JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1986. p. 11-16 Vol. 23, No.

1
0095-1137/186/010011-06$02.00/0
Copyright ©O 1986, American Society for Microbiology

Determinationt of Endotoxin in Injectable Antibiotic Preparations by

the Chromogenic Assay Method Using a Limulus Reagent
(Tachypleus Hemocyte Lysate) and a Chromogenic Substrate
SHIGEO YANO,* YAStJKO HOTTA, AND SAKIKO TAKAHASHI
Department of Antibiotics, National In stitiute of Health, Kainiosaki, Shlinagawi a-kia, Tokyo 141, Japan
Received 11 March 1985/Accepted 28 September 1985

The effects of 50 antibiotics on the detection and determination of bacterial endotoxins by the chromogenic
method using a Limulus reagent (Tachypleus hemocyte lysate) and a chromogenic substrate of p-nitroaniline
derivatives were tested, and the antibiotic concentration for 50% inhibition of the chromogenic reaction in the
presence of 0.5 ng of endotoxin (Escherichia coli 0111 :B4) per ml was estimated. All the antibiotic preparations
were depyrogenized by ultrafiltration treatment before they were subjected to the test. The reaction was
conducted in the presence of a high concentration (0.5 M) of Tris buffer to constantly maintain the pH of the
reaction mixture, and liberated p-nitroaniline was determined by high-pressure liquid chromatography.
Several aminoglycosides (amikacin, bekanamycin, kanamycin, and streptomycin sulfate), bleomycin hydro-
chloride, and fosfomycin disodium showed no inhibition of the reaction up to 20 mg/ml. However, other
antibiotics, including penicillins, cephalosporins, macrolides, and tetracyclines, inhibited the reaction concen-
tration dependently. Polymyxin B sulfate was the most potent inhibitor, with less than 8 ,ug/ml for 50%
inhibition. It was concluded that the chromogenic method can be applied to the detection and determination of
endotoxin in most of the antibiotic preparations. An application of this method to carbenicillin disodium
preparations was exemplified.

Because of high sensitivity of rabbits to pyrogen (4). all
injectable antibiotic preparations have been subjected to the
pyrogen test by rabbits specified in the pharmacopeias (7,
23). Although the pyrogen test is troublesome and expen-
sive, it has been shown to have adequate reliability except in
a rare case (19) in which a trace amount of pytogen contam-
ination could not be detected by the test. The most active
pyrogen is a bacterial endotoxin, lipopolysaccharide (10)
from gram-negative bacteria, Which has high molecular
weights. In recent years, the gelation method (1, 11, 16) and
the chromogenic method (3, 6), which use the Limululis
amoebocyte lysate (LAL) of a horseshoe crab to detect in
vitro trace amounts of bacterial endotoxin, have become
available. Recently it was reported that a polysaccharide,
(1-3)-3-D-glucan (8, 12), activates the LAL reagent as
strongly as does endotoxin, but it is not so common as
endotoxinl, and its contamination in antibiotic preparations is
unlikely. The gelation method, the so-called LAL test, is
simple and semiquantitative (22). On the other hand, the
chrormogenic method is more accurate in determining
endotoxin activity by measuring the concentration of p-
nitroaniline (PNA) which is enzymatically liberated from the
chromrogenic substrate; this enzymatic hydrolysis proceeds
in proportion to the amount of endotoxin present.
In the present study, we tested the effect of 50 antibiotic
preparations on the chromogenic reaction. The results will
be of importance when the chromogenic method is applied to
the determination of endotoxin contaminating the prepara-
tions.
Before the test, we determined the optimal pH for enzy-
matic reaction and the buffer concentration that would
prevent a pH change by antibiotics. Furthermore. some
antibiotics interfere with the spectrophotometric determina-
tion of the liberated PNA and we therefore used a high-pres-
* Corresponding author.
11
sute liquid chromatography (HPLC) niethod to isolate and
quantitate the liberated PNA from the reaction mixtures. In
addition, we used ultrafiltration treatment (18) of all the
antibiotic preparations before the test to remove trace
amounts of possibly contaminated endoioxin.
MATERIALS AND METHODS
Glassware and water. All glass pipettes and test tubes were
depyrogenized at 250°C for 2 h. The water used in this study
was the sterile water for injection recommended by the
Japanese Pharmacopeia (7).
Antibiotics. Commercial, injectable antibiotic preparations
certified suitable according to the Japanese Minimum Re-
quirements for Antibiotics (Ministry of Health and Welfare)
were used. Antibiotic preparations in dry form were recon-
stituted with water. All the antibiotic solutions were of the
highest concentrations and received ultrafiltration treatment.
Antibiotic concentration was represented by antibacterial
potency.
Endotoxin. Commercially available endotoxin (1 ,ug) from
Escherichia c oli O111:B4, obtained from Seikagaku Kogyo
Co. Ltd., Tokyo, Japan. was reconstituted with 10 ml of
sterile physiological saline (0.9% [wt/vol]) at room tempera-
ture with vigorous, intermittent mixing for 5 min with a
vortex mixer and sonicating for 3 min and diluted to the
required concentrations, providing control endotoxin solu-
tions. Each solution was sonicated again to prevent adsorp-
tion of endotoxin on a test tube before use. Endotoxin
activity of the control solutions was almost constant for 6 h
at room temperature.
Reagent for the chromogenic method. Pyrodick, the am-
poule kit of the chromogenic reagent composed of hemocyte
lysate from the Japanese horseshoe crab, Tachvpleius
triidentatius, that has the same biochemical properties as
LAL (15) and a chromogenic oligopeptide substate (tert-
butoxycarbonyl-Leu-Gly-Arg-p-nitroanilide) was obtained
12 YANO ET AL.
1.2
E were always performed in parallel.
c Liberated PNA in the acetic solution was assayed by
09
0ciU) 0.6
D
.40
c B, water-acetonitrile [1:1]; C, water-dioxane [4:1]) was ap-
nC
0.3 1
Ar- a I
0.0
40 50 60
Incubation Time (min)
FIG. 1. Time course of the chromogenic reaction in the presence
of 0.5 rig of endotoxin (E. coli O111:B4) per ml with Tris buffer (pH
8.0) of various concentrations at 37°C. Symbols: *, 0.05 M; 0, 0.25
M; *, 0.5 M; A, 0.75 M; A, 1.0 M.
from Seikagaku Kogyo Co. Ltd. The same lot of reagent was
used,in a serial inhibition test.
Tris buffer. Tris hydrochloride solutions of various con-
centrations containing 10 mM 0gCl2 were made with adjust-
ment to the required pH values at 22°C with 4 N HCI. Each
buffer was stored in sealed ampoules after ultrafiltration.
Endotoxin activity in buffers without ultrafiltration treat-
ment could not be abolished completely even at 120°C for 6
h.
Measurement of endotoxin. Ultrafiltration treatment was
done by using a Diaflo-cell (Amicon Far East Ltd., Tokyo,
Japan) of the stirring type (model 12) and a Diaflo-membrane
(PM 10; Amicon) with a fraction molecular weight of 10,000
and a diameter of 25 mm; the surface of the membrane was
washed with water after it was soaked in 0.1 N NaOH
overnight. After the cell was assembled, 10 ml of water was
charged and filtered under the pressure of nitrogen gas (3
kg/cm2). This procedure was repeated until no alkali in the
filtrate was detected by a pH test paper. Then 1 ml of the
control endotoxin solution (2 nglml) was charged and fil-
tered; the filtrate should contain less than 0.05 ng of
endotoxin per ml. After this was confirmed, Tris buffers and
antibiotic solutions were ultrafiltered; 2 ml of the first
fraction of the filtrate was discarded, and the following
fractions were collected; test solutions were made by mixing
control endotoxin solutions with these antibiotic solutions.
The contents ini each Pyrodick ampoule was reconstituted
with 0.1 ml of Tris buffer in an ice-water bath, and then 0.1
ml of either a test solution or a control endotoxin solution
was added to each ampoule. The ampoules were swirled
gently by a vortex mixer and placed in a water bath at 37°C
for the required period and cooled rapidly again in the
ice-water bath. A 1-ml sample of aqueous acetic acid (0.6 M)
was added to each mixture to stop the reaction. When
turbidity or a precipitate was present, the acetic solution was
passed through a Millipore membrane filter (HA; 0.45-,um
pore size). In the inhibition test, twofold, serial dilutions
(onefold to eightfold dilution) of the antibiotic solution of the
highest concentration were made, as test solutions, by
diluting the antibiotic solution with control endotoxin solu-
tions under conditions such that each test solution contained
J. CLIN. MICROBIOL.
1 ng of endotoxin per ml, and five experiments for four serial
test solutions and the control endotoxin solution (1 ng/ml)
HPLC with a Waters Associates model 6000A solvent deliv-
ery system and Lambda Max 480 LC spectrophotometer.
The analysis was performed on a Nucleosil SC18 column (4
by 150 mm; Macherey-Nagel Co.).
One of the three eluent systems (A, water-methanol [2:1];
plied (see Table 1 for the choice of system). A 5-,u1 sample
of the acetic solution was injected, and elution in 1 'ml/min
was monitored at 405 nm. The inhibition by antibiotics or the
amount of endotoxin was determined from the ratio of the
peak height of the PNA in the acetic solution to that in the
control PNA solution. The absorbancy of the PNA in the
acetic solution was measured at 405 nm in 1-cm cuvettes
(MT4; Carl Zeiss Co.) with a Carl Zeiss PQII spectropho-
tometer.
Determination of endotoxin in injectable carbenicillin
disodium preparatioiis. Six carbenicillin disodium prepara-
tions (1 g per vial) of three lots from the same batch were
used as test samples which were positive in the rabbit test by
the procedure of the Japanese Minimum Requirements for
Antibiotics (100 mg/ml per kg of body weight). Three sam-
ples from two lots which were negative in the test were used
as controls. These preparations were made by the same
manufacturer. Each sample was reconstituted with 5 ml of
water. These solutions were further diluted 10-fold and were
used as the test solutions; they contained about 17.5 mg/ml,
close to twice the antibiotic concentration for 50% inhibition
(IC50; 8 mg/ml). The liberated PNA was assayed by HPLC,
and the amounts of endotoxin were estimated from a cali-
bration curve (0 to 1 ng/ml) of the same antibiotic concen-
tration as in the test solutions; the antibiotic solution, after
ultrafiltration treatment, was used for the calibration curve.
RESULTS
The time course of the chromogenic reaction in the
presence of 0.5 ng of endotoxin per ml and various concen-
trations of Tris buffer (pH 8.0) is shown in Fig. 1. From these
experimental data and other preliminary data indicating that
1
(U)
cr
7.0 8.0 9.0
p H
FIG. 2. Effect of Tris buffer pH on the enzymatic activity in the
chromogenic reaction at 37°C for 50 tnin in the presence of 0.5 ng of
endotoxin (E. coli O111:B4) per ml. Tris buffers (pH 6.2 to 9.0) were
adjusted to a 0.5-M concentration.
VOL. 23, 1986 ENDOTOXIN DETERMINATION BY CHROMOGENIC ASSAY 13

100 The linear relationship between amounts of endotoxin and

0- liberated PNA was determined in the presence or absence of
0
1-
two different concentrations (5 and 10 mg/ml) of carbenicillin
4-.
80 disodium (Fig. 3). The calibration curves for a control and
C the experimental groups had a good linearity below 1.5 ng of
0 endotoxin per ml; however, after more than 70 to 80% of the
E 60 PNA was liberated, the two curves (0 and 5 mg/ml) tended to
level off. Also, carbenicillin disodium inhibited the
z chromogenic reaction concentration dependently (Fig. 3).
40 Because the three calibration curves gave linearity with
endotoxin concentrations between 0.5 and 1.5 ng/ml, 0.5 ng
0~ of endotoxin per ml was used for the subsequent inhibition
4., 20 test.
Typical examples of results of the inhibition test with
cloxacillin sodium, kanamycin sulfate, doxycycline hyclate,
0 and cefotaxime sodium, each from one of the four main
0 0.5 1 1.5 2 2.5 antibiotic groups, are shown in Fig. 4. Two inhibition curves
obtained by two different analytical methods (the HPLC and
Endotoxin ( ng/ ml) spectrophotometric methods) were similar in cloxacillin so-
dium and kanamycin sulfate, indicating either method can be
FIG. 3. Calibration curves of endotoxin (E. coli O111-B4) con- used for the assay. On the other hand, a yellow substance,
centration in the chromogenic reaction at 37°C for 50 min with or doxycycline hyclate of the tetracyclines, and other colored
without carbenicillin disodium with 0.5 M Tris buffer (pH 8.1). antibiotics, such as aclarubicin hydrochloride, should be
Concentration of carbenicillin disodium: 0, 0 mg/ml; 0, 5 mg/ml; A, applied to the HPLC method, owing to their intense spec-
10 mg/ml. The percentage of the liberated PNA obtained by the
HPLC method was the same as that obtained by the spectrophoto- trophotometric disturbance. The same result for tetracycline
metric method. was reported previously (H. Usami and K. Akiyama, Abstr.
Annu. Meet. Jpn. Pharm. Soc. 1982, 102, p. 654). However,
cefotaxime sodium, which is a colorless antibiotic as are
0.5 M and greater concentrations of Tris buffer were re- cloxacillin sodium and kanamycin sulfate, gave different
quired to maintain pH constantly in our inhibition test, 0.5 M inhibition curves in the two assay methods; the false-
Tris buffer and an incubation time of 50 min were chosen for inhibition curve in the spectrophotometric method could,
our experiments. possibly, have been due to color development by decompo-
The effect of various pHs of 0.5 M Tris buffer on the sition of the cefotaxime sodium itself during the chro-
enzymatic reaction is shown in Fig. 2, giving the optimal pH mogenic reaction. Color development which was not neces-
as 8.2. The pH value of 8.1 + 0.1 was chosen for 0.5 M Tris sarily proportional to antibiotic concentration was observed
buffer in the following chromogenic reaction. in most of the cephalosporins tested and less frequently in

Cloxacillin Kanamycin Doxycycli ne Cefotaxime

hyclate sodium
I+ 0.1 sodium sulfate
0.0- 0 W 1
Iq
-0.1

- 100
4-

.)
4-
Cu 50 -

a)
(a

0
Ia
0.625 1.25 2.5 5 2.5 5 10 20 0.250.5 1 2 2.5 5 10 20
Antibiotic Concentration (mg/mi)
FIG. 4. Effects of antibiotics on the chromogenic reaction in the presence of 0.5 ng of endotoxin (E. coli O111:B4) per ml at 370C for 50
min with 0.5 M Tris buffer (pH 8.1). Symbols: 0 and 0, percentage of relative activity obtained by the HPLC and spectrophotometric method,
respectively; 0, pH changes of 0.5 M Tris buffer (pH 8.1).
14 YANO ET AL. J. CLIN. MICROBIOL.

TABLE 1. Inhibition of the chromogenic reaction by antibiotics'

Relative activity (9/) at the following Init. highest
. .
Antibiotic. ~~~~~Eluent antibiotic dilution:
aniitcdlto:concn of Max. IC5,,
system" p(M/l
x 8 x 4 x 2 x 1 antibiotic (mg/ml) ApH (mgmi)
Penicillins
Ampicillin sodium A 75 62 29 11 20 +0.06 6.5
Carbenicillin disodium A 73 58 46 18 20 + 0.05 8
Cloxacillin sodium B 76 64 37 14 5 + 0.01 1.8
Hetacillin potassium A 81 58 35 9 20 +0.04 6
Methicillin sodium B 78 51 17 7 20 + 0.02 S
Meziocillin sodium B 52 29 14 7 20 + 0.01 2.5
Penicillin G potassium" A 79 67 38 11 12 + 0.01 4.5
Piperacillin sodium B 56 30 14 7 20 + 0.01 3
Sulbenicillin disodium A 100 74 51 21 20 + 0.03 10
Ticarcillin disodium A 82 71 48 22 20 + 0.02 9.5

Cephalosporins
Cefamandole sodium A 73 54 23 5 20 -0.02 5.5
Cefazolin sodium A 83 64 35 8 20 + 0.03 7
Cefmenoxime sodium B 70 51 18 7 20 + 0.01 S
Cefmetazole sodium A 74 42 29 12 20 + 0.03 4
Cefoperazone sodium B 63 22 5 4 20 + 0.02 3
Cefotaxime sodium B 89 65 34 10 20 + 0.01 7
Cefotetan sodium A 68 51 26 5 20 -0.03 5.5
Cefotiam hydrochloride C 79 60 32 7 20 -0.05 6.5
Cefoxitin sodium A 54 37 14 7 20 + 0.01 3
Cefsulodin sodium A 61 51 25 7 20 + 0.01 5.5
Ceftezole sodium A 101 81 56 14 20 + 0.02 11
Ceftizoxime sodium A 88 74 69 18 20 + 0.02 12.5
Cefuroxime sodium A 67 49 25 6 20 + 0.01 4.5
Cephacetrile sodium A 92 71 55 21 20 +0.04 11
Cephaloridine A 75 43 17 4 20 -0.01 4.5
Cephalothin sodium B 67 42 13 4 20 + 0.01 4
Cephapirin sodium A 70 52 24 5 20 + 0.04 5.5
Latamoxef sodium" A 85 75 50 17 20 + 0.05 10
Aminoglycosides
Amikacin sulfate A 105 97 96 99 20 -0.10 >20
Bekanamycin sulfate A 102 106 96 89 20 -0.18 >20
Dibekacin sulfate A 91 83 79 71 20 -0.14 >20
Gentamicin sulfate C 96 89 61 27 10 -0.14 6.5
Kanamycin sulfate A 103 98 96 94 20 -0.13 >20
Sisomicin sulfate A 95 92 77 56 12.5 -0.23 >12.5
Streptomycin sulfate A 99 106 95 102 20 -0.10 >20
Tobramycin sulfate A 94 84 76 53 10 -0.15 >10
Macrolides
Clindamycin phosphate A 92 73 21 7 5 -0.03 1.5
Erythromycin lactobionate A 92 65 35 18 5 -0.01 1.8
Leucomycin succinate A 104 98 65 4 5 -0.05 3
Lincomycin hydrochloride A 74 53 25 7 20 -0.10 5.5
Tetracyclines
Doxycycline hyclate A 78 58 33 17 + 0.01 0.6
Minocycline hydrochloride A 77 57 13 4 21 -0.04 0.3
Oxytetracycline hydrochloride A 92 84 44 8 2 + 0.00 0.9
Rolitetracycline nitrate A 10( 90 68 15 + 0.01 1.3
Others
Aclarubicin hydrochloride A 15 3 2 2 2.5 + 0.00 <0.312
Bleomycin hydrochloride A 104 98 96 79 20 -0.03 >20
Chloramphenicol sodium succinate C 63 40 10 5 20 + 0.03 4
Colistimethate sodium' C 61 46 35 26 0.835 + 0.00 0.16
Fosfomycin disodium A 97 94 94 97 20 + 0.15 >20
Polymyxin B sulfate' C 29 21 16 14 0.062 + 0.01 <0.008
" Abbreviations: Init. highest concn, antibiotic concentration at onefold dilution; max. ApH, maximum pH change of Tris buffer mixed with dilutions of
antibiotics.
'
Eluent systems: A. water-methanol (2:1); B, water-acetonitrile (1:1); C, water-dioxane (4:1).
Unit potencies were as follows: 1 U of penicillin G potassium. 0.600 jig; 1 U of colistimethate sodium, 0.0333 ,ug: 1 U of polymyxin B sulfate, 0.1274 ,ug.
d Latamoxcef sodium is an oxacephemcephalosporin.
VOL. 23, 1986 ENDOTOXIN DETERMINATION BY CHROMOGENIC ASSAY
the penicillins. The aminoglycosides tested showed no color
effect at all.
Because the effect of pH changes on enzymatic activity,
within + 0.15 pH unit of the Tris buffer (pH 8.1), was
negligible (Fig. 2), cloxacillin disodium, doxycycline hycl-
ate, and cefotaxime sodium were found to give concentra-
tion-dependent and pH-independent inhibition.
A good correlation between the extent of inhibition and
the logarithm of antibiotic concentration was obtained in a
number of the antibiotics tested (Fig. 4).
The results of the inhibition tests with various antibiotics
are summarized and the antibiotic concentration giving the
IC50 is estimated in Table 1; the percentage of relative
activity and the IC50 were variable to some extent, depend-
ing on the lot of antibiotic preparations. The inhibition may
be clf.rsified as folows. (i) The antibiotic was noninhibitory
when the IC50 was more than 20 mg/ml. (ii) The antibiotic
was moderately inhibitory when the IC50 was in the range of
5 to 20 mg/ml. (iii) The antibiotic was strongly inhibitory
when the IC50 was less than 5 mg/ml. The two ,B-lactam
antibiotic groups tested showed similar inhibition; the aver-
ages and the standard deviations of the IC50s of the penicil-
lins and the cephalosporins were 5.7 + 2.7 and 6.4 ± 2.8
mg/ml, respectively. The aminoglycosides tested could be
divided clearly into noninhibitory and moderately inhibitory
groups. In particular, the sulfates of amikacin, bekanamycin,
kanamycin, and streptomycin were noninhibitory up to 20
mg/ml. In sisomicin sulfate, some effect of the p1I change
could be considered. Clindamycin phosphate inhibited
nearly four times as strongly as lincomycin hydrochloride in
which only a chloride atom of clindamycin was replaced by
a hydroxy group. Polymyxin B sulfate was found to be the
strongest inhibitor among the antibiotics tested. The an-
thracycline antibiotic, aclarubicin hydrochloride, showed an
unlexpectedly strong inhibition. Strong inhibition was also
observed when precipitates were formed in the chromogenic
reaction (e.g., clindamycin phosphate, leucomycin suc-
cinate, and minocycline hydrochloride).
The results of the determination of endotoxin contents in
carbenicillin disodium preparations are shown in Table 2.
The average of 3.8 ± 1.2 ng of endotoxin was present in all
control samples, and on average about nine times greater
amounts of endotoxin contamination were found in the test
preparations.
DISCUSSION
To apply the LAL reagent to the detection and determi-
nation of endotoxin in antibiotic preparations, the interfering
effects of the antibiotics on the reaction must be established.
However, other factors, such as pH changes by antibiotics,
additives, and contaminating endotoxin _. --ntibiotic prepa-
rations may affect the results. In this study, we chose the
chromogenic reaction as a method mo?re quantitative and
sensitive than the Limulus gelation reaction, and we tried to
remove unfavorable factors by ti following procedures: (i)
ultrafiltration treatment of antibiotic preparations before the
tests to eliminate contaminating endotoxin, (ii) use of a high
concentration (0.5 M) of Tris buffer to prevent pH changes,
and (iii) measurement of the liberated PNA after HPLC
separation to exclude nonspecific colors derived from anti-
biotics. In this way, clear-cut data on the effects of antibiot-
ics on the chromogenic reaction were obtained. Without
ultrafiltration, some preparations (e.g., polymyxin B sulfate,
sisomicin sulfate, and leucomycin succinate) exhibited an
enhancement of the chromogenic reaction. However, this
was not observed after treatment. Thus, real enhancement of
15
TABLE 2. Endotoxin contents in injectable carbenicillin
disodium preparationsa
Endotoxin in
Endotoxin in
test samples control
samples
27.4 .
52.4 ...................................... 4.0
28.5 ...................................... 5.1
28.5 ...................................... 2.3
33.5 .
a Amounts of endotoxin (ng/1-g vial) were obtained as lipopolysaccharide
from E. coli 0111:B4. Test and control samples gave positive and negative
pyrogen tests, respectively. Average concentrations of endotoxin (+ standard
deviation) were 33.1 + 8.8 and 3.8 + 1.2 ng per vial in test and control
samples, respectively.
the chromogenic reaction was not found by the antibiotics
tested. Amphotericin B, which causes temperature rises in
rabbits, could not be applied to the ultrafiltration treatment,
because it made a colloidal solution, and no further attempt
with it was then made.
The reported maximum noninhibitory concentrations of
ampicillin sodium (11), cloxacillin sodium (16), methicillin
sodium (11), ticarcillin disodium (1), cephalothin sodium (1),
and tobramycin sulfate (1) in the LAL test were closely
consistent with the IC50s obtained in the present s,tudy by the
chromogenic method. However, this was not the case for
other antibiotics (1, 11, 16). This inconsistency was possibly
due to trace amounts of endotoxin contaminating the antibi-
otic preparations or to different sensitivities of the chro-
mogenic reaction and the gelation reaction, for instance, to
sodium chloride (6, 17) and EDTA (9, 14). Furthermore,
because magnesium or calcium ions play an important role in
the LAL test (21) and the chromogenic reaction (6), chela-
tion of alkaline earth metal ions with tetracyclines (24) can
be considered to be one of the inhibitory factors of tetracy-
clines. However, Nakamura et al. reported that EDTA at 25
mM does not inhibit the chromogenic reaction (14). Poly-
myxin B sulfate is a potent inhibitor of the LAL test (2)
owing to inactivation of endotoxin by the stoichiometric
bindirig to lipid A (13), and the IC50 was found to be less than
8 ,ug/ml, which was smaller than the reported concentration
(14). Bleomycin hydrochloride, having a drug-associated
pyrogenicity in humans, was proved to be noninhibitory up
to 20 mg/ml, whereas strong inhibition was found in the LAL
test (5).
The antibiotics which were noninhibitory can be applied
directly to this method. These antibiotics included bleomy-
cin hydrochloride, fosfomycin disodium, and several amino-
glycosides. In other antibiotics with moderate or strong
inhibition, endotoxin can be determined by diluting the
antibiotic solution to the noninhibitory level of the chro-
mogenic reaction; amounts of endotoxin in the diluted sam-
ple should be over the threshold limit of detection of the
chromogenic method. However, it will be onerous to deter-
mine the maximum noninhibitory concentrations of the
antibiotics as in the LAL test. Moreover, additives, such as
sodium deoxycholate (20) in amphotericin B preparations,
may interfere with the determination of maximum noninhib-
itory concentrations. A positive test spiked with a fixed
amount of endotoxin would possibly overcome these diffi-
culties. Providing the chromogenic method gives a linear
response to endotoxin over the range of 0 to 1 ng/ml, for
example, in an antibiotic solution with a concentration near
the IC50, endotoxin can be determined as follows. Two
antibiotic solutions of the same concentration near twice the
16 YANO ET AL.
IC50 are made, one of which is spiked in advance with
endotoxin, containing 1 ng/ml. The spiked and unspiked
antibiotic solutions are applied to the chromogenic method,
giving a and b for PNA concentration, respectively. The
amount of contaminating endotoxin in the antibiotic solution
is calculated from the formula bl(a-b) ng/ml. Thus, the
chromogenic method is found to be more applicable than the
gelation method to the determination of endotoxin amounts
in antibiotic preparations.
ACKNOWLEDGMENT
We thank S. Okamoto for many constructive comments and
suggestions during the course of this work.
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