Indian J. Pharm. Biol. Res. 2016; 4(4):32-38
CODEN (USA): IJPB07
ISSN: 2320-9267
Indian Journal of Pharmaceutical and Biological Research (IJPBR)
Journal homepage: www.ijpbr.in
Research article
Evaluation of Vitamin B1, B2 and B6 Tablets in Bangladesh by UV-Vis Spectrophotometer
Abida Sultana, Md. Sharear Saon, Mohammad Shoeb and Nilufar Nahar*
Department of Chemistry, University of Dhaka, Dhaka-1000
ARTICLE INFO:
Article history:
ABSTRACT
Received: 4 August 2016
Received in revised form:
1 October 2016
Accepted: 30 November 2016
Available online: 31 December 2016
Thiamine hydrochloride (vitamin B1), riboflavin (vitamin B2) and pyridoxine hydrochloride
(vitamin B6) were assayed in pharmaceutical dosages form. Thiamine hydrochloride of 100
mg dose of six different companies, riboflavin of 5 mg dose of five different companies and
pyridoxine hydrochloride 20 and 25 mg dosages of two different companies of Bangladesh
were extracted from the tablets, cleaned up and their active ingredients were evaluated by
UV-Vis spectrophotometer at 432, 445 and 292 nm, respectively. Evaluation was carried out
with respect to calibration curves of their standard reference samples. Amount of thiamine
hydrochloride in 100 mg tablets of six different companies were found to be in the range of
37.62±1.11 mg to 79.03±0.42 mg. For riboflavin in 5 mg tablets of five different companies,
active ingredient was found in the range of 6.78±0.19 mg to 8.23±0.15 mg. In case of
pyridoxine hydrochloride tablets, it was found that 20 mg tablet of one company contained
21.75±0.41 mg and 25 mg tablet of another company contained 29.72±0.59 mg of active
ingredient. Recovery experiments were done by spiking excipients of the respective
medicines at three different concentration levels with 5 replicate studies. Correlation
coefficients (r2) were found to be 0.99 for three vitamins and recoveries were 82.26± 0.10,
99.61± 0.06 and 109.91±0.12 for thiamine hydrochloride (vitamin B1), riboflavin (vitamin
B2) and pyridoxine hydrochloride (vitamin B6) respectively.
Keywords:
Thiamine hydrochloride,
Riboflavin,
Pyridoxine hydrochloride,
UV-Vis spectrophotometry
Introduction
Vitamins are essential nutrients for performing necessary
chemical and physiological functions in the human body
[1].On the basis of solubility, they are two types, solubility in
fat (A, D, E, and K vitamins)and water (C and B vitamins)[2].
The B vitamins are named as thiamine (vitamin B1),
riboflavin (vitamin B2), niacin (vitamin B3), pyridoxine
(vitamin B6), pantothenic acid (vitamin B5), biotin (vitamin
B7), folate (vitamin B9) and cyanocobalamin (vitamin
B12).The chemical structure of the thiamine hydrochloride
molecule is shown in Figure 1(a).It helps to prevent from
heart disease[3], brain disorder[4], mental illness[5, 6], autism
[7],to alleviate dysmenorrheal [8],protect against uremia[9],
renal disease[10], to the diabetic neuropathy[11 ], cures
Beriberi[12] and helps to the elderly population by improving
sleep and increase energy[13]etc. Vitamin B2 (Figure 1b) is
riboflavin whose IUPAC name is 7, 8-dimethyl-10-(1′-dribityl) isoalloxazine. It has been used in several clinical and
therapeutic situations including prevention of migraine[14,
15],gastroplasty[16],helps to ensure proper growth of unborn
*
babies[17],protect against gingivitis[18], beneficial for
premature babies[19], antiretroviral side effects[20]etc.
Vitamin B6 (Figure1c), chemically pyridoxine hydrochloride
is 5-hydroxy-6-methylpyridine- 3,4-dimethanol hydrochloride.
Its intake is necessary forrelieving the nausea and vomiting
associated with early pregnancy[21], help to alleviate
neuropathy[22],infant health growth[23], reduce stress and
anxiety[24],reduce risk of heart disease[25, 26], reduce blood
pressure in patients with hypertension[27], reduce risk of
lung cancer[28] etc.Vitamins have a variety of uses in
industrial and clinical sectors. They uses in foods as colorants,
antioxidants and especially nutritive additives [29]. Although
the vitamin requirement of the human body can be easily
satisfied by a balanced diet, but there is still a chance of
susceptibility to low micronutrient intakes and hence a higher
risk of vitamin deficiency. Enrichment and fortification of
vitamins in foods such as infant foods, fruit juices, milk,
cereal, etc. have helped address this issue. For planning a
healthy and balanced diet, the proper information on vitamin
Corresponding Author: Nilufar Nahar, Department of Chemistry, University of Dhaka, Dhaka
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Sultana et al Indian J. Pharm. Biol. Res., 2016; 4(4):32-38
content in food sources consumed frequently is critical to
assessing dietary adequacy. That is where the significance of
vitamin analysis comes into play. As many vitamins are
unstable and easily degraded, monitoring their loss during
processing is important in the development of appropriate
processing and storage schemes for optimal nutrient content in
the final food products [30]. Moreover, addition of vitamins
into food needs to be properly controlled to satisfy the
guidelines set by the governmental authorities [29].
Sometimes, the deficiency of vitamins in human body causes a
lot of diseases. Therefore, rapid and reliable analysis of
vitamins is in high demand by food manufacturers and drug
administrators. In continuation of our research group to work
on contaminants of food stuff, the objective of the present
work was to develop simple, precise, rapid, sensitive and
accurate methods to assay vitamin B1, B2 and B6 by UVvisible spectrophotometer.
Materials and Methods
Sample collection
Commercial tablets of thiamine hydrochloride (B1), riboflavin
(B2) and pyridoxine hydrochloride (B6) were collected from
different pharmacies in Dhaka city. Six different types of
commercial thiamine hydrochloride (coded as T1 to T6), five
different types of commercial riboflavin(coded as R1 to R5)
and 2 different types of commercial pyridoxine hydrochloride
(coded as P1 and p2) samples were collected for analysis. All
of them were in tablet form. Standard sample of vitamin B1,
B2, B6 and 4-aminophenol were obtained by courtesy of New
life and Co.Pvt.Ltd., Bangladesh.
Chemicals and Reagents
Methanol, acetone, acetic acid, ammonia solution, chloroform
(purchased from BDH England) and HPLC grade water were
used to carry out the study.
Instruments
A double beam Ultraviolet-visible spectrophotometer
(Shimadzu, UV-1800), an analytical balance (AL 104, Mettler
Toledo), a vortex mechine (Kebo LabReax-2000), anoven ( G1020, Salvis)and a centrifuge machine (Cowbell) were used.
Preparation of standards solutions
Thiamine hydrochloride (vitamin B1)
The primary standard solution (1000μg/mL) of riboflavin
(vitamin B2)was made by dissolving 100 mg of riboflavin in
100.0 mL volumetric flask by HPLC grade water. Primary
standard solution was diluted to 500, 100 and 50 μg/mLwith
HPLC grade water. Preparation steps were performed in the
subdued light condition using glass wares covered with foil to
prevent vitamins from degradation.20 mL of the thiamine
hydrochloride solution (50μg/mL) was taken in a separating
funnel. Then 10 mL of previously prepared 100 μg/mL 4aminophenol solution, 20 mL concentrated ammonia solution
and 40 mL of chloroform was added and mixed well. Then the
whole mixture was kept for settle down and a yellow colored
complex and two layers were formed. The yellow colored
complex was extracted in chloroform layer. Thiamine
hydrochloride in 50 μg/mL 20 mL solution is extracted in 40
Research Article
mL chloroform layer. So, the final concentration of thiamine
hydrochloride was 25μg/mL. The working standard solutions
were prepared by dilution of 25 μg/mL thiamine hydrochloride
and 4-aminophenol complex solution by chloroform. The
working standard solutions of thiamine hydrochloride and 4aminophenol complex were prepared in concentration 0.1, 0.5,
1, 2.5, 5, 10, 12.5, 15, 17.5, 20 and 25μg/mL. Absorptions of
working standard solutions of the respective medicines were
measured; calibration curves were drawn by plotting
absorption vs concentration graph (Fig. 2a) and limit of
detections were found out.
Riboflavin (B2)
The primary standard solution (1000μg/mL) of riboflavin
(vitamin B2) was made by dissolving 100 mg of riboflavin in
a 100 mL volumetric flask by HPLC grade water. The
working standard solutions of riboflavin (0.5, 1, 5, 10, 20, 40,
50, 100 and 500 μg/mL) were made from primary standard
solution by dilution with the same solvent. Absorbance
(deionized water was taken as reference) of these solutions
were measured by a double beam UV spectrophotometer to
draw a calibration curve of absorbance vs. concentration
(Fig2b).
Pyridoxine hydrochloride (B6)
The primary standard solution (500μg/mL)of pyridoxine
hydrochloride (vitamin B6) was made by HPLC grade water.
After dilution by the same solvent, working standard of
pyridoxine hydrochloride solutions (0.5, 1, 2.5, 5, 10, 12.5, 25,
50, 80, 100, 120, 150, 200 and 250 μg/mL)were made. 10 mL
from each solution was taken in 50 mL volumetric flask
separately. Then 25 mL of methanol was added followed by
the addition of 3.5 mL of glacial acetic acid. Then the solution
was up to the marked by deionized water. As 10 mL of each
solution was transferred and up to the marked in 50 mL
volumetric flask the final concentration of each solution was 5
times diluted. So the final concentration of 0.5, 1, 2.5, 5, 10,
12.5, 25, 50, 80, 100, 120, 150, 200, 250 μg/mL converted to
0.1, 0.2, 0.5 1, 2, 2.5, 5, 10, 16, 20, 24, 30, 40, 50 μg/mL
respectively. A blank solution was prepared in a 50 mL
volumetric flask by adding 25 mL of methanol, 3.5 mL of
acetic acid and then up to the marked by deionized water.
Absorbance (blank solution was taken as reference) of the
prepared working standard solutions were measured by a
double beam UV spectrophotometer to draw a calibration
curve (Fig.2c) of absorbance vs. concentrations.
Extraction and cleanup of active ingredients
Thiamine hydrochloride (vitamin B1) sample solutions
Ten tablets ofriboflavin of each company were weighted,
homogenized by making powder (using mortar and pestle).
Three replicate studies were done with the homogenized
powder of the medicine.By considering the actual weight of
tablet and written active constituent (100 mg of thiamine
hydrochloride) required amount of homogenized sample
powder was suspended in HPLC grade waterfor preparing50
μg/mL thiamine hydrochloride solution in 100.0 mL
volumetric flask. The suspended materials were vortex (2
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Sultana et al Indian J. Pharm. Biol. Res., 2016; 4(4):32-38
min), centrifuged (3000 rpm; 5 min) and the supernatants were
collected. The solution was filtered by passing through syringe
filter for removing any un-dissolved particles. Then 20 mL of
the 50 μg/mL thiamine hydrochloride tabletsolution was taken
in a separating funnel by the addition of 20 mL ammonia
solution, 10 mL previously prepared 4-aminophenol solution
(100 μg/mL) and 40 mL of chloroform. Couple of minute later
a nice yellow coloured solution was formed. The lower part of
the two layers was chloroform layer containing 1:1 complex of
thiamine hydrochloride and 4-amino phenol. 3 mL solution
from the chloroform layer was taken in a 10 mL volumetric
flask and then up to the marked by chloroform. The final
concentration was7.5μg/mL.The active ingredients present in
the supernatants were cleaned up by filtering through HPLC
grade syringe filters (0.45 µm). Absorptions of the cleaned
extracts (three replicates for each company of each medicine)
were measured and their concentrations were calculated using
calibration curves.
Relative Standard Deviations were
calculated and the data are presented in the Table 2.
Riboflavin (vitamin B2) sample solutions
Weight of ten tablets ofriboflavin of each company was
measured, homogenized by making powder (using mortar and
pestle). Three replicate studies were done with the
homogenized powder of the medicine. Considering the
average weight of riboflavin (5 mg)tablets, the required
amount of powder was suspended in HPLC grade water to
obtain 20 µg/mL active ingredients of the medicines. A
yellow-orange solution was formed. The solution was cleaned
up by filtering through HPLC grade syringe filters (0.45
µm).Three replicates for each brand were done andabsorptions
of the cleaned extracts were measured(Table 3).
Pyridoxine hydrochloride (vitamin B6) sample solutions
Weight of ten tablets ofpyridoxine hydrochloride of each
company was measured, homogenized by making powder
(using mortar and pestle). Three replicate studies were done
with the homogenized powder of the medicine. Considering
the average weight of pyridoxine hydrochloride (20 mg and 25
mg of pyridoxine hydrochloride) tablets, the required amount
of powder was suspended in HPLC grade water to obtain 10
µg/mL active ingredients of the medicines. The suspended
materials were vortex (2 min), centrifuged (3000 rpm; 5 min)
and the supernatants were collected. The active ingredients
present in the supernatants were cleaned up by filtering
through HPLC grade syringe filters (0.45 µm). 10 mL from
this solution was transferred in a 50 mL volumetric flask
where 3.5 mL of acetic acid and 25 mL of methanol were
added earlier. The solution was then up to the marked by
deionized water. A blank solution was also prepared in a 50
mL volumetric flask by adding 25 mL of methanol, 3.5 mL of
acetic acid and then up to the marked by deionized
water.Absorptions of the cleaned extracts (three replicates for
each company of each medicine) were measured and their
concentrations were calculated using calibration curves.
Relative Standard Deviations were calculated and the data are
presented in the Table 4.
Recovery experiment
Research Article
Recovery experiments were done by spiking the respective
medicines to the excipients at three different concentration
levels (6, 7.5 and 9 µg/mL of thiamine hydrochloride; 10, 20,
30 µg/mL of riboflavin; and 6, 10, 12µg/mL of pyridoxine
hydrochloride). Five Replicates were done for each
concentration. Relative Standard Deviations were calculated
and the results are presented in Table 1.
Results and Discussion
The active ingredients of thiamine hydrochloride, riboflavin
and pyridoxine hydrochloride were evaluatedby UV-Vis
spectrophotometer at 432, 445 and 292 nm, respectively. The
wavelength of maximum absorption (max)of the cleaned-up
extracts of thiamine hydrochloride, riboflavin and pyridoxine
hydrochloride tablets (Fig. 3b, 4b and 5b) were fitted very
well with their respective standard spectra (Fig. 3a,4a and 5a)
and overlain spectra (3c, 4c and 5c).In these studies the
methods were modified and validated by 5 replicate studies.
The correlation coefficients (r2) values 0.9993, 0.9941 and
0.9974 (Fig. 2a, 2b and 2c) of thiamine hydrochloride,
riboflavin and pyridoxine hydrochloride were showed
accuracy of experiments. Low limit of detection (LOD) were
0.1, 1 and 0.1 µg/mL (Fig. 3c, 4c & 5c) showed sensitivity of
the modified methods. The method was validated from
recovery experiments (Table 1) and their low respective
relative standard deviations were indicated that the method
was satisfactory. The results also showed acceptable
repeatability and reproducibility of the methods. The % of
mean recovery was 82.26± 0.10 for thiamine hydrochloride,
99.61± 0.06 for riboflavin and 109.91±0.12for pyridoxine
hydrochloride.
In case of thiamine hydrochloride the number of sample
analyzed was showed (Table 2) a range of results (37.62±1.11
to 79.03±0.42 mg). All samples were found to be contained
lower amount of active ingredients than the amount written on
the label (100 mg).For riboflavin tablets(Table 3), active
ingredient was found in the range of 6.78±0.19 to
8.23±0.15mg. All of them contained higher amount of active
ingredients than written value (5 mg). For pyridoxine
hydrochloride tablets (Table 4), it was found that sample P1
(20 mg, written on the label) contained 21.75±0.41 mg and
sample P2 (25 mg, written on the label) contained 29.72±0.59
mg of pyridoxine hydrochloride. The methodology for
determination of thiamine hydrochloride [31] and riboflavin
[32]were partially according to the published methods with
some modifications but for pyridoxine hydrochloride, the
method was new. The described methods for the determination
of thiamine hydrochloride, riboflavin and pyridoxine
hydrochloride were satisfactory with a wide range of
concentration. Our goal was to determine thiamine
hydrochloride, riboflavin and pyridoxine hydrochloride easily
in dosage forms with sufficient precision and accuracy. As the
relative standard deviation values (0.53-7.51 for thiamine
hydrochloride, 0.43-5.56 for riboflavinand1.89- 1.99 for
pyridoxine hydrochloride) obtained from the analysis were
below 20, so the methods were satisfactory. The method was
sufficiently sensitive and reproducible with high accuracy and
precision, analysis of thiamine hydrochloride, riboflavin and
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Sultana et al Indian J. Pharm. Biol. Res., 2016; 4(4):32-38
pyridoxine hydrochloride of pharmaceutical dosage forms can
be done within a short period of time.
Table 1: Accuracy studies in standard thiamine hydrochloride, riboflavin and pyridoxine hydrochloride
Standard with matrix
Thiamine hydrochloride
Spiking concentration (ppm, n= 5)
6
7.5
9
10
20
30
6
10
12
Riboflavin
Pyridoxine hydrochloride
% Recovery
84.47± 0.04
75.69± 0.11
86.62± 0.14
91.43± 0.05
103.90±0.06
103.49±0.06
115.07±0.07
106.70±0.21
107.95±0.08
Mean recovery
82.26± 0.10
99.61± 0.06
109.91±0.12
Table 2: Assay of thiamine hydrochloride (vitamin B1)
Sample ID
Amount of active ingredients
written in tablet (mg)
T1
T2
T3
100
T4
T5
T6
Estimated amount,
(mg; n=3)
60.62±0.84
79.03±0.42
37.62±1.11
Relative standard
deviation (%)
1.38
0.53
2.95
66.83±1.22
71.02±1.22
48.42±3.63
1.82
1.72
7.51
Percentage of active
ingredient (%)
60.62
79.03
37.62
66.83
71.02
48.42
Table 3: Assay of riboflavin
Sample ID
Amount of active ingredients
written in tablet (mg)
R1
R2
R3
R4
R5
5
Estimated average amount
(mg; n=3)
6.78±0.19
7.73±0.03
7.60±0.42
7.74± 0.09
8.23±0.15
Relative standard
deviation (%)
2.77
0.43
5.56
1.16
1.80
Percentage of active
ingredient (%)
135.6
154.6
152
154.8
164.6
Table 4: Assay of pyridoxine hydrochloride
Sample ID
Amount of active ingredients
written in tablet (mg)
Average amount, (mg; n=3)
Relative standard
deviation (%)
Percentage of active
ingredient (%)
P1
P2
20
25
21.75±0.41
29.72±0.59
1.89
1.99
108.75
118.87
Research Article
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Sultana et al Indian J. Pharm. Biol. Res., 2016; 4(4):32-38
Fig 1: Structures of vitaminB1; thiamine hydrochloride(a), B2; riboflavin (b) and B6; pyridoxine (c).
Fig 2: Calibration curves of standard thiamine hydrochloride (a), riboflavin (b) and pyridoxine hydrochloride (c).
Fig 3: Spectra of standardthiamine hydrochloride (a), cleaned extract of tablet (b) and overlain.
Fig 4: Spectra of standard riboflavin(a), cleaned extract of tablet (b) and overlain spectra (c).
Research Article
36
Sultana et al Indian J. Pharm. Biol. Res., 2016; 4(4):32-38
Fig 5: Spectra of standard pyridoxine hydrochloride (a), cleaned extract of tablet (b) and overlain spectra (c).
Conclusion
Vitamin B1 (thiamine hydrochloride), vitamin B2 (riboflavin)
and B6 (pyridoxine hydrochloride) are very common
medicines for the treatment of vitamin deficiency diseases.
Though vitamins are essentials, but insufficient or excessive
amount can bring harmful situation. The present
spectrophotometric method for the determination of thiamine
hydrochloride (vitamin B1), riboflavin (vitamin B2) and
pyridoxine hydrochloride (vitamin B6) was successful for a
wide range of concentration and was cheap, easy and rapid. As
the standard deviation values obtained from the analysis were
satisfactory, it can be concluded that the method is sufficiently
sensitive and reproducible in the routine analysis of vitamin
B1, B2 and B6.
6.
7.
8.
9.
10.
Acknowledgements
The authors are grateful to the International Program in
Chemical Science (IPICS), University of Uppsala, Sweden for
financial support. New life and Co. Pvt. Ltd., Bangladesh is
thanked for supplying standard reference samples and
excipients.
11.
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Cite this article as: Abida Sultana, Md. Sharear Saon, Mohammad Shoeb and Nilufar Nahar. Evaluation of Vitamin B1, B2 and
.
B6 Tablets in Bangladesh by UV-Vis Spectrophotometer Indian J. Pharm. Biol. Res.2016; 4(4):32-38
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