{"id":505,"date":"2015-01-22T06:50:05","date_gmt":"2015-01-22T06:50:05","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=505"},"modified":"2020-04-25T06:26:03","modified_gmt":"2020-04-25T06:26:03","slug":"hptlc-method-for-estimation-of-metformin-hydrochloride","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol1no2\/hptlc-method-for-estimation-of-metformin-hydrochloride\/","title":{"rendered":"HPTLC Method for Estimation of Metformin Hydrochloride"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Metformin, an hypoglycemic drug, official in IP,Chemically 1,1-dimethyl biguanide hydrochloride, widely used to enhanced peripheral glucose uptake and utilization, \uf020inhibition of hepatic gluconeogenesis, \uf020increased muscle glyconeogenesis and reduction of net glucose absorption by the small intestine, indicated for patients with non-insulin dependent diabetes mellitus.IP reports UV method for estimation of metformin HCl. Literature survey reveals many HPLC method2-7<\/sup> for the estimation of metformin HCl in biological fluids and formulations. So far no HPTLC method has been reported for estimation of metformin HCl in biological fluids. In our present study, a HPTLC method for estimation of Metformin HCl in single and combination dosage form was developed and validated.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Instruments <\/strong><\/p>\n

HPTLC was performed with a Camag Linomat V (Switzerland),twin-trough TLC chamber (10×10),a Camag TLC Scanner-3, a Wincats \u2013version 1.3.3 software, Hamilton HPTLC syringe (100mcL), Acculab ALC 210.3 weighing balance and ultrasonicator were used during study.<\/p>\n

Chemicals and Reagents<\/strong><\/p>\n

Metformin standard was procured as a gift sample from Franco-Indian Remedies Pvt. Ltd. Methanol, chloroform, ammonium acetate, glacial acetic acid and ammonia solution all are Analytical Reagent (AR) grade. Tablets containing metformin, single dosage form Glyciphase (500mg\/tablets) and Obimet (500mg\/tablets)\u00a0 and combination dosage form Dionorm, Diabetrol and Dibizide were purchased from local market.<\/p>\n

Chromatographic Conditions<\/strong><\/p>\n

TLC was performed in the form of bands of width 6 mm with a Camag microlitre syringe on precoated silica gel aluminium plate 60 F-254, (10\u00d710 cm) with 250 \u03bcm thickness; E. Merck, (Germany) using a Camag Linomat V (Switzerland). A constant application rate of 15 \u03bcl\/s was<\/p>\n

employed. The mobile phase consisted of methanol: chloroform: ammonium acetate (6:3:1 v\/v\/v). Linear ascending development of chromatogram was carried out in a Camag twin trough glass chamber saturated with the mobile phase. The chamber saturation time for mobile phase was optimized at 30 min. The length of chromatogram run was 7.5 cm. Subsequent to the<\/p>\n

development; the TLC plates were dried with the help of a Camag TLC plate dryer. Densitometric scanning was performed on a Camag TLC scanner III in the absorbance mode at 236 nm. The plate was analysed on a TLC scanner 3\u00ae densitometer driven by the Wincats \u2013version 1.3.3 software, dried by using TLC plate heater and scanned at 236nm with D2 lamp<\/p>\n

Preparation of standard Metformin Hydrochloride Solution<\/strong><\/p>\n

50 mg of \u00a0standard metformin HCl was accurately weighed and dissolved in methanol and the volume made up to 100 ml from this 0.5ml was withdrawn and diluted upto 10 ml to give concentration 25 \u03bcg\/ml.<\/p>\n

Estimation of Metformin HCl in single dosage form <\/strong><\/p>\n

The tablets were powdered and powder weight equivalent to 50 mg of drug was extracted in methanol. To ensure complete extraction of the drug it was shaken for 15min and the volume was made up to 100 ml. The resulting solution was filtered. 0.5 ml of the filtrate was taken and diluted up to 10 ml with methanol to get 25 ng\/\u03bcl solution. Different volumes of this solution ranging from 5, 6 and 7 \u03bcl were spotted onto the plate, the chromatogram was developed and scanned at 236nm.(Glyciphase and Obimet label claim 500 mg\/tablets respectively).<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 1:<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Estimation of<\/strong> Metformin HCl in combination dosage forms<\/strong><\/p>\n

The tablets were powdered and powder weight equivalent to 50 mg Metformin HCl of was extracted in methanol. To ensure complete extraction of the drug it was shaken for 15min and the volume was made up to 100 ml. The resulting solution was filtered. 0.5 ml of the filtrate was taken and diluted up to 10 ml with methanol to get 25 ng\/\u03bcl solution.10 \u03bcl of these three solutions were spotted on a the plate. The chromatogram was developed and scanned as 236 nm.<\/p>\n

( Dibizide (glipizide + metformin HCl) coded as glpm, \u00a0Dianorm (gliclazide + metformin HCl) coded as gclm and Diabetrol (glibenclamide +metformin HCl) coded as mgl.<\/p>\n

Method Validation<\/strong><\/p>\n

The HPTLC method was developed and validated by using various parameters like limit of detection, limit of quantification, linearity, range, specificity, intra day precision, inter day precision, robustness, ruggedness and recovery studies.<\/p>\n

Results and discussion<\/strong><\/p>\n

The linearity range was found to be in the concentration of 100 \u2013 300ng\/spot. The LOQ \u00a0and LODwas found to be 100 ng\/spot and 25 ng\/spot respectively. The RSD for intra and inter day was found to be 0.828-0.3318% and 1.093-0.5114% respectively. The percentage recovery in solution was found to be 100.24-101.58%. The percentage purity of metformin HCl in single dosage was found to be 99.2-100.6% w\/w and in combination dosage form 98.9-103.2% w\/<\/p>\n

 <\/p>\n\n\n\n
\"Table<\/td>\nTable 1:<\/strong>\u00a0Summary of Validation Parameters.<\/strong><\/p>\n

 <\/p>\n

Click here to View Table<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Table<\/td>\nTable 1a: Data showing % recovery \u00a0of metformin HCl (In solution).<\/strong><\/p>\n

 <\/p>\n

Click here to View Table<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Table<\/td>\nTable 2: Data showing % purity in Metformin HCl.<\/strong><\/p>\n

 <\/p>\n

Click here to View Table<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

 <\/p>\n

Average of three readings<\/strong><\/p>\n

Conclusion<\/strong><\/p>\n

The developed HPTLC method is found to be precise, specific accurate and reproducible for estimation for the metformin HCl in single dosage form and in combination dosage forms.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Indian Pharmacopoeia. Ministry of Health and Family Welfare, New Delhi. 1996; I: P.469.<\/li>\n
  2. Charles BG, Jacobsen NW, Ravenscroft PJ. Rapid liquid-chromatographic determination of metformin in plasma and urine. J Clinical Chem. 1981;27;434-6.<\/li>\n
  3. Benzi L, Marchetti P, Cecchetti P. Navalesi R. Determination of metformin and phenformin in human plasma and urine by reversed-phase high-performance liquid chromatography. J Chromatogr Biomed Appl. 1986;48(1):184.<\/li>\n
  4. Keal J, Somogyi A. Rapid and sensitive high-performance liquid-chromatographic assay for metformin in plasma and urine using ion-pair extraction techniques. J Chromatogr Biomed Appl. 1986;51(2):503-8.<\/li>\n
  5. Tanabe S, Kobayashi T, Kawanabe K. Determination of oral hypoglycaemic biguanides<\/li>\n
  6. by high-performance liquid chromatography with fluorescence detection. J Anal Sci. 1987;3(1):69.<\/li>\n
  7. Huupponan R, Ojala KP, Rouru J, Koulu M, J. Determination of metformin in plasma by high-performance liquid chromatography. J Chromatogr Biomed Appl. 1992;121:270-3.<\/li>\n
  8. Ohta M, Iwasaki M, Kai M, Ohkura Y. Determination of a biguanide, metformin, by<\/li>\n
  9. high-performance liquid chromatography with pre-column fluorescence derivatization.<\/li>\n
  10. J Anal Sci. 1993;9(2):217.<\/li>\n
  11. ICH Guidelines (Q2A): validation of analytical procedure:terminology and definition.Geneva.Oct 1994 (CPMP\/ICH\/381\/95).<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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