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

RESEARCH ARTICLE

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.

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

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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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

RESEARCH ARTICLE

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%

Mean Peak Area

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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

RESEARCH ARTICLE

3. PrecisionThe 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

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.

Tablet 2: Preparation Solution B

S. No. Volume of Solution A Taken Final Volume Prepared Final Concentration

(Solution B) 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% RSD =1.11%

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

Mecobalamin

Intraday Assay 100.37% RSD: 0.16%

Interday Assay 99.35% RSD: 0.08%

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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

RESEARCH ARTICLE

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.

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