Development, Validation and Application of HPLC Method for Metformin in Rabbit Plasma

Author(s): Muhammad Ubaid, Mahmood Ahmad, Farhan Ahmad Khan, Ghulam Murtaza*

Journal Name: Current Pharmaceutical Analysis

Volume 15 , Issue 6 , 2019

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Objective: This study was aimed at conducting a pharmacokinetic evaluation of metformin in rabbit plasma samples using rapid and sensitive HPLC method and UV detection.

Methods: Acetonitrile was used for protein precipitation in the preparation of plasma samples. Reverse phase chromatography technique with silica gel column (250 mm × 4.6 mm, 5 μm) at 30°was used for the separation purpose. Methanol and phosphate buffer (pH 3.2) mixture was used as a mobile phase with flow rate 0.8 ml/min. The wavelength of UV detector was adjusted at 240 nm.

Results: The calibration curve was linear in a range of 0.1-1 µg/ml with R² = 0.9982. The precision (RSD, %) values were less than 2%, whereas, accuracy of method was higher than 92.37 %. The percentage recovery values ranged between 90.14 % and 94.97 %. LOD and LOQ values were 25 ng/ml and 60 ng/ml, respectively. Cmax and AUC0-t values were found to be 1154.67 ± 243.37 ng/ml and 7281.83 ± 210.84 ng/ml.h, respectively after treating rabbits with a formulation containing 250 mg metformin.

Conclusion: Based on the above findings, it can be concluded that present method is simple, precise, rapid, accurate and specific and thus, can be efficiently used for the pharmacokinetic study of metformin.

Keywords: Metformin, rabbit plasma, HPLC method, validation, protein precipitation, pharmacokinetics.

Rahnama, E.; Mahmoodian-Moghaddam, M.; Khorsand-Ahmadi, S.; Saberi, M.R.; Chamani, J. Binding site identification of metformin to human serum albumin and glycated human serum albumin by spectroscopic and molecular modeling techniques: A comparison study. J. Biomolecule. Structure. Dynamic., 2015, 33(3), 513-533.
Ahmad, S.; Shahab, U.; Baig, M.H.; Khan, M.S.; Khan, M.S.; Srivastava, A.; Saeed, M. Inhibitory effect of metformin and pyridoxamine in the formation of early, intermediate and advanced glycation end-products. PloS One, 2013, 8(9)e72128
Setter, S.M.; Iltz, J.L.; Thams, J.; Campbell, R.K. Metformin hydrochloride in the treatment of type 2 diabetes mellitus: A clinical review with a focus on dual therapy. Clin. Ther., 2003, 25(12), 2991-3026.
Robert, F.; Fendri, S.; Hary, L.; Lacroix, C.; Andrejak, M.; Lalau, J. Kinetics of plasma and erythrocyte metformin after acute administration in healthy subjects. Diabet. Metabolism., 2003, 29(3), 279-283.
Tahara, K.; Yonemoto, A.; Yoshiyama, Y.; Nakamura, T.; Aizawa, M.; Fujita, Y.; Nishikawa, T. Determination of antihyperglycemic biguanides in serum and urine using an ion‐pair solid‐phase extraction technique followed by HPLC‐UV on a pentafluorophenylpropyl column and on an octadecyl column. Biomed. Chromatogr., 2006, 20(11), 1200-1205.
Lin, J.; Ying, N.; Wu, Y.; Lin, G. Determination of Imatinib and CGP74588 in Rat Plasma by HPLC and Its Application to a Pharmacokinetic Study. Curr. Pharm. Anal., 2018, 14(2), 133-138.
Zarghi, A.; Foroutan, S.; Shafaati, A.; Khoddam, A. Rapid determination of metformin in human plasma using ion-pair HPLC. J. Pharm. Biomed. Anal., 2003, 31(1), 197-200.
Heinig, K.; Bucheli, F. Fast liquid chromatographic-tandem mass spectrometric (LC-MS-MS) determination of metformin in plasma samples. J. Pharm. Biomed. Anal., 2004, 34(5), 1005-1011.
Van de Merbel, N.; Wilkens, G.; Fowles, S.; Oosterhuis, B.; Jonkman, J. LC phases improve, but not all assays do: Metformin bioanalysis revisited. Chromatogr, 1998, 47(9-10), 542-546.
Zhang, M.; Moore, G.A.; Lever, M.; Gardiner, S.J.; Kirkpatrick, C.M.; Begg, E.J. Rapid and simple high-performance liquid chromatographic assay for the determination of metformin in human plasma and breast milk. J. Chromatogr. B , 2002, 766(1), 175-179.
Charles, B.G.; Jacobsen, N.W.; Ravenscroft, P.J. Rapid liquid-chromatographic determination of metformin in plasma and urine. Clin. Chem., 1981, 27(3), 434-436.
David, V.; Barcutean, C.; Sora, I.; Medvedovici, A. Determination of metformin in plasma samples by HPLC-DAD based on plasma derivatization and precipitation with acetic anhydride. Revue. Roum. de Chim., 2005, 50(4), 269-276.
Wang, Y.; Tang, Y.; Gu, J.; Fawcett, J.P.; Bai, X. Rapid and sensitive liquid chromatography-tandem mass spectrometric method for the quantitation of metformin in human plasma. J. Chromatogr. B, 2004, 808(2), 215-219.
Yuen, K.H.; Peh, K.K. Simple high-performance liquid chromatographic method for the determination of metformin in human plasma. J. Chromatogr. B Biomed. Sci. Appl., 1998, 710(1), 243-246.
Cheng, C.L.; Chou, C.H. Determination of metformin in human plasma by high-performance liquid chromatography with spectrophotometric detection. J. Chromatogr. B Biomed. Sci. Appl., 2001, 762(1), 51-58.
Huupponen, R.; Ojala-Karlsson, P.; Rouru, M.K.J. Determination of metformin in plasma by high-performance liquid chromatography. J. Chromatogr., Biomed. Appl., 1992, 121, 270-273.
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. B Biomed. Sci. Appl., 1986, 378, 503-508.
Tache, F.; David, V.; Farca, A.; Medvedovici, A. HPLC-DAD determination of Metformin in human plasma using derivatization with p-nitrobenzoyl chloride in a biphasic system. Microchem. J., 2001, 68(1), 13-19.
Moore, K.A.; Levine, B.; Titus, J.M.; Fowler, D.R. Analysis of metformin in antemortem serum and postmortem specimens by a novel HPLC method and application to an intoxication case. J. Anal. Toxicol., 2003, 27(8), 592-594.
Bonfigli, A.R.; Manfrini, S.; Gregorio, F.; Testa, R.; Testa, I.; De Sio, G.; Coppa, G. Determination of plasma metformin by a new cation-exchange HPLC technique. Therap. drug Monitor., 1999, 21(3), 330.
Vesterqvist, O.; Nabbie, F.; Swanson, B. Determination of metformin in plasma by high-performance liquid chromatography after ultrafiltration. J. Chromatogr. B Biomed. Sci. Appl., 1998, 716(1), 299-304.
Amini, H.; Ahmadiani, A.; Gazerani, P. Determination of metformin in human plasma by high-performance liquid chromatography. J. Chromatogr. B , 2005, 824(1), 319-322.
Ahmed, S.; Khan, A.; Sheraz, M.A.; Bano, R.; Ahmad, I. Development and validation of a stability-indicating hplc method for the assay of carvedilol in pure and tablet dosage forms. Curr. Pharm. Anal., 2018, 14(2), 139-152.
Razzaq, R.; Ranjha, N.M.; Rashid, R.; Rashid, Z.; Hanif, M. Determination of diltiazem hcl by reverse phase high performance liquid chromatography in rabbit plasma. Curr. Pharm. Anal., 2018, 14(2), 153-156.
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. B Biomed. Sci. Appl., 1986, 375(Supplement. C), 184-189.
Hu, L.D.; Liu, Y.; Tang, X.; Zhang, Q. Preparation and in vitro/in vivo evaluation of sustained-release metformin hydrochloride pellets. Eur. J. Pharm. Biopharm., 2006, 64(2), 185-192.
Tatiparthi, R.; Duraiswamy, D.; Kottapalli, C.B. Method development and validation of metformin and repaglinide in rabbit plasma by RP-HPLC. Fabad J. Pharm. Sci., 2010, 35, 69-75.
He, W.; Li, Y.; Zhang, R.; Wu, Z.; Yin, L. Gastro-floating bilayer tablets for the sustained release of metformin and immediate release of pioglitazone: Preparation and in vitro/in vivo evaluation. Int. J. Pharm., 2014, 476(1), 223-231.
Polagani, S.R.; Pilli, N.R.; Gajula, R.; Gandu, V. Simultaneous determination of atorvastatin, metformin and glimepiride in human plasma by LC-MS/MS and its application to a human pharmacokinetic study. J. Pharm. Anal., 2013, 3(1), 9-19.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Published on: 17 July, 2019
Page: [574 - 579]
Pages: 6
DOI: 10.2174/1573412914666180308124858
Price: $65

Article Metrics

PDF: 61
PRC: 1