VALIDATION OF A HPLC METHOD FOR THE
SIMULTANEOUS ANALYSIS OF METFORMIN
AND GLICLAZIDE IN HUMAN PLASMA.
MARIA-CRISTINA RANETTI^1*, MIHAELA IONESCU¹, LAVINIA
HINESCU¹, ELENA IONICĂ¹, VALENTINA ANUŢA^2,3, AURELIAN E.
RANETTI⁴, CAMELIA ELENA STECOZA², CONSTANTIN MIRCIOIU^2,3
1Army Center for Medical Research, 37 C.A. Rosetti Str., Bucharest, Romania
2UMF Carol Davila, Faculty of Pharmacy, 6 Traian Vuia Str., Bucharest,
Romania
3Biopharmacy&Pharmacol Res, 23 Pitar Mos Str., Bucharest, Romania,
4UMF Carol Davila, Faculty of Medicine
*corresponding author: cranetti@yahoo.com
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Abstract:

The study presents the development and validation of a simple HPLC method for the simultaneous determination of metformin (MTF) and gliclazide (GCZ) in the presence of glibenclamide, in human plasma, for the clinical monitoring of MTF and GCZ after oral administration or for bioequivalence studies.
Ion-pair separation followed by UV detection performed on deproteinised plasma samples was chosen for the determination of metformin and gliclazide. The internal standard was glybenclamide. The HPLC method used a Zorbax Eclipse XDB-C18 150x4.6 mm i.d. (5μm) column and analytical guard column 12.5x4.6 mm (5μm), with a gradient elution (1 mL/min) at 40°C column temperature. The mobile phase was acetonitrile:
methanol (1:1v/v) and sodium dodecylsulphate 5mM, pH=3.5 with H₃PO₄ 85% and gradient elution. The eluent was monitored at 236 nm.
The calibration curve was linear within the range of 0.05-5.00 μg/mL (r²=0.99, n=6). The lowest limit of quantification (LLOQ) was 50 ng/mL for metformin and 49 ng/mL for gliclazide.
The proposed method was validated and proved to be adequate for metformin and gliclazide clinical monitoring, bioavailability and bioequivalence studies.

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