A rapid, simple, sensitive and selective LC-MS/MS method was developed and validated for simultaneous quantification of montelukast (MT) and fexofenadine (FF) in human plasma (200 μL) using montelukast-d6 (MT-d6 ) and fexofenadine-d10 (FF-d10 ), respectively as an internal standard (IS) as per the US Food and Drug Administration guidelines. The chromatographic resolution was achieved on a Chromolith RP18e column using an isocratic mobile phase consisting of 20 mm ammonium formate-acetonitrile (20:80, v/v) at flow rate of 1.2 mL/min. The LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. The total run time of analysis was 4 min and elution of MT, FF, MT-d6 and FF-d10 occurred at 2.5, 1.2, 2.4 and 1.2 min, respectively. The standard curve found to be linear in the range 2.00-1000 ng/mL with a coefficient of correlation of ≥0.99 for both the drugs. The intra- and inter-day accuracy and precision values for MT and FF met the acceptance as per FDA guidelines. MT and FF were found to be stable in a battery of stability studies viz., bench-top, auto-sampler and repeated freeze-thaw cycles. The validated assay was applied to an oral bioequivalence study in humans.

A simple, rapid, sensitive, reliable and selective ultra high performance liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS) method was developed, for quantification of linagliptin (LGN) in human plasma. LGN and its deuterated internal standard (IS), LGN-d4 was extracted from low plasma sample volume (300 µL) by simple liquid-liquid extraction protocol. Efficient estimation of analyte and IS at mean retention time (RT) of 1.75 and 1.74 min respectively, with a rapid 3.5 min run time per sample was chromatographically established on Gemini C18 (100 mm × 4.6 mm, 3µ) column under simple isocratic elution conditions, using a mixture of 10 mM ammonium formate: methanol [20:80 (v/v)] delivered at a flow rate of 0.5 mL/min. Following separation of compounds, protonated precursor → product ion transitions were monitored, for LGN (m/z: 473.3 → 420.1) and IS (m/z: 477.5 → 424.3) on a triple quadrupole mass spectrometer, operating in multiple reaction monitoring (MRM) mode. Most recent regulatory guidelines were adopted during method validation. Method demonstrated very good analyte and IS recovery (not less than 71.0%), precision (≤ 8.6 %CV), accuracy (range: 86.7% to 95.6%) and linearity (r>0.99) across clinically relevant LGN plasma concentration range: 50.3 to 12115.5 pg/mL. Validated method was successfully applied to pharmacokinetic study samples for measuring linagliptin plasma levels.

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