference sus-
pension II (V.6.1) and not more intensely coloured
than intensity 6 of the range of reference solutions of
the most appropriate colour (Method II, V.6.2).
pH (V.6.3.1) Dissolve 0.5 g in carbon dioxide-free
water R and dilute to 10 ml with the same solvent.
The pH of the solution is 4.5 to 6.5.
O PHARMEUROPA Vol. 6, Ne 1 , March 1994
 immunoglobulina

Related substances Exa VII. 1.1REAGENTS

graphy (V.6.20.4).
Test solution Dissolve O.
Triethylamine. - C,H,,N (M,101.2).
be examined in the m
100.0 ml with the mobile phase. Liquid, with a strong ammonia odour, slightly soluble in water,
miscible with alcohol and ether.
Reference solution Dilute 1
d:: about 0.725.
to 100.0 ml with the mobil
$: about 1.40.
The chromatography m
- a stainless steel column, 0.25 (1)Cphensorb ODS 1 has been found to be suitable.
in diameter, packed with oct
for chromatography
- as the mobile phas PA/PH/Exp. 6B/T (94) 5 ANP
minute, a mixture . .
and 7 volumes of
ammonium acetat Exceptionnuly, comments on these monographs
triethylamine R, are to be sent by 15 May 1994.
- as the detector, IMMUNOGLOBULINUM HUMANUM
265 nm. ANTI-D
Inject separately a sui
Human Anti-D Immunoglobulin
In the chromatogram
the area of any peak
is not greater than 2 IMMUNOGLOBULINUM HUMANUM
in the chromatogra HEPATITIDIS A
and the total area
principal peak is not more th Human Hepatitis A immunoglobulin
of the peak in the
test solution. IMMUNOGLOBULINUM HUMANUM
HEPATITIDIS B
Heavy metals (V.3.2.8) 1.0 g co
test C for heavy metals (20 ppm). Human Hepatitis B immunoglobulin
dard using 2 ml of lead
(10 ppm Pb) R. IMMUNOGLOBULINUM HUMANUM
MORBILLICUM
Loss on drying (V.6.22) Not mo
cent, determined on 1.000 g by Human Measles immunoglobulin
100 "C to 105 "C.
IMMUNOGLOBULINUM HUMANUM
Sulphated ash (V.3.2.14) Not RABICUM
cent, determined on 1.0 g.
Human Rabies Immunoglobulin
ASSAY I IMMUNOGLOBULINUM HUMANUM

i
Dissolve 0.250 $3 in 50 ml of dimet ylformamide R. RUBELLAE
Add 0.25 ml of thymol blue solutio R. Titrate with
O. 1 N tetrabutylammonium hydroxide (V.3.5.5),under Human Rubella Immunoglobulin
nitrogen. Carry out a blank titratio .
IMMUNOGLOBULINUM HUMANUM
1 ml of 0.1N tetrabutylammoniu hydroxide is TETANKUM
equivalent to 29.63 mg of C,,H,, 4010S2.
Human Tetanus immunoglobulin

STORAGE I IMMUNOGLOBULINUM HUMANUM

VACCINICUM

"
Store in an airtight container. ~

Human Vaccinia immunoglobulin

impurities limited by the requi
onograph are: IMMUNOGLOBULINUM HUMANUM
p(pamidinophenoxypentamethy1e eoxybenzene-
carboxamide isethionate. I VARICELLAE
Human Var iceIla immunog1obuIin

O PHARMEUROPA Vol. 6, NQ1, Markh 1994 11

Felodi pinum
The monographs on the above immunoglobulins all
require compliance with the monograph on Normal
Human Immunoglobulin, with the exception of the
requirement for a minimum of 1000 donors for the
plasma pool. A minimum content of 10 per cent
m/V of protein is therefore implied. It is now
considered that where there is a limit for the activity
of the specified antibody, a lower limit for the protein
content is no longer needed. Stable preparations can
be prepared with a lower protein content and such
stability has to be demonstrated for licensing
purposes. If a minimum protein content is required,
then a manufacturer has to add normal immuno-
globulin to reach the limit, thus wasting valuable
starting material. It is therefore proposed no longer
to require a minimum protein content and the second
paragraph of each of the above monographs is to be
modified as shown below.
It complies with the monograph on Immuno-
globulinum Humanum Normale except for the mini-
mum number of donors and the minimum total
protein content.
PA/PH/P3 (94) 1
This monograph has been prepared by the
European Pharmacopoeia in collaboration with
the Swedish Pharmacopoeia Authority and the
man ujac t u re r.
FELODIPI NUM
Felodi pine
\ dry in air and examine in ultraviolet light at
H3C’ c=o
I 254 nm. The principal spot in the chromatogram
O obtained with the test solution is similar in posi-
I
CH3
M, 384.3
Felodipine contains not less than 99.0 per cent and
not more than the equivalent of 101.0 per cent of
ethyl methyl (R,S)4-(2,3dichlorophenyl)-1,4-dihydro-
2,6-dimet hylpyridine-3,5-dicarboxylate,calculated
with reference to the dried substance.
12
CHARACTERS
A white to light yellow, crystalline powder, insoluble
in water, freely soluble in acetone, in ethanol, in
methanol and in methylene chloride, practically in-
soluble in heptane.
IDENTIFICATION
identification tests A, C and D may be omitted if
identification test B is carried out. identification
test B may be omitted if identification tests A ,
C and D are carried out.
A. Dissolve 50 mg in methanol R and dilute to
100 ml with the same solvent. Dilute 3 ml to
100 ml with methanol R. Examined between
220 nm and 400 nm (V.6.19),the solution shows
two absorption maxima, at 238 nm and 361 nm.
The ratio of the absorbance measured at the
maximum at 361 nm to that measured at the
maximum at 238 nm is 0.34 to 0.36.
B. Examine by infrared absorption spectro-
photometry (V.6.18). The absorption maxima in
the spectrum obtained with the substance to be
examined correspond in position and relative
intensity to those in the spectrum obtained with
felodipine CRS. Examine the substances prepared
as discs.
C. Examine by thin-layer chromatography
(V.6.20.2), using as the coating substance a
suitable silica gel with a fluorescent indicator
having an optimal intensity at 254 nm.
Test solution Dissolve 10 mg of the substance to
be examined in methanol R and dilute to 10 ml
with the same solvent.
Reference solution (a) Dissolve 10 mg of
felodipine CRS in methanol R and dilute to 10 ml
with the same solvent.
Reference solution (b)Dissolve 5 mg of nifedipine
CRS in reference solution (a) and dilute to 5 ml
with the same solution.
Apply separately to the plate 5 pl of each solu-
tion. Develop over a path of 15 cm using a
mixture of 40 volumes of ethyl acetate R and
60 volumes of cyclohexane R. Allow the plate to
tion, fluorescence and size to the principal spot
in the chromatogram obtained with reference
solution (a). The test is not valid unless the
chromatogram obtained with reference solution
(b) shows two clearly separated principal spots.
D. Dissolve 150 mg in a mixture of 2 5 ml of 2-
methyl-2-propanol R, and 2 5 ml of perchloric
acid solution R. Add 10 ml of 0.1N cerium
sulphate and neutralise with dilute sodium
O PHARMEUROPA Vol. 6, Ne 1 , March 1994

hydroxide solution R. Shake
25 ml of methylene chloride
lower layer to dryness on a
nitrogen. (The residue,
the test for related
20 mg of the
TESTS
Appearance of solution Dis
methanol R and dilute to 10 ml with
The solution is clear (V.6.1).
Related substances Exami
tography (V.6.20.4).
Test solution Dissolve 25.0
be examined in the mobile pha
with the mobile phase.
Reference solution (a) Dilu
solution to 100.0 ml with th
Reference solu tion (b) Dilu
solution (a) to 10.0 ml with
Reference solution (c) Disso
A (obtained in identificatio
phase and dilute to 50.0 m
Dilute 1.0 ml to 100.0 ml
The chromatographic p
using:
- as mobile phase a
a mixture of 20
Inject 20 y1 of reference solution
recorder, adjust the sensitivity of
the height of the peak
valid unless there is a
the peak due to
(felodipine).
O PHARMEUROPA Vol. 6, Ne 1, Match 1994
Felodipinum
corresponding to impurity B and to impurity C. The
substances elute in the following order: impurity B,
impurity A, felodipine and impurity C.
Inject 20 yl of the test solution, 20 yl of reference
solution (a) and 20 yl of reference solution (b). Con-
tinue the chromatography for twice the retention
time of felodipine, which is about 1 2 min. In the
chromatogram obtained with the test solution: the
sum of the areas of any peaks corresponding to
impurity B and impurity C is not greater than the
area of the principal peak in the chromatogram
I obtained with reference solution (a) (1.0 per cent);
the area of any other peak apart from the principal
peak and any peaks corresponding to impurity B
and impurity C, is not greater than the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.1 per cent) and the sum of
their areas is not greater than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.3per cent). Disregard any
peak with an area less than 0.2 times that of the
principal peak in the chromatogram obtained with
reference solution (b).
Loss on drying (V.6.22) Not more than 0.5 per
cent, determined on 1.000 g by drying in an oven at
100 "C to 105 "C for 3 h.
Sulphated ash (V.3.2.14) Not more than 0.1 per
cent, determined on 1.0 g.
ASSAY
Dissolve 0.160g in a mixture of 2 5 ml of 2-methyl-
2-propanol R and 25 ml of perchloric acid solution R.
Add 0.05ml of ferroin R. Titrate with 0.1N cerium
sulphate until the pink colour disappears. Titrate
slowly towards the end of the titration.
1 ml of 0.1N cerium sulphate is equivalent to
19.21mg of C1,H1,Cl,NO~.
STORAGE
Store protected from light.
The impurities limited by the requirements of this
monograph include:
ethyl methyl 4-(2,3-dichlorophenyl)-2,6-dimethyl-
pyridine-3,5-dicarboxylate,
dimethyl 4-(2,3-dichlorophenyl)-l,4-dihydro-2,6-
dimethylpyridine-3,5-dicarboxylate,
diethyl 4-(2,3-dichloropheny1)-1,4-dihydro-2,6-
dimethylpyridine-3,5-dicarboxylate
13
Zopiclonum
WI.1.1. REAGENTS
-
Cerium sulphate. Ce(S04),,4H,0 (Mr 404.3).Cerium (IV)
sulphate, ceric sulphate.
ViI.2.2. VOLUMETRIC SOLUTIONS
Cerium sulphate 0.1N.Dissolve 40.0 mg of cerium sulphate
R in a mixture of 500 ml of water and 50 ml of sulphuric acid
R. Allow to cool and dilute to 1000.0 ml with water.
Standardisation To 25.0 mi of the cerium sulphate solution,
add 2.0 g of potassium iodide R, 150 ml of water and 1 ml of
starch solution R. Titrate immediately with 0.1N sodium
thiosulphate.
(1)Hypersil ODS,Nucleosil C18 and Lichrosorb RF-18are suitable
., PA/PH/P3 (94)Z 6
I
This monograph has been prepared by the
European Pharmacopoeia in collaboration with
the French Pharmacopoeia Authority and the
manufacturer. An alternative method for the
determination of solvents is proposed using a
capillary column (see text in annex).
ZOPICLONUM
Zopiclone
Zopiclone contains not less than 98.5 per cent and
not more than the equivalent of 100.5 per cent of
(RS)-6-(5-chloropyridin-2-yl)-7-oxo-6,7-dihydro-5H-Solution S Dissolve 1.0 g in dimethylformamide R
pyrrol0[3,4-b]pyrazine-5-y14-methylpiperazine-l-
14
carboxylate, calculated with reference to the solvent-
free substance.
CHARACTERS
A white to slightly yellowish powder, practically inso-
luble in water, freely soluble in methylene chloride,
sparingly soluble in acetone and practically insoluble
in alcohol. It dissolves in dilute mineral acids.
It melts at about 177 OC, with decomposition.
IDENTIFICATION
Identification test B may be omitted if identifica-
tion tests A and C are carried out. Identification
tests A and C may be omitted if identification test
B is carried out.
A. Dissolve 10.0 mg in a 0.35 per cent m/V solu-
tion of hydrochloricacid R and dilute to 100.0 ml
with the same solvent. Dilute 10.0ml to 100.0 ml
with a 0.35 per cent flsolution of hydrochloric
acid R. Examined between 220 nm and 350 nm
(V.6.19),the solution shows an absorption maxi-
mum at 303 nm. The specific absorbance at the
maximum is 340 to 380.
B. Examine by infrared absorption spectro-
photometry (V.6.18). The absorption maxima in
the spectrum obtained with the substance to be
examined correspond in position and relative
intensity to those in the spectrum obtained with
zopiclone CRS. Examine the substances prepared
as discs.
C. Examine by thin-layer chromatography
(V.6.20.2), using silica gel G F R as
~ the
~ coating
~
substance.
Test solution Dissolve 10 mg of the substance to
be examined in methylene chloride R and dilute
to 10 ml with the same solvent.
Reference solution Dissolve 10 mg of zopiclone
CRS in methylene chloride R and dilute to 10 ml
with the same solvent.
Apply separately to the plate 10 pi of each
solution. Develop over a path of 15 cm using a
mixture of 2 volumes of triethylamine R, 50
volumes of acetone R and 50 volumes of ethyl
acetate R. Allow the plate to dry in air and
examine in ultraviolet light at 254 nm. The prin-
cipal spot in the chromatogram obtained with
the test solution is similar in position and size to
the principal spot in the chromatogram obtained
with the reference solution.
TESTS
and dilute to 20 ml with the same solvent.
O PHARMEUROPA Vol. 6, N" 1 , March 1994

~
Appearance of solution
opalescent than reference
not more intensely
range of reference
colour (Method II,
Related substances Exami
tography (V.6.20.4).
Test solution Dissolve 40.0
be examined in the mobile ph
with the mobile phase.
Reference solution (a) Di
Reference solu tion (c)Dissolve 1
with the mobile phase.
The chromatographic p
using:
a stainless steel co
in internal diamet
as mobile phase at a flow
minute a mixture of 380 volu
and 620 volumes
acid R,
30 OC.
the mobile phase (increasin
decreases the retention time
concentration increases the
adjust the apparent pH of the
with a 10 per cent V/Vsolution o
O PHARMEUROPA Vol. 6, N9 1, Ma(ch 1994
Zopiclonum
Inject 20 yl of the test solution, 20 y1 of reference
solution (a) and 20 yl of reference solution (b). Con-
tinue the chromatography for 1.5 times the retention
time of zopiclone. In the chromatogram obtained
with the test solution: the area of any peak, apart
from the principal peak, is not greater than the area
of the principal peak in the chromatogram obtained
with reference solution (a) (0.3 per cent) and not
more than two such peaks have an area greater than
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.1 per cent).
Disregard any peak with an area less than O. 1times
that of the principal peak in the chromatogram
obtained with reference solution (b).
2-Propanol Not more than 0.7 per cent m/m,
determined by gas chromatography (V.6.20.3) using
ethanol R 1 as internal standard.
Internal standard solution Dilute 2 ml of ethanol
R1 to 100 ml with ethylene chloride R. Dilute 1 ml
to 10 ml with ethylene chloride R.
Test solution (a) Dissolve 0.5 g of the substance to
be examined in ethylene chloride R and dilute to
5.0 ml with the same solvent.
Test solution (b)Dissolve 0.5 g of the substance to
be examined in ethylene chloride R, add 0.5 ml of
the internal standard solution and dilute to 5.0 ml
with ethylene chloride R.
Reference solution Dilute 3.0 ml of 2-propanol R
to 100.0 ml with ethylene chloride R. Dilute 1.0 ml
to 10.0 ml with ethylene chloride R. To 1.0 ml of
this solution, add 1.0 mi of the internal standard
solution and dilute to 10.0 ml with ethylene
chloride R.
The chromatographic procedure may be carried out
using:
- a glass column 2 m long and 2 mm in internal
diameter, packed with ethylvinylbenezene-
divinylbenzene copolymer R (150 pm to
180 ym)(2),
- nitrogen for chromatography R as the carrier gas
at a flow rate of 30 ml per minute,
- a flame-ionisation detector,
maintaining the temperature of the column at 140 "C
for 10 min, then raising the temperature at a rate of
20 "C per minute to 200 "C and maintaining at
200 "C for 5 min, and maintaining the temperature
of the injection port and that of the detector at
225 "C.
Inject 1 y1 of each solution. In the chromatogram
concentration obtained with test solution (a), verify that there is no
peak with the same retention time as the internal
standard. Calculate the percentage content of
2-propanol (per cent m/m)taking its density to be
0.785 g per millilitre at 20 "C.
Heavy metals (V.3.2.8) 1.0 g complies with limit
test C for heavy metals (20 ppm). Prepare the stan-
15
Zopiclonum
dard using 2 ml of lead standard solution
(10 ppm Pb) R.
Sulphated ash (V.3.2.14) Not more than 0.1 per
cent, determined on 1.0 g.
ASSAY
Dissolve 0.300 g in a mixture of 10 ml of acetic
acid R and 40 ml of acetic anhydride R. Titrate with
O. 1 N perchloric acid determining the end-point
potentiometrically (V.6.14).
1 ml of 0.1N perchloric acid is equivalent to 38.88
mg of C,,H,,ClN60,.
STORAGE
Store protected from light.
The impurities limited by the requirements of this
monograph include:
6-(5-chloropyridin-2-yl)-7-oxo-6,7-dihydro-[5H]- Inject 1 pl of the test solution and 1 yl of reference
pyrrolo[3,4-blpyrazine-5-yl-4-methylpiperazine-
1- solution (b). In the chromatogram obtained with the
carboxylate-1-oxide (zopiclone oxide)
6-(5-chloropyridin-2-yl)-7-hydroxy-6,7-dihydro-
[5H]-pyrrolo[3,4-b]pyrazine-5-one,
6-(5-chloropyridin-2-yl)-6,7-dihydro-[5H]-pyrrolo-
[3,4-blpyrazine-5-one
VII. 1.1REAGENTS
Sodium lauryl sulphate R1.Complies with the requirements
prescribed in the monograph on Sodium lauryl sulphate and
contains not less than 99 per cent m/m of C12N25Na04S,
determined by gas Chromatography.
ANNEX
ALTERNATIVE METHOD FOR THE
DETERMINATION OF SOLVENTS
Solvents Not more than 0.7 per cent m/m of
2-propanol and not more than 0.02 per cent of any
other solvent, determined by gas chromatography
(V.6.20.3).
Test solution Dissolve 0.25 g of the substance to be
examined in ethylene chloride R and dilute to 5.0 ml
with the same solvent.
16
Reference solution (a)Dilute 4.5 ml of 2-propanol R
to 100.0 ml with ethylene chloride R.
Reference solution (b) Dilute 1.0 ml of reference
solution (a) to 100.0 ml with ethylene chloride R.
The chromatographic procedure may be carried out
using:
- a fused silica capillary column 10 m long and
about 0.53 mm in internal diameter, lined with a
film of styrene-divinylbenzene copolymer R,
20 pm thick(,),
- helium as the carrier gas at a flow rate of 30 ml
per minute,
- a flame-ionisation detector,
maintaining the temperature of the column at 50 OC
for 5 min, then raising the temperature at a rate of
4 "C per minute to 70 "C and maintaining at 70°C
for 4 min and raising the temperature at a rate of
20 "C per minute from 70 OC to 200°C and
maintaining at 200 "C for 7 min, and maintaining
the temperature of the injection port at 150 "C and
that of the detector at 250 "C.
Carry out a blank injecting the dissolution solvent.
blanck verify that there is no peak with the same
retention time as the 2-propanol. From the
chromatograms obtained with the test solution and
reference solution (b), calculate the content (per cent
m/m) of 2-propanol taking its density to be 0.785 g
at 20°C.
(1) Kromasil Cl,is suitable.
(2) PORAPAK Q is suitable.
(3) PORAPAK Q is suitable
O PHARMEUROPA Vol. 6, N* 1 , March 1994
 Ticlopidini Hydrochloridum

examined correspond in position and relative

PA/PH/P3 (94) 3 intensity to those in the spectrum obtained with
ticlopidine hydrochlorideCRS. Examine the subs-
tances prepared as discs.

C. Examine the chromatograms obtained in the test

for related substances. The principal spot in the
chromatograms obtained with test solution (b) is
This monograph has been prc pared by the similar in position, colour and size to the princi-
European Pharmacopoeia in col rboration with pal spot in the chromatograms obtained with
the French Pharmacopoeia Authoi ty and the ma- reference solution (b).
nufacturer.
D. Dissolve about 5 mg in 0.3 ml of a 2 per cent
Wsolution of citric acid R in acetic anhydride R.
Heat on a water-bath at 80 OC. A red colour
develops.
TICLOPIDINI H YDROCHI 3RIDUM
E. About 20 mg gives reaction (a) of chlorides
Ticlopidine Hydrochlc ride (V.3.1.1).

,HCl
CI
C14H15C12NS M, 300.2
Ticlopidine hydr chloride ontains n t less than 99.0
per cent and-not more than the eqL Jalent of 101.0
per cent of 5-(2-~hlorobenzyl)-4,E6,7-tetrahydro-
thieno[3,2-~]pyridinehydrochloride, calculated with
reference to the anhydrous substan e.
TESTS
Appearance of solution Dissolve 0.5 g in a 1 per
cent V/Vsolution of hydrochloric acid R and dilute to
20 ml with the same acid solution. The solution is
clear (V.6.1) and not more intensely coloured than
reference solution B, (Method II, V.6.2).
pH (V.6.3.1) Dissolve 0.5 g in carbon dioxide-free
water R and dilute to 20 ml with the same solvent.
The pH of the solution is 3.5 to 4.0.
Related substances Examine by thin-layer
chromatography (V.6.20.2), using two plates with
silica gel G R as the coating substance.

CHARACTERS Test solution (a) Dissolve 0.50 g of the substance to

A white or almost white, crystallineF bwder, sparingly
soluble in water and in ethanol, ver slightly soluble
in ethyl acetate, practically insolubl in ether.
IDENTIFICATION
Identification tests A, C and D ml y be omitted if
identification tests B and E are cc .ried out. Iden-
tification test B may be omitted identification
tests A, C,D and E are carried c it.
A. Dissolve 40.0 mg in water and d ute to 100.0 ml
with the same solvent. Dilute 10. 1ml to 100.0 ml
with water. Examined betwee 200 nm and
400 nm (V.6.19),the solution s. ows an absorp-
tion maximum at 214 nm, a shc ilder at 232 nm
and two absorption maxima, t 268 nm and
275 nm. The specific absorban e at 232 nm is
230 to 250.
B. Examine by infrared absor Ition spectro-
photometry (V.6.18).The absor Ition maxima in
the spectrum obtained with the substance to be
be examined in a 0.03 per cent V/Vsolution of dilute
hydrochloric acid R in methanol R and dilute to
20 ml with the same solvent.
Test solution (b) Dilute 1 ml of test solution (a)and
dilute to 10 ml with a 0.03 per cent V/V solution of
dilute hydrochloric acid R in methanol R.
Reference solution (a) Dissolve 10 mg of
2-chlorobenzylamine hydrochloride CRS in a 0.03
per cent V/V solution of dilute hydrochloric acid R in
methanol R and dilute to 20 ml with the same
solvent. Dilute 1 ml to 20 ml with a 0.03 per cent
V/V solution of dilute hydrochloric acid R in
methanol R. Dissolve 2 5 mg of ticlopidine
hydrochloride CRS in 1 ml of this solution.
Reference solution (b) Dilute 1 ml of reference
solution (a) to 10 ml with a 0.03 per cent V/V
solution of dilute hydrochloric acid R in methanol R.
Reference solution (c) Dissolve 10 mg of bis-
ticlopidine dihydrochloride CRS in a 0.03 per cent
V/v solution of dilute hydrochloric acid R in
methanol R and dilute to 20 ml with the same solvent.
Dilute 1 ml to 20 ml with a 0.03 per cent V/V
solution of dilute hydrochloric acid R in methanol R.

O PHARMEUROPA Vol. 6, N* 1, Ma ch 1994 17

Ticlopidini Hydrochloridum
A. Apply separately to the first plate 10 y1 of test
solution (a), 10 yl of test solution (b), 10 y1 of
reference solution (a) and 10 yl of reference solu-
tion (b). Develop over a path of 15 cm using the
upper layer from a mixture of 10 volumes of
glacial acetic acid R, 40 volumes of butanol R and
50 volumes of water. Allow the plate to dry in air
and spray with ninhydrin solution R1. Heat at
100 "C for 20 min. In the chromatogram obtained
with test solution (a): any spot corresponding to
2-chlorobenzylamine hydrochloride is not more
intense than the secondary spot in the chroma-
togram obtained with reference solution (a) (O. 1
per cent); any spot, apart from the principal spot
and any s p o t corresponding t o 2-chloro-
benzylamine hydrochloride is not more intense
than the secondary spot in the chromatogram
obtained with reference solution (a) (0.1per cent).
The test is not valid unless the chromatogram
obtained with reference solution (a) shows two
clearly separated spots.
B. Apply separately to the second plate 10 y1 of
test solution (a), 10 y1 of test solution (b), 10 y1 of
reference solution (b) and 10 y1 of reference solu-
tion (c). Develop over a path of 15 cm using the
upper layer from a mixture of 10 volumes of
glacial acetic acid R, 40 volumes of butanol R and
50 volumes of water. Allow the plate to dry in air
and spray with iodoplatinate reagent R. In the
chromatogram obtained with the test solution (a):
any s p o t corresponding t o bis-ticlopidine
dihydrochloride is not more intense than the spot
in the chromatogram obtained with reference so-
lution (c) (0.1 per cent); any spot, apart from the
principal spot and any spot corresponding to bis-
ticlopidine dihydrochloride, is not more intense
than the spot in the chromatogram obtained with
reference solution (c) (0.1 per cent).
Methanol Not more than 100 ppm, determined by
head-space gas chromatography (V.6.20.3,
Method II).
Test solution In a 20 ml vial introduce 0.20 g of the
substance to b e examined. Add 1.0 ml of
diethyleneglycol R and close with a butyl rubber
membrane stopper; secure the stopper with an alu-
minium cap.
Reference solution (a) In a 20 ml vial introduce
1.0 ml of diethyleneglycol R. Close and seal.
Reference solution (b) Mix 0.20 ml of methanol R
with diethyleneglycol R and dilute to 25.0 ml with
the same solvent. Dilute 0.1 ml of the solution to
20.0 ml with diethyleneglycol R. In a 20 ml vial
introduce 0.5 ml of this solution and 0.5 ml of
diethyleneglycol R. Close and seal.
The chromatographic procedure may be carried out
using:
- a column 2 m long and 2 mm in internal diameter,
packed with ethylvinylbenzene-divinylbenzene
copolymer R (180 ym)(l),
18
- helium for chromatography R as the carrier gas
at a flow rate of 30 ml per minute,
- a flame-ionisation detector,
maintaining the temperature of the column at 170 OC,
and that of the injection port at 200 "C and that of
the detector at 250 "C. Maintain each solution at
115 "C for 15 min, pressurise for 1 min and transfer
onto the column.
Calculate the content (per cent m/m)of methanol
taking its density to be 0.792 g per millilitre at
20 "C.
Formaldehyde Dissolve 0.200 g in 4.0 ml of water.
Add 0.4 ml of dilute sodium hydroxide solution R.
Centrifuge, filter the supernatant liquid through cotton
previously impregnated with water and dilute to
5.0 ml with water. Transfer into a test-tube. Add
5.0 ml of acetylacetone reagent R1. Place the test-
tube in a water-bath at 40 "C for 40 min. The test
solution is not more intensely coloured than a stan-
dard prepared at the same time and in the same
manner using 5.0 ml of a 0.8 ppm solution of
formaldehyde (CH20), obtained by dilution of
formaldehyde standard solution (5 ppm CH20) R in
water (20 ppm). Examine the tubes down their ver-
tical axis.
Heavy metals (V.3.2.8) Dissolve 1.0 g in an 85 per
cent Vflsolution of methanol R and dilute to 20.0 ml
with the same solvent. 1 2 ml of the solution com-
plies with limit test B for heavy metals (20 ppm).
Prepare the standard using 10 ml of lead standard
solution (1ppm Pb) R.
Water (V.3.5.6) Not more than 1.0 per cent,
determined on 0 . 5 0 0 g by the semi-micro
determination of water.
Sulphated ash (V.3.2.14) Not more than 0.1 per
cent, determined on 1.0 g.
ASSAY
Dissolve 0.150 g in 15 ml of anhydrous acetic
acid R. Add 35 ml of acetic anhydride R. Titrate
with 0.1N perchloric acid, determining the end-
point potentiometrically (V.6.14). Disregard the
inflection point at the beginning of titration. Carry
out a blank titration.
1 ml of 0.1N perchloric acid is equivalent to 30.02
mg of Cl,Hl,C12NS.
The impurities limited by the requirements of this
monograph include:
2-chlorobenzylamine hydrochloride,
bis-ticlopidine dihydrochloride.
O PHARMEUROPA Vol. 6, N' 1 , March 1994

ViI.l.l. REAGENTS
i
Methyleneglycol.-C4H,003 (Mr 106.1). -(2-Hydroxyethoxy)-
ethanol. Contains not less than 99.5 per ce t m/m of C,HlOO3.
A colourless liquid, hygroscopic, miscible wi water and alcohol,
practically insoluble in carbone tetrachlori e.
bp : 244 OC to 246 OC.
d:': 1.115 to 1.119.
(1)Poropak Q column tias been found to be suitable.
FRANGULAE EXTRACTU
NORMATUM
Standardized Frangula Bark
Standardized frangula bark dry extr ct contains not
less than 20.0 per cent and not mo e than 30.0per
cen of glucofrangulins,calculated as lucofrangulin A
(M,578.5)and with reference to th dried drug. The
content is to be labelled. The measu ed content does
not deviate from the labelled value y more than f
5 per cent.
The extract is produced from dried and cut bark of
Rhamnus frangula L. (Frangula a nus Miller) and
ethanol 80 per cent V/V with n appropriate
procedure according to the mon graph Extracts.
The ratio between drug and extract is 5-6:lfor the
extract before adjustment. The inert ateria1 used for
the adjustment does not exceed 30 per cent in the
standardized extract.
CHARACTERS
The extract is a yellowish-brown
faintly aromatic, characteristic
in cold water, soluble in 80 per
slightly soluble in ether and
IDENTIFICATION
A. Examine the chromatogram obt
Other species of Rhamnus,
chromatogram obtained with t
shows the zones of the glucofran
and frangulaernodine.
B. To 25 mg of the extract add
hydrochloric acid R and heat t
O PHARMEUROPA Vol. 6, Ne 1, Madch 1994
Frangulae Extractum Siccum Normatum
1 water-bath for 15 min. Allow to cool, shake with
20 ml of ether R and discard the aqueous layer.
Shake the ether layer with 10 ml of dilute
ammonia R1. The aqueous layer becomes
reddish-purple.
TESTS
Other species of Rhamnus, anthrones Exa-
mine by thin-layer chromatography (V.6.20.2),
using
silica gel G R as the coating substance.
~
Test solution To 0.05g of the extract, add 5 ml of
alcohol (70 per cent V/v) and heat to boiling. Cool
and centrifuge. Decant the supernatant solution
immediately and use within 30 min.
Reference solution Dissolve 20 mg of aloin R in
alcohol (70 per cent V , and dilute to 10 ml with
the same solvent.
Apply separately to the plate, as bands 20 mm by not
more than 3 mm, 10 yl of each solution. Develop
over a path of 10 cm using a mixture of 13 volumes
of water, 17 volumes of methanol R and 100 volu-
dry Extract mes of ethyl acetate R. Allow the plate to dry for
5 min, spray with a 5 per cent m/V solution of
potassium hydroxide R in alcohol (50 per cent V ,
i
and heat at 100 "C to 105 "C for 15 min. Examine
immediately after heating. The chromatogram
obtained with the reference solution shows a reddish-
brown zone in the median third corresponding to
aloin. The chromatogram of the test solution shows
two orange-brown zones (glucofrangulins)in the lower
third and two to four red zones (frangulins, not
always clearly separated, and above them frangul-
i
aemodine) in the upper third. Examined in ultraviolet
light at 365 nm, the chromatogram obtained with
the test solution does not show zones of intense
yellow or blue fluorescence (other species of
Rhamnus).
Apply to another plate as a band 20 mm by not more
than 3 mm, 10 p1 of the test solution and develop as
described above. Allow the plate to dry for not longer
than 5 min and spray immediately with a 0.5 per
I cent m/V solution of nitrotetrazolium blue R in
methanol R. Examine the chromatogram immedi-
ately. No violet or greyish-blue zones appear.
Loss on drying Not more than 5 per cent. The test
is carried out as described for dry extracts in the
monograph Extracts.
I Microbial contamination Total count of viable
aerobic organisms (V.2.1.8.1, V.2.1.8.2)not more
i
ined in the test than lo1 per gram extract determined by colony
nthrones. The counting on agar-plates; not more than 10' yeasts
e test solution and fungi, no Salmonella, no Escherichia coli.
ulins, frangulins
ASSAY
Carry out the assay protected from bright light.
I
19
Frangulae Extractum Siccum Normatum

In a round-bottomed flask with a ground-glass neck, Calculate the percentage content of glucofrangulin A
weigh 0.100 g of the drug. Add 25.0 ml of methanol from the expression:
(70 per cent V/V), mix and weigh again. Immerse the
flask in a water-bath and heat under a reflux conden- A x 306
ser at 70" C for 15 min. Allow to cool, weigh and m
adjust to the original mass with methanol (70 per
cent V/v. Filter and transfer 5.0 ml of the filtrate to i.e. taking the specific absorbance of glucofrangulin
a separating funnel. Add 50 ml of water and 0.1 ml A to be 204, calculated on the basis of the specific
of hydrochloric acid R. Shake with 5 quantities, each absorbance of barbaloin,
of 20 ml, of light petroleum R1. Allow the layers to
separate and transfer the aqueous layer to a 100 ml A = absorbance at 515 nm,
volumetric flask. Combine the petroleum layers and
wash with 2 quantities, each of 15 ml, of water. Use m = mass of the substance to be examined in grams.
this water for washing the separating funnel and add
it to the aqueous solution in the volumetric flask. Add
5 ml of a 5 per cent flsolution of sodium carbo- STORAGE
nate R and dilute to 100 ml with water. Discard the
petroleum layer. Transfer 40.0 ml of the aqueous Store in an air-tight container, protected from light
solution to a 200 ml round-bottomed flask with a and moisture.
ground-glass neck. Add 20 ml of a 20 per cent m/V
solution of ferric chloride R and heat under a reflux
condenser for 20 min in a boiling water-bath with LABELLING
the water level above that of the liquid in the flask.
Add 3 ml of hydrochloric acid R and continue heating The drug is labelled as indicated for dry extracts in
for 40 min, shaking frequently, until the precipitate the monograph Extracts.
is dissolved. Allow to cool, transfer the mixture to a
separating funnel and shake with three quantities,
each of 2 5 ml, of ether R, previously used to rinse
VII. 1.1.REAGENTS
the flask. Combine the ether extracts and wash with
two quantities, each of 15 ml, of water. Transfer the
ether extracts to a volumetric flask and dilute to
100.0 ml with ether R. Evaporate 20.0 ml carefully Nitrotetrazolium blue - C,oH30C,,N,006(M~818~
to dryness and dissolve the residue in 10.0 ml of a 3,3'-(3,3'-Dimethoxybiphenyl-4,4'-diyl)bis[2-(4-nitrophenyl)-5-
0.5 per cent m/V solution of magnesium acetate R phenyl-2H-tetrazolium] dichloride.
in methanol R. Measure the absorbance (V.6.19) at
515 nm using methanol R as the compensation Crystals, soluble in methanol, giving a clear, yellow solution.
liquid. mp: about 189"C, with decomposition.

20 O PHARMEUROPA Vol. 6, Ne 1 , March 1994

 I Bumetanidum
ADAPTATION IOF NATIONAL MONOGRAPHS
During the last few years, the
European Pharmacopoeia has f
on ways to speed up the
monographs. Since n
European countries adhering
the Elaboration of a European
the same methodology, it was t
be possible to transfer some mo
national pharmacopoeias to the
copoeia.
A working party (consisting
number of Expert Groups)
approach by selecting a num
which monographs were requir
for which no draft document had
The following procedure has be
1. Modern monographs of
from national pharmac
by the working party.
are considered adequate and re
amendment will be chosen for
in the European Phar
party needs more i
will then request th
copoeia authority to supply
study performed for the
monograph.
A number of such
I Bumetanide contains not less than 99.0 per cent and
PA/PH/GNM (93)80 eNp
I calculated with reference to the dried substance.
NOTE ON THE MONOGRA ’H
BUMETANIDUM
This monograph has been adapted,
Pharmacopoeia (FU) taking in tc
monographs of the BP (93) ana
complete the series of identificatioi
to introduce a TLC test replacir
point which is now referred under C
Under TESTS an “appearance of
“heavy metals” have been introdu
BUMETANI DUM
Bumetanide
O PHARMEUROPA Vol. 6, NP1, Mar h 1994
2. The Secretariat will rewrite the monographs in
the editorial style of the Ph.Eur. and samples of
the substance will be obtained and checked
against the monograph by the Ph.Eur. Laboratory.
3. The text and the findings of the laboratory will
be considered by the working party and should
no difficulties be encountered, the monograph
will be published in Pharmeuropa and submitted
to the national pharmacopoeia authorities for
comment.
4. The working party will examine the comments
received from the national pharmacopoeia
authorities and in the light of the comments
decide to:
- send the monograph to the Commission for adop-
tion (no objections by the NPAs);
- send the monograph to the Commission for adop-
tion but with a recommendation for a future
revision of the monograph: for example, where
an improvement in methodology or harmonisa-
tion with an existing monograph may be thought
desirable;
report of any - send the monograph to a competent expert group
for study. This would be the case where there is
a fundamental objection to the monograph.
completed Stage 3 are published below.
upon in the usual way.
not more than the equivalent of 101.5per cent of
3-butylamino-4-phenoxy-5-sulphamoylbenzoicacid,
CHARACTERS
‘om the Italian
account the A white or almost white, crystalline powder,
the USP. To practically insoluble in water, soluble in acetone and
it is proposed in alcohol, slightly soluble in methylene chloride. It
the melting dissolves in solutions of alkali hydroxides. It exhibits
+ARACTERS. polymorphism.
solution” and
ed. It melts at about 233 OC.
IDENTIFICATION
identification test B may be omitted i f identifica-
tion tests A, C, and D are carried out. Identifica-
tion tests A, C and D may be omitted i f identifi-
cation test B is carried out.
COOH A. Dissolve 0.10 g in a 0.4 per cent m/V solution
of sodium hydroxide and dilute to 100 ml with
the same alkaline solution. Dilute 1 ml of the
solution to 100 ml with a 0.4 per cent m/V
solution of sodium hydroxide. Examined between
M, 364.4 210 nm and 350 nm (V.6.19), the solution shows
~~
21
Cyclizini Hydrochloridum

three absorption maxima, at 224 nm, 259 nm Heavy metals (V.3.2.8) 1.0 g complies with limit
and 320 nm. The ratio of the absorbance test C for heavy metals (20 ppm). Prepare the stan-
measured at the maximum at 224 nm to that dard using 2 ml of lead standard solution
measured at the maximum at 259 nm is 2.7 (10 ppm Pb) R.
to 3.0.
Loss on drying (V.6.22) Not more than 0.5 per
B. Examine by infrared absorption spectro- cent, determined on 1.000 g by drying in an oven
photometry (V.6.18). The absorption maxima in at 100 "C to 105 OC for 4 h.
the spectrum obtained with the substance to be
examined correspond in position and relative Sulphated ash (V.3.2.14) Not more than 0.1 per
intensity to those in the spectrum obtained with cent, determined on 1.0 g.
bumetanide CRS. If the spectra obtained shows
differences,dissolve the substance to be examined
and the reference substance separately in ASSAY
acetone R, evaporate to dryness and record new
spectra using the residues. Dissolve 0.300g in 50 ml of alcohol R. Add 0.1 ml
of phenol red solution R. Titrate with 0.1N sodium
C. Examine the chromatograms obtained in the test hydroxide.
for related substances. The principal band in the
chromatogram obtained with test solution (b) is 1 ml of 0.1N sodium hydroxide is equivalent to
similar in position and size to the principal band 36.44mg of C1,HzoNzO,S.
in the chromatogram obtained with reference
solution (a).
STORAGE
D. To about 20 mg add 0.1g of anhydrous sodium
carbonate R. Add 0.1 ml of water and heat. Store in a well-closed container, protected from light.
Vapours of ammonia are evolved, which turn red
litmus paper R blue. ~~ ~

The impurities limited by the requirements of this

TESTS monograph include:
acid
3-nitro-4-phenoxy-5-sulfamoylbenzoic
Appearance of solution Dissolve 50 mg in 10 ml
of a 0.6 per cent m/V solution of potassium
3-amino-4-phenoxy-5-sulfamoylbenzoicacid
hydroxide. The solution is clear (V.6.1)
and colourless butyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-
(Method II, V.6.2). benzoate
3-(2-ethylhexylamino)-4-phenoxy-5-sulfamoyl-
Related substances Prepare the solutions benzoic acid
immediately before use. Examine by thin-layer
chromatography (V.6.20.2),using silica gel G F R ~ ~ ~
as the coating substance.
Test solution (a) Dissolve 0.10 g of the substance
to be examined in methanol R and dilute to 10 ml
with the same solvent.
Test solution (b)Dilute 1 ml of test solution (a) to
10 ml with methanol R.
Reference solution (a) Dissolve 10 mg of bumetanide
CRS in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (b) Dilute 1ml of test solution (b)
to 20 ml with methanol R.
Apply separately to the plates as bands 10 mm by
2 mm 10 p1 of each solution. Develop over a path of
15 cm using a mixture of 10 volumes of concentrated
ammonia R, 30 volumes of methanol R and 60 vo-
lumes of methylene chloride R.Allow the plate to dry
in air and examine in ultraviolet light at 254 nm. Any
band in the chromatogram obtained with test solu-
tion (a), apart from the principal band, is not more
intense than the band in the chromatogram obtained
with reference solution (b) (0.5per cent).

22 O PHARMEUROPA Vol. 6, Ne 1, March 1994

 Cyclizini Hydrochloridum

measured at the maximum at 258 nm is 1.0 to

1.1. Dilute 10.0 mi of solution A to 100.0 ml
with a 0.5 per cent f l solution of sulphuric
acid R (solution B). Examined between 210 nm
and 240 nm, solution B shows an absorption
maximum at 225 nm. The specific absorbance
at the maximum is 370 to 410.
B. Examine by infrared absorption spectro-
photometry (V.6.18). The absorption maxima in
the spectrum obtained with the substance to be
examined correspond in position and relative
intensity to those in the spectrum obtained with
cyclizine hydrochloride CRS. Examine the subs-
tances prepared as discs.
C. Examine the chromatograms obtained in the test
for related substances. The principal spot in the
chromatogram obtained with test solution (b) is
similar in position, colour and size to the princi-
CYCLIZINI HYDROCHLbRIDUM pal spot in the chromatogram obtained with
reference solution (a).
Cyclizine Hydrochlhride D. Dissolve 0.5 g in 10 ml of alcohol (60 per cent
V/v)using heat, if necessary. Cool in iced water.
Add 1ml of dilute sodium hydroxide solution R
and 10 ml of water. Filter, wash the precipitate
with water and dry it in an oven at 60 "C at a
pressure not exceeding 0.7 kPa for 2 hours. The
melting point (V.6.11.1) is 105 "C to 108 "C.
E. It gives reaction (a) of chlorides (V.3.1.1).

TESTS

Cyclizine hydrochloride
I 302.8 pH (V.6.3.1) Dissolve 0.5 g in a mixture of 40 vo-
lumes of alcohol R and 60 volumes of water and
98.0 per cent and not dilute to 25 ml with the same mixture of solvents.
101.0 per cent of The pH of the solution is 4.5 to 5.5.
piperazine
Related substances Prepare the solu tions
immediately before use. Examine by thin-layer
chromatography (V.6.20.2) using silica gel G R as
CHARACTERS I the coating substance.
Test solution (a) Dissolve 0.10 g of the substance
A white, crystalline powder, slightly soluble in water
to be examined in methanol R and dilute to 10 ml
and in alcohol, practically insoluble in ether.
with the same solvent.
Test solution (b) Dilute 1 ml of test solution (a) to
IDENTIFICATION I 10 ml with methanol R.
identification tests A, C and D Reference solution (a) Dissolve 10 mg of cyclizine
identification tests i3 and E are hydrochloride CRS in methanol R and dilute to 10 ml
tification test U may be with the same solvent.
tests A, C, D and E are Reference solution (b) Dissolve 25 mg of
methylpiperazine R in methanol R and dilute to
A. Dissolve 20.0 mg in a 50 ml with the same solvent. Dilute 1 ml to 10 ml
with methanol R.
Reference solution (c) Dilute 1ml of test solution (b)
to 20 ml with methanol R.
Reference solution (d) Dissolve 10 mg of
hydroxyzine hydrochloride CRS and 10 mg of
I

O PHARMEUROPA Vol. 6, NQ 1, Madch 1994 23

Tolnaftatum
cyclizine hydrochlorideCRS in methanol R and dilute
to 10 ml with the same solvent.
Apply separately to the plate 20 p1 of each solution.
Develop over a path of 15 cm using a mixture of
2 volumes of concentrated ammonia R, 8 volumes of
methanol R and 90 volumes of methylene chloride R.
Allow the plate to dry in air for 30 min. Expose the
plate to iodine vapour for 10 min. In the
chromatogram obtained with test solution (a): any
spot corresponding to methylpiperazine is not more
intense than the spot in the chromatogram obtained
with reference solution (b) (0.5 per cent); any spot,
apart from the principal spot and any spot
corresponding to methylpiperazine, is not more in-
tense than the principal spot in the chromatogram
obtained with reference solution (c) (0.5 per cent).
The test is not valid unless the chromatogram obtained
with reference solution (d)shows two clearly separated
principal spots.
Loss on drying (V.6.22) Not more than 1.0 per
cent, determined on 1.O00 g by drying in an oven at
130 OC.
Sulphated ash (V.3.2.14) Not more than 0.1 per
cent, determined on 1.0 g.
ASSAY
Dissolve 0.200 g in 15 ml of anhydrous formic
acid R and add 40 ml of acetic anhydride R. Carry
out a potentiometric titration (V.6.14) using 0.1N
perchloric acid.
1ml of 0.1N perchloric acid is equivalent to 15.14 mg
of C,,H,,ClN,.
STORAGE
Store in a well-closed container, protected from light.
The impurities limited by the requirements of this
monograph include:
methylpiperazine.
24
'R.
PA/FW/ANM (93) 72 ANP
NOTE ON THE MONOGRAPH TOLNAFTATUM
This monograph has been adapted from the BP
(93) taking into account the monographs of the
DAC and USP. I t is proposed to have two series of
identity tests. For the assay the giuen specific
absorbance values differ. Examination of the
batches at our disposal would support the value
of 720.
TOLNAFTATUM
Tolnaftate
M, 307.4
C19HlFS
Tolnaftate contains not less than 97.0 per cent and
not more than the equivalent of 103.0 per cent of
û-(Z-naphthyl) N-methyl-N-(3-methyIphenyl)thio-
carbamate, calculated with reference to the dried
substance.
CHARACTERS
A white or yellowish-white powder, practically inso-
luble in water, freely soluble in acetone and in
methylene chloride, sparingly soluble in ether, very
slightly soluble in alcohol.
It exhibits polymorphism.
IDENTIFICATION
identification test B may be omitted if identifica-
tion tests A, C and D are carried out. identifica-
tion tests A, C and D may be omitted if identifi-
cation test B is carried out.
A. Melting point (V.6.11.1): 109 OC to 112 OC.
B. Examine by infrared absorption spectro-
photometry (V.6.18). The absorption maxima in
the spectrum obtained with the substance to be
examined correspond in position and relative
intensity to those in the spectrum obtained with
tolnaftate CRS. Examine the substances prepared
as discs. If the spectra obtained show differences,
O PHARMEUROPA Vol. 6, N" 1, March 1994
 I Selenii Sulfic

Sulphated ash (V.3.2.14) Not more than 0.1 per

cent, determined on 1.0 g.
evaporate to
using the residues.
ASSAY
C. Examine the chromatograms
for related substances. The Dissolve 50.0 mg in methanol R and dilute to 250.0
chromatogram obtained ml with the same solvent. Dilute 2.0 ml to 100.0 ml
similar in position and with methanol R. Measure the absorbance (V.6.19)
in the chromatogram at the maximum at 257 nm. Calculate the content of
solution (a). C H NOS taking the specific absorbance to be
7%. l7

f
D. Mix about 1mg with 0.5 ml of ulphuric acid R.
Add 0.05 rnl of formaldehyd solution R. A
greenish-blue colour develops. STORAGE

Store in a well-closed container, protected from light.

TESTS I
Appearance of solution Dissolve
acetone R. The solution is clear The impurities limited by the requirements of this
intensely coloured than monograph include:
II, V.6.2). P-naphthol (or 2-nathphol),
Related substances O,Odi(2-naphthyl) thiocarbonate,
O(2-naphthyl) chlorothioformate,
methyl(3-methylpheny1)amine.

the same solvent. PA/PH/ANM (93) 73 ANP

Test solution (b) Dilute 0.5 ml *..I.
. L I

10 ml with acetone R.
Reference solution (a) Dissolv
NOTE ON THE MONOGRAPH SELEN11 SULFIS
This monograph has been adapted from the Bri-
Reference solution (b)Dilut tish Pharmacopoeia (BP 1993).
to 10 ml with acetone R.
R e f e r e n c e solution (c) SELENII SULFIS
P-naphthol R in 1ml of test
10 ml with acetone R.
Selenium sulphide
Apply separately to the plate 5 pl
Develop over a path of 12 cm using

ses2 M, 143.1
is not more intense tha Selenium sulphide contains not less than 52.0 per
chromatogram obtained cent and not more than 55.0 per cent of Se.
(0.5 per cent). The t
chromatogram obtaine
shows two clearly sep CHARACTERS
Chlorides (V.3.2.4) To 1.0 g a A bright orange to reddish-brown powder, practically
and boil for 5 min. Cool and filter. insoluble in water.
dilute the combined
with water. The solu
for chlorides (50 ppm). IDENTIFICATION

A. Gently boil about 50 mg with 5 ml of nitric

acid R for 30 min. Dilute to 50 ml with water and
filter. To 5 ml of the filtrate add 10 ml of water
I

O PHARMEUROPA Vol. 6, N" 1, Marbh 1994 25

Nitrofuralum
and 5 g of urea R. Heat to boiling, cool and add
1.5ml of potassium iodide solution R. A yellow
to orange colour is produced which darkens
rapidly Ön standing. This solution is used in the
identification test B.
B. Allow the coloured solution to stand for 10 min
and filter through kieselguhr H R. 5 ml of the
filtrate give reaction (a) of sulphates (V.3.1.1).
TESTS
Soluble selenium compounds To 10 g add
100 ml of water, mix well, allow to stand for 1h with
frequent shaking and filter. To 10 ml of the filtrate
add 2 ml of an 11.5 per cent m/V solution of
anhydrous formic acid R, dilute to 50 ml with water
and adjust the pH between 2.0 and 3.0 with a
11.5 per cent m/V solution of anhydrous formic
acid R. Add 2 ml of a 0.5 per cent m/V solution of
3,3'-diaminobenzidine tetrahydrochloride R. Allow
to stand for 45 min and then adjust the pH between
6.0 and 7.0 with dilute ammonia R1. Shake the
solution for 1min with 10 ml of toluene R and allow
the phases to separate. The absorbance (V.6.19) of
the upper layer measured at 420 nm is not greater
than that of a standard prepared at the same time
and in the same manner using 5 ml of selenium
standard solution (1ppm Se)R (5ppm, calculated as
Se).
ASSAY
To 0.100 g add 25 ml of fuming nitric acid R, heat
on a water-bath for 1 h, cool and dilute to 100.0 ml
with water. To 25.0 ml add 50 ml of water and 5 g
of urea R and heat to boiling. Cool, add 7 ml of
potassium iodide solution R, 3 ml of starch solution R
and titrate immediately with 0.1N sodium thio-
sulphate. Carry out a blank titration.
1 ml of 0.1N sodium thiosulphate is equivalent to
1.974 mg of Se.
STORAGE
Store in a well-closed container.
VII.l.l. REAGENTS
3-3'-Diaminobenzidine tetrahydrochloride.-
C,,H &I4N4, 2H,O (Mr396.2).
An almost white or slightly pink powder, soluble in water.
Selenious acid.- H,Se03 (M,129.0).Selenous acid; selenic
(iv) acid.
Deliquescent crystals, freely soluble in water.
26
v11.1.2.STANDARD SOLUTIONS FOR LIMIT TESTS
Selenium standard solution (1 ppm Se). Immediately before
use, dilute with water to 40 times its volume a solution containing
selenious acid R equivalent to 0.00654g of H,SeO, in 100.0 ml.
NOTE ON THE MONOGRAPH ON
NITROFURALUM
This monograph has been adapted from that of
the British Pharmacapoeia (BP 93), taking into
account that of the Italian Pharmacopoeia (FU).
The identification tests have been extended so
that there is a simple alternative identification
series, which includes an UV absorbance ratio test
and a thin-layer chromatographic test. Published
as an annex is a thin-layer test to limit the content
of semicarbazide to a level of 0.2 per cent. Also
published as an annex is a liquid chromatographic
method for the control of related substances which
is considered to be an improvement to the TLC
method published in t h e text itself. At the
wavelength of detection chosen (31O nm), which is
not the wavelength of maximum absorption of
nit rof ura I, the impurities, nit rof u rf urylidene azine
and nitrofurfural diacetate, absorb to a similar
extent but more strongly than nitrofural. This
difference is taken into account when setting the
limits.
NITROFURALUM
Nitrofu ral
O
02N*CH= Il
N-NH-C-NH2
M, 198.1
Nitrofural contains not less than 97.0 per cent and
not more than the equivalent of 103.0 per cent of
5-nitro-2-furaldehyde semi-carbazone, calculated with
reference to the dried substance.
O PHARMEUROPA Vol. 6, NQ1, March 1994
 I Nitrofuralum

CHARACTERS I Related substances Examine by thin-layer

chromatography (V.6.20.2), using silica gel G R as
A yellow to brownish-yellow, crystal1 ne powder, very the coating substance.
slightly soluble in water, slightly souble in alcohol,
practically insoluble in ether. Test solution Dissolve 0.10 g of the substance to be
examined in a mixture of equal volumes of acetone R
and dimethylformamide R and dilute to 10 ml with
IDENTIFICATION I the same mixture of solvents.

r
identification test B may be omitt d if identifica-
tion tests A, C and D are carried out. identifica-
tion tests A, C and D may be om tted if identifi-
cation test B is carried out.
Reference solution (a) Dissolve 2.0 mg of impurity
A CRS (5-nitrofurfurylidene azine) in a mixture of
equal volumes of acetone R and dimethylformamideR
and dilute to 100 ml with the same mixture of
solvents.

i
A. Carry out the test protected fr m bright light. Reference solution (b) Dissolve 5.0 mg of impurity
Use the solution prepared for the ssay. Examined B CRS (nitrofurfural diacetate) in a mixture of equal
between 220 nm and 400 n (V.6.19), the volumes of acetone R and dimethylformamide R and
solution shows two absorption axima, at 260 dilute to 50 ml with the same mixture of solvents.
nm and 375 nm. The ratio of the absorbance
measured ai: the maximum at 7 5 nm to that Apply separately to the plate 10 pl of each solution.
measured at the maximum at 2 O nm is 1.15 to Develop over a path of 15 cm using a mixture of
5 volumes of dioxan R and 95 volumes of toluene R.
1.30. Dry the plate at 100 "C to 105 "C for 5 min and
spray with phenylhydrazine hydrochloride solution R.
B. Examine by infrared absor In the chromatogram obtained with the test solution:
photometry (V.6.18). The absor
the spectrum obtained with the any spot corresponding to impurity A is not more
examined correspond in intense than the spot in the chromatogram obtained
intensity to those in the with reference solution (a) (0.2 per cent); any spot
corresponding to impurity B is not more intense than
nitrofural CRS. Examine
the spot in the chromatogram obtained with reference
as discs.
solution (b) (1.0 per cent).
C. Examine by thin-layer chromatography
Loss on drying (V.6.22) Not more than 0.5 per
(V.6.20.2), iising silica gel G R as the coating
cent, determined on 1.000 g by drying in an oven at
substance.
100 "C to 105 OC.
Test solution Dissolve 10 mg of the substance
to be examined in methanol €3 and dilute to Sulphated ash (V.3.2.14) Not more than 0.1 per
10 ml with the same solvent. cent, determined on 1.0 g.
Reference solution Dissolve 10 mg of nitrofural
CRS in methanol R and dilute to 10 ml with the ASSAY
same solvent
Apply separately to the plate 5 1 1 of each solu- Carry out the assay protected from bright light
tion. Develop over a path of 15 cm using a Dissolve 60.0 mg in 20 ml of dimethylformamide R
mixture of 10 volumes of metl-anol R and 90 and dilute to 500.0 ml with water. Dilute 5.0 ml to
volumes of nitromethane R. AllDw the plate to 100.0ml with water. Measure the absorbance (V.6.19)
dry in air and spray with pienylhydrazine at the maximum at 375 nm. Calculate the content of
hydrochloride solution R. The principal spot in C,H,N,O, taking the specific absorbance to be 822.
the chromatogram obtained wit I the test solu-
tion is similar in position, colour and size to the
principal spot in the chromat gram obtained STORAGE
with the reference solution.
Store in a well-closed container, protected from light.
D. Dissolve about 1 mg in 1 ml
amide R and add 0.1 ml of
hydroxide solution R. A red

TESTS
monograph include:
pH (V.6.3.1) To 1g add 100 ml of
free water R. Shake and filter. The p nitrofurfurylidene azine,
is 5.0 to 7.0. nitrofurfural diacetate.

O PHARMEUROPA Vol. 6, N" 1, Markh 1994 27

Nitrofuralum
APPENDIX
Related substances Examine by liquid chromato-
graphy (V.6.20.4).
Test solution Dissolve 0.10 g of the substance to be
examined in the mobile phase and dilute to 100.0 ml
with the mobile phase.
Reference solution Dilute 1.0 ml of the test solution
to 200.0 ml with the mobile phase.
The chromatographic procedure may be carried out
using:
- a stainless steel column 0.25 m long and 4.6 mm
in diameter pack with octadecylsilyl silica gel for
chromatography R (5 yrn)(l),
- as mobile phase at a flow rate of 1ml per minute
a mixture of 50 volumes of water and 50 volu-
mes of acetonitrile R,
- as detector a spectrophotometer set at 310 nm.
Inject 20 yl of the reference solution. When using a
recorder, adjust the sensitivity of the system so that
the height of the principal peak in the chromatogram
obtained is not less than 50 per cent of the full scale
of the recorder. Inject 20 y1 of the test solution.
Continue the chromatography for four times the
retention time of nitrofural, which is about 2.5 min.
In the chromatogram obtained with the test solution:
the area of any peak (corresponding to impurity A)
with a relative retention time of 3.0 in respect with
the principal peak, is not greater than the area of the
principal peak in the chromatogram obtained with
the reference solution (0.2 per cent); the area of any
peak, apart from the principal peak and any peak
corresponding to impurity A, is not greater than 2.5
times the area of the principal peak in the
chromatogram obtained with the reference solution
(0.5 per cent).
Disregard any peak with an area less than 0.05
times that of the principal peak in the chromatogram
obtained with the reference solution.
28
Semicarbazide Examine by thin-layer chromato-
graphy (V.6.20.2), using silica gel G R as the coating
substance.
Test solution Dissolve 0.20 g of the substance to be
examined in a mixture of equal volumes of acetone
R and dimethylformamide R and dilute to 10 ml with
the same mixture of solvents.
Reference solution Dissolve 10 mg of semicarbazide
hydrochloride R in a mixture of equal volumes of
acetone R and dimethylformamide R and dilute to
200 ml with the same mixture of solvents.
Apply separately to the plate 20 yl of each solution.
Develop over a path of 15 cm using a mixture of 1
volume of diethylamine R, 10 volumes of methanol
R and 90 volumes of nitromethane R. Dry the plate
at 100 "C to 105 OC for 5 min and spray first with
a saturated solution of sodium naphthoquinone-
sulphonate R in alcohol (50 per cent VW),then with
a 4.2 per cent m/V solution of sodium hydroxide R.
Any spot corresponding to semicarbazide in the
chromatogram obtained with the test solution, is not
more intense than the spot in the chromatogram
obtained with the reference solution (0.2 per cent).
ViI.1.1.REAGENTS
Semicarbazide hydrochloride.- CH,CIN,O (Mr 111.5).
A white, crystalline powder, freely soluble in water, insoluble in
ether.
mp: about 175 "C, with decomposition.
(1)Hypersil ODS column has been found to be suitable.
O PHARMEUROPA Vol. 6, NP1, March 1994
 ~
International Harmonisation - the first three years

International Harmonisation
Harmonisation of onographs and General Analytical Methods of
the United Japanese and European Pharmacopoeias

m
Review of t e First Three Years of Operation of the
Phar acopoeial Discussion Group(1)

Dr. A. ARTIGESU)
~ Dr. P.J. SCHORNW

The Pharmacopoeial Discussion For texts that have already been published and
informal group whose implemented, the “retrospective” harmonisation
procedure takes place in seven steps; for the
elaboration of new texts (“prospective”harmonisa-
United States, tion), notably in the area of biological products, the
first three steps have been adapted and condensed.

Step 1: Identification of the Pharmacopoeia

responsible for a subject selected for harmonisation
(monograph, general analytical method etc).
registration authorities.
The Pharmacopoeial Discussion Group distributes by
Its activity after the first seven consensus the work amongst the three pharma-
summarised as follows: copoeias; in general, one of the pharmacopoeias
- development of a procedure volunteers (lead pharmacopoeia) but at the same
- more than sixty items now time the Pharmacopoeial Discussion Group seeks a
balanced distribution of the subjects.
- comments on the
an example the Within the framework of “prospective” harmoni-
implemented on 1/1/1994. sation, the PDG selects the subjects. For each
subject, each pharmacopoeia designates a “con-
Each of these points will be tact” expert.
later on:

Step 2: Investigation - preparatory work for

I. PROCEDURE the responsible Pharmacopoeia

After a pilot programme in The pharmacopoeia responsible for a subject to be

procedure was identified*, at harmonised takes any measures it deems appropriate
lando in October 1993, to develop the harmonised draft. This entails
procedure in niore detail collecting all the information on the existing
changing its working specifications in the three pharmacopoeias on the
characteristics of grades of products on the world market and on the
(already published potential analytical methods that are available. For
pharmacopoeias) this, each pharmacopoeia operates according to its
monisation were own internal organisation.

(1) Director of the European Pharmacopoeia. (2)

i ecretay to the Commission.
‘Extracts of articles published in Pharmeuropa, Vol. 5, no 3 (Sept. 1993)and Vol. 5, no 4 (Dec. 1993).

O PHARMEUROPA Vol. 6, NQ 1, Madch 1994 29

International Harmonisation - the first three years

THE HARMONISATION PARTNERSHIP

-
Pharmacopoeial Responsible Forum of the other comments
itep Procedure Discussion Pharmacopoeia Responsible Forums from the
h u p Pharmacopoeia UserS
-
identification of the
1 responsible Decision
pharmacopoeia

Establishment
2 Investigation of a report and
a proposal

PropOSad To be sent to the

3 PropOsal monogmph Announcement Pharmacopoeia
+ comments responsible(')
-
Publication Pubiication To be sent to the
4 of the of the national
monograph monograph
-

Search for consensus

5 consensus and proposai for a
date for common
implementation

END OF WORK BY THE PHARMACOPOEIAL DISCUSSION GROUP

-
Ph.Eur. JP USP

6 Adoption European Pharmacopoeia Japanese Executive Committee

Commission Pharmacopoeia Commission of Revision

7 Date of
implementation
Proposed by the
European Pharmacopoeia
Commission
Ministry of Health
Publiihed in a
Ydopted by the Public Health
anrl supplement of the USP
Committee (Resolution) Welfare

Implementation in each
member state

(1) If this is the European Pharmacopoeia send to the Director of the Ph. Eur
(2)In Europe, send to the Secretariat of each natiod pharmacopoeia (see addresses on last page of Pharineuropa).

30 O PHARMEUROPA Vol. 6, NP1, March 1994

 ~
International Harmonisation - the first three years
For example, within the European its entirety (sometimes after the corresponding
secretariats have added national information needed
for the understanding or implementation of the texts,
eg, the addition of the description of an analytical
method or of reagents that did not exist in this
procedure for pharmacopoeia, sometimes also necessitated by
translation from English into Japanese or from English
into French).
Information or proposals This type of monograph is published in the
leads to the establishment of a “Previews” section of Pharmacopeial Forum (if it
draft monograph or chapter. was originally published in the “Stimulito the Revision
Process” section; it appears in the “In-process
Within the framework of Revision” section if it was originally published in the
sation, the “contact” “Pharmacopeial Previews” section). The users send
informed on reciprocal their comments to their respective pharmacopoeias.
common draft monographs.
In Europe, as for all other monographs subjected to
Step 3: Proposal
This draft harmonised mon
I public survey, the comments should be sent within
four months to the national pharmacopoeia
authorities, which will send them to the European
by comments which explai Pharmacopoeia Secretariat.
test, method or limit propose
the secretariats of the three Readers on the other continents send their proposals
then presented to all intere to the responsible pharmacopoeia.
tion in the forum of the ph
Questions are sometimes a The pharmacopoeias examine the comments received
whose opinions are sought. and prepare, if necessary, a proposal for modifica-
tion of the monograph, with justification, which is
The responsible pharmac then sent to the responsible pharmacopoeia.
the three Pharmacopoeias
the other two pharmac Step 5: Consensus
draft prepared by the responsible
however, they announce in their ow The responsible pharmacopoeia collects and exami-
responsible pharm nes all the comments and seeks to reach a consensus
and all the readers with its two partners on the text, preferably by
send their comments correspondence, but if necessary by organising a
copoeia. special meeting of experts.

In Europe, the comments at this A common date for implementation is proposed.

survey are to be sent within 4
European Pharmacopoeia Within the framework of “prospective” harmon-
and for certain countries ization, the “contact” experts try to reach a con-
national secretariat. sensus. if necessary, a n open conference is
organised.
Within the framework of
sation, the draft In this step, the Pharmacopoeial Discussion Group
published in the ends its work, of scientific and technical collabora-
send their tion by the experts of the three pharma-copoeias.
forum. The search for consensus has been pushed as far as
possible. Nevertheless, the specifications in the
pharmacopoeias are also legally binding texts which
Step 4: Official public survey fit into a special administrative and regulatory context.
Hence, the last two steps of the implementation of
The responsible pharmacopoeia
the “harmonised”monographs take place individually
the information received. If according to the procedures established by each
hold an open session to
pharmacopoeia organisation.
partners and identify
problems. It prepares
will be subjected to Step 6: Adoption
The three pharmacopoeias The draft monographs are submitted to the organi-
the draft harmonised sations responsible for each pharmacopoeia. In the
responsible case of the European Pharmacopoeia, this is the

O PHARMEUROPA Vol. 6, N”1, M a r i h 1994 31

International Harmonisation - the first three years
European Pharmacopoeia Commission, which adopts
submitted texts by a unanimous vote of the national
delegations of the countries that are parties to the
Convention(’) .
If necessary, the monograph may be adopted with
some amendments corresponding to general policy
in the area. The monographs will therefore not be
totally harmonised, and this will only be a first step
towards completely identical texts.
Step 7: Date of implementation
In general, the three pharmacopoeia organisations
will try to choose the same date for implementation.
It seems that so far, the European Pharmacopoeia
and United States Pharmacopeia have legislation
that is sufficiently flexible in this respect (frequent
publication of supplements of the USP and possibility
in Europe of “rapid implementation”between publi-
cations of annual fascicules). The Japanese
Pharmacopoeia, which was initially published every
five years, now publishes supplements annually, but
does not yet have the possibility of intermediary
publication.
To ensure the best transparency for the work, the
Pharmacopoeias would like to identify the
monographs either by using a logo (USP proposal) or
by using a characteristic mark accompanied if
necessary, by a note explaining the residual
differences (study in progress at the Ph. Eur.).

(1) Twenty-one countries at present: Austria, Belgium, Cyprus, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,
Luxembourg, the Netherlands, Norway, Portugal, Slovenia, Spain, Sweden, Switzerland, United Kingdom and Turkey.

II. WORK IN PROGRESS

As regards the international harmonisation of general test methods, the following allocation has been made by
the PDG:
1 Organic volatile impurities (OVI): I USP/Ph. Eur.
~~~

Efficacy of antimicrobial preservation: Ph. Eur. (Status report in: Pharmeuropa 5.4)
Heavy metals: USP
Residue on ignition/Sulphated ash: JP
Test for sterility: Ph. Eur. (Status report in Pharmeuropa 5.4 and extended present-
ation in 6.1)
Inhalations: Ph. Eur. (a proposal for a revised version of the Ph. Eur. mono-
graph was published in Pharmeuropa 5.4)
Hygroscopicity: Ph. Eur. (Pharmeuropa 4.3)
Particle size/Surface area USP/Ph. Eur. (a proposal for a revised version of the Ph. Eur. test
was published in Pharmeuropa 5.2)
Dissolution testAlisintegration test: USP
Particulate matter: Ph. Eur: before proceeding to the harmonisation procedure, the
Ph. Eur. test (subvisible parts) is intended to be enlarged by includ-
ins also a test for visible parts.
~~ ~

Test for arsenic: Ph. Eur.

Analytical validation Ph. Eur. (in relation with ICH)
Flowabilit y: USP (published in Fascicule 17)
Bulk density/tapped density: Ph. Eur. (published in Fascicule 17 as “Apparent volume”)
~~~

Endotoxins/pyrogens : JP

32 O PHARMEUROPA Vol. 6, Ne 1, March 1994

 International Harmonisation - the first three years
As regards the International harm nisation of monographs, the progress on each subject on the work-
programme is summarised in the fc owing table.

-
Title of monograph Leat Group ol 1st Europear Ph. Eur. Foreign Step Decision
Bharm Experts Draft Forum

Lactose (monohydrate) USP 11 7 implementation:

1.1.94;
USP: suppl. 9;
Ph. Eur.Fasc.18
(AP-CSP (93) 31
Magnesium stearate USP 9 9/T (92) 69 3.4 'F 17 (5) 2419 4
'F 18 (4) 3591
'F 19 (4) 5755
IP Vol. 1, No 1
IP Vol. 1, No 3
Cellulose, rnicrocrystalline USP 11c 1 1 C P (93) 5 5.3 IP Vol. 2, No 4 4
'F 18 (2) 3194
'F 19 (4) 5596
Cellulose, powdered USP 11c 11C/T(93) 4 5.3 IP Vol. 2, No 4 4
'F 18 (2) 3194
'F 19 (4) 5598
Sucrose Ph. E 11 11/T (92) 5 4.4 IP Vol. 2, No 1 4
'F 19 (4) 5328
Polyvidone JP 11 11/T (93) 36 5.3 IP Vol. 1, No 1 4
IP Vol. 2, No 2
Methyl parahydroxybenzoate Ph. E 10A
5.4 4
Sodium chloride Ph. E 9
5.4 4
Citric acid Ph. E 11 5.2 3
Starch, potato Ph. E 13 5.2 >F19(5)6167 3
Starch, wheat Ph. E 13 5.2 'F19(5)6167 3
Calcium phosphate, dibasic JP 9 IP Vol. 2, No 3 3
Stearic acid Ph. E 13H 13H/T (92) 10 5.3 3
Calcium disodium edetate JP 9 IP Vol. 2, No 4 3
Ethylcellulose Ph. E 11c 11C/T (93) 6 5.4 3
Hydroxyethylcellulose Ph. E 11c 11c/T (93) 2 5.4 3
Polysorbate 80 Ph. E 11 11/T (93) 37 5.3 3
Cellulose acetate phthalate USP 11c 'F 18 (4) 3746 3
Hydroxypropylmethylcellulose JP 11c 'F 18 (4) 3746 3
Starch, maize USP 13 'F 19 (5)6167 2-3
Starch, rice JP 13 'F 19 (5)6167 2-3
Starch, tapioca USP 13 'F 19 (5)6167 2-3
Lactose (anhydrous) USP 11 11/T (93) 71 2
Water(s) USP 9 4- 12 >F 18 (6) 4388
'F 19 (5)6179
'F 19 (6) 6633 2
Benzyl alcohol Ph. E 11 11/T (92) 53 >F 15 (4) 5375 2
Silicon dioxide, colloidal JP 9 2
Talc Ph. E 9 9/T (92) 71 2
Titanium dioxide JP 9 2
Methylcellulose JP 11c 2
Alcohol(s) Ph. E ANM 2
Carboxymethylcellulose calcium USP 11c 2
Carboxymethylcellulose sodium USP 11c 2
Cellulose acetate USP 11c 2
Cellulose acetate butyrate USP 11c 2
Hydrochloric acid USP 9 2
Hydroxypropylcellulose USP 11c 2
Petrolatum USP ANM ANM (93)55-57 5.4 2
Polyethylene glycols USP 10A 2
Sodium starch glycolate USP 11 2
Saccharin sodium USP 1OA 2
Sodium Hydroxide Abandoned

O PHARMEUROPA Vol. 6, NP1, Mai h 1994 33

International Harmonisation - the first three years

Following the Interpharmacopoeial Open Conference mandatory, but for information

on Harmonisation of Standards for Biotechnology only.
Products on 20-22 April 1993 in Verona, Italy, the
PDG has selected the following monographs on IDENTIFICATION: The three identification tests
Biotech Products and test methods in decreasing are virtually the same. As in
order of priority for international harmonisation. In other cases of monographs of
most cases there will be a prospective harmonisation the Ph. Eur., an alternative
as compared with the retrospective harmonisation of series of identification is
the excipients. prescribed.

Monographs Methods TESTS

Insulin Peptide mapping Appearance of The requirements “not more

solution intensely coloured than refer-
Hepatitis B vaccine SDS-PAGE ence solution BY ” (Ph. Eur.)
t-PAs (alteplase and others) Amino acid analysis and “nearly coloriess” USP)
are considered to be equivalent.
Somatropin (HPLC; capillary
electrophoresis) Acidity or alkalinity: Experimental conditions and
Factor VIII criteria are identical; end point
Isoelectric focusing
in Ph. Eur: pink colour; in
Interferons USP: red.

Specific optical Identical.

rotation:

Absorbance: Identical, except that in USP

one part of the requirements
are quoted under “Clarity and
III. COMMENTS ON color of solution”, the other
THE FIRST HARMONISED part in a new chapter: “Protein
MONOGRAPH and light absorbing impurities”.
LACTOSUM MONOHYDRICUM
Heavy metals: Identical limit of 5 ppm.

A comparison of the harmonised monograph on Water: Identica1.

Lactose monohydrate, published in PF 19 (4) 5753
- 5754 (1993)and in Pharmeuropa Vol. 5, No. 3 Sulphated ash: This test is considered to be
241-242 (1993)shows that the texts are not identical equivalent to the residue on
but are reasonably well-harmonised. The following ignition test of USP: same
slight differences exist, as compared with the text limits.
which has been implemented by means of Resolution
A P K S P (93) 3 on 1January 1994 in Europe.

The harmonised monograph on Lactose monohydrate
will be implemented in Japan during 1994.
DEFINITION: The Ph. Eur. does not describe
the origin of the substance
whereas USP indicates that it
is obtained from milk.
The introduction of a Section
on Production, at present
discussed by the European
Pharmacopoeia Commission
and applying in principle to a
large number of monographs,
might by helpful in this context.
CHARACTERS: No such Section in USP. This
Section of the Ph. Eur. is not
Tests not introduced into the Ph. Eur.
Loss on drying: This test discriminates be-
tween lactose monohydrate
and a modified monohydrate
form.
These two qualities only differ
in their galenical properties:
such properties are at present
not controlled in the Ph. Eur.
and consequently this test was
not included.
Microbial In the Ph. Eur., the test for
contaminat ion: microbial contamination is not
included in the legally binding
parts of the monograph but as
a footnote.

34 O PHARMEUROPA Vol. 6, N” 1, March 1994

 International Harmonisation - the first three years
When comparing the footnote
in the Ph. Eur. monograph and
the test for microbial limits in
USP, the only difference exists
in the additional requirement
in USP for the total combined
molds and yeasts count (not
more than 50 per gram).

Organic volatile Since during production of

impurities lactose no organic solvents are
used, no such test is included
be reached.
in the Ph. Eur. It is the pol-
icy of the European Pharma-
copoeia Commission to des-
cribe specifications for active
ingredients and excipients in-
cluding impurities in relation
to the method of manufacture.

USP seems to prefer to include

systematically a test for organic
volatile impurities as a means
of verifying adequate trans-
portation and storage of bulk
criteria. material.

O PHARMEUROPA Vol. 6, N" 1, Madch 1994 35

International Harmonisation - Harmony in Sterility Tests

International Harmonisation - texts

Users are invited to submit their comments, in relation to the stage the monograph
has reached and in accordance wit the procedure outlined in the Table on page 30,
before 30 June 1994.

E3.3 Effectiveness of culture media in the

presence and absence of the product to be
PA/PH/SG (93) 139 ANP,R examined

E4 Test of sterility of the product to be examined

Step 3: Proposal E4.1 Membrane filtration
E4.1.1 Aqueous solutions
HARMONY IN STERILITY TESTS E4.1.2 Soluble solids
E4.1.3 Oils and oily solutions
Int roductory No te E4.1.4 Ointment and creams
Since publication of the status report ((Harmony E4.2 Direct inoculation of the culture medium
in Sterility Tests) in Pharmeuropa Vo. 5, No. 4 E4.2.1 Oily liquids
(December 1993), the text has been further E4.2.2 Ointments and creams
reviewed and completed. It consequently replaces
the previous text. E5 Observation and interpretation of results
Comments should be sent to the Secretariat of the E5.1 First retest
European Pharmacopoeia Commission in Stras- E5.2 Second retest
bourg before 30 June 1994.
E6 Application of the test to parenteralia (with table I)

E7 Application of the test to ophthalmic and other

A comparison of the sterility tests in
the European Pharmacopoeia (2nd ed.),
the Japanese Pharmacopoeia (XII) and
the United States Pharmacopeia (XXII)
non-injectable preparations required to comply
with the test for sterility (with table II)
E8 Application of the test to surgical dressings
E8.1 Test sample
E8.2 Inoculation of the culture media
E8.3 Elimination of antimicrobial properties
E8.4 Membrane filtration

1. DOCUMENTS REFERRED TO E9 Application of the test to catgut and other surgical

sutures
The individual paragraphs in the three texts have E9.1 Test sample
been coded and they are referred to in this document E9.2 Inoculation of culture media
as follows.
E9.3 Effectiveness of culture media in the
presence and absence of the test sample
E9.4 Incubation
E = European Pharmacopoeia, 2nd edition,
V.2.1.1. Sterility (1991)
NON-MANDATORY ANNEXES
E l Introduction

E2 Precautions against microbial contamination

VIII.3.1 Minimum number of items recommended
E3 Choice of culture media to be tested in relation to the number of
E3.1 Sterility items in the batch

E3.2 Nutritive properties VIII.3.2 Suggested culture media

36 O PHARMEUROPA Vol. 6, Ne 1, March 1994

 International Harmonisation - Harmony in Sterility Tests
J= Japanese Pharmacopoe I XII, Chapter U4 Growth Promotion Test (with table of suitable
45, Sterility Test microorganisms)
51 Introduction U 5 Bacteriostasis and Fungistasis
52 Media and their preparation U6 General Procedure
53 Growth promotion test U6.1 Opening containers
U6.2 Selection of test specimens and incubation
54 Direct transfer method (with table of quantities for liquid articles)
54.1 Preparation of sample so ition
U7 Test Procedures for Direct Transfer to Test Media
54.2 Opening of container
U7.1 Liquids
54.3 Choice of culture mediun
U7.2 Ointments and oils insoluble in isopropyl
54.4 Containers for test myristate
54.4.1 Bacteria U7.3 Solids
54.4.2 Fungi
U7.4 Purified cotton, gauze, surgical dressings,
54.5 Procedure sutures, and related articles
54.6 Culture procedure U7.5 Sterilized devices
54.7 Determination of antimic >bialactivity U7.6 Sterile empty or prefilled syringes
54.8 Determination U8 Test Procedures Using Membrane Filtration;
54.8.1 Microscopical ex; iination Apparatus
54.8.2 Transfer to fresh iedium U8.1 Liquids miscible with aqueous vehicles
55 Membrane filtration method U8.2 Liquids immiscible with aqueous vehicles
(less than 100 ml per container)
55.1 Preparation of sample s ution
55.2 Opening of containers U8.3 Filterable solids
55.3 Choice of culture media U8.4 Ointments and oils soluble in isopropyl
myristate
55.4 Containers for test
U8.5 Non-filterable solids
55.4.1 Bacteria test
U8.6 Devices
55.4.2 Fungi test
U9 Interpretation of Sterility Test Results
55.5 Apparatus for filtration
U9.1 First stage
55.6 Rinsing fluids
U9.2 Second stage
55.7 Procedure
55.8 Culture procedure
Note: Reference is made to chapter (1211) Steriliz-
55.9 Adsorption test for anti ,icrobial agents ation and Sterility Assurance of Compendia1 Ar-
55.10 Determination ticles in case sterility testing is used as part of an
assessment of a production lot or as one of the
quality control criteria for release of such lot or
batch.
U = United States Pharmaco eia XXII (71)
Sterility tests
2. GENERAL STRUCTURE OF THE STERILITY
U 1 Introduction TEST
U2 Media A test for sterility is valid only when certain condi-
tions are met, and these are usually described first.
U 3 Diluting and Rinsing Fluids They include:
U3.1 Fluid A
- media must be used with which the presence of
U3.2 Fluid D aerobic and anaerobic bacteria and fungi in the
U3.3 Fluid K sample under test can be demonstrated;

O PHARMEUROPA Vol. 6, N" 1, h irch 1994 37

International Harmonisation - Harmony in Sterility Tests
- the media themselves must be sterile;
- when the media are inoculated with aerobic and
anaerobic bacteria and fungi a copious growth
must be obtained in a shorter time than the
shortest observation period in the actual test;
- inoculation of the media with the sample under
test must not inhibit the growth of aerobic or
anaerobic bacteria or fungi; if inhibition occurs,
measures to neutralize or remove this must be
taken, and it must be shown that they are effec-
tive;
- extraneous contamination of the media etc used
in the test must be avoided;
- the size of the sample and the number of
recipients tested must be adequate to allow extra-
polation of the result of the test;
- the incubation period must be long enough to
allow growth of bacteria or fungi present in the
sample;
- if growth of bacteria or fungi is observed at the
end of the observation period, so that the sample
does not pass the test directly, a repeat test may
be performed only under well defined conditions.
Two methods are available for testing for sterility: the
membrane filtration method (MF) and the direct ino-
culation method (DI). The details of the application
depend in many cases on the pharmaceutical of the
product to be tested; hence theoretically a test for
sterility should contain the information of an n x 2
table, the 2 columns being used for the MF method
and the DI method and the rows being taken by the
different pharmaceutical forms (e.g. aqueous solu-
tions, solids, devices).
3. COMPARISON OF THE THREE STERILITY
TESTS AND PROPOSALS FOR HARMONI-
SATION
The three texts of the test for sterility are compared
item by item in the following paragraphs, and
proposals for a harmonised text are given.
3.1 Introduction
E l contains a reminder that adherence to Good
Manufacturing Practice and efficient monitoring of
autoclaving provide greater assurance for the sterility
of the product than the sterility test; the sterility test
however is the only means of control in the case of
aseptical preparation, and the only tool available for
checking the sterility of a sample.
U l refers to the informational chapter (1211) for the
concepts and principles involved in the quality control
of articles that must be sterile.
38
51 prescribes the DI method unless indicated
otherwise.
3.2 Precautions against microbial contamina-
tion
E2 describes these, and means for checking them.
51 has a similar text. U1 mentions the possibility of
such contamination as an argument for a repeat test
(see under Interpretation of results).
3.3 Culture media
E3 refers to the informational Annex VIII.3.2. for
examples of suitable media, and announces the
criteria of validity. Examples are given in 52 and in
u2.
Note: although the three texts give practically the
same composition for a Fluid thioglycollatemedium,
they suggest different applications:
VIII.3.2: “primarily for anaerobic bacteria, but will
also detect aerobic bacteria.”
54.3: “(This medium) is used for the test of
bacteria. When it is difficult to use (this
medium) because of the turbidity or
viscosity of the sample, fluid thiogly-collate
medium II for sterility test can be used
alternatively.” (This does not contain agar
and resazurin solution). 55.3 gives similar
directions.
u2: “Use Fluid thioglycollate medium by
incubating it under aerobic conditions.”
“Use Alternative Thioglycollate Medium
in a manner that will ensure anaerobic
conditions for the duration of the incuba-
tion period.” (This latter medium is
practically identical with the fluid
thioglycollatemedium II of 54.3). See also
the table under U4.
Soya-bean casein digest medium is recommended
for detecting fungi by Annex VIII.3.2 and U2, but
54.3 recommends for this purpose the glucose-pep-
tone medium described in 52.
All three texts allow the use of media with another
composition, provided they have been validated.
Proposa I: Suggest f 1uid th ioglycol la te medium wi th
a pH of 7.1 f o r anaerobic and aerobic bacteria,
and a Soybean-casein digest medium with a p H of
6.0 for aerobic bacteria and fungi.
3.4 Composition of the rinsing fluid for the
MF method
Extensive information in U3, including the direction
to add sterile penicillinase in tests on preparations of
O PHARMEUROPA Vol. 6, NB1, March 1994
 ~
International Harmonisation - Harmony in Sterility Tests
a penicillin or cephalosporin. indications for Proposal: Include general instructions including
making a fluid corresponding the objective (avoiding microbial con tamina tion)
in E4.1.1and 55.6. and how to monitor the process, as worded in EZ.

Proposal: Use a 0.1%peptone olution pH 7.1;

add polysorbate 80 to a concen
case of oily products.
3.5 Control of the sterility
E3.1;under the heading
in 53 and U4.
of 0.1%in 3.10 Sample size
This depends on the physical nature of the
preparation for aqueous solutions. see under 3.21;
for solids, see under 3.23;for oils, see under 3.25
and ointments, see under 3.27.
3.1 1 Number of containers to be tested

fungi for seven days. t

Proposal: Test both the media fo bacteria and for
No indication in J. For aqueous solutions see under
3.22,for solids under 3.254,for oils under 3.26.

3.6 Nutritive properties of thb medium

3.12 Rinsing volume per filter in the MF
E3.2,with the names of 4suitabl method
for testing this. 53only mentions
and aerobic bacteria and fungi. All three texts prescribe 3 X 100 ml; EP and USP
test and gives a table of test prescribe this for all products with antimicrobial
activity in EP and USP, but the 5 prescribes this for
Proposal: Name the following all products.

Staph. aureus Proposal: 3 X 100 m l f o r products w i t h

Bac. subtilis antimicrobial activity.
Clostr. sporogenes
Candida albicans
3.13 Incubation temperature (OC)

i
3.7 Nutritive properties in t e absence and E4.1 54.6 U8.1
presence of the sample under test Fluid thioglycollate 30-34 30-32 30-35
Soybean-casein peptone 20-25 20-25 20-25
E3.3and U5;54.7includes this te t for preparations
which are inherently bacteriostati or fungistatic or Proposal: For the fluid thioglycollate medium
which contain antimicrobial agent , in the descrip- 30-35 OC,for the soybean-casein digest medium
tion of the DI method. 55.9menti ns it briefly in the 20-25 “C.
description of the MF method. H wever, this test is
not required categorically, as it is n E3.3and U5.
3.14 Incubation period
3.8 Check of the of the removal Requirements for the DI and the MF method differ.
of bacteriostatic activity of the
preparation under test
E4.1 specifies in the case of the MF method: “not
less than 7 days unless otherwise prescribed.” A
explicitly. P
In E3.3and U5.54.7and 55.9d not require this
footnote authorizes national authorities to impose a
longer period if the nature of the product or its
treatment give reason to suspect the presence of
Proposal: Make such a check mdndatory.
micro-organisms of impaired viability.

3.9 Containers for the test E4.2prescribes in the case of the DI method: “not
less than 14 days unless otherwise prescribed.”

e
~

54.4defines these explicitly. No m ntion of this in E

or U. E9 specifies for catgut and other sutures: “not less
than 14 days.”
Detailed instructions for the of the contai-
ners are given in 54.2 54.6specifies in the case of the DI method: “not less
requiremnts in EZ. than 7 days for bacteria and not less than 10 days for

O PHARMEUROPA Vol. 6, N” 1, Mbrch 1994 39

International Harmonisation - Harmony in Sterility Tests
fungi.” 55.8 specifies the same periods in the case of
the MF method.
U6.2 specifies: “not less than 14 days unless
otherwise directed in the individual monograph or in
a section of this chapter.”
For the MF method U 8 specifies: “not less than 7
days.”
Proposal: For the DI method 14 days. For the MF
method in the case of water-soluble products
without inherent antimicrobial activity 10 days,
but 12 days in all other application of the MF
method.
3.15 Frequency of ready the samples:
E5 during and at the end of the incubation period.
No indication in J. U9: Examine the samples on day
3, 4 or 5 , 7 or 8 and on the last day.
Proposal: two to three times during the incubation
period and again at the end of the incubation
period.
3.16 Procedure in case of turbidity
No mentioned in E. 54.8 prescribes microscopical
examination; transfer equal aliquots to a suitable
fresh medium and incubate three days at 30-32 “C
in case of testing for bacteria and for five days at 20-
25 “C in case of testing for fungi. U7.1 prescribes
transfer of suitable aliquots to a suitable fresh me-
dium on day 3-7 from the beginning and incubation
of the original samples and subculture at least until
day 14 from the beginning.
Proposal: Microscopical examination at the end
of the incubation period, transfer to a fresh me-
dium and incubate as indicated above under «In-
cubation period)).
3.17 Interpretation of results
The three texts agree that the sample passes the test
when no growth is observed after the minimum
observation period. They differ in what has to be
done when growth is seen.
E5.1 First retest: Repeat the test with the same
number of items.
If no growth: the sample passes. If there is growth try
to determine the contaminant organism. If it cannot
be determined the sample fails the test. If it can
determined, do the second retest.
Second retest: use twice the number of items of the
original test. If there is growth, the sample fails; if not
it passes.
40
54.8 (DI method): The sample fails when there is
macroscopically visible growth. If the determination
is difficult try microscopical examination directly or
after staining. If determination remains difficult,
transfer adequate portions of the contents of the
tubes to fresh medium, and continue incubation for
at least 3 days for bacteria and at least 7 days. (Note:
the text uses “Determination” as the title of the
paragraph, so “determination” is used presumably
in the sense of “decision” and not in the sense of
“identification of the species” as €5.1.)
55.10: same directions for the MF method.
U9: First stage: if growth is found but there is evidence
that it is due to faults in the test itself, the first stage
is invalid and the test may be repeated. If there is no
evidence of faults in the test, proceed to the Second
Stage.
Second Stage: Perform the test with twice the number
of items used in the original test. If no growth is
found the sample passes. If growth is found but there
were faults in the technique the test may be repeated.
If growth is found and there were no faults in the
technique the sample fails.
3.18 Number of items in repeat tests
No mention in J. E5.1 specifies the same number of
items in the first repeat test as in the original one. If
a second retest is called for twice the number of
items are taken (from each item the same quantity is
taken as in the original test). In case U9 allows a
repetition of the first stage the same number of items
is used. In case U9 allows a second stage twice the
number of items is used and the same minimum
volume is taken from each item as in the original test.
Proposal: Apply the E5.1 procedure in case of
overkill sterilisation and equivalent procedures:
apply the U9 procedure in all other cases.
3.19 Method of testing and pharmaceutical
form.
Different pharmaceutical forms may require slightly
different ways of handling in the sterility test; as
stated in paragraph 2 the texts should ideally contain
the information of an n x 2 table with the
pharmaceutical forms as the rows and the informa-
tion how to handle this in the DI and in the MF
method in two columns.
In practice the three texts differ in the way of grouping
different pharmaceutical forms; besides some texts
have extra paragraphs for important subgroups of
products. The following table indicatesthe paragraphs
where this information is given in the three
pharmacopoeial texts.
O PHARMEUROPA Vol. 6, N” 1, March 1994
 ~

International Harmonisation - Harmony in Sterility Tests

E J U
MF DI MF DI MF DI
General Technique E4.1 E4.2 55 54 U8 u7
Aqueous solutions(') E4.1.1 E4.2 55.1 54.1 U8.1 U7.1
Solids(2) E4.1.2 55.1 54.1 U8.3 U7.3
Oils and oily E4.1.3 E4.2.1 55.1 54.1 U8.2 U7.2
Ointments and creamd4) E4.1.4 E4.2.2 54.1 U8.4 U7.2
Parenteralia E6 E6 55.1 54.1 U8.1 U7.1
Ophthalmic prepar. E7 E7
Surgical dressings E8.4 E8.2 u7.4
Sutures E9 u7.4
Sterilised devices U8.6 U7.5
Sterile syringes U7.6
(1)See 3,20-22, (2) See 3.23-24, (3) Sec 3.25-26, (4) See under 3.27

3.20 Minimal volume of each medium in case of aqueous solutions

E.42 only specifies a ratio of produc: volume: medium volume of 1:lO. 54.5 gives the following specification for DI:
Type of Test Preparation mi per Content of tube Number of tubes
container ml. of prep. ml. of medium
Test for bacteria 1420 15 1
2(D-<100 40 1
100-<500 40 2
500 and over 40 3
Test for fungi 1420 15 1
20-<100 15 2
100-<500 2 15 5
500 and over 2 15 7
U5.2 prescribes 100 ml of medi
following volumes of medium for $: for the MF method irrespective of the volume per container, and the

ml of medium

Proposal: 100 ml of each as a minimum, for all methods.

3.21 Minimal volume for solution to be examined per container for each culture medium
Volume in eich
container (mi)
E I 54.5 (DI) U6.2 Proposal

Bacteria
<1 idem
I
idem idem
1-(4
4420
1410 1 ml lm1
1-<20 1 mi
10-50 5 ml 5 ml
20-ao0 10%of cbntents 5 ml
>50 10%
50-100 10 ml, but whole cont.
if intraven. applic.
100-500
>500 t
10% of conten s min 50 ml
10%of conten s min 50 ml
5 ml
5 ml
whole contents
500 ml
Fungi
(1 whole contents
1420 1 ml
20-<100 2 ml
100 - 500 2 mi
>500 2 ml

O PHARMEUROPA Vol. 6, NQ 1, Mdrch 1994 41

International Harmonisation - Harmony in Sterility Tests
5.5.7 dealing with MF prescribes the use of the
whole of the labelled volume, but not more than 500
ml whrn the labelled volume exceeds 500 ml.

Number of cont. Minimal number of containers

in the batch to be tested Number of cont. Minimum number of cont.

I
per batch to be tested
E(VIII.3.1) USPXXII * 1-4 each container
<20 at least 2 5-50 20% or 4, whichever is more
20-200 10% >50 2% or 10, whichever is more
(100 10%or 4,
whichever is
U6.2: test 20 units per medium
greater Proposal: to be discussed.
100-500 10
>500 2% or 20, 3.25 Minimal mass of liquid oils
whichever is
the less No data in J. E6 Table I and U6.2 prescribe the same
* USP General information: at lest 20 containers per delivery quantitites per culture medium as for aqueous solu-
line (10 for each culture medium), but not more than 20 tions (see 3.21)
containers per batch.
3.26 Minimal number of containers containing
However, U6.2 relates the minimum number of con- oils to be tested with each medium:
tainers to the volume of fluid per container in the
following table. No data in J. E (VIII.3.1) refers to parenteralia
ml per Number of cont. to be tested per without distinguishing aqueous and non-aqueous
container medium (U6.2) ones: see above under 3.22.
4 0 20 U6.2 similarly refers to liquid articles when the number
10-50 20 of containers to be tested is prescribed dependent on
50-100 10 the volume per container: see above under 3.22.
100-500 10 3.27 Minimal mass of ointments to be tested
>500 10 with each culture medium:
Proposal: adopt the procedure of the Ph. Eur.
(E V111.3.1) No data in J. E7 Table II prescribes making a sample
of 1-10 g by combining containers as required, and
3.23 Minimal mass of solids to be tested using 0.5-1.0 g of this for each culture medium.

No data in J. U7.2: Select 20 representative containers, assign

Table 1 (E6) specifies the following masses to be
used, depending on the quantity in each container:
Quantity per Minimum quantity
container to be used per medium
(50 mg the whole contents of the cont
50-<200 mg half the contents
200 mg or more 100 mg
For MF these quantities are dissolved in a suitable
solvent (see rinsing fluid) and tested in the same way
as aqueous solutions. In DI the ratio of product to
medium must be about 1:lOO.
For DI U7.3 prescribes the use of 300 mg per
container, or the total contents when each container
contains (300 mg. This quantity is transferred to at
least 40 ml of culture medium. For MF U8.3
prescribes taking about 6 g of preparation or not less
than 300 mg from each container, or the whole
them to 2 groups of 10 containers. Transfer 100 mg
from each of the 10 containers to a flask containing
100 ml of a sterile aqueous vehicle containing a
dispersing agent. Mix an aliquot of 10 ml of this
mixture with 80 ml of each medium and incubate as
in the case of aqueous preparations (hence 100 mg
of the preparation per medium).
Proposal: to be discussed.
Note: No attention has been giuen in this
comparison to surgical dressings and medical
deuices. No proposals have been giuen for:
- control of the methods,
- testing for antimicrobial activity
- maximal uolume of the preparation per mem-
brane filter,
- filtration pressure and flow rate in MF,
- composition of the filters used in MF.

42 O PHARMEUROPA Vol. 6, N" 1, March 1994

 Scientific Notes

Scientific Notes
CQNTAMIN BY HEAVY METALS IN DRUGS
FROM COMMERCIAL SOURCES
R. De Pasquale(l), M.P. Germano(')
Abstract. - Contamination by metals of medicinal plants can induce toxic effects not only in humans
but also in plants themselves. we observed the induction of qualitative and
quantitative modifications of content in the plants. In this report we attempt to verify the
heavy metais contamination commercial sources. Cadmium, Copper, Lead and Zinc
content was measured by Results show that Cu and Pb content sometimes go
beyond the range of the The Cu content was the highest in drugs made from
leaves, fruits and some might be due to spraying with pesticides.

INTRODUCTION I Cape Aloe [3],Aniseed [3], Star Anise [i],Belladonna

leaves [2], Chamomile flowers [6],Anthemidis flowers [4],
Heavy metal pollution in plants Cinnamon [3],Cinchona [5],Digitalis leaves [i], Frangula
vation in the neighbourhood of [4], Cloves [Z]Gentian [4], Hyoscyamus 121, Ipecacuanha
with intense traffic. Many [i],Linseed [5],Liquorice [7],Peppermint [7],Rhubarb [3],
how the level of Rhatany [i], Senega [21, Senna leaves (51, Senna fruits [41,
the distance of Stramonium [i],Valerian [3].The number of samples is
indicated in brackets.

active principles.
EXPERIMENTAL
Plant material
The samples of drugs anaiysed and
from different firms importing
country where the different drugs
instrumentation
Heavy metal content was measured with a Perkin Elmer
Model 372 atomic absorptionspectrophotometerequipped
with an HGA 500 graphite furnace. A deuterium
background corrector was used for Cd and Pb determination.
Absorbance measurements were made in the peak height
mode, and single-cathode lamps were used.
Table I gives the optimum instrumental conditions for
measurement of Cd,Cu, Pb and Zn.
I Reagents
Standard solutions of Cd, Cu, Pb and Zn (ig/L BDH) were
used to prepare intermediate reference solutions of various
below were bought concentrations. Nitric acid (65 %, analytical grade) was
used for treatment of all biological materials under exami-
nation. All solutions were prepared with de-ionised water.

Table I - Temperature Program for determination of heavy metais in vegetable drugs

Hold (s)
Step Cd Cu P/b Zn Cd Cu Pb Zn Cd Cu Pb Zn
1 Drying 100 105 I20 10 10 10 20 30 30 20 30
2 Charring 350 900 15 10 10 10 40 30 50 20
3 Atomi- 2100 2500 23 O 2500 1 1 1 1 5 6 6 6
zation
4 Cleaning 2600 2600 25bO 2600 1 1 1 1 3 4 4 5

Perkin-Elmer Model 372 and HGA-500 with normal graphite tubes, purge gas argon, internal flow stopped during atomization.

(i)Dept.Pharmaco-Biological,School of Pha acy,, University of Messina, Italy. 'Dept. of Occupational Medicine, School of Medicine, University of
Messina, Italy. Î"

43
Scientific Notes
Plant analysis
10 ml of 50 % HNO, were added to 1g of powdered dry
drug. After overnight digestion, the residue was filtered
through Whatman nQ42 filter paper into 50 ml volumetric
flask; then 1ml of 10 % NH H2P04 (Matrix modifier) was
added and diluted to the m z i with de-ionised water 18-91.
Cd,Cu, Pb and Zn content is expressed in ppm of dry
matter.
Calculations :
CxVxdf
PPm =
W harvest.
C = p.s/ml in sample solution,
V = final diluted volume in ml,
W = sample weight in grams,
df = dilution factor.
Each sample was analysed three times.
RESULTS
Heavy metal content in drugs from different commercial
sources is shown in Table II.
Whereas Cd and Zn concentrations are within the range
of the CA (seeTable below),those of Cu and Pb can go
beyond it.
Leaves. Different samples of Senna leaves generally show
a lower content of Cd,Cu and Pb in comparison with other
leaves. In the two samples of Belladonna leaves there are
the highest content of the elements analysed, in fact in
sample “C”,as also in Stramonium leaves, the Cu content
goes beyond the range of the CA. In the Digitalis sample
and in sample A4 of Peppermint, the limit indicated from
CA for Pb is just exceeded.
Flowers.The content of heavy metals in the flowers never
reaches a high level, even if in sample “D” of Chamomile,
the Cd and Cu contents are double in comparison with the
content of sample “D extra” of the same commercial
sources.
Fruits. In all the fruits the content of heavy met& is low.
Senna fruits particularly show the lowest content of Cu,
whereas that of Pb is a little higher.
Seeds.In linseed there is the highest level of Cu and in
Belladonna and Stramonium leaves and Aniseed.
Barks. All the barks, specially Frangula, show a low content
of Cu, however all the Frangula samples have a Pb content
that goes a little beyond the range of the Ca, while Cinchona
has the highest content of Cd.
Roots and Rhizomes.in Liquorice the content of heavy
metals is higher in whole drugs than in the decorticated
ones (samples B1 dec and D2 dec)of the same commercial
sources.
Juice. In Aloe samples there is a limited content of all the
elements analysed.
44
DISCUSSION
Results show that heavy metal concentration can vary in
a significantway, not only in drugs from different commercial
sources, but also in samples of the same commercial origin,
such as in Linseed samples “Dl”, “DZ”, “D3”, and in
Chamomile samples “Drand “D extra”. Our observations
that the Cu content was the highest in drugs made from
leaves, fruits and linseed (this level increase might be due
to spraying with pesticides), and that in the whole drugs
the content of heavy metals is higher than in the decorticated
ones, suggest that the presence of heavy metals is mostly
a consequence of external tissue contamination before
REFERENCES
Codex Alimentarius, Eds. FAOWHO, 1981.
CottenieA., Dhaese A., Camerlink R., “Plantsquality response to
uptake of polluting elements”, Qualitas Plantarum 26, 293-
319, 1976.
De Pasquale R., Ragusa S., lauk L., Barbera R., Galati E.M.,
“Effect of Cadmium on germination, growth and active principle
contents of Achillea millefolium L.”, Pharm. Res. Comm. 20,
SUPPI.V, 145-149, 1988.
De Pasquale R., Ragusa S., lauk L., Barbera R., Galati E.M.
“Effect of Cadmium on germination, growth and active principle
contents of Matricaria recutita. L.”, Pharm. Res. Comm. 20,
SUPPI.V, 151-154, 1988.
De Pasquale R., Ragusa S., Iauk L., Barbera R., Galati E.M.
“Effect of Cadmium on germination, growth and active principle
contents of Datura metel L.”, Toxicologicaland Environmental
Chemistry 23,121-127, 1988.
Galati E.M., lauk L., Ragusa S., Barbera R., De Pasquale R.
“Effects of Copper on the growth and active principle content of
Datura metel L.”, Int. J. Crude Drug Res. 27, 2, 113-117,
1989.
Galati E.M., Iauk; L., Germano M.P., De Pasquale R. “Effettidel
Cadmio e di Fitofarmaci sul contenuto di silibina nei semi di
Silybum marianum Gaertn.” Atti del V Congresso Nazionale
della Società I t a l i a di Fitochimica, Ischia 30 May - 2 June 1990.
Kahn H.L., Fernandez F.J., Slavin S. “The determination of Lead
and Cadmium in soils and leaves by atomic absorption
spectroscopy”.Atomic Absorption Newsletter 2,4242, 1972.
Slavin S., Camrick G.R., Manning D.C., Pruszkowska E. “Recent
experiences with the stabilized temperature platform fumace and
Zeeman background correction”.Atomic Spectroscopy 4,3,69-
86, 1983.
Range of Tollerability of Metals
and Metalloids from Codex Alimentarius
I Metals and Metalloids I Max ppm
Aluminium 250
Antimony 2
Arsenic 0.1
Barium 500
Boron 80
Cadmium 5
Copper 10
Fluorine 1.5
Lead 2
Mercury 0.05
Selenium 0.05
Silver 1
Tin 300
Zinc 100
O PHARMEUROPA Vol. 6 , N” 1, March 1994
 Scientific Notes

TABLE II
Heavy metal contents in Jegetable drugs of different commercial sources (ppm of dry drug)
~

Drugs Cd cu Pb Zn
Leaves
Digitalis B* 0.067 2.03 2.08 79.12
Belladonna B 0.122 9.19 1.O5 60.09
Belladona C 0.126 11.20 1.33 73.84
Hyoscyamus B 0.036 9.18 1.45 43.25
Hyoscyamus C 0.043 7.46 1.15 53.67
Stramonium B O. 047 11.80 1.14 52.77
Peppermint Al** 0.038 6.52 1.10 29.06
Peppermint A2 0.046 5.70 0.98 30.10
Peppermint A3 0.050 5.31 1.10 34.00
Peppermint A4 0.037 4.76 2.56 25.04
Peppermint B 0.052 4.22 1.18 32.03
Peppermint C 0.050 5.76 1.78 36.15
Peppermint D 0.051 6.84 1.70 36.18
Senna A 0.019 3.38 0.30 39.77
Senna B 0.012 3.87 0.40 46.55
Senna c1 0.016 4.06 0.29 50.38
Senna c2 0.014 5.77 0.25 50.64
Senna D 0.020 3.55 0.40 45.32
Flowers
Anthemidis A 0.045 2.35 0.54 38.64
Anthemidis B O. 128 2.53 0.43 42.56
Anthemidis C 0.036 2.59 0.39 31.08
Anthemidis D 0.068 4.25 0.31 32.14
Chamomile A 0.129 2.51 0.61 50.29
Chamomile A extra O. 147 3.77 0.61 46.33
Chamomile B 0.086 4.93 0.47 36.16
Chamomi1e C 0.098 3.03 0.58 57.73
Chamomile D 0.103 5.12 0.50 39.77
Chamomile D extra O. 054 2.54 0.35 41.23
Cloves C 0.010 4.75 0.39 18.81
Cloves D 0.014 3.38 0.67 32.39

Fruits and seeds

Aniseed B 0.016 6.40 0.32 44.42
Aniseed C 0.022 6.73 0.39 64.37
Aniseed D 0.015 7.44 0.41 54.22
Linseed A 0.056 7.88 0.45 49.65
Linseed C 0.093 9.64 0.54 40.50
Linseed D1 0.132 5.85 0.56 47.21
Linseed D2 0.038 8.50 0.52 54.76
Linseed D3 0.025 7.81 0.30 57.88
Senna A 0.019 3.86 0.82 33.14
Senna B 0.011 3.98 0.74 30.05
Senna C 0.010 3.74 0.47 30.62
Senna D 0.010 4.29 0.63 36.01
Star Anise D 0.013 6.48 0.57 12.70

O PHARMEUROPA Vol. 6, Na 1, I ,arch 1994 45

Scientific Notes

TABLE II (continued)

Drugs Cd cu Pb Zn
Barks
Cinchona A 0.502 2.64 0.95 56.84
Cinchona B 0.727 2.64 0.98 48.01
Cinchona C 0.750 5.52 0.76 54.29
Cinchona D1 0.137 3.18 0.80 41.68
Cinchona D2 0.435 4.42 0.77 38.75
Cinnamon A 0.093 2.56 0.78 19.21
Cinnamon B 0.101 2.97 0.58 15.94
Cinnamon C 0.029 2.87 0.37 7.10
Frangula A 0.029 1.89 2.05 6.59
Frangula B 0.026 0.90 2.42 7.67
Frangula C 0.022 0.80 2.35 12.04
Frangula D 0.032 0.67 2.11 9.92
Roots and Rhizomes
Gentian A 0.082 0.46 0.75 32.23
Gentian B 0.046 0.66 0.52 27.89
Gentian C 0.089 0.47 0.43 16.06
Gentian D O. 046 0.63 0.58 19.70
Ipecacuanha B 0.051 1.28 0.31 47.33
Liquorice Al 0.023 1.23 2.11 44.53
Liquorice A2 0.016 3.36 0.35 31.83
Liquorice B1 dec 0.018 1.63 0.36 19.84
Liquorice B2 0.014 1.75 0.48 26.80
Liquorice C 0.023 1.19 0.60 30.15
Liquorice D1 dec 0.018 1.92 0.41 19.44
Liquorice D2 0.022 2.09 0.69 26.94
Rhatany B O. 046 1.76 0.25 9.25
Rhubarb A 0.028 0.76 0.46 18.62
Rhubarb B 0.028 0.76 1.O8 27.98
Rhubarb D 0.021 1.10 0.33 29.59
Senega A 0.082 0.69 0.85 26.45
Senega B 0.085 0.69 0.67 28.86
Valerian B 0.080 3.42 2.65 34.18
Valerian C 0.025 4.06 0.99 27.94
Valerian D 0.060 3.81 1.22 33.25

Juice
Cape Aloe B 0.015 1.10 0.56 34.44
Cape Aloe C 0.010 1.46 0.72 69.90
Cape Aloe D 0.014 1.33 0.39 31.15

* The letters A, B, C, D indicate the different commerical sources.

** The numbers 1,2,3,4and the name extra indicate the different samples of the same commercial sources.

46 O PHARMEUROPA Vol. 6, N" 1 , March 1994

 Scientific Notes
~

A NOTE ON HE MICROBIAL CONTAMINATION

J F MEDICINAL PLANTS
Alonzo V.(')*, Monfbt-te M.T.('), Tumino G.(l), Ragusa S.('), Bisignano G.(l)*
Key words: Microbial Plants, Charge Limit

Abstract. - plant material were checked for mesophilic microorganisms.

gram-positive, gram-negative and spore-formers, while total
Moulds and yeast were recovered from Sabouraud
0.5 X 104 to 3.6 X 105 per gram of vegetable matter.
bacterial species are discussed with reference to the

of same samples were studied with the Scanning

INTRODUCTION I MATERIALS AND METHODS

Vegetable matter is easily conta
micro-organisms (Salunke 1974).
et al. (1980)there are four main
the contamination level:
a) physiological leaf condition,
by several Medicinal plant samples
The following samples of fresh medicinal plants belong-
ing to different species cultivated in the garden of the
Pharmaco-Biological Department of University of
1 Messina (Italy) have been assayed: leaves of Datura
innoxia Mill., D. stramonium L., D.metel L., Laurus

I
b) vector activity, nobilis L., Nerium oleander L., Nicotiana tabacum
L., N. glauca, R. Grah, Rosmarinus officinalis Mill.,
c) micro-climate, Ruta graueolens L., and flowers of Lavandula angus-
tifolia Mill. To avoid too heavy contamination, the leaves
d) accessibility of leaves to micro- rganisms. picked were those standing as far as possible from the
soil. Immediately after picking, each sample was divided
Other factors we have to be conci into two lots: one lot was assayed within 8 hours of
tamination due to hu harvesting to determine microbial charge; the other lot
Westhoff 1976; Romond was weighed and dryed under an air-stream at room tempe-
from soil (Graham et al. 1977; rature protected from dust and direct light and then
the microbiological quality of t weighed to determine the percentage loss of water and
kind of manure (Robinson et submitted to microbial evaluation as for the fresh drug.
possibility of diffusion of Table 1lists the number of samples, the species names
mycetes, through the air and the minimal distance from soil of the picked parts.
Andrews et Kenerley 1978; All samples were picked between November and Febru-
factors are the climate, th ary and transported to the laboratory at room tempe-
presence of water. It has bee rature.
retaining a higher humidity
easily support bacterial g Homogenates and culture

Moreover the presence o Preliminary research was performed to ascertain whether

sition of mucous (often the investigated parts could exert some inhibitory effect
the leaf surface can p on bacterial growth, in order to establish the dilution
survival. rate. For this purpose, plant homogenates, at 5 differ-
ent dilution values, were added in several plates con-
taining melted Bacto Plate Count (Difco) agar medium

P
Medicinal plants used in pharmac utical or cosmetic
products should thus be carefully c ntrolled for micro-
bial, pesticide or heavy metais con amination.

The aim of the present is to evaluate the

and, after solidification, inoculated with known amounts
of bacterial suspension, either of gram-positive
(Staph y lococcus, St rep tococcus) or gram-negative
(E. coli, Pseudomonas) bacterial. After 48 hours incu-
bation at 37 OC,the colony count was compared with
charge of mesophilic s in some medici- that of control plates. The best dilution rate, which does
nal plants at the time well as after the not give any statistically significant colony count reduc-
drying procedure tion, was found to be 1:20. 2 g of each sample were

(1)Pharmaco-Biological Department, Pharmadognosy Section, *Microbiology Section, University of Messina (Italy),

O PHARMEUROPA Vol. 6, N" 1, M&h 1994 47

Scientific Notes

TABLE 1 - Microbial charge (lo4per g)

of fresh crude plant material

Mesophilic bacteria
Species listance from rotal count Micetes
soil (cm) mean S.E. Yeasts gram- gram- lacto- spore-
negative positive bacillus formers
~~

Flowers:
Lavandula angustifolia 30-40 246 f 3.6 !O0 (82.3)** 11.5 (4.7) 5.47 (2.2) 10.5 (4.26) 16.8 (6.8)
Mill. (8)*

Leaves:
Datura innoxia 50-60 0.87 f 0.7 0.08 (9.2) 0.25 (28.7) 0.055 (6.3) O 0.48 (55.2)
Mill. (5)
Datura metei L. (5) 50-70 1.7 I O . l 0.01 (0.58) 1.1 (64.7) 0.07 (4.1) O 0.49 (28.8)

Datura stramonium (5) 30-40 48.8 f 2.5 2 (4.09) 32 (65.5) 8.8 (18) O 6.02 (12.4)
Laurus nobilis L. (10) 100 28.6 f 1.1 8.24 (28.8) 13.4 (46.8) 2.2 (7.7) O 2.4 (8.4)
Nerium oleander L. (6) 60 18.3 I 0.45 4.8 (26.2) 9.5 (51.9) 0.32 (1.84; O 3.65 (19.9)
Nicotiana glauca 30-40 1.4 f 0.15 3.072 (5.14) 0.12 (8.5) 0.031 (2.21 1.01 (0.71) 1.14 (81.4)
Grah. (8)
Nicotiana tabacum L. (7 80-100 0.91 f 0.1 3.018 (1.97) 0.4 (43.9) 0.095 (10.4 O 0.39 (42.8)
Rosmarinus offici- 30 9.1 f 0.4 1.64 (18.02) 3.8 (41.7) 0.3 (3.3) O 3.3 (36.3)
nalis L. (12)
Ruta graveolene L. (2) 40 1.12 tr 0.1 3.032 (2.85) 0.76 (67.8) 0.15 (13.4 O 0.17 (15.2)

* ( ) n. samples.
**( ) percentage of total count.

blended in 20 ml of sterile peptonewater 0.1 % by means
of an Ultra-Turrax (Janke-Kunkel) homogenizer.
Peptonewater was chosen as suspending medium in
order to avoid membrane damages as well as to pro-
mote resumption of metabolic activities by stressed cells.
1ml from the homogenate or from its dilutions in sterile
peptonewater (from 1 : l O to 1:lOOO) was inoculated,
within 60 min, onto agarized media in Petri dishes and
uniformly streaked over the surface. Before inoculating
spore-formers media, the homogenate was kept at 80 "C
for 10 min.
Cultura medía
The following culture media were used: Plate count agar
(Difco) for total bacterial counts; Sabouraud destrose
agar medium (Difco) and Malt extract agar (Difco) for
enumeration of moulds and yeast. To detect gram-nega-
tive bacteria, Hektoen Enteric Agar (Difco),Levine EMB
Agar (Difco), Desoxicholate Agar (Difco) and Pseudo-
monas Isolation Agar (Difco)were used. For isolation of
gram-positive bacteria, Azide Blood Agar (Difco),
Enterococci Confirmatory Agar, Chapman Stone Me-
dium (Difco) and Bile Esculin Agar (Difco) were used,
while for growth of lactobacilli the Lactobacilli Agar
AOAC (Difco) and Rogosa SL Agar (Difco) were em-
ployed. For sporeformers, Tripticase Agar Base (Difco)
with Manganous sulphate 0.03 % supplied, Liver Infu-
sion Agar (Difco), Tripticase Soy Agar (BBL) and SPS
Agar (BBL) were used.
Viable counts were made after 24 and 48 hours incu-
bation at 30 "C. For moulds, incubation time was pro-
tracted to 7 days. The media for anaerobes were incu-
bated in Gaspak Anaerobic System under a mixed at-
mosphere of CO2 and hydrogen.
Bacterial colonies grown on selective media were counted
and separated into distinct groups having the same
morphology and biochemical reactions. For this pur-
pose, five to ten colonies of each group were screened
for their main reactions: Gram staining, oxidase, catalase,
O/F activity on glucose, anaerobic growth, gas produc-
tion on glucose and motility.
Out of those having the same characteristics at this first
screening, some were further characterized either by
several single tests, according to the scheme of Bergey's
Manual of Determinative Bacteriology (1984, 1986)or,
if necessary, by multiple tests with the API System
galleries.
When the tests performed were similar for all tested
colonies, the identified species were attributed to the

48 O PHARMEUROPA Vol. 6, Na 1, March 1994

 Scientific Notes

totality of the colonies having identi
the primary screening. With some
secondary screeningwas applied to I
colonies belonging to the same spl
on different selective media the mc
culated only on those found on the
medium for that species.
Scanning Electron Microscopy
Fragments about 5 mm square o
were placed for 24 hours in 5 % glu
with 0.1M cacodylate at pH 7..
sequentially for 15 min each time ir
and 95 % ethyl alcohol solutions, i
ethanol, critical point dried with liq
Critical Point dryer”, coated with
E 5100 Spatter Coater” and obseri
500.
RESULTS
The mean numbers of mesophilic bacteria and yeasts
per gram of vegetable tissue a z summarized on
11 characteristics in Table 1.As far as moulds are concerned, only names of
*esultsin contrast, the species are reported as it is impossible to quantify
we colonies. When the real number, owing to mycelium fragmentation; the
:ies were identified prevailing species which belonged to Alternaria were
n number was cal- found in Lauandula angustifolia, Cladosporium in
ost suitable growth Datura innoxia and Fusarium in Rosmarinus
officina lis; Aspergillus, Penicillum and Rhizopus spe-
cies were found in the examined samples. The same
table carries the height above soil at which aerial parts
were picked. On Tables 2 and 3 are listed the main
the fresh samples gram-negative and gram-positive species, respectively,
aaldehyde buffered found on the examined samples. The genus Erwinia is
then submerged listed separately from other Enterobacteria, being the
30,45,60,75,90 most common genus present on vegetable matter. Spore-
id finally in 100 % formers are listed on Table 4.
d CO, by “Balzer’s
!old in a “Polaron
:d in a Philips SEM Out of ten vegetable drugs examined, eight were also
tested by the same methods, after desiccation at room
temperature. Table 5 reports: I. The loss on drying; II.
Values of total viable counts referred to one gram dry
weight; III. Values of total viable counts calculated for
one gram of fresh vegetable material, i.e. to the gram of
fresh tissue necessary to make up 1 gram dry weight;
IV. Percentages of the prevailing bacterial species.

TABLE 2 - Spec s of gram-positive cocci (as percent of those listed in table 1)

found on crude plant material

Staphylococcus Micrococcus
Sêcoes
meus saprophiticus luteus-varians

Flowers:
iavandula angustifolia Mill. O 91.7 O 8.3 O

Leaves:

Datura innoxia Mill. O 68 4 28 O

Datura mete1 L. O 100 O O O

Datura stramonium L. 32 5 O 3 O

Laurus nobilis L. O O 59.7 40.3 O

Nerium oleander L. O 20 20 40 20

Nicotiana glauca Grah. O 50 O 50 O

Nicotiana tabacum L. 35 15 O O O

Rosmarinus officinalis L. O 4.76 94.24 1 O

Ruta graveolens L. O 45.5 O 54.5 O

O PHARMEUROPA Vol. 6, N” 1, M rch 1994 49

Scientific Notes

O 0 0 0 ~ 0 0 0 0 0

"! 0 0 d 0 0 0 0 0 0
l-l

"!
b
d

O
o o o o o o o ?
~ m
N e

c'
m
l-l

O
o o o o x ?
a o o o
e m

c'
2

i
i
i E i
i o;
a a
([I
6
d
a
ci

!i
-8
O
%
ci

([I s9,
d

3a
e so;
.- .-
([I
ci

8 ([I
ci

z E i?

50 O PHARMEUROPA Vol. 6, NQ1, March 1994

 Scientific Notes

TABLE4-1 kin spore-formers species found on crude plant material

(as percent of those listed in table 1)

Bacillus 210s tridium

Species leaaterium Pumilus Licheni- Cereus Antracis Subtilis Polymixa Species

formis

Flowers:
Lavandula angustifolia 15.87 33.57 10 7.94 24.62 8
Mill.

Leaves:
Datura innoxia Mill. 13.64 18.2 6.09 12.34 3 45.43 1.3
Datura metel L. 55.37 12.5 16.5 8.33 2 5.3
Datura stramonium L. 2 37.5 8.5 4 46.5 1.5
Laurus nobilis L. 26.07 6.7 6.13 60.5 0.6
Nerium oleander L. 43.47 32 7.69 16.76 0.08
Nicotiana glauca Grah. 1 80 15 0.9 3 0.1
Nicotiana tabacum L. 73.27 13.04 8.19 5.5
Rosmatinus offici- 66.17 7.18 6.15 20 0.5
nalis L.
Ruta graveolens L. 40 50 10

TABLE 5 - Microbial charge of dried crude plant material (104/g)

Loss on Total ïhoretical Gram-

leng % munts :harge for positive Spore-
Species mean g of fresh cocci others formers Yeast
S.E. tissue

Flowers:
Lavandula angustifolia 27 210.36 2 1.68(14)
I
1.42(11.8) 0.9(7.5) 7.56(63) 0.3(2.5)
Mill. (6)*

Leaves:
Datura innoxia Mill. (4) 10.04 78k0.05 15
I
0.05(6.4) ).18(23.07) 0.05(6.4)
~~

0.47(60.2) O.OOZ(2.5)
Datura metel L. (5) 15.82 .7+0.10 23 3.08(2.96) 0.25(9.25) 0.15(5.55) Z.ll(78.1) O.Ol(0.37)
Datura stramonium L. (5) 16.17 1612.5 36 1.05(0.9) 65.4(56.3) 27.6(23.7) 17.2(14.8) 0.4(0.34)
Laurus nobilis L. (8) 51.96 .9&0.10 5 D.36(18.9) 0.13(6.8) 0.05(2.6) 1.3(68.4) 0.003(0.15)
Nerium oleander L. (5) 50 .2k0.10 4 D. 13(10.8) 0.31(25.8) 0.065(5.4) 0.66(55) O.OOl(0.83)
Nicotiana tabacum L. (7) 6.05 .9+0.15 25 0.4(10.5) 1.33(34.1) 0.48(12.3) 1.54(39.4) O.Ol(2.5)
Rosmatinus offici- 64.4 .1kO.15 17 0.12(5.7) 0.04(1.9) O. 03(1.4) 1.85(88.1) 0.004(0.19)
nalis L. (8)

( ) %oftotal count C.E. standard error

( )' n. samples

~~~~

O PHARMEUROPA Vol. 6, N" 1, iV ch 1 9 9 4 51

Scientific Notes

The main groups of gram-negative and prevailing species of gram-positive and spore-formers are listed on Tables
6, 7, 8, respectively.

TABLE 6 - Microbial charge of dried crude plant material. Species of gram-positive cocci
(as percent of those listed in table 5)

r- Species

Flowers:
Staphylococcus Micrococcus
luteus-varians
Aerococcus

Lavandula angustifolia 83.34 8.33 8.33 O

Mill.
Leaves:
Datura innoxia Mill. 83.87 O 16.13 O
Datura metel L. 100 O O O
Datura stramonium L. 96.87 O 3.13 O
Laurus nobilis L. O 80 20 O
Nerium oleander L. 24.3 15.15 36.35 24.2
Nicotiana tabacum L. 1O0 O O O
Rosmarinus officinalis L. 20 40 5 35

TABLE 7 - Microbial charge of dried crude plant material. Main gram-negative species
(as percent of those listed in table 5)

Species Erwinia Enterobacteria Pseudomonas Acinetobacter

Flowers:
Lavandula angustifolia

Leaves:
40.1 1 7.7
I
52.2

Datura innoxia Mill. 20 12.77 46.67 20.56

Datura metei L. 37.33 13.34 49.33
Datura stramonium L. 29.73 32.05 3.57 34.65
Laurus nobilis L. 28 14 58
Nerium oleander L. 17.62 67.62 13.33 1.43
Nicotiana tabacum L. 26.76 12.32 60.92
Rosmarinus officinalis L. 38.57 44.79 8.64 8

TABLE 8 - Microbial charge of dried crude plant material, spore-formers species

(as percent of those listed on table 5)

Bacillus Clostridium
Pumilus ìicheni- Cereus Antracis Subtilis 'oly mixa Species
Species formis
~

Flowers:
Lavandula angustifolia 35 13.33 20 6.67 20 5
Mill.
Leaves:
Datura innoxia Mill. 24.04 14.28 26.3 4.16 2.5 28.57 0.6
Datura metel L. 12.5 43.3 20.2 17 1 6
Datura stramonium L. 10.1 30.1 13.1 6 39 1.7
Laurus nobilis L. 22.73 32.7 9.1 36.36 0.1
Nerium oleander L. 28.57 43.76 14.28 14.28 0.1
Nicotiana tabacum L. 4 26 54.9 15 0.1
Rosmatinus offici- 47.54 4.42 3.28 44.26 0.5
nalis L.

52 O PHARMEUROPA Vol. 6, NQ1, March 1994

 Scientific Notes

The identified moulds belonged to the same species The most polluted vegetable drug was Lavandula
found on fresh material. angustifolia (Fig. i),probably because specimens were
picked near to the soil and because the tubular

to 5. I
The microscopical characteristics of he leaf and flower
surfaces and the microbia are rep rted in Figures 1
structure of flowers facilitatesthe permanence of micro-
organisms of exogenous origin. The high yeast counts
can be ascribed to contamination by passive insect trans-
port, as described for other flowers (Frazier et Westhoff,
1976; Speck 1984). Generally, the harvesting area
seems to be a less important factor than others, speci-
mens picked in the same area showing different degrees
of contamination, as already demonstrated by other
authors (Geerson 1979).

The morphology and anatomical structure of leaves

seems to be more important for bacterial charge. Those
rich in covering trichomes or glandular hairs with revolute
margins (as Rosmarinus officinalis, Fig. 2), or with a
large surface (as Datura stramonium, Fig. 3), or with
hairs at the pit entrances (as Nerium oleander, Fig. 4))
are more heavily contaminated. On the contrary, being
small and glabrous, the leaflets of Ruta graueolens
(Fig. 5) are less contaminated.

Figure 1
Budding Yeast on Lauandula angustifolia Mill.: FLOWERS

DISCUSSION I
The high content of carbohydrates plants should im-
prove the growth of epiphytic such as Erwinia,
Lactobacillus and 1976) as well
as those of

Figure 3
A yeast on upper surface of Datura stramonium L.: LEAVES

Figure 2
A yeast attached to a covering trichomes Figure 4
Rosmarinus officinalis L.: LEAVES Upper surface of Nerium oleander L.: LEAVES

O PHARMEUROPA Vol. 6, N" 1, MJrch 1994 53

Scientific Notes
Figure 5
Bacterial cells of the upper surface of
Ruta graveolens L.: LEAVES
This is particularly true for the charge in spore-formers.
Moreover, among those species belonging to the same
genus, higher charges in Bacillaceae were found on
those picked nearer the soil. Enterobacteria and gram-
positive cocci are likely to depend on the bacteriological
quality of the irrigation water as well as on the post-
harvested spoilage due to handling and storage. For
these species, bacterial count is independent of the
distance from soil but the morphological features may
contribute to the permanence of the polluting microbial
charge.
Therefore, in order to keep the contamination level low,
it would be advisable to pick specimens as far as pos-
sible from the soil and to check the microbiological quality
of the irrigation water (Andrews et al. 1980, Bovalius et
al. 1978). The use when possible of a mechanized sys-
tem of harvesting could reduce the contamination by
humans, as suggested by some authors (Lenoble et al.
1980).
The incidence of predominant micro-organisms on dried
specimens is shown in Table 5. The great influence that
initial contamination level has on number of survivors is
evident.
Among the gram-negative bacteria, it should be noted
that the drying procedure seems to cause a more in-
tense reduction in counts for Erwinia than for other
Enterobacteria. This can be explained by the fact that,
due to manipulation, secondary contamination occurred
during the drying procedure with further pollution with
Enterobacteria other than Erwinia.
Therefore it is necessary to fix some limits for bacte-
rial total charge and for different groups of bacteria,
on dried medicinal plants which have to be used in
-
54
pharmaceutical preparations. Even if the procedures in-
volved in making such preparations could reduce the
contamination level (Lenoble et al. 1980), it has been
pointed out that several vegetable drugs are heavily con-
taminated (Negretti 1983). Moreover, a high level of
bacterial contamination could be dangerous both be-
cause of the presence of pathogenic micro-organisms as
for the possible presence of toxins or microtoxins in the
pharmaceutical form (Hitokoto et al. 1978). The possi-
bility that bacteria might transform the active principle
should also be kept in mind.
While there are microbiological standards fixed for non-
sterile medicaments, upper limits have not been applied
to medicinal plants for which separate standards have
been proposed (Lenoble et al. 1980; Cingolani et Aureli
1983).
For fresh crude plant material we think that only the
total count should be performed, because the various
factors already discussed can make it vary from one
specimen to another. All specimens having total counts
not higher than 1 X 106 could be accepted.
For the dried commercial product, beside the total count
and the search for pathogens, upper limits should be
fixed for Enterobacteria, Pseudomonas, Staphylo-
coccus and Streptococcus. While faecal coliforms should
be absent in 1 g of dried product, among total Entero-
bacteria, it could be advisable to count separately the
E. agglomerans species. As reported in the last edition
of Bergey's Manual of Determinative Bacteriology
(1984),E. agglomerans is not easily distinguishable or
can be synonymous with Erwinia herbicola, a largely
epiphytic micro-organism (Abdelnoor V. et al. 1983)
which lives on plant surfaces or on lesions caused by
plant pathogens. It has been proposed to designate as
E. agglomerans the strains isolated from human or
animal sources (Ewing W.M. et al. 1980)and as Erwinia
those of phytopathological interest.
To count Erwinia herbicola (as E. agglomerans) among
total Enterobacteria could result in an error, because
this species, according to our results also, accounts for
a large percentage of the gram-negative population
present on leaves. Moreover, a strong reduction in
Erwinia, with respect to the fresh material, could indi-
cate a correct drying procedure for the reduction of the
epiphytic flores.
Among Pseudomonas it would be advisable to distin-
guish between phythopathogens and pathogens for
humans (e.g. Pseudomonas fluorescens and Pseudo-
monas aeruginosa) which should be respectively less
than 1 X 103/g and lO/g.
Among spore-formers, the search for Bacillus cereus
and Clostridium perfrigens should be limited to
those products to be used in preparations for
oral use, with the same criteria adopted for food ana-
lysis.
O PHARMEUROPA Vol. 6, N" 1, March 1994
 Scientific Notes
~

I REFERENCES

i
Salunke, D.K. Developments in Technology of
Storage and Handling of Fres Fruits and vegeta-
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Andrews, J.N., Kenerley C. . The effects of a

11) Geeson, J.D. The fungal and bacterial flora of
stored white cabbage. Journal of Applied Bacte-
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12) Bovallius, A., Bucht ,B., Roffey, R., Anas, P. Three-

pesticide program on epiphytic micro-
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24, 1058 (1978).
Frazier' w.c.,
Mac Graue Hills B.C., 194
Romond, Ch.
Graham, D.C., Quin, C.E.,
tive studies on the
nia carotouora
year investigation on the natural airborne bacterial
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Microbiol. 35,847 (1978).
Microbio'ogy 13) Lenoble, M., Fourniat, J., Bourlioux, P., Paris, M.,
Maghami, P., German, A. Contrôle de la qualité
microbiologique d'échantillons de la Mentha
piperita de diverses origines géographiques.
Annales Pharmaceutiques Françaises 38, 333
(1980).
L.F. Quantita-
14) Negretti, F. Ricerche microbiologiche sulla
contaminazione microbica dei prodotti fitoterapici.
Boll. Chim. Fram. 122, 440 (1983).

Weeb, T.A., Mundt, J.O.
the time of harvest.
35,655 (1978).
Robinson, I.,
on vegetables at 15) Hitokoto, H., Morozumi, S., Wauke, T., Sakai S.,
Microbiology Kurata, H. Fungal contamination and mycotoxin
detection of powdered herbal drugs. Appl.
Enuiron. Microbiol. 36,252 (1978).
16) Cingolani, E., Aureli, P. Carica batterica delle piante
medicinali: aspetti legislativi. Notiziario U. TI.
FAR. 5, 15 (1983).

Flannigany B. Hui, occurrence Of 17) Williams & Wilkins, Baltimore. Bergey's. Manual
aflatoxin-producingstrains of flavus in of systematic bacteriology 1, 409 (1986).
the mould floras of ground

I
Applied Bacteriology 41, 4 1 (1976).
18) Abdelnoor, A.M., Batshoun, R. Roumani, B.M.
Andrews, J.H., Kenerley, C.M., Nordheim V. The bacterial flora of fruits and vegetables in Leba-
Positional variation in Phy loplane microbial non and the effects of washing on the bacterial
populations within an apple tr e canopy. Microb. content. Zbl. Bakt. Hys. 177, 342 (1983).
Ecol. 6, 71 (1980).
19) Ewing, W.H., Farner, J.J. III, Brenner, D.J.
10) Speck, M.L. Compendium of hods for the Micro- Proposal of Enterobacteriaceae nom. rev. to repiace
biological examination of American Public Enterobacteriaceae Rahn. 1938. Int J. Syst.
Health Association, Bacteriol. 30,664 (1980).

O PHARMEUROPA Vol. 6, NQ1, March 1994 55

Scientific Notes

A TWO-STEP GAS CHROMATOGRAPHIC

A N D CHIRAL LIQUID CHROMATOGRAPHIC M E T H O D FOR
THE SEPARATION O F CHLOR- A N D BROMPHENIRAMINE
MALEATE A N D THE ASSESSMENT OF THE ISOMERIC PURITY
OF DEXCHLORPHENIRAMINE MALEATE

L. Borka and A. Lönmo(l)

A monograph on chlorpheniramine maleate was pub-
lished in the European Pharmacopoeia in 1985(NQ386).
Since it is a racemate and the only member of the
pheniramine group published, there was no need for a
test for angle of optical rotation or a test for separation
from other pheniramines. In 1993, however, a draft
monograph on racemic brompheniramine maleate was
published in Pharmeuropa [i].Furthermore a mono-
graph on dexchlorpheniramine maleate is in prepara-
tion.
During preparatory work for dexchlorpheniramine, it
became apparent that the melting points, the IR and
UV spectra and the results of the other identification
tests for chlor- and brompheniramine maleate were very
similar. The introduction of a test for chlorine and bro-
mine might identify the two substances. The melting
point of dexchlorpheniramine maleate is lower than the
melting point of the two racemates, but the other iden-
tification tests again yielded similar results. A test for
specific optical rotation further identified the dextro-
compound, although a small amount of the levo-com-
pound can be present without influencing the results.
Chiral HPLC
A 10 cm Chiralpak ADO (Amylose derivative) column
was used at ambient temperature for the chiral separa-
tion of the dextro- and levo-isomers, with n-hexane/
2-propanol/diethylamine (490/10/1.5) as a mobile
phase and UV detection at 254 nm. The maleic acid
was removed by dissolving the substance in water, add-
ing a few drops of concentrated ammonia and extract-
ing the brompheniramine with chloroform. The chloro-
form extract was injected.
RESULTS
First analytical step: Good separation of the pheni-
ramines by gas chromatography and, using chemical
reference substances, good identification can be
achieved. (Fig. 1).

In order to introduce a fast and clear-cut identification

of these compounds, a two-step chromatographic analy-
sis was proposed. For the racemic chlor- and brompheni-
ramine pair, a gas chromatographic method was intro-
duced in the draft monograph of brompheniramine
maleate. (The existing monograph of racemic chlor-
pheniramine maleate should be revised accordingly).
Actually two gas chromatographic methods of the USP
XXII for the pheniramines were combined [2]. A chiral
liquid chromatographic method is proposed for the as-
sessment of the isomeric purity of dexchlorpheniramine
maleate.

EXPERIMENTAL

Gas chromatography
The analysis was carried out on a 2.3 m X 2 mm
o 414
minutes
column filled with 3%OV 17 on ChromosorbO W AW-
DMSC at 105 OC.Injection port and detector were at Figure 1
250 OC. It was not necessary to remove the maleic acid. Gas ch roma tographic separa tion
of pheniramine maleates
The substances were dissolved in methylene chloride.

(1) The Norwegian Medicines Control Authority, Sven ORedals vei 6, 0950 Oslo, Norway.

56 O PHARMEUROPA Vol. 6, NP1, March 1994

 Scientific Notes

Second analytical step: Chiral liqi 3 chromatography REFERENCES

separates the R- and S-isomers anc
ure of the isomeric ratio or isomer
ample, the separation of a mixture 94.2 Per cent (S)- (2) United States Pharmacopeia, XXII Edition, United
dexbrompheniramine and 5.2 per ent (R-)-levobrom- States Pharmacopeia1 Convention Inc., Rockville
pheniramine is presented (Fig. 2). MD. USA, pp. 189 and 209 (1990).

Waters
O 10 20 30 minutes
I . . . . I I . . < . 1 . . . . 1 . . . , 1 . . . . 1 . . . ,

MI
(S)-dex

........................

SOLVENT

Figure 2
Chiral LC s 3aration of the stereo-isomers of brompheniramine

DECONTAMINA- ION OF A SOLUTION OF ENDOTOXIN

BY R, DIATION AND BY HEATING
- A TRIAL EXPERIMENT -
J.W. /an der Laan(1) and B. van Klingeren(2)

INTRODUCTION to establish zero-growth (as the ultimate purpose) and

the stability of the endotoxin (both with the LAL-test
At the 15th meeting of expert grc p dealing with the and the Rabbit-pyrogen test). We should like to present
LAL test, ampoules containing the 'irst Biological Ref- the results because they may be useful for those who
erence Preparation for Endotoxin ere reported to be want to remove contamination with endotoxin from e.g.
contaminated with Pseudomonas ialtophila and two medical appliances.
other species of micro-organisms. 1 mce the European
Pharmacopoeia Commission decidc to replace this ref-
erence preparation by a new one free from bacterial MATERIALS AND METHODS
contamination.
Ampou les
We have used the First Reference Preparation to ex-
plore the possibility of decontamini on by non-invasive Forty ampoules were received from i..s Secretariat of
methods, without influencing the ndotoxin potency. the European Pharmacopoeia for the decontamination
Initially gamma radiation was studi 1 and subsequently by gamma radiation. For the heat sterilisation, another
heating in a water-bath. Experime s were carried out 20 ampoules were received.

(1)Laboratory for Medicines and Medical Devic , National Institute for Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The
Netherlands.
(2) Laboratory for Bacteriology and Antimicrc al Agents, National Institute for Public Health and Environmental Protection, P.O. Box 1, 3720 BA
Bilthoven, The Netherlands.

O PHARMEUROPA Vol. 6, N" 1, M ch 1994 57

Scientific Notes

Gamma radiation made to a concentration of 2 EU/ml. This concentra-

Radiation was carried out by GAMMASTER B.V. (Ede,
The Netherlands) in two experiments. Experiment 1:
nominal value: 5, 10 and 15 kGy, each 5 ampoules.
Experiment 2:nominal value: 2,3,4 kGy, each 5 am-
poules.
Water-ba th
A standard laboratory water-bath was used, thermo-
statically set at 80 OC. This temperature was validated
with a calibrated Hg-thermometer.
Standard
USP standard EC5 2 EU/ml.
LAL test
A gel-clot test was carried out according to the EP using
Pyroquant Lot NQ 99-84-427with a sensitivity of
0.125EU/ml.
The ampoules contained 1 ml of endotoxin solution.
After opening 0.1 ml portions were sampled for micro-
biological testing. The ampoule was then vortexed for
5 min. Nominal content: 1000 EU/ml. Dilutions were
tion was used as the highest concentration on the test
plate. The EC5 (2500EU/ml) was also diluted down to
2 EU/ml. Each ampoule was tested in duplicate. All
standards (EC5) showed the expected concentration,
2 EU/ml.
Rabbit pyrogen test
This test was carried out as described in the European
Pharmacopoeia Rabbit Pyrogen Test (V.2.1.4) except
that only three rabbits were used per sample.
Microbiologica I testing
From each ampoule 0.1 ml portions (undiluted and 10-
fold diluted) were plated out on CSA agar. Incubation
was carried out at 24 "C and 32 OC. Colonies were
counted after 48 hours.
RESULTS
DECONTAMINATION BY RADIATION
The treatments and the results of the microbiological
and endotoxin test may be summarised as follows:

Table 1 .- Decontamination by gamma radiation

1
Bacterial count Endotoxin level
ampoule Radiation in kGy cfu * 10.000/ml

o
nominal real ~ 1; (meanhange)

~ lloc EU/ml

1-5 1.30
(1.2-2.1) (1.6-2.1)
I I I

6-10 5
I I no viable bacteria 0.013

11-15 10 I I no viable bacteria 0.013

16-20 15 I l no viable bacteria O

21-25 O 33.44 2.44 1.90

(1.8-85) (2.1-2.9)

26-30 2 I 2.42 1 no viable bacteria o. 19

31-35 3 no viable bacteria 0.15

36-40 4 4.64 no viable bacteria O

Thus, radiation (5-15kGy) destroyed the endotoxin. Using a dose of 2-3kGy, the endotoxin was strongly decreased
but not totally absent.

58 O PHARMEUROPA Vol. 6, N Q 1, March 1994

 Scientific Notes

Rabbit pyrogen test These data indicate that heating to 80 OC even for only
3 min leads to the bactericidal effect which was desired,
The contents of the ampoules after microbio-
whereas the endotoxin activity appears to be unaffected.
logical and LAL-testing, were
A definite proof of the stability of endotoxin is difficult
dose of 20 EU/ml, 1 ml/kg
using the gel-clot test because of the large confidence
three rabbits as described in
interval which is a result of the quantal response.
poeia Rabbit Pyrogen Test
Endotoxin is known to be a heat stable compound in
ampoules 1-5 and 21-25
however.
whereas all radiated samples
was concluded that the
confirmed the LAL-test.

OVERALL CONCLUSION
DECONTAMINATION BY HEAT 1
Heat decontamination was carried out in a water-bath The data on the influence of radiation on endotoxin
at 80 OC. The incubation lasted 3 to 6 min. content confirm earlier findings by Koppensteiner et al
(1976) and Czsako et al (1983) indicating that this
method may be useful to eliminate the bacterial contami-
Table 2. - LAL iest nation on endotoxin reference preparation.

control 3 min 5 min 6 min Obviously, the method is not useful to decontaminate an
endotoxin reference preparation. In contrast, heat treat-
ampoule EU/ml EU/ml EU/ml EU/ml ment in a water bath at 80 OC appears to be useful in
removing bacteria without influencing endotoxin con-
tent.
0.125 0.125 0.25 0.125

0.125 0.25 0.125 0.25 It has to be emphasised that the data are no longer
relevant for the First Endotoxin Reference Preparation,
since this standard is no longer distributed. However, the
0.25 0.25 0.25 0.125 experiments gave information on the differences between
two approaches to the decontamination of parenterals.
0.125 0.125 0.125 0.125

0.125 0.125 0.125 0.125

Microbiological test
From each ampoule 0.1 ml portio (undiluted and 10- REFERENCES
fold diluted) were plated out on agar. Incubation
was carried out at 32 OC. counted after
72 hours. 1) Czsako G., Elin R.J., Hochstein H.D., Tsai C.M.
Physical and biological properties of US standard
endotoxin EC after exposure to ionising radiation.
Table 3. - Microbiol&cal test Infect. Immun. 41, 190 (1983).

ampoule
control

cfu/ml

1.8 * 104
3 min

cfu/ml L 5 min

cfu/ml
6 min
cfu/ml
2) Koppensteiner G., Kruger D.,Osmers K.,Pault W.,
Woog H., Zimmerman G. An experimental investi-
gation of the elimination of pyrogens from parenteral
medicines. Drugs Made Germ. 19, 113 (1976).

1.7 * 104

1.5 * 104

2.2 * 104
Acknowledgement
G. Weick and W. Puilen gave valuable technical
1.7 * 104 - ((10) assistance.

O PHARMEUROPA Vol. 6, NQ1, h, rch 1994 59

International Workshop on Somatropin
INTERNATIONAL WORKSHOP
Standardization and labelling of somatropin
Report of an International Workshop
on assays, standardization and
labelling requirements of somatropin
AF Bristow, D Schulster and SL Jeffcoate(l)
I . INTRODUCTION
Dose regimens of pituitary growth hormone, adminis-
tered to children for the treatment of hypopituitary
dwarfism, were described in international units, the unit
of growth hormone activity being measured by in vivo
bioassay in hypophysectomised rats. With the introduc-
tion of recombinant-DNA-derived growth hormone
(somatropin), in the early 1980's, this practice was
continued in many markets, including Europe and Ja-
pan. As a consequence, the therapeutic use of
somatropin depended on the continued widespread use
of the in vivo bioassay, a procedure known to be costly,
invasive and imprecise.
In 1988, the National Institute for Biological Standards
and Control, in collaboration with the European Phar-
macopoeia, initiated a collaborative study designed to
investigate whether the in vivo bioassay could, with
security, be removed from the routine batch control of
therapeutic somatropin (Pharmeuropa, March 1991,
vo1.3, special issue; human growth hormone; AF. Bristow
and SL. Jeffcoate, Biologicals, 1992, 20, 221-231). In
this study participants were provided with normal, and
variously degraded somatropin preparations, and re-
quested to analyze them using physicochemical tech-
niques in comparison with the in vivo bioassay. Partici-
pants in the study, and other representatives of manu-
facturers and National Control Authorities met at an
International workshop in July 1990, at the National
Institute for Biological Standards and Control, which
concluded that:
- the in vivo bioassay for somatropin could be re-
moved from the routine batch control with security.
- to support the international change to assaying
somatropin in mass units, an International standard
for somatropin should be established.
- the collaborativestudy of the proposed International
Standard should determine:
1.the ampoule content in mg
2. the specific biological activity in I.U./mg
3. the specific UV absorbance
(1) The National Institute for Biological Standards and Control, South Mimms, UK.
60
Since 1990 there have been a number of consequences
of this initiative. The collaborativestudy described above
has been carried out, and forms the basis of the present
workshop. In addition to this collaborative study, the
Ph. Eur. Monograph for Somatropin with no in vivo
bioassay has been drafted and adopted. The last two
years have also seen the approval within the EEC of
variations to delete in vivo bioassay from release speci-
fications for various preparations of somatropin.
The imminent availability of an internationally available
standard for somatropin calibrated in mass units has led
to calls for a further re-evaluation of the standardization
and expression of potency of somatropin, in particular
to include a discussion of whether it is appropriate to
consider changing the labelling of therapeutic somatropin
preparations in Europe, Japan and other markets from
International Units to mg. In September 1993 partici-
pants in the collaborative study on the International
Standard for somatropin, together with representatives
of manufacturing industry, National Control Authorities
and clinicians met at an International Workshop at The
National Institute for Biological Standards and Control,
to discuss the following agenda:
i) The collaborative study of the proposed Interna-
tional Standard for somatropin, 88/624, and the
recommendations that should be made to the World
Health Organization concerning the ampoule con-
tent, the specific biological activity and the purity.
ii) The present and future status of biological assays
and biological identity tests in control of soma-
tropin.
iii) Possible future revisions to the analytical specifica-
tion for somatropin, including the introduction of
new analytical methodology.
iv) The proposed future conversion from labelling
somatropin in International Units to labelling in
mg, and possible strategies for effecting this change.
O PHARMEUROPA Vol. 6, N" 1, March 1994
 International Workshop on Somatropin
~

2. THE CO4LABORATIVE STUDY O F THE PROPOSED

INTERNATIONAL STANDARD FOR SOMATROPIN, 88/624

2.1 Interim report of the collaborative study a) That the preparation coded 88/624 be established
as the International Reference Reagent for

i
The purpose of this study was to c aracterize a prepa-
ration of somatropin as a candidat WHO International
Standard. This would provide a r ference material for
physicochemical tests of identity a d purity and an in-
ternationally agreed figure for its s ecific biological ac-
tivity. This would provide a mean of relating mass or
molar units to the biological unit of activity of the
current International Standard (IS) of human pituitary
origin.
Somatropin (recombinant DNA-derived human
Growth Hormone).
b) That the preparation 88/624 be assigned a defined
ampoule content of 2.0 mdprotein per ampoule.
c) That the following additional information be pro-
vided:
- that the preparation coded 88/624 has an EI%

design of the study: L

The followinganalytical objectives ere included in the
-
at 276nm of 8.21, and
that the specific in vivo biological activity is con-
sistent with a value of 3 IU/mg for the specific
i) to measure the activity of
bioassay in terms of Units of the biological activity of somatropin.
pituitary derived IS
2.2 Report of the Discussion and consensus-
ii) to measure the mass of the ampouled forming session on the collaborativestudy
candidate preparation in absolute of somatropin (88/624)
terms and to determine
ficient. 2.2.1 Proposed defined protein content of 88/624
iii) to assess the purity of the mpouled candidate The proposed defined protein content of 2.0mg/am-
preparation of somatropin an its stability in order poule was considered acceptable, and no manufacturer
to determine its suitability as International ref- reported that its in-house standardization would be in-
erence material. consistent. Dr Riggin (Eli Lilly) felt that, although the
proposed protein content was acceptable, he still had

i
A complete report of the collabo ative study, and its reservations concerning the use of amino-acid analysis
conclusions and recommendation is to be published to quantify a preparation containing glycine and
elsewhere (Bristow,Gaines-Das, Je fcoate and Schulster, mannitol, and that the possibility ought to be recognised
1994). that data may subsequently become available which will
necessitate re-evaluation of the figure.
In summary, it was reported to thc? workshop that:
2.2.2 Definition of somatropin content of 88/624
1. Determination of the protein of 88/624 by
quantitative amino-acid It was noted that the collaborative study had yielded a
a mean estimate of reasonably precise figure for the % monomer, deter-
a relative standard mined by SE HPLC. Since this procedure is currently
used as the assay in most regulatory areas, it would, in
2. The mean (and fiducial in viu0 biological principle, be possible to define the somatropin content
activity (10 laboratories) (6.2- of 88/624, as 2.0 x % monomer/100. Most speakers
7.3). In vitro bioassay cautioned against this step, and it was recognised that
the figure would inevitably depend on the methodology
receptor assays tended employed. Ms Rabouhans (BP)noted that whilst it would
be inappropriate for the primary, WHO standard to be
3. The overall study so defined, secondary, regional standards such as the
(276nm, assuming EP standard could be established with a defined
8.21, with a somatropin content, they would be used with a pre-

m
4. The mean % monomer, dete mined by high-per-
formance size-exclusion chro atography (HP SEC,
10 laboratories) was 97.2% k 0.8.
cisely defined assay method. The monomer content of
88/624, derived from the collaborative study, will be
offered as additional information.

c
Based on the presented data, the ollowing provisional
recommendations were put to the orkshop to form the
basis of the discussion:
2.2.3 Biological activity of 88/624
It was noted that the mean in vivo biological potency,
obtained using the two compendia1bioassays (tibial width

O PHARMEUROPA Vol. 6, Ne 1, darch 1994 61

internationalWorkshop on Somatropin
and weight gain) was 6.7 IU/ampoule. This figure, to-
gether with the defined ampoule content of 2.0mg/
ampoule, yielded a derived specific activity of 3.35 IU/
mg protein for the somatropin preparation in 88/624.
Separately derived estimates have ranged from 2.6 to
3.3IU/mg, with most current estimates being around
3.0, and this had lead to the earlier suggestion that 88/
624 should be assigned a specific activity of 31U/mg
(one Significant figure), in order to avoid any incompat-
ibility, and that this should only be offered as additional
information. This proposal was put to the workshop.
Professor Morimoto (NIHS,Japan) argued that 88/624
should have a formally assigned biological activity based
on the results of the study, for the following reasons:
i) Measurement of biological activity was one of the
primary aims of the study; and the large body of
bioassay data obtained in the study should be re-
corded.
ii) The need for an International Somatropin Stand-
ard for bioassays will not disappear.
iii) Assignation of potency should be based on the
data obtained in the study.
iv) That the derived specific activity of 3.35IU/mg
was not very different from values reported by
manufacturers, which are in the range 3.0-3.2
IU.mg.
V) The proposal to assign a value of 31U/mg is based
purely on practical and political reasons and would
be unacceptable.
3. THE PRESENT AND FUTURE STATUS OF BIOLOGICAL ASSAYS
AND BIOLOGICAL IDENTITY TESTS IN CONTROL OF SOMATROPIN
3.1 The Determination of the Potency
and/or Bioidentity of Somatropin
Dr D. Parikh, Genentech
This presentation focused on the replacement of the in
uivo rat weight gain bioassay for biological activity and
bioidentity of somatropin with acceptable in vitro alter-
natives.
The determination of biological activity as a measurable
physiological response in living organisms was neces-
sary for highly impure biologicals and blood products.
However, the determination of biological activity of
highly purified and well-characterized recombinant
proteins, whose impurities are also well characterized,
should be possible by in vitro biochemical or
physicochemical assays validated against in uivo
bioassays.
62
This viewpoint was supported by Dr Yokoya (Toranomon
Hospital, Japan), who considered that the international
collaborative study was a scientifically sound exercise
which may never be repeated, and to assign a value to
1 significant figure would lose the benefit of the work.
Dr Yokoya also noted that in the event of an analogue
of somatropin being developed as a therapeutic agent,
it would be necessary to have an International Standard
for bioassays available and it would not be appropriate
to return to the pituitary standard.
On the basis of these arguments, the meeting reached
a clear consensus that 88/624 should have a formally
assigned a biological activity of 6.7International Units
per ampoule.
It was suggested that WHO should also be informed
that this figure is not inconsistent with widely used
conversion factors of around 3.0 IU/mg for somatropin,
and should be asked to make such a statement in its
formal report in order to avoid any regulatory authori-
ties concluding that a product having a specific activity
of 3.0IU/mg is inconsistent with the WHO International
Standard.
2.2.4 UV absorbance
That the proposed assigned value for Al% of 8.21, to
be offered as additional information, was considered to
be acceptable.
It was agreed that the draft report of the collaborative
study should be revised in view of the consensus reached
by the meeting on each of the above topics, before the
final document was submitted to the Expert Committee
on Biological Standardization of WHO.
The determination of biological activity by the widely
accepted classical hypophysectomized rat weight gain
bioassay is accurate but highly imprecise. It requires
several replicate analyses to obtain an accurate determi-
nation of biological activity. Moreover, it is extremely
costly, time-consuming, and requires sacrifice of ani-
mals. For these reasons, several in vitro alternatives are
currently being considered to replace the rat weight gain
or rat tibia width assays. Physicochemical assays such
as reversed-phase high-performance liquid chromatog-
raphy (RPHPLC) and high-performance size-exclusion
chromatography (HPSEC),and more biomimetic assays
such as high-performance receptor binding chromatog-
raphy (HPRBC) and cell proliferation assays have been
proposed or are under development.
A chromatography-based in vitro assay that combines
the accuracy and precision of size-exclusion chromatog-
raphy with the specificity of receptor binding was pre-
sented as a modern alternative to the rat weight gain
O PHARMEUROPA Vol. 6, N" 1, March 1994

~
bioassay. The theoretical
ance receptor binding
on the recently
using fluorescence detection.
The HPRBC assay was shown t
specific for active forms of gro
forms of growth h
specific mutants of growt
ing sites to the receptor, were
of the complex. The specificity
was demonstrated by performin
dissociation constant (Kd)of the
4 x 10-10~). Excellent correi
gain bioassay. Deamidat
and oxidized and thermal1
to be fully potent both by
rat weight gain bioassay w
considerable loss of potency
des-Phel-Pro2-hGH. It was
ance size-exclusion chromato
assay whereas the rat
of relative potency o
surprisingly, showed
the HPRBC assay was c
gain bioassay with t
effective and noninvasive.
A cell proliferation assay
Genentech was also
to the rat weight
proprietary cell
In conclusion, the HPRBC assay
tion assay were presented as
the hypophysectomized rat
HPRBC assay was shown
both for biological activity
international Workshop on Somatropin
3.2 Biological assays and identity tests for
somatropin: report of discussion and con-
sensus-forming session
3.2.1 The bioidentity test for somatropin
The meeting noted the standpoint of the FDA, that the
bioidentity test (essentiallyan in vivo bioassay with fewer
animals and a lower statistical precision) should remain
a requirement for somatropin. Dr Riggin explained the
basis of this requirement. In the CSFR ‘points to con-
sider’ document, point 6.10, it specifically states that a
biological will be tested by an in vivo or in vitro bioassay.
Even though somatropin is not treated as a biological in
the USA, it has been decided that these criteria will be
applied to somatropin, and therefore it remains neces-
sary to apply some sort of growth promotion test to
somatropin preparations. In support of this standpoint,
several speakers emphasised that somatropin is a com-
plex macromolecule, and physicochemical techniques
alone cannot guarantee the absence of all possible
mutations exhibiting altered biological activity. Whilst
wer area counts recognising this reservation, the meeting was essen-
tially unanimous in agreeing that the bioidentity test is
unlikely to detect forms of somatropin having altered
biological activity which are not detected by the battery
of physicochemical tests currently employed. Professor
Morimoto presented results of a Japanese collaborative
study comparing physicochemical and biological analy-
sis of variously degraded somatropin preparations, and
concluded that physicochemical assays alone can guar-
antee the security of somatropin preparations.
In summary, the majority of speakers at the meeting
agreed that there is no available scientific evidence to
suggest that any additional security would be gained by
the inclusion of a bioidentity test. Both the FDA and the
USP however remain committed to the concept of the
bioidentity test, although it was noted that this need not
necessarily be an in vivo procedure, and a properly
validated in vitro method could be acceptable.
The meeting also considered the future development of
bioassays for somatropin, and noted a divergence in
approach between the United States and other regula-
tory authorities. Whilst the European Pharmacopoeia is
satisfied that a bioassay is not required for this product,
the USP appears committed to the introduction of an
r development at in vitro receptor assay, based on receptor dimerisation
possible alternative measured by SE HPLC, as described by Dr Parikh. It
is assay utilizes the was noted that this assay is based on a cloned receptor
a myeloid cell line which has not yet been made available to other labora-
however, tories, and has not therefore been validated between
laboratories. A number of speakers expressed concern
as to the future international availability of this reagent
should this procedure be adopted in the USP. For the
USP DR Feyns noted these concerns, but felt that it
should be possible to find a solution to the practical
the cell prolifera- aspects of distributing a single source reagent.
In summary, although there is at present insufficient
information available to reach a consensus on the appli-
cability of the proposed USP assay for somatropin, there
International Workshop on Somatropin

was near unanimous concern at the approach being
taken.
The meeting finally considered the question of applica-
tion of bioassays to stability testing. It was noted that a
general response to regulatory authorities to application
to vary product licenses has been to require full bioassay-
based stability testing, even for relatively minor vari-
ations such as altered vial contents or even altered
labelling. There was unanimous agreement that such
biological stability testing is often not justified, and
the meeting recommended that regulatory authori-
ties should give very careful consideration when re-
quiring additional bioassay based stability testing.

4. THE CURRENT ANALYTICAL SPECIFICATION FOR SOMATROPIN

A N D POSSIBLE FUTURE DEVELOPMENTS

4.1 Analytical methods for the routine batch The current specification in the European Pharma-
control of somatropin copoeia for somatropin for injection is summarised
Dr P. Gellerfors, Kabi Pharmaceuticals below.

TESTS LIMITS

IMPURITIES:

Host cell derived proteins

Host cell and vector derived DNA

IDENTIFICATION:

Isoelectric focusing migration rel. stand.

RP-HPLC migration rel. stand.
Tryptic mapping migration T-frag. rel. stand.

PURITY:

Absorbance (A276nm, 0.1%) 0.72-0.88

Related proteins (RP-HPLC) less than 13.3%
Dimers and sub.of higher mol.mass 6%
Isoelectric focusing (deamidation) no band apart from major band
more than 5 %
Water 10%

ASSAY:

Size-exclusion chromatography 89% - 105%

OTHER:

Sterility sterile
Bacterial endotoxin Not more than 2OIU/mg somatropine

(1)Limit approved by relevant authority

64 O PHARMEUROPA Vol. 6, N" 1, March 1994

 international Workshop on Somatropin
~

b
In considering future improvemen s to this analytical
specification we may consider a umber of possible
Figure 1
developments, summarised below.

PURITY: SDS-PAGE/Silver Istain Column Asahipak ODP, 250 X 4mm, HP

Advantage:

d
Sensitive method o detect all
polypeptides pres nt in the
sample of both h H and host origin
Mobile phase A

Mobile phase B
35.5mM Tris HC1, pH 8.5, 20%
1-propanol

35.5mM Tris HC1, pH 8.5, 40%

1-propanol
ASSAY: HI-HPLC
RP-HPLC Flow rate 0.5 ml/min
Improvement: Quantitation of somatropin will be Temp +55 c
based on the nativemolecular form
and excluding other hGH variants Inj vol 251.11
such as:
- oxidized hGH Detection 210nm
- deamidated hGH
- cleaved hGH Components: RT (min)
- des Phe hGH Oxidised GH 18.5
- hGH dimers low.mol.wt GH 22.5
- polymers and aggregated forms of Clip 2 26.0
hGH
GH 33.O
Variants can Clip 1 41.0

The current pharmacopoeial test r related proteins is Figure 1

a reverse-phase HPLC method at
illustrates the improved RP-HPLC analysis of hGH variants
a refinement of the method Method Kabipharmacia

-
ml: OX.CH, LMWGH, C L I P 2 , C H , CLIP1
4 00-

300-

3
a 200-
E

100-
I

a+==
Ttma (rntn,)

O PHARMEUROPA Vol. 6, N' 1, March 1994 65

International Workshop on Somatropin
4.2 Revision of the analytical specification
for somatropin; report of discussion and consen-
sus-forming session
As an introduction to the discussion of this topic it was
noted that the adoption of EP monograph for somatropin
by the EP Commission had been subject to an undertak-
ing to begin immediately a process of revision of the
monograph to bring the analytical methodology in line
with the most recent developments. During the discus-
sion therefore each of the pharmacopoeial analytical
methods covered by Dr Gellerfors in his presentation
were considered by the meeting with a view to possible
future refinements and revision. In summary, the follow-
ing conclusions were reached:
4.2.1 The current statement on DNA contamination
in the Production section need not be changed.
4.2.2 Although a value for A I % (276nm) for
somatropin has been derived from the collaborative study,
it was felt that this test is of little value, and there was
agreement that it need not be re-introduced into the
monograph.
4.2.3 The reverse phase HPLC procedure was con-
sidered to perform adequately, and although alternate
methodologies have been described, it is not clear that
the improvements in performance are significant enough
at this stage to warrant adopting a different procedure.
There was agreement however that the current proce-
dure adopted by the EP for method validation could be
improved. Limited oxidation with hydrogen peroxide is
a difficult reaction to control precisely, and it was sug-
gested either that a reagent is made available by the EP,
5. THE PROPOSED FUTURE CONVERSION FROM LABELLING SOMATROPIN
IN INTERNATIONAL UNITS TO LABELLING IN mg,
AND POSSIBLE STRATEGIES FOR EFFECTING THIS CHANGE
This topic was introduced by Dr Jens Fogh, of Novo/
Nordisk, who outlined some of the problems and pos-
sible consequences of making the change, and also
presented a possible strategy for effecting this change in
labelling. The subsequent discussion centred on four
main aspects of the question.
5.1 The current labelling situation for thera-
peutic somatropin preparations
The workshop noted that in the United States,
somatropin is labelled in mg, and has never, since the
introduction of the drug onto the market, been labelled
in units. In Europe, before January Ist 1994, labelling
of somatropin is in International Units. After January
1st 1994, when the somatropin monograph comes into
force, the labelling statement will require that the label
66
~
or that deamidation (an easier reaction to control) is
used. Notwithstanding these comments however, the
meeting noted that there is some evidence of different
performance in different laboratories when using this
test, and some further studies to improve the reproduc-
ibility appear to be indicated.
4.2.4 The peptide mapping test was also felt to be a
powerful tool, and not to require significant alteration.
There was some debate as to whether, when interpret-
ing the chromatograms obtained, the analyst should be
required to consider peak heights as well as peak reten-
tion times. Although no clear conclusion was reached,
it was agreed that this point should be re-examined
when revising the monograph.
4.2.5 Introduction of new analytical methodology. It
was noted that, during revision of the monograph, the
opportunity exists to introduce a new analytical proce-
dure, based on a different mechanism of separation.
Three such procedures exist as candidates, hydrophobic
interaction HPLC, high performance ion-exchange
HPLC and capillary zone electrophoresis.Although there
is some experience in individual laboratories with each
of these methods, none have been tested in a multi-
centre study for the analysis of somatropin. It was sug-
gested however that high performance ion-exchange
chromatography does not separate any variants of
somatropin that are not resolved by the current RP
HPLC test in the EP monograph, that capillary zone
electrophoresis remains at present too variable for
consideration as a pharmacopoeial test, and that whilst
hydrophobic interaction HPLC appears to offer some
promise, it needs to be validated in a multi-centre study.
states both the number of mg of somatropin and the
equivalent in international units. In Japan labelling is
in International Units.
Representing the European Pharmacopoeia,
M. Spieser explained to the meeting two implica-
tions of the EP monograph. Firstly, whilst requiring
dual labelling, the labelling statement makes no re-
quirement as to which is to be predominant. A manu-
facturer may continue to label predominantly in Units,
with a small statement on the number of mg, or may
choose to have the predominant label statement in
mg. Secondly, the monograph makes no statement
on the specific activity (or conversion factor) to be
used, and manufacturers may continue to use in-
house estimates of the number of IU/mg for
somatropin when calculating the Unit equivalent. The
O PHARMEUROPA Vol. 6, N" 1, March 1994

second implication is particul
meeting noted that, whilst it
actually necessary to have an
ure on the number of units
labelling change. Despite thi
of speakers expressed the
agreed figure for the conver
and would both facilitate t
mg, and would also help to elimi
differences in mg/unit th
somatropin preparations
the labelling change?
5.2 Is there sufficient justifi ation for making
The meeting recognised that to
belling change would requi
might result in varying d
manufacturer, patient and
to be important to identify the
to assay a substance in
It was also noted that t
material is formulated
in units using an in-h
significant differences
of mg of somatropin that the
arising from the use of differe
it was recognised tha
on a common International Stand
problem. Dr Yokoya presented c
that the differences i
have clinical signific
Professor Morimoto indicated
nese market this
5.3 What problems will nee to be overcome,
or avoided in making change from la-
belling in units to
5.3.1 It was recognised that,
factor of 3IU/mg, many of the
formulations would be
tions (16IU, for
manufacturer.
5.3.2 As a corollary to the
that recommended dose
established for “mg”
numbers of mg.
O PHARMEUROPA Vol. 6, N” 1, M rch 1994
international Workshop o n Somatropin
5.3.3 Although it was recognised that it may not be
necessary for an internationally agreed figure for the
conversion factor for somatropin to be adopted, it was
the opinion of most speakers that the adoption of the
figure of 3IU/mg would both facilitate the transition and
also eliminate variations in the mg somatropin/labelled
unit that exist on the market today.
5.3.4 Leading up to, and during, any transition pe-
riod (see below) there will need to be a process of re-
education, for both clinicians and patients.
5.3.5 Any change, from a “unit” product range to a
C
“mg” product range, must not preclude development of
new products during the transition period. Dr. Rouiller
(Serono) stressed, for example, that current develop-
ment of new products, labelled in units, must not be
rendered obsolete by the introduction of a “mg” product
range.
5.3.6 A “mixed market” situation should be avoided,
should be done. where clinicians are faced with a choice of some manu-
facturers providing products labelled in mg, and some
in units, or where different dose regimens in mg or units
co-exist.
5.3.7 As had been previously noted, the change to a
“mg” product range would require require submission of
new market authorization applications. It was generally
cts in the number agreed by the meeting that, if possible, a facilitated
receives per dose, registration procedure should be adopted by regulatory
ersion factors, and authorities for approval of these submissions. The need
for extensive repetition of clinical trials, toxicology or
stability studies would severely hinder the process.
5.4 A proposed strategy for the change
Dr. Fogh, introducing the topic to the workshop, pro-
posed that the change to labelling only in mg should be
effected in 1998, and that the year 1998 should be seen
as a one year transition period, such that the process of
change is initiated in January, and must be completed
by the end of the year. Companies will therefore have
one year to run down existing stocks, install stocks of
the new product lines, and complete the process of re-
education of clinicians and patients. It should be noted
however that this date was not accepted by all manufac-
turers present, and the topic gave rise to extensive de-
bate, which was not completely resolved by the end of
the meeting.
ming a conversion
5.5 Further action
available “unit”
It was noted that the meeting has no official status, and
was not therefore in a position to formulate a policy to
be implemented. Nonetheless it was agreed that the
proceedings of the meeting should be publicised in or-
der to lay the foundation for a meeting having official
status, to be held in 1994, to formulate and implement
it was also noted strategy for making the labelling change. In particular,
need to be it was felt appropriate that steps should be taken to
bring the report of the workshop to the attention of
relevant regulatory authorities such as CPMP.
I
67
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 ADDRESSES OF THE NATIONAL PHARMACOPOEIA AUTHORITIES
parties to t h e European Pharmacopoeia Convention

C o r r e s p o n d e n c e relating to th t draft m o n o g r a p h s s u b m i t t e d f o r c o m m e n t in t h e p r e s e n t issue are to

b e s e n t to t h e relevant A u t h o r i t y whose a d d r e s s is s h o w n below.

O AUSTRIA 0 ITALY
Bundeskanzleramt Segretario Generale
Sekt. VI (Volksgesundheit) Commissione Permanente della Farmacopea Ufficiale
Radetzkistrasse Istituto Superiore di Sanità
A 1031 WIEN Viale Regina Elena, 299
I00161 ROME
0 BELGIUM 0 LUXEMBOURG (GRAND DUCHY OF)
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Direction de la Santé
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Division de la Pharmacie et des Médicaments
Cité Administrative de I'Etat - Quartier Vésale
Rue CM Spoo, 10
L 2456 LUXEMBOURG
0 CYPRUS 0 NETHERLANDS
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Ministry of Health Pharmacopoeia
CY NICOSIA Rijksinstituut voor geneesmiddlenonderzoek
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et d e la Ville O SPAIN
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O PHARMEUROPA Vol. 6,
(i
N" 1, Mar h 1 9 9 4 71
PHARMEUROPA
March 1994 6.1

READERS' TRIBUNE
Endotoxin Testing in Pharmaceuticals Industry:
An example for an external quality control scheme ................................................. 3

DRAFT MONOGRAPHS FOR COMMENT

Alcohol polyvinylicus .......................................................................... ..... 6
Felodipinum ...................... ................................................................. 1 2
Frangulae extractum siccum .................... 19
Immunoglobulinum humanum ..................................................... 11
Irrigation Solutions ............
Methylprednisoloni hydrogenosuccinas ....................... ................................. 7
Pentamidini diisetionas ................... ........................................................ 10
...................................................................................... 5
Tamponae ................................................................ ................... 5
Ticlopidini hydrochloridum ....................................
...............................

ADAPTATION OF NATIONAL MONOGRAPHS

Bumetanidum ....................................... .................................... 21
Cyclizini Hydrochloride ......................................................
Nitrofuralum ............................................. ........................................ 26
Selenii sulfas .................. ........................................................
Tolnaftatum . .....................................................................

INTERNATIONALHARMONISATION
Harmonisation of Monographs and General Analytical Methods of
USP, JP and Ph. Eur. - A review of the first three years ........................................ 29
A comparison of sterility tests .............................................................................. 36

SCIENTIFIC NOTES
Contamination by heavy metals in drugs from different commercial sources ........... 43
A note on the microbial contamination by medicinal plants ................................... 47
A two-step gas chromatographic and chiral liquid chromatographic method
for the separation of chlor- and brompheniramine maleate and the assessment
of the isomeric purity of dexchlorpheniramine maleate ....................................... 56
Decontamination of a solution of endotoxin by radiation and by heating ................ 57

INTERNATIONALWORKSHOP ON SOMATROPIN .................................... 60

NATIONAL PHARMACOPOEIA AUTHORITIES ........................................... 71

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