A Simple and Validated RP-HPLC Method for the Estimation of Methylcobalamin in Bulk and Solid Dosage Form

1. A Simple and Validated RP-HPLC Method for the Estimation of
Methylcobalamin in Bulk and Solid Dosage Form
Inventi Rapid: Pharm Analysis & Quality Assurance Vol. 2014, Issue 2
[ISSN 0976-3813]
2014 ppaqa 1373, CCC: $10 © Inventi Journals (P) Ltd
Published on Web 29/03/2014, www.inventi.in
R E S E AR C H A R T I C LE
INTRODUCTION
Mecobalamin occurs as dark red crystals or crystalline
powder. Vitamin B12, a water-soluble vitamin, occurs in
the body mainly as methylcobalamin (mecobalamin) and as
adenosylcobalamin (cobamamide) and hydroxocobalamin.
Mecobalamin and cobamamide act as coenzymes in nucleic
acid synthesis. Mecobalamin is also closely involved with
folic acid in several important metabolic pathways. Vitamin
B12 deficiency can occur in strict vegetarians with an
inadequate dietary intake, although it may take many years
before a deficiency is produced. Deficiency is more likely in
patients with malabsorption syndromes or metabolic
disorders, nitrous oxide-induced megaloblastosis or after
gastrectomy or extensive ileum resection. Deficiency leads
to the development of megaloblastic anaemias and
demyelination and other neurological damage. A specific
anaemia known as pernicious anaemia develops in patients
with an absence of the intrinsic factor necessary for good
absorption of the Vitamin from dietary sources. Vitamin
B12 preparations are used in the treatment and prevention
of vitamin B12 deficiency.
No official isocratic RP HPLC estimation method has
been reported for analysis of mecobalamin in
pharmaceuticals preparation. An attempt has been made to
develop a simple, economical, precise, accurate and
reproducible HPLC method for estimation of mecobalamin
in bulk as well as pharmaceutical formulations. [1-2]
MATERIALS AND METHODS
Instrumentation
Column C8 ODS (250 × 4.6 mm, 5 micron). A Merck UV/
Visible spectrophotometer detector model L-7400, Merck
pump model L-7110, Integrator Winchrom Software
Window base was employed, A Shimadzu electronic
analytical balance (AX-200) was used for weighing the
sample.[3]
Reagents and Chemicals [4]
1. H.P.L.C. grade water.
2. Acetonitrile (HPLC Grade).
3. Sodium Dihydrogen Phosphate Dihydrate.
1Zee Laboratories, Uchani, G. T. Road, Karnal-132001, Haryana, India.
E-mail: Aloksinghlab@gmail.com
*Corresponding author
4. Dilute Ortho Phosphoric Acid (10%v/v).
5. Sodium 1-Hexane Sulfonate (Hexane-1-Sulfonic acid
Sodium salt).
6. Mecze Tablets contains 500 mcg mecobalamin per
tablets.
7. Working standard of mecobalamin.
Preparation of Mobile Phase [5-9]
Mobile phase was consisting of 1.0% w/v solution of
Sodium Dihydrogen Phosphate Dihydrate and
Acetonitrile in the ratio of (735:265) respectively to
obtained 1000 ml. Adjusted pH 3.50 With 10% Ortho
Phosphoric Acid and then added 3.76 gm of Sodium 1-
Hexane Sulfonate (Hexane-1-Sulfonic acid Sodium salt)
and filtered of 0.45 micron use the solution within twelve
hours.
Preparation Standard Stock Solution A
50.0 mg of Mecobalamin working standard was accurately
weighed and transferred in 50 ml volumetric flask. It was
dissolved in 30 ml mobile phase and diluted to 50 ml with
mobile phase.
Method Validation
Validation of the developed method was done according to
the USP35, Asian edition and ICH guideline.
1. Linearity
Graph of standard linearity was plotted between
Concentration (in ppm) on x-axis and Mean peak area.
Coefficient of correlation (i.e. r²) was found 0.9998 hence
the linearity between concentration range of 16 ppm to 24
ppm was established.
2. Recovery Study
In order to ensure the reliability and suitability of the
proposed method, recovery studies were carried out. It was
done by mixing known quantity of standard drug with
formulation sample and the content was reanalyzed by the
proposed method. The dilution of Mecobalamin of 18 ppm,
20 ppm, 22 ppm prepared separately with addition of 200
mg of placebo. This was extracted, diluted and reanalyzed
as per the formulation procedure. Area was noted at
respective wavelength. Recovery studies results are shown
in the Table 4.
Alok Kumar Singh1*, Pushpendra Jain1
Abstract: A simple, rapid, specific, accurate and precise reverse phase high performance liquid chromatographic method was
developed for the estimation of methylcobalamin in bulk and solid dosage form. The determination was carried out on a C-8
(250 × 4.6 mm, 5 micron) column using mobile phase 1.0 %w/v solution of sodium dihydrogen phosphate dihydrate and
acetonitrile in the ratio of 735:265 adjusts pH 3.50 with 10% ortho phosphoric acid and then added 3.76 gm/liter of sodium 1-
hexane sulfonate (hexane-1-sulfonic acid sodium salt). The flow rate and runtime were 1 ml/min and 10 min, respectively. The
eluent was monitored at 318 nm. The retention time of methylcobalamin was 2.88 mins. The method was reproducible, the
detector response was found to be linear in the concentration range of 16 ppm to 24 ppm.
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2. Mean Peak Area
Inventi Rapid: Pharm Analysis & Quality Assurance Vol. 2014, Issue 2
[ISSN 0976-3813]
2014 ppaqa 1373, CCC: $10 © Inventi Journals (P) Ltd
Published on Web 29/03/2014, www.inventi.in
R E S E AR C H A R T I C LE
a. Sample Preparation and Assay
20 tablets of Mecze Tablet (Claim 500 mcg mecobalamin
per tablets) were crushed and powdered. This powder was
accurately weighed equivalent to 50.0 mg of Mecobalamin
and transferred in 50 ml volumetric flask. Dissolve with 30
ml mobile phase and diluted to 50 ml with mobile phase.
Then filter with 0.45 μ filter paper Dilute 1 ml of this
solution to 50 ml with mobile phase. This solution were
injected and run on HPLC instrument. Results are shown in
the Table 5.
Figure 1: Mecobalamin
Figure 2: Standard linearity curve
Tablet 1: Optimized Chromatographic Conditions
Instrument Merck HPLC Integrator Parameters
Pump : L-7110 Peak Width : 4.0
Detector : L-7400 Peak Threshold : 30
Integrator : Winchrom Software
Window base
Area Reject
Height Reject
: 5
: 4
Column : C8
(250 mm x 4.6 mm) (5 micron)
Integrator
Base/ Base
: ON
Detection wavelength : 318 nm
Flow rate : 1.0 ml/min
Injection volume : 20 μl Acceptance criteria for System Suitability
Max. Pressure : 400 kg/cm² Theoretical plates: NLT 2500
Run time : About 10 mins. RSD NMT 2.0%
2

3. Tablet 2: Preparation Solution B
S. No. Volume of Solution A Taken Final Volume Prepared
1. 0.80 ml 50 ml 16 ppm
2. 0.90 ml 50 ml 18 ppm
3. 1.00 ml 50 ml 20 ppm
4. 1.10 ml 50 ml 22 ppm
5. 1.20 ml 50 ml 24 ppm
Table 3: System Suitability Parameter
Parameter Mecobalamin
Calibration Range 16 ppm to 24 ppm
Intercept 118269
Regression Coefficient (r2) 0.9998
Retention Time 2.88 mins
Table 4: Recovery Study
Mecobalamin
S. No. Conc. of Active Recovery
1 18 ppm 99.25%
2 20 ppm 100.8%
3 22 ppm 98.64%
Table 5: Result of Tablets Analysis
Drug Name Area Retention Time Label Claim Assay in % Relative Standard Deviation
Mecobalamin 2352614 2.88 min 500mcg/Tablets 100.39% 0.29%
Table 6: Intermediate Precision Study of Mecobalamin
Inventi Rapid: Pharm Analysis Quality Assurance Vol. 2014, Issue 2
[ISSN 0976-3813]
Final Concentration
(Solution B)
RSD =1.11%
2014 ppaqa 1373, CCC: $10 © Inventi Journals (P) Ltd
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R E S E AR C H A R T I C LE
3. Precision
The precision of an analytical method is determined by
assaying a sufficient number of aliquots of a homogeneous
sample to be able to calculate statistically valid estimate of
% Relative Standard Deviation (%RSD). Intermediate
precision was done to express within laboratory variation,
on different days. Six replicates of 20 ppm concentration of
the working standard mixture and sample solution were
analyzed % RSD was found to be less than 2%.
4. Robustness
Robustness of the method was studied by changing the
λmax from 318 nm to 315 nm and the mobile phase
composition of organic phase changed by ±2% and pH±0.5
The results showed that the retention time and peak area
of methylcobalamin is remains almost unchanged and no
significant degradation was observed.
RESULT AND DISCUSSION
The proposed method for estimation of mecobalamin in
tablets dosage form was found to be simple, accurate,
economical and rapid. The % RSD was found to be less than
Mecobalamin
2% in the developed method. Hence proposed method may
be used for routine analysis of these drugs in solid dosage
forms.
REFERENCES AND NOTES
1. Martindale. The Complete Drug Reference, 36th Edition,
Pharmaceutical Press, London, 1983
2. Japanese Pharmacopeia. Ministry of Health, Japan, 16th
Edition, 1067, 2003.
3. Bently and Driver’s. Textbook of Pharmaceutical Chemistry.
8th ed. Oxford University Press. London, 876, 2003.
4. Sethi P D. Quantitative Analysis of Pharmaceutical
formulations. 1st ed. CBS Publishers and Distributors, New
Delhi, 212, 879, 1985.
5. Takeuru Higuchi. Pharmaceutical Analysis. 1st ed. CBS
Publishers and Distributors. New Delhi, 671-680, 2004.
6. Code Q2A. Text on Validation of Analytical procedures. Step III
Consensus guidelines. ICH Harmonized Tripartite Guidelines,
153, 1994.
7. Code Q2B. Text on Validation of Analytical procedures. Step IV
Consensus guidelines. ICH Harmonized Tripartite Guidelines,
1-8, 1994.
8. Beckette A H and Stenlake J B. Practical Pharamceutical
Chemistry, 4th ed. Vol II. CBS Publishers, New Delhi, 275, 1997.
Intraday
Assay 100.37%
RSD: 0.16%
Interday
Assay 99.35%
RSD: 0.08%
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4. Inventi Rapid: Pharm Analysis Quality Assurance Vol. 2014, Issue 2
[ISSN 0976-3813]
2014 ppaqa 1373, CCC: $10 © Inventi Journals (P) Ltd
Published on Web 29/03/2014, www.inventi.in
R E S E AR C H A R T I C LE
9. British Pharamcopeia. British Pharmacopeal Commision. Vol
III, London, 23-34, 2004.
Acknowledgments
The authors are thankful Zee Laboratories India for providing
mecobalamin tablets as gift samples and opportunities for this
work. And also thanks to Mrs. Anil Kumar Jha Q. C. Manager of
Zee Laboratories, Karnal, India for providing the required
facilities for my research work. I am also grateful of Mrs.
Rakesh Kumar for supporting me.
Cite this article as: Alok Kumar Singh, Pushpendra Jain.
A Simple and Validated RP-HPLC Method for the
Estimation of Methylcobalamin in Bulk and Solid Dosage
Form. Inventi Rapid: Pharm Analysis Quality
Assurance, 2014(2): 1-4, 2014.
4