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Analisis SEDIAAN Farmasi

Golongan Vitamin
Generic name Vitamer chemical name(s) (list not complete) Solubility logP
Vitamin B
1

Thiamine Water -2.96
Vitamin B
12

Cyanocobalamin, hydroxycobalamin, methylcobalamin Water -3.52
Vitamin B
2

Riboflavin Water -0.85
Vitamin B
3

Niacin, niacinamide Water -0.61
Vitamin B
5

Pantothenic acid Water -1.31
Vitamin B
6

Pyridoxine, pyridoxamine, pyridoxal Water -1.05
Vitamin B
7

Biotin Water 0.20
Vitamin B
9

Folic acid, folinic acid Water 0.16
Vitamin C
Ascorbic acid Water -2.34
Vitamin A
Retinol, retinal & 4 carotenoids including beta carotene Fat 4.32
Vitamin D
Cholecalciferol Fat 6.58
Vitamin E
Tocopherols, tocotrienols Fat 9.60
Vitamin K
phylloquinone, menaquinones Fat 8.43
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VITAMIN LARUT AIR (water soluble)

Vitamin C dan B
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Methods from literature
1. Capillary electrophoresis: Vitamin B
1
, B
2
, B
6
dan nikotinamida (S. Boonkerd,
M.R. Detaevernier, dan Y. Michotte, 1994; J. Chromatography A, 670: 209-214)
2. Cyclic voltammetry: Vitamin B
6
(Teixeira et al., 2003; J. Braz. Chem. Soc., 14(2):
316-321)
3. Fluorimetric: Vitamin B
12
(Li dan Chen, 2000; Fresenius J Anal Chem, 368: 836-
838)
4. HPLC-DAD: Vitamin B (Thiamine, Nicotinic acid, Pyridoxal, Pyridoxine,
Nicotinamide, Pantothenic acid, Cyanocobalamin, Riboflavin dan Thioctic acid)
dan Vitamin C (Klejdus et al., 2004; Analytica Chimica Acta 520: 57-67)
5. Catalytic voltammetric: Vitamin C (M.H. Pournaghi-Azar dan R. Ojani, 1996;
Talanta 44: 297-303)
6. Spectrophotometric: Vitamin C (Backheet et al., 1991; Analyst 116: 861-865)
7. Non-spectrophotometric methods: Vitamin C (S.P. Arya, M. Mahajan, dan P.
Jain, 2000; Analytica Chimica Acta, 417: 114)
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Vitamin C (asam askorbat)
Human Glutathione Transferase O1-1 C32S Mutant in Complex with
Ascorbic Acid (3VLN)
created by Chimera and POV-Ray
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Pilihan Metode
No Metode Sampel Sumber
1. Catalytic voltammetric pharmaceutical
preparations & complex
matrices of fresh
fruit juices
Talanta 44: 297-303
(1997)
2. HPLC-DAD pharmaceutical
preparations
Analytica Chimica Acta
2004 520: 57-67
J. Agric. Food Chem.
2005, 53, 1370-1373
3. Spectrophotometric pharmaceutical
preparations & fresh
fruit juices
Analyst 1991 116:
861-865
4. Non-spectrophotometric natural and fortified
food samples &
pharmaceuticals
Analytica Chimica Acta
2000 417: 1-14
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Catalytic voltammetric
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Sample
1. tablets containing ascorbic acid as a single component
(chewable tablets);
2. tablets containing sodium carbonate combined with
ascorbic acid (effervescent tablets);
3. powdered vitamin C;
4. ampoules containing ascorbic acid as the single
component;
5. multivitamin capsules containing vitamins A, B complex,
D
2
and E, nicotinamide, calcium pantothenate, and folic
acid together with minerals (Fe
2+
, Ca
2+
, Mg
2+
, Mn
2+
, Cu
2+
,
Zn
2+
and Mo
2+
);
6. multivitamin tablets containing vitamins A, B complex, D
2

and E, nicotinamide, calcium panthothenate, folic acid.
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Instrumentasi
EG&G potentiostat/galvanostat model 273 coupled
with an IBM personal computer connected to an Epson
model FX-850 printer was used.
A conventional three-electrode cell thermostatted at
25+0.1C with calomel electrode as reference
electrode, a platinum wire as auxiliary electrode and
a glassy carbon disk as working electrode
(A=0.126 cm
2
from EG&G) was used.
The working electrode was polished with alumina
powder (0.05 /tm) and then washed with water and
acetone in turn before each voltammetric
measurement.
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Procedure
VitC tablets, capsules or powders
An accurately weighed portion of finely
powdered sample equivalent to about
100 mg of ascorbic acid was transferred to
a 10 mL assay tube and ascorbic acid was
extracted with two 5 mL portions of 0.5%
citric acid in bidistilled water. The extracts
were combined in a 50 mL flask and diluted
to volume. A 1 mL portion of extract was
diluted with 10 mL of glycine buffer pH 4,
containing 0.1 M LiCIO
4
and 0.1 mM
ferrocenecarboxylic acid, in a voltammetric
cell and a cyclic voltammogram was
recorded using a well polished glassy
carbon (GC) electrode. The amount of
ascorbic acid was determined by means of
a calibration graph.
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Sejumlah sampel (serbuk) yang
ditimbang seksama setara dengan
100 mg asam askorbat dipindahkan ke
dalam tabung 10 mL dan dilarutkan
dengan 5 mL asam sitrat 0,5% dalam
akuabides. Larutan ini dipindahkan ke
dalam labu takar 50 mL dan ditambahkan
pelarut hingga tanda. Diambil 1 mL dan
dilarutkan dengan 10 mL dapar glisin
pH 4 (larutan mengandung 0,1 M LiCIO
4

dan 0,1 mM ferrocenecarboxylic acid)
kemudian diukur dengan voltammeter
sehingga diperoleh voltammogram.
Konsentrasi asam askorbat ditentukan
dari rerata dari grafik kalibrasi.
electrochemical oxidation
Some electrogenerated ferricinium derivatives (ferriciniumcarboxylic acid)are
able to catalyse the electrochemical oxidation of ascorbic acid in buffered
aqueous solutions at pH 4-5 via a homogeneous process when the ferrocene
derivatives (ferrocenecarboxylic acid) are present in dissolved forms
(see Scheme 1)

The catalytic peak current is linearly dependent on the ascorbic acid
concentration and the range of linearity depends on the amount of mediator
(ferrocenecarboxylic acid) in the solution
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Cyclic voltammograms
(a) 2 mM ferrocenecarboxylic acid in
0.5 M glycine buffer + 0.1 M LiCIO
4
at
pH 4,
(b) as for (a) but with addition of 5 mM
ascorbic acid,
(c) 5 mM ascorbic acid in buffer
solution pH 4.
Scan rate: 5 mV s
-1
.

anodic peak current for cyclic
voltammograms with a scan rate of
10 mV s
-1
was proportional to the
ascorbic acid concentration within the
range 5 x 10
-5
s/d 1 x 10
-3
M with only
a small intercept.

The regression equation was:
i (mA) = - 1.28 + 9757C (M);
r = 0.999, n = 6.
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Procedure
Injection
An accurate ampoule volume
equivalent to about 100 mg of
ascorbic acid was transferred to a
50 mL flask and diluted to volume
with 0.5% citric acid solution. A 1
mL portion of the solution was
diluted in a voltammetric cell to
10 mL as described for tablets and
the cyclic voltammogram was
recorded using the GC electrode
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Sejumlah volume larutan dari
ampul diambil dengan pipet
volume yang setara dengan
100 mg asam askorbat,
dipindahkan ke dalam labu
takar 50 mL dan ditambahkan
larutan asam sitrat 0,5%
hingga tanda. Diambil 1 mL
dan diperlakukan seperti yang
telah dijelaskan untuk tablet.
Voltammogram direkam
menggunakan elektroda GC.
Result from article
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Interference
Some reducing ions, such as
1. Fe(II) exceeded 3 mM,
2. Cu(I),
3. Sn(II) and
4. Sulphite

The experimental investigation showed that these ions did not interfere in
buffered solution up to 50, 20, 100 and 100 mM respectively

The results of the analysis of some pharmaceutical preparations using the
proposed method compared favourably with those obtained by the USP
method confirming that no interference was observed from concomitant
mineral ions, other vitamins, glucose, sucrose, nicotinamide and tablet
excipients
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Metode Pembanding
A 0.05 M iodine solution was standardized in the
usual way with a primary standard of As
2
0
3
or
titrisol thiosulphate solution. For pharmaceutical
analysis an iodimetric procedure described in the US
Pharmacopeia (USP) was used.

Larutan standar Iod 0,05 M yang telah dibakukan
dengan As
2
0
3
atau larutan titrisol tiosulfat
digunakan dalam metode pembanding seperti yang
tercantum dalam US Pharmacopeia (USP).
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HPLC-DAD
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Sample
Vitamin drink (Penko, Krnov, Czech Republic)
consisted of vitamins (thiamine, riboflavin,
pyridoxine, pantothenic acid, nicotinamide, -
tocopherol, cyanocobalamin, l-ascorbic acid,
folic acid) and adjuvants (saccharose, glucose,
E330, maltodextrine, natural identical aroma,
milk proteins, lemon pectin, soy lecithin, ZnO,
MnSO
4
and Na
2
SeO
3
).

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Preparasi sampel
The homogenised samples of Vitamin drink (1 g
of powder) were dissolved in 10 mL of solution
consisted of 0.010% trifluoroacetic acid
(TFA):methanol (50:50) and stirred on Vortex for
15 min.
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Instrumentasi
An HP 1100 liquid chromatographic system (Hewlett-
Packard, Waldbronn, Germany) was equipped with a
vacuum degasser (G1322A), a binary pump (G1312A),
an auto sampler (G1313A), a column thermostat
(G1316A), and a UV-Vis diode array detector (model
G1315A) working at 190690 nm.

The ChemStation software (Rev. A 08.01) controlled
the whole liquid chromatographic system. Spectra were
registered in the range of 190400 nm (SBW 100 nm).
Chromatograms were registered at 280 nm.
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Sistem Kromatografi
Reversed-phase chromatographic columns Zorbax AAA
(150mm4.6 mm, 3.5 m particle size, Agilent
Technologies, USA), Atlantis dC18 (150mm2.1 mm, 3
m particle size, Waters, Milford, MA, USA) and
MetaChem Polaris C18-A (150mm 4.6 mm, 3m
particle size, Ansys Technologies, Torrance, CA, USA)
were tested for the separation of vitamins.
The flow rate was 0.7 mLmin
1
. Auto sampler injection
was 3 L.
Gradient of mobile phase and its composition was
tested, too. See next slide.
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Gradient profile
(A, mobile phase methanol) for
simultaneous vitamin separation that
started at 95:5 (0.010% TFA:methanol)
and was constant in the first 4 min,
then decreased to 2:98 during the next
10 min and finally linearly increased up
to 95:5 from 32 to 35 min is shown on
(A); pH value was 3.9.
Simultaneous HPLCUV
chromatograms of water- and fat-
soluble vitamins at three detection
wavelengths: 230 nm (B), 266 nm (C)
and 280 nm (D). Vitamin
concentrations were 100 gmL
1
.
Other conditions are the same as in
Fig. 2.
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Simultaneous HPLCUV determination
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Dependences of retention
(A) and symmetry (B)
factors
Dependences of retention (A) and
symmetry (B) factors of water- and fat-
soluble vitamins (100 gmL
1
of each)
on pH values, respectively.
MetaChem Polaris C18-A column,
mobile phase consisted of 0.010% TFA
and methanol; gradient profile started
at 92:8 (TFA:methanol; (v/v)) and was
constant in the first 2 min, then
decreased to 2:98 during the next 10
min and finally linearly increased up to
92:8 from 32 to 35 min. Vitamin
concentrations were 100 gmL
1
.
Autosampler injection was 3 L. HPLC
UV parameters: MetaChem Polaris
C18-A (150mm 4.6 mm, 3 m
particle size, Ansys Technologies,
Torrance, CA, USA; isocratic flow rate
of 0.7 ml min
1
; column temperature
20C; detection wavelength of DAD is
280 nm.
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Kromatogram
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Validation
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a
Retention times in min.
b
Regression coefficients.
c
Limits of detection (3.S/N).
d
Limits of quantitation (10.S/N).
e
Relative standard
deviations.
f
Non-vitaminous substances.
Result from article
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Spectrophotometric
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Sample
1. tablets containing ascorbic acid as a single component: Vitamin-C (Alexandria) , Vitascorbol (Specia)
and Vitacid-C effervescent tablets (Cid);
2. tablets containing ascorbic acid in combination with salicylamide: Cidal-C (Cid) ; with phenylephrine,
paracetamol and caffeine: Rhino-C (Cid); with rutin: Ruta-C-60 (Kahira); and with menadiol dibutyrate
and rutin: Styptobion (Nile/Merck);
3. tablets containing ascorbic acid with vitamins A, B complex, D2 and E, nicotinamide and calcium
pantothenate together with minerals: Chewa-vit (Pfizer-Egypt), Totacid (Cid) , Optical Compound
(Eipico/ Leo) and Thereagran Hematic (Squibb-Egypt);
4. effervescent tablets containing calcium carbonate combined with ascorbic acid: Vitacid-calcium (Cid);
5. multivitamin capsules containing vitamins A, B complex, D
2
and E with minerals: Mineravit (Eipico) ,
Obron (Pfizer-Egypt) and Viterra (Pfizer-Egypt);
6. syrups containing vitamins A, B complex, D
2
and E, nicotinamide and calcium pantothenate: Beco-C
(Misr), Medivit (Adco) and Fruital (Cid);
7. ampoules containing either ascorbic acid alone: Cevarol (Memphis); or combined with novalgine:
Cevagine (Memphis); with menadion sodium diphosphate and rutin: Styptobion (Nile/Merck); with
calcium glubionate: Calcium-C Sandoz (Sandoz); or with other vitamins and nicotinamide: Polyvit
(Misr);
8. Drops containing ascorbic acid as a single component: Ceviline (Kahira).
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Reaction
The procedure is based on the
reaction of ascorbic acid with the
zinc chloride salt of diazotized I-
aminoanthraquinone (Fast Red AL
salt) in an acidic medium,
followed by development of a
blue colour (
max
630 nm) in
alkaline solution.

The mechanism of the reaction
between the diazonium salt of 1-
aminoanthraquinone (I) and
ascorbic acid (II) to form the
lactone (III), which in an alkaline
medium, yields the xalohydrazide
derivative (IV) , is shown in
Scheme 1
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General Procedure
In a 10 mL calibrated flask were placed 1 mL of
the standard or the sample preparation, 1 mL of
the reagent solution and 2 mL of 10% sodium
hydroxide solution. The volume was made up to
the mark with distilled water, the solution mixed
and the absorbance at 630 nm measured
against a reagent blank prepared similarly
(absorbance of the reagent blank = 0.002).
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Ampoules, syrups & drops procedure
An accurately measured volume, equivalent to
about 20 mg of ascorbic acid, was transferred
into a 100 mL calibrated flask, 0.5 g of citric acid
was added and the volume was made up to the
mark with distilled water
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Procedure for tablets
An accurately weighed amount of powder
obtained from 20 tablets, equivalent to about
20 mg of ascorbic acid, was transferred into a
100 mL calibrated flask, followed by 50 mL of
extracting solution. The flask was shaken for
15 min and the contents were made up to the
mark with distilled water
The solution was filtered, the first portion of the
filtrate being rejected
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Procedure for multi-vitamin capsules
Ten capsules were completely disintegrated by
adding 50 mL of the extracting solution to a
200 mL beaker containing the capsules and
gently heating the mixture.
The suspension was transferred quantitatively
into a 500 mL calibrated flask and rapidly cooled
to room temperature. The volume was made up
to the mark with distilled water
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Absorption Spectra
Fast Red AL salt (dioazotized 1-
aminoanthraquinone stabilized as the
zinc chloride salt) reacts with ascorbic
acid in an acidic medium after which a
blue colour develops in alkaline
solution with a maximum absorbance
at 630 nm (Fig. 1); the reagent and
ascorbic acid have no absorption in
this region. The blue colour formed in
the alkaline medium is dispelled on
acidification.
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Stability of Colour
The colour developed immediately,
attained a maximum within 1 min and
remained stable for at least 2 h.

1. Berapakah OT reaksi dalam hal ini?
2. Kenapa air dapat mengurangi
stabilitas dari produk yang
terbentuk antara asam askorbat dan
pereaksi?

Stability of Ascorbic Acid
Fifty percent of the reagent-reducing
activity of a solution of ascorbic acid
(200 g.mL
-1
) disappeared within 2 h
(Fig. 2). When ascorbic acid was
prepared in 5% trichloracetic acid,
0.5% oxalic acid or 0.5% citric acid, the
reducing activity was stable for 2 h but
it decreased steadily after that time
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Concentration of Reagent
The colour developed strongly as the
amount of Fast Red AL salt in the
reaction mixture was increased from
0.75 to 1.25 mL of a 0.25% solution
(Fig. 3).

Info:
Oxalic and citric acids have been
reported to be efficient stabilizers of
ascorbic acid solution,
16,17
but the
resulting final solutions were turbid
when oxalic acid was used; therefore,
citric acid, which produces no turbidity,
was used throughout this work



16 Ponting, J. D., Ind. Eng. Chem. Anal. Ed., 1943, 15,
389.
17 Wills, R. B. H., Wimalasiri, P., and Greenfield, H., J.
Assoc. Off.A nal. Chem., 1983, 66, 1377.
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Type and Concentration of
Alkali
In acidic and neutral media, no
development of colour was observed,
whereas in an alkaline medium the
blue colour developed rapidly.
Different alkalis were tested (Table 1)
and all produced virtually identical
absorbance readings but with small
shifts in the maxima. Sodium
hydroxide was preferred as the
max

observed with this alkali was shifted to
the longest wavelength. In addition,
the use of an excess of sodium
hydroxide is required in order to
dissolve any precipitated zinc
hydroxide (resulting from the zinc
chloride present in the reagent)
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Effect of Diluting Solvents
Methanol, ethanol, water, propan-2-ol,
dimethyl sulphoxide and
dimethylformamide were tested as
diluting solvents (Table2). The results
revealed that water and ethanol were
the best solvents. However, water was
used throughout this work; this is an
additional advantage of the proposed
method. Carbon tetrachloride,
chloroform and 1,2-dichIoroethane
were not suitable as the colour
produced could not be extracted into
these solvents.

Table 3 represent the recovery result of
ascorbic acid with other substance

Kenapa molekul lain dalam sampel
tidak mengganggu perolehan kembali
dari asam askorbat?
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Result from article
At 630 nm, the absorbance was
proportional to the amount of ascorbic
acid within the range 5-25 g with only
a small intercept; the regression
equation was:
A
630
= -0.0324 + 0.024% (g mL
-1
);
r = 0.9990, n = 8. The molar absorptivity
was 4.07 x 1031 mol
-1
cm
-1
.
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Dimethoxydiquinone
Dimethoxydiquinone
gives a violet-colored
product with
ascorbic acid in a
phosphate buffer (pH
6.6). The reduced
indigoid
quinhydrone form is
perhaps responsible
for the formation of
violet-colored
product
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After diluting with dioxane, absorbance of the
colored solution which is stable over 24 h only
under dark conditions is measured at 510 nm.
Heating leads to a decrease in color intensity.
Beers law holds good up to 80 g ml
1
with a
detection limit of 10 g ml
1
.
DMDQ Interferences
Riboflavin and copper interfere.
The interference of iron(II) sulfate responsible
for precipitation can be removed by
centrifugation.
Though the method is not sufficiently
sensitive (=1.62x10
3
), it can still be applied to
the analysis of citrus fruits after extracting the
colored product into chloroform
(
max
=530 nm).
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DCIP as dye
The blue dye DCIP is reduced to the colorless
form on addition of ascorbic acid but it gives a
pink color to the acidic solutions.
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Color extraction
Using the dye, ascorbic acid present in sample
has been determined. The excess dye can be
extracted with xylene or butanol.
Many substances which are capable of reducing
the dye resulting from the preparation and
processing of samples interfere.
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Tetrachlorobenzoquinone as dye
Ascorbic acid is determined at 336 nm
(=535 cm
2
mol
1
) via a decrease in absorbance
of 7.104 M tetrachlorobenzoquinone (chloranil)
in 80% acetonewater (v/v) medium.
With these methods, mixtures of ascorbic acid
with thiols like o-mercaptobenzoic acid,
mercaptosuccinic acid, 3-mercaptopropionic acid
can not be resolved.
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Methylene Blue
Methylene Blue has been used to determine
ascorbic acid in food products.
The colorless form of the dye is extracted into
chloroform after its reduction with ascorbic
acid; back oxidation of the dihydro derivative
to Methylene Blue has been used for the assay
of ascorbic acid (
max
=653 nm). The method is
reported to be highly sensitive.
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Ninhydrin & Resorcinol
The reaction of ascorbic acid with ninhydrin carried
out on a boiling water bath using 80% aqueous
solution as a medium in 0.01 M NH
4
OH is used for
its determination in pharmaceuticals (
max
=415 nm),
but without added advantages.

Methanolic solution of resorcinol gives a pale yellow
color (
max
=425 nm) with ascorbic acid in
hydrochloric acid medium, obeying Beers law for
80-400 g ml
1
.
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Diazotized dye
A purplish or blue colored species is produced by these salts with
ascorbic acid in alkaline medium. Diazotized-4-methoxy-2-nitroaniline
couples with ascorbic acid in oxalic acid medium in the presence of
ethanol or isopropanol, giving a purplish color in alkaline solutions.

Though Fe(II), Sn(II) and dehydroascorbic acid (DHAA) do not interfere,
the presence of reductones and reductic acid requires formaldehyde
condensation. Low contents of vitamin C in the presence of flavanoids
and pectic substances are also detected.

The reaction of ascorbic acid with 4-nitrobenzene diazonium
fluoroborate in acetic acid medium was used for its determination at

max
415 nm. But the mixture has to be kept for 25 min in the dark,
followed by the addition of sodium hydroxide.
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4-Chloro-7-nitrobenzofurazane
4-Chloro-7-nitrobenzofurazane forms a bluish green colored
species with ascorbic acid in presence of 0.2 M NaOH.

The absorbance is measured at 582 nm after diluting the
reaction contents with 50% (v/v) aqueous acetone solution.
Beers law is obeyed in the concentration range 5 20 gmL
1
.
The colored product is stable for 30 min only when kept away
from direct sunlight or artificial day light.

The method is reported free from the interference of all other
vitamins and minerals present in multivitamin preparations
and can be applied to the analysis of pharmaceuticals, fresh
fruit juices and vegetables.
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2,3,5-triphenyltetrazolium chloride
Hashmi et al. proposed a method based on the
reaction of 2,3,5-triphenyltetrazolium chloride with
ascorbic acid in alkaline medium. The pink solution
is allowed to stand in the dark for 30 min at 25C; it
obeys Beers law over the range 5-25 g mL
1
.
Sugars (>15 g mL
1
) except sucrose interfere by
forming a similar color to that of the reagent.
Riboflavin, cyanocobalamin and folic acid interfere
due to their own color.
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Phenylhydrazinium chloride
Phenylhydrazinium chloride produces a yellow color
(
max
=395 nm) when treated with ascorbic acid in 0.1 M
HCl medium.
The reaction contents are kept for 1 h in an incubator or
water bath at 502C, thus making the method time-
consuming. Beers law is obeyed in the range 25100 g
of ascorbic acid.
No interference is observed from other vitamins,
minerals, glucose, sucrose, excipients and reducing
agents. However, the presence of excessive amounts of
riboflavin requires the addition of 0.5 g talc, which
imparts a yellow color to the solution.
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3-Methyl-2-benzothiazolone hydrazone
3-Methyl-2-benzothiazolone hydrazone reacts in
the presence of sodium metaperiodate to form
a blue colored solution (
max
=630 nm) which
helps in the determination of ascorbic acid over
the range 6-14 meq mL
1
.
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LEUCOMALACHITE GREEN (LMG)
AA reacts with potassium iodide-iodate solution under acidic conditions
to liberate iodine & this compound selectively oxidizes LMG to MG dye
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Principle
AA reacts with potassium iodide-iodate solution
under acidic conditions to liberate iodine and the
liberated iodine selectively oxidizes LMG to MG dye.
The colour of the dye was measured at 620 nm.
Beers law is obeyed over the concentration range
of 0.8-8 g AA per 25 mL of final solution (0.032-
0.32 ppm). The apparent molar absorptivity and
Sandells sensitivity of the method were found to be
2.98x10
5
l mol
-1
cm
-1
, 0.0042 g cm
-2
, and
respectively.
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reaction
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pharmaceuticals
All drug samples tested were fresh and purchased from
local pharmacy. An AA tablet or the content of a capsule
were weighed, ground in to a fine power and stirred for 2-
3 min with 50 mL of deionized water. 1 mL of 5% EDTA
was added and filtered through Whatman No. 41 filter
paper.
The insoluble mass was washed with three successive 5
mL portions of water and the filtrate plus washings were
diluted to volume in a 250 mL calibrated flask. A known
volume was further diluted depending on the AA content
and the colour of the sample. 1 mL aliquot was analyzed
as recommended above.
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Procedures
An aliquot of sample solution containing 0.8-8.0 g AA was transferred
in to a series of 25 mL graduated tube. To this 0.4 mL of potassium
iodide-potassium iodate mixture solution and 1 mL of 0.02-mol l
-1

hydrochloric acid solution were added, and the mixture was gently
shaken until the appearance of yellow colour, indicating the liberation
of iodine. Then 1 mL of 0.05% LMG solution was added to it followed
by addition of 2 mL of acetate buffer (pH-4.5).

The contents were heated (~ 40C) in a water bath for 5 min, cooled to
room temp and diluted to the mark with distilled water. The mixture
was kept for 10 min for completion of the reaction. The absorbance of
the formed dye was measured at 620 nm against the reagent blank.
The concentration of AA content was established from the calibration
graph.
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Comparison
It offers a sensitivity, selectivity,
simplicity and cost-effectiveness
of the method. The method
involves no extraction steps,
thereby the use of organic
solvents, which are generally
toxic in nature are avoided. The
stability of formed MG dye is an
added advantage of the method.
The sensitivity in terms of molar
absorptivity and precision in
terms of relative standard
deviation of the present method
indicated it to be very reliable for
the determination of AA in
various samples. This method is
good alternative to some
reported costly instrumental
method.
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Non-spectrophotometric
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Titrimetric methods
1. 2,6-Dichlorophenolindophenol (DCIP)
2. tetrachlorobenzoquinone
3. N-bromosuccinimide
4. iodine, potassium iodate, potassium bromate & iodine
monochloride
5. chloramine T
6. o-Iodosobenzoate & o-diacetoxyiodobenzoate
7. thallium(III) perchlorate & copper(II) sulfate
8. cerium(IV) sulfate
9. potassium hexacyanoferrate(III)
10. ferricinium trichloroacetate
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2,6-dichlorophenolindophenol (DCIP)
the reduction of DCIP (Tillimans Reagent) with
ascorbic acid in acidic solution.
Official method: the solution containing ca. 2 mg
of ascorbic acid and 5 mL of a mixture of
metaphosphoric acid and acetic acid is titrated
with a standard solution of DCIP. The titrant acts
as a self indicator.
it is not applicable to many pharmaceutical
preparations containing Fe(II), Sn(II), Cu(I), SO
2
,
SO
3
2
and S
2
O
3
2
ions which are usually associated
with mineral or liver preparations.
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reaction
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J Pharmaceutical Sciences Vol. 72, No. 2, February 1983
Analytical Sciences October 1998, Vol. 14
Kekurangan DCIP
It is not applicable if:
there are substances naturally present in fruits or
biological materials such as tannins, betannins and
sulfhydryl compounds are oxidized by the dye
the concentration of dehydroascorbic acid is negligible
the alkalinity of the sample also hinders the
determination
natural or added colors render the end point difficult to
judge visibly such as edible dyes like amaranth,
indigotine and tetrazine present in pharmaceutical
preparations need removal either by ion exchange or
by addition of charcoal

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Modified DCIP
titrating the citrate buffered solutions (pH 3.5)
with alcoholic solution (ethanol)
the alcoholic solution is claimed to be superior to
official solutions mainly because of its stability,
but it requires storage in a refrigerator
it can avoid or reduce interferences from colors,
iron, reductones and reductic acid
but the dye is useful only for solutions containing
not more than negligible amounts of interfering
substances and dehydroascorbic acid
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Anal. Chem., 1982, 54 (4), pp 793796
Tetrachlorobenzoquinone
the presence of EDTA which acts as an indicator as well
as a masking agent for associated metal ion impurities
the end point is detected by the appearance of a
golden yellow color
applicable for the interferences of citric acid, oxalic
acid, tartaric acid, glucose, sucrose and maltose
mixtures of ascorbic acid with thiols like cysteine, o-
mercaptobenzoic acid, mercaptosuccinic acid and 3-
mercaptopropionic acid cannot be resolved
the interference of thiols is reported to be avoidable by
masking with acrylamide
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Reaction TCBQ
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Remove the thiols
Resolutions of mixtures of vitamin C with thiols has been
successfully carried out by first titrating the vitamin C
content with tetrachlorobenzoquinone till the orange-red
color appears.
Upon dilution of the contents, thiols can be titrated with
standard chloramine-T solution. Thiols are quantitatively
oxidized to their corresponding disulfides with chloramine-
T in the presence of potassium iodide

CH
3
C
6
H
4
SO
2
NCl
-
Na
+
+ 2I
-
+ 2H
+
CH
3
C
6
H
4
SO
2
NH
2
+ Cl
-
Na
+
+ I
2

2RSH + I
2
RSSR + 2HI
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N-bromosuccinimide
N-bromosuccinimide was introduced as a reagent for the
determination of ascorbic acid using starch as an indicator
Reductones, reductic acid and iron salts do not interfere with this
titration, but it was found to give unsatisfactory results with the
samples of preserved juices and squashes containing metabisulfite
as a preservative
Quinoline yellow solution has been used for detecting the end point
in the titrations with N-bromophthalimide and N-bromosaccharin
These reagents were also used in the potentiometric titration of
Vitamin C. Cysteine and glutamic acid interfere
N-bromosaccharin was used for the microdetermination of ascorbic
acid in pure solutions and pharmaceutical preparations
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iodine, potassium iodate, potassium
bromate & iodine monochloride
starch cannot be used in such titrations because it decreases the
reaction rate between ascorbic acid and iodine
variamine blue, carbon tetrachloride or chloroform in the presence
of mercuric chloride and p-ethoxychrysoidine as indicators have
been recommended. naphthol blue black, amaranth or Brilliant
Ponceau 5R as alternative indicators
Cu(II), As(III), Hg(II), cysteine, thiourea, thioglycollic acid, sulfide and
sulfite interfere seriously
Seetharampa and Shubha proposed a titrimetric method based on
the oxidation of ascorbic acid with pyridinium chlorochromate and
estimating the unconsumed oxidant iodometrically
perphenazine, 1-amino-4-hydroxyanthraquinone, azine and oxazine
dyes, phenosafranine, safranine T, wool fast blue and aposafranine
have also been used in the bromatometric estimation of ascorbic
acid
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cerium(IV) sulfate
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Lebih detail lihat di
Analytica Chimica Acta 2000 417: 1-14

Non-spectrophotometric methods for the
determination of Vitamin C
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Riboflavin
Human Liver Glycogen Phosphorylase a Complexed with Riboflavin, N-2
Acetyl-beta-D-Glucopyranosylamine and CP-403,700 (1L5R)
created by Chimera and POV-Ray
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Sample- Analytica Chimica Acta 2004 520: 57-67
1. B-Komplex tablets (Lciva, Prague, Czech Republic)
consisted of vitamins (thiamine, riboflavin, pyridoxine,
pantothenic acid, nicotinamide) and adjuvants (lactose
mohydrine, may amylose, gelatine, calcium stearate,
talcum, saccharose, titanium dioxide, sodium caramel,
paraffin, silica gel anhydride, natural identical aromas,
fibres and additives).
2. Vitamin drink (Penko, Krnov, Czech Republic) consisted of
vitamins (thiamine, riboflavin, pyridoxine, pantothenic
acid, nicotinamide, -tocopherol, cyanocobalamin,
l-ascorbic acid, folic acid) and adjuvants (saccharose,
glucose, E330, maltodextrine, natural identical aroma,
milk proteins, lemon pectin, soy lecithin, ZnO, MnSO
4
and
Na
2
SeO
3
).

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Kromatogram
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Validation
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a
Retention times in min.
b
Regression coefficients.
c
Limits of detection (3.S/N).
d
Limits of quantitation (10.S/N).
e
Relative standard
deviations.
f
Non-vitaminous substances.
Result from article
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FIA
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Reaction
Riboflavine forms a 1:1 complex with Ag(I)
within pH 6.5 and 7.1
Pyridoxine reacts with N,N-diethyl-p-
phenylenediamine and potassium
hexacyanoferrate(III) in phosphate buffer
yielding an indophenol dye
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Konsumsi pereaksi
The reagent consumption was in media 25-fold lower
than those required in the determination of the vitamins
by flow-based procedures with continuous reagent
addition (Table 4)
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Hasil
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Application Note 251
Determination of Water- and Fat-Soluble
Vitamins in Nutritional Supplements by HPLC
with UV Detection

using
Dionex UltiMate 3000 HPLC system
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Samples
Five vitamin supplement
samples (Brands 1 to 5)
were analyzed. The
ingredients are listed in
Table 1. Brands 1 and 2 were
from two different
pharmaceutical companies,
and Brands 3, 4, and 5 were
from a third company that
produces special vitamin
tablets for women, children,
and the elderly
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Sistem Kromatografi
Sistem gradien eluen Panjang gelombang
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Kondisi KCKT
Vit Water-soluble Vit Fat-soluble
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Kromatogram
V
C
& V
B
group vitamins fat-soluble vitamins & carotene
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Hasil-method detection limit (MDL)
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Sample preparation
One gram of the pulverized dried thiamine
hydrochloride powder was dissolved in the least
necessary amount of water, transferred to a 100-mL
standard flask and made up to the mark with water.
Ten tablets were ground and the powder was mixed,
dried, and dissolved in the minimum of water, then the
solution was filtered into a 100-mL standard flask and
made up to the mark with water.
The contents of 10 vials were transferred to a 100-mL
standard flask and made up to the mark with water.
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Potentiometric titration
Aliquots of thiamine hydrochloride sample solution (0.50-
5.0 mL) were transferred to a 100-mL beaker and diluted to
10 mL with water.
Ten ml of 1M sodium hydroxide were added and the
solution was stirred for 2 min. The silver-silver sulphide ion-
selective electrode and the double-junction silver-silver
chloride reference electrode were introduced into the
solution and standard 0.01M silver nitrate was slowly
added from a dark-glass burette with its tip immersed in
the solution.
The electrode potential was monitored as a function of the
titrant volume added. Titration end-points were calculated
from first or second derivative titration curves (1 mL of
O.OlM silver nitrate 1.535 mg of thiamine hydrochloride).
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Kurva & perolehan kembali
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Hasil
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Metode lainnya
Derivatisasi KCKT: Analytical Biochemistry, 333
(2004) 336344 VitB
6
& B
12

Spektofotometri derivatisasi: J.Pharm. Biomed.
Analysis, 22 (2000) 915923 VitB
1
& B
6

Voltammetri: J. Braz. Chem. Soc., 14_2 (2003)
316-321 & Talanta, 61 (2003) 743/753 VitB
6
&
B
12
Fluorometri: Fresenius J Anal Chem, 368 (2000)
836838 VitB
12

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VITAMIN TIDAK LARUT AIR (fat soluble)

Vitamin A, D, E dan K
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Retinol
Crystal Structure of Human Holo Cellular Retinol-binding Protein II (CRBP-
II) from 2RCT
created by Chimera and POV-Ray
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Metode Analisis
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Flow Injection- chemiluminescence
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Tris(2,2-bipyridyl)ruthenium(II) [Ru(bpy)
3
2+
]
Hasil
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Interference studies
The influence of commonly used excipients and additives
in pharmaceutical formulations was investigated by
analyzing solutions containing 1.0 10
6
mol/L vitamin A.
The tolerable foreign species were taken as a relative
error not greater than 8%.
No interference could be found when 750-fold sorbitol
and mannitol, 500-fold glycerol and cellulose, 200-fold
sucrose, glucose, starch, gum acacia, magnesium stearate
and glycerol monostearate, 100-fold stearic acid and
tocopherol, 10-fold triglyceride and 1-fold vitamin D3 and
cholesterol were added to 1.0 10
6
mol/L vitamin A.
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Prosedur dan hasil
From each standard solution of ethanol/retinol prepared in water and
methanol respectively, 0.1 mL was mixed with 1.3 mL of pyrophosphate
(50mM, pH 10.5) and 1.5 mL of NAD+ (10 mM).
The reaction mixture was incubated for selected time periods (e.g. 5 min) in
the thermostated water bath at 37C. At zero time, 0.1mL of alcohol
dehydrogenase enzyme was added and the reactants were mixed gently.
The liberated NADH wasmeasured at 340 nm for approximately 10 min (at
37C) using UVvis spectrophotometer equipped with 10mm silica cuvettes. A
thermostated water bath was used to maintain the temperature during
incubation.
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Retinol
Crystal Structure of Human Holo Cellular Retinol-binding Protein II (CRBP-
II) from 2RCT
created by Chimera and POV-Ray
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Analyst 1975 100: 238-242
A colorimetric method for the determination of vitamin
D, based upon the use of anisaldehyde - sulphuric acid
as a colour reagent, has been developed.
The method enables 0.1-0.5 mg of calciferol to be
determined with a mean percentage recovery of 100.2
1.44 percent, and it avoids the difficulties met with in
the antimony(II) chloride colour reaction.
Both methods, as applied to oily injections of vitamin D,
are compared, and a statistical analysis of the results
reveals that the proposed method is the more precise,
and has an accuracy equal to that of the antimony(II)
chloride method.
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Prosedur kerja
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Hasil
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Hasil pada minyak ikan
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Tocopherol
Crystal Structure of Human Alpha-tocopherol Transfer Protein Bound to
Its Ligand (1R5L)
created by Chimera and POV-Ray
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Preparasi
Transesterifikasi Sampel
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standard additions method
For the standard additions method three separatory funnels were used. To the
first was added a 5.0-mL aliquot of the EDTA-vitamin solution.
A mixture of a 5.0-mL aliquot of the EDTA-vitamin solution and 0.10 mL of
standard solution (measured with a micropipette) was placed in the second.
The third contained 100 mL of the standard solution alone. The tocopheryl
acetate in each solution was extracted with two 10-mL portions of petroleum
ether and the extracts were treated as just described for the samples.
The standard additions method was repeated with 10-mL aliquots.
The tocopheryl acetate content in mg/g of dry multi-vitamin (I.U.) is calculated
from the standard additions method results as follows:
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Reaction
The rate of reaction is
indicated by the rate of
colour development, which
depends on the rate of
reduction of tetrazolium
blue by tocopherol. To
optimize conditions, we
have investigated a number
of parameters such as
alkalinity, temperature,
time, reagent concentration,
solvent, transesterification
and order of additions
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Validasi
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Hasil
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Hasil1
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Hasil2
The method proposed for
determining vitamin K1
combines the advantages
of photochemical
reactions (e.g. selectivity,
sensitivity, cleanliness and
easy manipulation) with
those associated with the
use of flow-injection
systems (e.g. simplicity,
rapidity and low cost)
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Vitamin K
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Terima Kasih
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