A rapid and sensitive liquid chromatography–mass spectrometry method was developed, optimized, and validated for simultaneous quantification of empagliflozin and metformin in human plasma using empagliflozin D4and metformin D6 as an internal standard. Analytes and internal standard were extracted from plasma by optimized solid-phase extraction technique using Strata X polymeric reverse phase (30 mg-1cc) solid-phase extraction cartridges. The prepared samples were chromatographed on Orosil C18 column (150 × 4.6 mm, 3 µ). Separation was done by pumping isocratic mobile phase consisting of methanol and 10 mM ammonium trifluoroacetate (90:10, v/v) in positive ion mode at a flow rate of 0.8 mL/min. The API 3200 liquid chromatography–mass spectrometry system having turbo ion spray as an ion source coupled with Shimadzu Prominence ultrafast liquid chromatography system was operated under the selected reaction monitoring mode. Turbo ion spray ionization was used for mass transition of m/z 468.070/355.100 and m/z 130.072/71.200 for empagliflozin and metformin, respectively. A method was successfully validated for concentration range of 10.09–5013.46 ng/mL for both the analytes and according to the United States Food and Drugs Administration guidelines. The linearity was found to be in the range of 10.09–403.46 ng/mL for empagliflozin and 25.44–5013.46 ng/mL for metformin. The limit of quantification was found to be 10.09 ng/mL for empagliflozin and 25.44 ng/mL for metformin. Intra- and inter-day/between batch precision determination for empagliflozin and metformin, expressed as coefficient of variation were within the acceptance limits and ranged below 13.16%. A short run time of 3.3 min allows analysis of more than 400 plasma samples per day. The developed method was successfully applied to fasting pharmacokinetic study in healthy human volunteers. Results of incurred sample re-analysis were within the acceptance range of ±20% of original value, for 97.2% of samples reanalyzed for empagliflozin and 100% of samples reanalyzed for metformin.

A high-performance liquid chromatography tandem mass spectrometric method for the determination of Rivaroxaban in human plasma has been developed and validated using Rivaroxaban D4 as an internal standard. The extraction of analyte and internal standard was accomplished by solid phase extraction technique. The method has been validated over a concentration range of 5.96–801 ng/mL. Chromatographic separations were achieved using Gemini C18, 150 mm × 4.6 mm, 5 µm, column eluted at flow rate of 1.5 mL/min with mobile phase (acetonitrile: ammonium acetate buffer (80:20 v/v)). The overall run time of method was about 1.8 min with elution times of Rivaroxaban and its internal standard Rivaroxaban D4 at around 1.18 min. The multiple reaction monitoring transitions were set at 436/145 (m/z) and 440/145 (m/z) for Rivaroxaban and Rivaroxaban D4, respectively. The calibration curves were linear (r2 ≥ 0.99) over the range of 5.96–801 ng/mL with lower limit of quantitation validated at 5.96 ng/mL. Extraction recoveries were >88% for both rivaroxaban and its stable labeled internal standard rivaroxaban D4. The inter-day/between run precisions were ranged from 1.08% to 3.75%, while accuracy ranged from 96.3% to 102%. The presented method was used in pharmacokinetic study in healthy volunteers. Results of incurred sample reanalysis were within the acceptance range of ±20% of original value, for 98.3% of samples reanalyzed. This indicated good assay precision of target analytes in their real matrix at the employed experimental conditions. The applicability of the assay for the determination of the pharmacokinetic parameters was demonstrated.

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Apixaban is a novel oral anticoagulant intended to treat and prevent blood clots and to prevent strokes in patients with nonvalvular atrial fibrillation.