LC–tandem mass spectrometry method for the simultaneous determination of metformin and sitagliptin in human plasma after ion-pair solid phase extraction

• A sensitive method for the simultaneous determination of metformin and sitagliptin in plasma.
• Ion-pair SPE using sodium lauryl sulphate afforded quantitative recovery for metformin.
• Chromatography on a cyano column provided adequate retention for both the drugs.
• The method was applied for a bioequivalence study and its reproducibility was checked by ISR.

A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was established for simultaneous determination of two oral hypoglycemic drugs metformin (MET) and sitagliptin (STG) in human plasma. The analytes were extracted from 50 μL human plasma by ion-pair solid phase extraction using sodium lauryl sulphate on Phenomenex Strata-X (30 mg/1 mL) cartridges. The chromatographic separation was accomplished on XSelect HSS CN (150 × 4.6 mm, 5 μm) column using mobile phase consisting of methanol-8.0 mM ammonium formate in water, pH 4.5 (80:20, v/v) under isocratic condition. Tandem MS detection was performed on a triple quadrupole spectrometer equipped with an electrospray ionization source, operated in the positive mode. Multiple reaction monitoring (MRM) was used to quantify the analytes following transitions, m/z 130.1 → 60.1 and m/z 408.3 → 235.1 for MET and STG respectively. The method displayed acceptable linearity in the concentration range of 4.00–3200 ng/mL for MET and 1.00–800 ng/mL for STG. The intra-batch and inter-batch precisions were ≤5.1% and accuracy ranged from 96.5 to 103.3% for both the drugs. The mean recovery of MET and STG obtained from spiked plasma samples was 82.5% and 90.4% respectively with minimal matrix interference. Both the drugs were found to be stable under all mandatory storage conditions. The validated method was successfully applied to a clinical pharmacokinetic study for a fixed-dose tablet formulation containing 500 mg MET and 50 mg STG in 16 healthy volunteers.

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