AnFar 2013 Vitamin

Analisis SEDIAAN
Farmasi
Golongan Vitamin
Solubilit
y
logP
Thiamine
Water
-2.96
Vitamin B12
Cyanocobalamin, hydroxycobalamin,
methylcobalamin
Water
-3.52
Vitamin B2
Riboflavin
Water
-0.85
Vitamin B3
Niacin, niacinamide
Water
-0.61
Vitamin B5
Pantothenic acid
Water
-1.31
Vitamin B6
Pyridoxine, pyridoxamine, pyridoxal
Water
-1.05
Vitamin B7
Biotin
Water
0.20
Vitamin B9
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
Generic name
Vitamer chemical name(s) (list not complete)
Vitamin B1
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broto_santoso@yahoo.com
2 Mei 2013
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Vitamin C dan B
VITAMIN LARUT AIR (water soluble)
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Methods from literature

1. Capillary electrophoresis: Vitamin B1, B2, B6 dan
nikotinamida (S. Boonkerd, M.R. Detaevernier, dan Y. Michotte,
1994; J. Chromatography A, 670: 209-214)
2. Cyclic voltammetry: Vitamin B6 (Teixeira et al., 2003; J. Braz.
Chem. Soc., 14(2): 316-321)
3. Fluorimetric: Vitamin B12 (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)
2
2013
6
7.Mei
Non-spectrophotometric
methods: Vitamin C (S.P. Arya, M.
created by Chimera and POV-Ray
Vitamin C (asam askorbat)

Human Glutathione Transferase O1-1 C32S Mutant in
Complex with Ascorbic Acid (3VLN)
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Pilihan Metode
No
Metode
Sampel
Sumber
1. Catalytic voltammetric pharmaceutical

preparations &
complex matrices
of fresh
fruit juices
Talanta 44: 297303

(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. Nonspectrophotometric
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natural and
fortified food
samples &
Analytica Chimica
Acta 2000 417: 1- 8
14
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,
D2 and E, nicotinamide, calcium pantothenate, and folic
acid together with minerals (Fe2+, Ca2+, Mg2+, Mn2+, Cu2+,
Zn2+ and Mo2+ );
6. multivitamin tablets containing vitamins A, B complex,
D2 and E, nicotinamide, calcium panthothenate, folic acid.
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10
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 cm2 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 LiCIO4 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 LiCIO4
dan 0,1 mM ferrocenecarboxylic
acid) kemudian diukur dengan
voltammeter sehingga diperoleh
voltammogram. Konsentrasi asam
askorbat ditentukan dari rerata dari
grafik kalibrasi.
12
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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13
Cyclic
voltammograms
(a) 2 mM ferrocenecarboxylic
acid in 0.5 M glycine buffer +
0.1 M LiCIO4 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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14
Procedure
Sejumlah volume larutan

dari ampul diambil
dengan pipet volume
Injection
yang setara dengan 100
An accurate ampoule volume
mg asam askorbat,
equivalent to about 100 mg
dipindahkan ke dalam
of ascorbic acid was
labu takar 50 mL dan
transferred to a 50 mL flask
and diluted to volume with
ditambahkan larutan
0.5% citric acid solution. A 1
asam sitrat 0,5% hingga
mL portion of the solution
tanda. Diambil 1 mL dan
was diluted in a voltammetric
diperlakukan seperti
cell to 10 mL as described for
yang telah dijelaskan
tablets and the cyclic
untuk tablet.
voltammogram was recorded
using the GC electrode
Voltammogram direkam
menggunakan elektroda
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15
GC.
Result from article
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16
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 203 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 As203 atau larutan titrisol tiosulfat
digunakan dalam metode pembanding seperti
yang tercantum dalam US Pharmacopeia (USP).
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18
HPLC-DAD
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19
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, MnSO4 and Na2SeO3).
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20
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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21
Instrumentasi
An HP 1100 liquid chromatographic system (HewlettPackard, 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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22
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 mLmin1. Auto sampler
injection was 3 L.
Gradient of mobile phase and its composition
was tested, too. See next slide.
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23
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 gmL1. Other
conditions are the same as in
Fig. 2.
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24
Simultaneous HPLCUV
determination
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25
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.
HPLCUV 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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26
Kromatogram
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27
Validation
Retention times in min. b Regression coefficients. c Limits of detection (3.S/N).

Relative standard deviations. f Non-vitaminous substances.
a
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Limits of quantitation (10.S/N).
28
Result from article
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29
Spectrophotometric
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30
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31
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 (PfizerEgypt), 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, D2 and E with
minerals: Mineravit (Eipico) , Obron (Pfizer-Egypt) and Viterra (Pfizer-Egypt);
6. syrups containing vitamins A, B complex, D2 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 Iaminoanthraquinone (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 1aminoanthraquinone (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 1aminoanthraquinone 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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38
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 reagentreducing 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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39
Concentration of
The
colour developed strongly
Reagent
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
Diluting
Methanol, of
ethanol,
water,
propan-2-ol,
Solventsdimethyl
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 1,2dichIoroethane 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?
2
Mei 2013
42
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:
A630 = -0.0324 + 0.024% (g mL1
);
r = 0.9990, n = 8. The molar
absorptivity was 4.07 x 1031
mol-1 cm-1.
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Dimethoxydiquinone
Dimethoxydiquinon
e gives a violetcolored product
with ascorbic acid
in a phosphate
buffer (pH 6.6). The
reduced indigoid
quinhydrone form
is perhaps
responsible for the
formation of violetcolored 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 ml1 with a
detection limit of 10 g ml1 .
45
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 (=1.62x103), 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 cm2 mol1) 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 4OH
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 ml1.
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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-2nitroaniline 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-7nitrobenzofurazane
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
gmL1. 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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53
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 mL1.
Sugars (>15 g mL1) 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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54
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 mL1.
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AA reacts with potassium iodide-iodate solution under

acidic conditions to liberate iodine & this compound
selectively oxidizes LMG to MG dye
LEUCOMALACHITE GREEN
(LMG)
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57
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
2.98x105 l mol-1cm-1, 0.0042 g cm-2, and
respectively.
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reaction
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59
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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60
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.
2 Mei 2013
61
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
2
method
Mei 2013 is good alternative
to some reported costly
62
Non-spectrophotometric
2 Mei 2013
63
Titrimetric methods
1.
2.
3.
4.
2,6-Dichlorophenolindophenol (DCIP)
tetrachlorobenzoquinone
N-bromosuccinimide
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
2 Mei 2013
64
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), SO2,
SO32 and S2O32 ions which are usually
associated with mineral or liver preparations.
2 Mei 2013
66
reaction
J Pharmaceutical Sciences Vol. 72, No. 2, February 1983

Analytical Sciences October 1998, Vol. 14
2 Mei 2013
67
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
2 Mei 2013
68
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
2 Mei 2013
69
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, omercaptobenzoic acid, mercaptosuccinic acid and 3mercaptopropionic acid cannot be resolved
the interference of thiols is reported to be avoidable
by masking with acrylamide
2 Mei 2013
71
Reaction TCBQ
2 Mei 2013
72
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 chloramineT in the presence of potassium iodide
CH3C6H4SO2NCl-Na+ + 2I- + 2H+ CH3C6H4SO2NH2 + Cl-Na+ +
I2
2RSH + I2 RSSR + 2HI
2 Mei 2013
73
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 Nbromophthalimide and N-bromosaccharin
These reagents were also used in the potentiometric
titration of Vitamin C. Cysteine and glutamic acid
interfere
2 Mei 2013
75
N-bromosaccharin was used for the
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
2 Mei 2013
77
cerium(IV) sulfate
2 Mei 2013
78
Lebih detail lihat di

Analytica Chimica Acta 2000 417: 1-14
Non-spectrophotometric methods for
the determination of Vitamin C
2 Mei 2013
79
Riboflavin
Human Liver Glycogen Phosphorylase a Complexed with
Riboflavin, N-2 Acetyl-beta-D-Glucopyranosylamine and
CP-403,700 (1L5R)
2 Mei 2013
80
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
Na2SeO3).
2 Mei 2013
81
Kromatogram
2 Mei 2013
82
Validation
Retention times in min. b Regression coefficients. c Limits of detection (3.S/N).

Relative standard deviations. f Non-vitaminous substances.
a
2 Mei 2013
Limits of quantitation (10.S/N).
83
Result from article
2 Mei 2013
84
FIA
2 Mei 2013
86
Reaction
Riboflavine forms a 1:1 complex with

Ag(I) within pH 6.5 and 7.1
Pyridoxine reacts with N,N-diethyl-pphenylenediamine and potassium
hexacyanoferrate(III) in phosphate
buffer yielding an indophenol dye
2 Mei 2013
87
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)
2 Mei 2013
88
Hasil
2 Mei 2013
89
Application Note 251

Determination of Water- and FatSoluble Vitamins in Nutritional
Supplements by HPLC with UV
Detection
using
Dionex UltiMate 3000 HPLC
system
2 Mei 2013
90
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
2 Mei 2013
91
Sistem Kromatografi
Sistem gradien eluen
2 Mei 2013
Panjang gelombang
92
Kondisi KCKT
Vit Water-soluble
2 Mei 2013
Vit Fat-soluble
93
Kromatogram
VC & VB group vitamins
2 Mei 2013
fat-soluble vitamins &

carotene
94
Hasil-method detection limit

(MDL)
2 Mei 2013
95
2 Mei 2013
96
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 100mL standard flask and made up to the mark with
water.
2 Mei 2013
97
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 darkglass 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).
2 Mei 2013
98
Kurva & perolehan kembali
2 Mei 2013
99
Hasil
2 Mei 2013
100
Metode lainnya
Derivatisasi KCKT: Analytical Biochemistry,
333 (2004) 336344 VitB6 & B12
Spektofotometri derivatisasi: J.Pharm.
Biomed. Analysis, 22 (2000) 915923
VitB1 & B6
Voltammetri: J. Braz. Chem. Soc., 14_2
(2003) 316-321 & Talanta, 61 (2003)
743/753 VitB6 & B12
Fluorometri: Fresenius J Anal Chem, 368
(2000) 836838 VitB12
2 Mei 2013
101
Vitamin A, D, E dan K
VITAMIN TIDAK LARUT AIR (fat

soluble)
2 Mei 2013
102
Retinol
Crystal Structure of Human Holo Cellular Retinol-binding
Protein II (CRBP-II) from 2RCT
2 Mei 2013
103
2 Mei 2013
104
Metode Analisis
2 Mei 2013
105
Flow Injectionchemiluminescence
Tris(2,2-bipyridyl)ruthenium(II)
[Ru(bpy)32+]
2 Mei 2013
106
Hasil
2 Mei 2013
107
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.
2 Mei 2013
108
2 Mei 2013
109
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.
2 Mei 2013
110
Retinol
Crystal Structure of Human Holo Cellular Retinol-binding
Protein II (CRBP-II) from 2RCT
2 Mei 2013
111
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.
2 Mei 2013
112
Prosedur kerja
2 Mei 2013
113
Hasil
2 Mei 2013
114
Hasil pada minyak ikan
2 Mei 2013
115
Tocopherol
Crystal Structure of Human Alpha-tocopherol Transfer
Protein Bound to Its Ligand (1R5L)
2 Mei 2013
116
2 Mei 2013
117
Preparasi
Transesterifikasi
2 Mei 2013
Sampel
118
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:
2 Mei 2013
119
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
2 Mei 2013
120
Validasi
2 Mei 2013
121
Hasil
2 Mei 2013
122
2 Mei 2013
123
Hasil1
2 Mei 2013
124
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 flowinjection systems (e.g.
simplicity, rapidity and
low cost)
2 Mei 2013
125
Vitamin K
2 Mei 2013
126
Terima Kasih
2 Mei 2013
127

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Analisis SEDIAAN

Pyridoxine, pyridoxamine, pyridoxal

Folic acid, folinic acid

Retinol, retinal & 4 carotenoids including beta

Vitamer chemical name(s) (list not complete)

VITAMIN LARUT AIR (water soluble)

Methods from literature

created by Chimera and POV-Ray

Vitamin C (asam askorbat)

1. Catalytic voltammetric pharmaceutical

Talanta 44: 297303

Analyst 1991 116:

Sejumlah sampel (serbuk) yang

Some electrogenerated ferricinium derivatives (ferriciniumcarboxylic

Sejumlah volume larutan

Result from article

Retention times in min. b Regression coefficients. c Limits of detection (3.S/N).

Limits of quantitation (10.S/N).

Result from article

Ampoules, syrups & drops

Procedure for tablets

Procedure for multi-vitamin capsules

Fast Red AL salt (dioazotized 1aminoanthraquinone stabilized

Ponting, J. D., Ind. Eng. Chem.

Result from article

At 630 nm, the absorbance was

After diluting with dioxane, absorbance

Ninhydrin & Resorcinol

AA reacts with potassium iodide-iodate solution under

J Pharmaceutical Sciences Vol. 72, No. 2, February 1983

Anal. Chem., 1982, 54 (4), pp 793796

Remove the thiols

iodine, potassium iodate, potassium

Lebih detail lihat di

created by Chimera and POV-Ray

Analytica Chimica Acta 2004

1. B-Komplex tablets (Lciva, Prague, Czech Republic)

Retention times in min. b Regression coefficients. c Limits of detection (3.S/N).

Limits of quantitation (10.S/N).

Result from article

Riboflavine forms a 1:1 complex with

The reagent consumption was in media 25-fold lower

Application Note 251

fat-soluble vitamins &

Hasil-method detection limit

Kurva & perolehan kembali

VITAMIN TIDAK LARUT AIR (fat

created by Chimera and POV-Ray

Prosedur dan hasil

created by Chimera and POV-Ray

Analyst 1975 100: 238-242

Hasil pada minyak ikan

created by Chimera and POV-Ray

standard additions method

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