Abstract

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE DETERMINATION OF METFORMIN IN HUMAN PLASMA

Ahmed Yusuf, Syed N. Alvi and Muhammad M. Hammami*

ABSTRACT

We developed and validated simple, rapid, and precise reversed-phase high performance liquid chromatographic (RP-HPLC) assay for the determination of metformin level in human plasma. HCl-acidified plasma samples (0.5 ml) were deproteinized with acetonitrile and centrifuged, and the supernatant was washed with dichloromethane and injected into the HPLC system. Separation was achieved within 10 min on a Resolve CN cartridge and an isocratic mobile phase consisting of 0.03 M dibasic ammonium phosphate (pH 7) and acetonitrile (28:72, v:v) and delivered at a flow rate of 2.0 ml/min. Metformin and atenolol (internal standard, IS) were detected by a photodiode array detector operating at 240 nm. No interference in blank plasma or by commonly used drugs was observed. The method was validated in the range of 0.05 – 5 µg/ml with acceptable recovery, specificity, and intra- and inter-run precision and accuracy. It was used to assess the stability of metformin in human plasma under various clinical laboratory conditions. Further it was successfully employed to measure metformin level in samples obtained from a healthy volunteer.

Keywords: Metformin, Atenolol, Human plasma, HPLC.