IJP International Journal of Pharmagenesis

2(1), January-June 2011, pp. 43-48

To Develop a Simple (UV-VIS Spectrometric) Method for the Estimation of

Multivitamin with Special Reference to Capsules & Tablets

Kumar ASHOK 1* and Rajput Gaurav Kumar2

1
Dept. of Biotechnology, Himachal Institute of Life Sciences, Paonta Sahib-173025 (HP), India
2
Department of Quality Control, Syncom Healthcare Limited Selaqui, Dehradun-248002 (UK), India

Abstract: Fat-soluble vitamin regulation is of particular significance in cystic fibrosis. A vitamin is an organic
compound required as a nutrient in tiny amounts by an organism. In the present study, we had developed a
simple method to estimate the fat soluble vitamin by Spectrophotometer. The vitamin A, D, E and K were estimated
by spectrophotometer at 325 nm, 264 nm, 525 nm and 635 nm respectively. After calibration of the curves for the
absorbance, the data were validated against label claimed on the packaging material. The precision, repeatability,
intermediate precision and accuracy were tested. The accuracy of the method was determined by spiking working
standards of the four vitamins into the placebo at different concentration levels: 80, 100 and 120% of target
concentration of each of the vitamins. The mean recoveries (%) of vitamins A, D3, E and K3 were found to be 99.32,
98.68, 99.10 and 99.28 respectively which are within the acceptance limit. The spectrophotometer estimation is
easier than other existing techniques in the pharmaceutical laboratories.
Keywords: Fat soluble vitamins, UV-visual spectroscopy, Estimation, Tablets, Capsule.

Introduction human diet only in certain circumstances. The term

Vitamins are non-energy producing organic vitamin does not include other essential nutrients
compound, essential for normal human such as dietary minerals, essential fatty acids, or
metabolism that must be supplied in small essential amino acids, nor does it encompass the
quantities in the diet.[1] Fat-soluble vitamins are large number of other nutrients that promote
absorbed through the intestinal tract with the health but are otherwise required less often[3, 4, 5].
help of lipids (fats). Because they are more likely Vitamins are classified by their biological and
to accumulate in the body, they are more likely chemical activity, not their structure. Thus, each
to lead to hypervitaminosis than are water- vitamin may refer to several vitamer compounds
soluble vitamins. Fat-soluble vitamin regulation that all show the biological activity associated with
is of particular significance in cystic fibrosis[2]. A a particular vitamin. Such a set of chemicals are
vitamin is an organic compound required as a grouped under an alphabetized vitamin generic
nutrient in tiny amounts by an organism. A descriptor title, such as vitamin A, which includes
compound is called a vitamin when it cannot be the compounds retinal, retinol, and many
synthesized in sufficient quantities by an carotenoids. Vitamins have diverse biochemical
organism, and must be obtained from the diet. functions, including function as hormones (vitamin
Thus, the term is conditional both on the D), antioxidants (vitamin E), and mediators of cell
circumstances and the particular organism. For signaling and regulators of cell and tissue growth
example vitamins D and K are required in the and differentiation (vitamin A) [6].
Vitamin A acetate chemically, 15-apo-β-
Corresponding Author: Kumar Ashok
caroten-15-ol,3,7-Dimethyl-9-(2,6,6-
E-mail: asokumr@gmail.com trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraen-1-
44 International Journal of Pharmagenesis, 2(1) 2011

ol[7,8]. It is a yellowish powder. Vitamin A occurs vitamin D is to increase the absorption of calcium
in nature in several forms[9]. Retinal (Vitamin A1) and phosphorus from the intestines, and also to
is an unsaturated alcohol containing an ‘ionone’ reciprocally increase the renal excretion of
ring. It is present in marine fish (cod, shark and phosphorus. Such an action is needed for proper
halibut) liver oil, egg yolk, milk and butter. mineralization of bone, and for the regulated of
Dehydroretinol (Vitamin A2) is present in fresh normal plasma calcium levels. It is known that
water fishes. Carotenoids (α, β and γ) and large of vitamin D cause decalcification of bone.
cryptoxanthin, which are fat soluble lipochromes A deficiency is rickets (in children) and
found in plants are converted into vitamin A osteomalasia (adult rickets) [10].
(active form) in the intestinal mucosa or liver.
Vitamin E acetate chemically, (2’RS, 4’RS,
Retinal generated by reversible oxidation of
8’RS)-6-acetoxy-2, 5, 7, 8-tetramethyl-2-(4’,8’,12’-
retinol is a component of the light sensitive
trimethyltridecyl)chroman(all-rec-á-tocopherol
pigment Rhodopsin which is synthesized by rods
acetate)[12]. It is a clear, slightly greenish yellow
during dark adaptation. This pigment gets
viscous oily liquid [9]. Vitamin E is absorbed from
bleached and split into its components by dim
intestine through lymph with the help of bile; it
light and in the process generates a nerve impulse
circulates in plasma in association with â-
through a G-protein called Transducin. Retinal so
lipoprotein, is stored in tissue and excreted slowly
released is synthesized in the cones- responsible
for vision in bright light, colour vision and in bile and urine as metabolites. Vitamin E acts
primary dark adaptation. It maintains the as antioxidant, protecting unsaturated lipids in
functional and structural integrity of epithelial cell membranes, coenzyme Q, etc. from free
cells throughout the body by preventing its radical oxidation damage and curbing generation
metaplasia to stratified squamous type. This of toxic peroxidation products. No clear-cut
vitamin A probably does by stabilizing the –SH vitamin E deficiency syndrome has been
groups in proteins, maintaining the normal described in humans, but vitamin E deficiency has
amounts of mucoprotein, glycoprotein and been implicated in certain in hepatobiliary disease
keratoprotein in epithelial structures. Vitamin A and some cases hemolytic anaemia [10].
is also required for bone growth. A deficiency is Vitamin K 3 chemically , 2-methyl-1,4-
night blindness (nyctalopia) i.e. inability to see naphthaquinone [13]. It is a pale yellow crystalline
in poor light. Increased tendency to urinary stone powder [9]. It is a fat-soluble dietary principle
formation may be due to shedding of ureteric required for the synthesis of clotting factors.
epithelial lining which acts as a nidus [10]. Vitamin K has a basic naphthoquinone structure,
Vitamin D3 is chemically, (5Z, 7E)-(3S)-9, 10- with or without aside chain (R) at position 3. The
secoholesta-5, 7, 10(19)-triene-3-ol[11]. It is a white side chain in K1 is phytyl, in K2 prenyl, while in
crystalline compound9. Vitamin D is the collective K3 there is no side chain. Daily requirement is
name given to antirachitic substances synthesized uncertain, because a variable amount os
in the body and found in foods activated by menaquinone (vitamin K2) produced by colonic
ultraviolet radiation. Vitamin D1, which mixture bacteria becomes available. Vitamin K acts a
of antirachitic substances found in food-only of cofactor at late stage in the synthesis by liver of
historic interest. Vitamin D2 (calciferol), present coagulation protein-prothrombin, factors VII, IX
in irradiated food-yeasts, fungi, bread and milk. and X. The vitamin K dependent change (beta
Vitamin D3 (cholecalciferol) is synthesized in the carboxylation of glutamate residues of these
skin under the influence of UV rays. Vitamin D zymogen proteins) confers on them capacity to
in food requires bile and acids for its proper bind Ca 2+ and to get bound to phospholipid
absorption from the small intestines. Vitamin D surfaces-properties essential for partition in the
itself is inactive. In the body it is converted by coagulation cascade. Vitamin K deficiency is an
hydroxylation to calcifediol and 1, 2, 5- increased bleeding tendency. Epistaxis,
dihydroxycholecalciferol (calcitriol). Alfacalcidol haematuria, ecchymoses, gastrointestinal
is a precursor of calcitriol (one of the two active bleeding, postoperative haemorrhage and
metabolites of vitamin D). The primary action of intracranial haemorrhage are common [14].
To Develop a Simple (UV-VIS Spectrometric) Method for the Estimation of Multivitamin... 45

Material and Methods % w/v), 10 ml glycerol and 25 ml methanol was

added and mixed well, then reflex for 45 minutes
Instrumentation on boiling water bath, cool transfer the solution
Shimadzu-1700 UV-Visible double beam into separating funnel and extract with 50 ml
spectrophotometer with 1 cm matched quartz cell. ether for 5 minutes. Discard the water layer and
ether layer filter through anhydrous sodium
Vitamin A Acetate (Retinol) sulphate. After that evaporate the ether layer to
dryness and dissolved with methanol in 100 ml
Weigh equivalent to 500 IU of sample was taken
volumetric flask.
into round bottom flask. In bottom flask, 2 ml of
potassium hydroxide solution (50% w/v), 10 ml Procedure: 5 ml of the standard, sample and
glycerol and 50 ml methanol added and mixed blank solution was taken in 25 ml volumetric
well, then reflex for 45 minutes on boiling water flask. In each volumetric flask, 2 ml of 0.1% 2, 2
bath and cool. Wash the flask with distil water bilyridil solution (in methanol) and 1 ml of 0.1 %
and taken washing in separator then extract with ferric chloride solution (in water) added and
4×25 ml diethyl ether, combined ether extract mixed well. It was diluted 25 ml with methanol
washed it with water. Discard the water layer and absorbance recorded at 525 nm against blank.
then taken ether layer in dry 100 ml volumetric
flask by passed through anhydrous sodium Vitamin K3 (Menadione)
sulphate and make up to 100 ml with diethyl Standard preparation: Weigh accurately 25 mg of
ether, mixed well. It was absorbance recorded at menadione working standard was taken into 100
325 nm against blank. ml volumetric flask and 50 ml of chloroform was
added to dissolve. The volume was made up to
Vitamin D3 (Cholecalciferol) the mark with chloroform. After that filter and
Standard preparation: Weigh accurately 25 mg further 1 ml was taken into 50 ml volumetric flask
vitamin D3 working standard was taken 25 ml made up to the mark with chloroform.
volumetric flask with solution mixture Sample preparation: Weigh equivalent 250 mcg
(chloroform and methanol in ratio 1:9) dissolved of sample was taken into separator. In separator,
and dilute with solution mixture and make up to 5 ml of water was added, mixed well and extract
the mark mix well. with 4×10 ml chloroform. Discard the water layer
Sample preparation: Weigh accurately then taken chloroform in dry 50 ml volumetric
equivalent to 40, 00000 IU vitamin D3 of sample flask by passed through anhydrous sodium
was taken 25 ml volumetric flask with solution sulphate and made up to 50 ml with chloroform.
mixture (chloroform and methanol in ratio 1:9) Procedure: 5 ml of the standard, sample and
dissolved and dilute with solution mixture and blank solution was taken into test tube. In each
make up to the mark mix well. test tube, added 2 ml of 0.2% solution of 2, 4-
It was absorbance recorded at 264 nm against dinitrophenyl hydrazine (in hydrochloric acid
blank. and alcohol in ratio of 1:5 v/v) and mixed well.
After that heat on water bath until to almost
Vitamin E Acetate (Tocopherol) dryness and cool at room temperature. 15 ml
Standard preparation: Weigh accurately 25 mg solution mixture (ammonia and alcohol in
vitamin E acetate working standard was taken ratio of 1:1) was added in each test tube.
into 100 ml volumetric flask and 50 ml of It was absorbance recorded at 635 nm against
methanol was added to dissolve. The volume was blank [15].
made up to the mark with methanol. Statistical Analysis
Sample preparation: Weigh accurately The proposed methods were successfully applied
equivalent to 25 IU sample of vitamin E acetate to the analysis of vitamins (vitamin A, vitamin
was taken in round bottom flask. In round bottom D3, vitamin E and vitamin K 3) in tablets and
flask, 2 ml of potassium hydroxide solution (50 capsule. These data were subjected to ANOVA
46 International Journal of Pharmagenesis, 2(1) 2011

test to see any significant difference between the Sample Absorbance Standard Weight
Amount of vitamins = × ×
data sets as calculated for p = 0.05. Standard Absorbance Standard Deilution
Sample Dilution
× Standard potancy × Average Weight
Sample Weight
Calculation

∑( x1 + x2 +  xn ) Results and Discussion

Mean =
Number of item( N )
Calibration Curves
∑( x − mean )2 The linearity for the four vitamins were
Standard Division =
( n − 1) determined solutions with concentrations of 5-20
µg/ml of vitamin A, 3-20 µg/ml of vitamin E, 5-
Standard Deviation 25 µg/ml of vitamin D3 and 5-20 µg/ml of vitamin
RSD = × 100 K3 prepared using working standards of each of
Mean
the four vitamins. The linear regression data for
The amount of vitamin A was calculated by the calibration curves indicate that the response
the following formula: is linear over the concentration range studies with
1000 Sample Dilution coefficient of correlation (r) value as 0.9993 of
Amount of vit( IU ) = Sample Absorbance × Factor (1830) × × × Average Weight
100 Sample Weight vitamin A, 0.9998 of vitamin E, 0.9981 of vitamin
The amount of vitamins (D3, E and K3) was D 3 and 0.9989 of vitamin K 3 . Results are
calculated by the following: summarized in Table 1.

Table 1
Parameter Value
Vitamin A Vitamin D3 Vitamin E Vitamin K3
λ max (nm) 325 264 525 635
Beer’s law limits (µg/mL) 5-35 3-20 100-350 3-10
Correlation coefficient (r) 0.9993 0.9981 0.9998 0.9989
Regression equation (Y) a

Slope, b 0.0694 0.1265 0.0603 0.1043

Intercept, c 0.0943 0.0652 0.0539 0.0749
a
Y = bX + c, where X is the concentration of drug in µg/ml.

Validation of the Method: The amount found in repeatability (intraday-assay precision) and
the claiming the label and their precision for intermediate precision are listed in the table 2.

Table 2
Results of Assay and Precision Studies
Ingredient Label claim Amount Percentage* Standard Precision**
Found* Deviation* Repeatability Intermediate
(intra-assay Precision
precision)
Retinol (Vitamin 5000 IU 6352.7 IU 127.05 0.3631 0.2858 0.4063
A Acetate)
Cholecalciferol 200 IU 344.53 IU 172.26 0.5326 0.3092 0.7542
(Vitamin D3)
Tocopherol 25 IU 27.09 IU 108.36 0.3268 0.3016 0.7375
(Vitamin E Acetate)
Menadione (Vitamin K3) 70 mcg 74.89 mcg 106.98 0.8533 0.7976 0.9723
*Mean of six determinations. **%RSD of six determination Precision
To Develop a Simple (UV-VIS Spectrometric) Method for the Estimation of Multivitamin... 47

The validity of the methods for the assay of Intermediate precision is usually
vitamin was examined by determining precision demonstrated by repeated measurements of the
and accuracy [16]. In this study, we had validated sample used in the repeatability experiment
the protocol for UV-visual spectrophotometer and within the same laboratory. Usually the
four vitamins were estimated by the same repeatability experiment is repeated on the same
instrument. These were determined by analyzing sample by a different analyst, on a different day,
six replicates of the drug within the Beer’s law using different equipment if possible.
limits. The results of analysis of dosage forms are
given in Table 2. Precision was measured in terms Accuracy
of repeatability of application and measurement The accuracy of the method was determined by
date. Repeatability of a standard sample was spiking working standards of the nine vitamins
carried out six sample of the same batch. Precision into the placebo at different concentration levels:
is usually expressed as the relative standard 80, 100 and 120% of target concentration of each of
deviation (co-efficient of variation). Precision the vitamins. The resulting solutions were assayed
should be considered at different levels as in triplicate and results obtained were compared
follows: with the expected results and expressed as
percentage [18, 19]. The mean recoveries (%) of
Repeatability (Intra-assay Precision) vitamins A, D3, E and K3 were found to be 99.32,
Repeatability expresses the precision under the 98.68, 99.10 and 99.28 respectively which are within
same operating conditions over a short interval the acceptance limit.
of time. Repeatability is also termed intra-assay
precision. Repeatability of the sample preparation Table 3
Accuracy of Vitamins in Percentage
from same homogenous of marketed sample (10,
1000, 250 and 5 µg/ml of vitamin A, vitamin D3, S. No. Vitamin A Vitamin D3 Vitamin E Vitamin K3
vitamin E and vitamin K3 respectively). The low 1 99.12 99.42 98.36 99.27
values of relative standard deviation (R.S.D.) 2 100.02 98.23 99.26 98.84
indicate good precision of the methods. 3 98.82 98.41 99.69 99.74
The RSD for repeatability of standard Mean 99.32 98.68 99.10 99.28
preparation is 0.4431, 0.3438, 0.5915 and 0.3216 for ±SD 0.6244 0.6414 0.4697 0.4501
vitamin A, vitamin D3, vitamin E and vitamin K3 RSD 0.6286 0.6499 0.4739 0.4533
respectively, whereas the RSD for repeatability of
sample preparation is 0.2858, 0.3092, 0.3016 and Conclusion
0.7976 vitamin A, vitamin D 3, vitamin E and
A simple, sensitive, highly accurate UV-visual
vitamin K 3 respectively. This show that the
spectrophotometric method for determination of
method is precise as RSD is below 2.0% (RSD is
vitamins (vitamin A, vitamin D3, vitamin E and
not more than 2.0% as a suggested in IP 2010) [17].
vitamin K3) in pharmaceutical capsule and tablet
Table 2 represents the precision data obtained for
dosage form was developed and validated. The
the method.
study proved that the UV-VIS Spectrophotometer
was the best instrument for estimation of fat
Intermediate Precision soluble vitamin in easier way.
Intermediate precision expresses within-
laboratory variations: different days, different Acknowledgements
analysts and equipment. Intermediate precision The authors are grateful to Syncom Healthcare Limited
of the method was determined by same sample (Labs), Dehradun for providing gift sample of the vitamins
and Mrs. K.P. Rathoure for technical help.
blend by two different analysts, using different
instruments and on different days. The results are References
included in Table 2. As the value of RSD is below
[1] Bender, David A. Nutritional biochemistry of the
2% for the nine vitamins, intermediate precision vitamins. Cambridge, U.K.: Cambridge University
of the method is established. Press. 2003.
48 International Journal of Pharmagenesis, 2(1) 2011

[2] Lieberman, S., Bruning, N. The Real Vitamin and [11] The Marck Index; Monograph No. 10079. 13 th edn.
Mineral Book. NY: Avery Group, 3. 1990. Marck and Co., White House Station, NJ, USA, 2001.
[3] Bolander F. F. Vitamins: Not Just for Enzymes. Curr pp. 1786.
Opin Investig Drugs 2006, 7(10): 912–5. [12] The Marck Index; Monograph No. 10081. 13 th edn.
[4] Maqbool A., Stallings V. A. Update on Fat-soluble Marck and Co., White House Station, NJ, USA, 2001.
Vitamins in Cystic Fibrosis. Curr Opin Pulm Med. 2008, pp. 1787.
14(6): 574–81. [13] The Marck Index; Monograph No. 10082. 13 th edn.
[5] Maton, Anthea; Jean Hopkins, Charles William Marck and Co., White House Station, NJ, USA, 2001.
McLaughlin, Susan Johnson, Maryanna Quon Warner, pp. 1788.
David LaHart, Jill D. Wright; Human Biology and [14] Rohde L. E., Assis M. C., Rabelo E.R. Dietary Vitamin
Health. Englewood Cliffs, New Jersey, USA: Prentice K intake and Anticoagulation in Elderly Patients. Curr
Hall. 1993. Opin Clin Nutr Metab Care, 2007: 10 (1): 1–5.
[6] Barar F. S. K.; Essentials of Pharmacology. 4 th edn. S. [15] Sethi P. D. Quantitative Analysis Drug Pharmaceutical
Chand and Company Ltd., New Delhi, 2008, pp. 376- Formulation. CBS Publishers and Distributors, Darya
382. Ganj, New Delhi. 2006, pp. 603.
[7] Martindale. The Complete Drug Reference. 34 th edn. [16] Analytical Methods Committee. Recommendations for
Pharmaceutical Press. 1996. the Definition, Estimation and use of the Detection
Limit. Analyst 1987, 112: 199-204.
[8] The Merck Index; Monograph No. 10073. 13 th edn.
Marck and Co., White House Station, NJ, USA, 2001. [17] Indian Pharmacopoeia. Government of India, Ministry
pp. 1785. of Health and Family welfare. 6 th edn. The controller
of publication, Civil Lines, Delhi, India. 2010.
[9] Indian Pharmacopoeia. Government of India, Ministry
[18] ICH. Draft Guidelines on Validation of Analytical
of Health and Family Welfare. 5 th edn, The Controller
Procedures, Definitions and Terminology, Federal
of Publication, Civil Lines, Delhi, India. 2007.
Register, 60, IFPMA, Switzerland 1995: 1260.
[10] Tripathi K. D.; Essentials of Medical Pharmacology. 6th
[19] ICH. Guidelines Q2BValidation of Analytical
edn. Jaypee Brother Medical Publication (P) Ltd., New
Procedures: Methodology. International Conference on
Delhi; 2008. pp. 869-878.
Harmonization. Geneva 1996.