Challenges in simultaneous extraction and chromatographic separation of metformin and three SGLT-2 inhibitors in human plasma using LC-MS/MS.

Optimization of extraction and chromatographic conditions is essential for the simultaneous analysis of drugs having different physico-chemical properties, especially for in vivo applications. The present work describes concurrent estimation of metformin (MET) and three sodium-glucose co-transporter 2 (SGLT-2) inhibitors namely canagliflozin (CANA), dapagliflozin (DAPA) and empagliflozin (EMPA) in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample clean-up was optimized using ion-pair solid-phase extraction with sodium lauryl sulphate on Strata-X extraction cartridges. Consistent recoveries were obtained by critically optimizing parameters such as washing and elution solvents during extraction. The extraction recovery ranged from 79 to 88% for all the analytes. Chromatographic separation were accomplished on a Cyano (150 × 4.6 mm, 5 µm) column within 5.0 min using acetonitrile and 10 mM ammonium formate buffer (75:25, v/v) as the mobile phase. The resolution factors between MET-EMPA, MET-DAPA, MET-CANA, DAPA-EMPA and CANA-DAPA were 4.92, 5.85, 7.13, 1.01 and 1.44, respectively. Calibration curves were linear in the concentration range of 2.00-2000, 3.00-3000, 0.20-200 and 1.50-1500 ng/mL for MET, CANA, DAPA and EMPA, respectively. Mass spectrometric analysis was performed using a polarity switching approach to achieve high sensitivity in multiple reaction monitoring mode. The method was validated using current regulatory guidelines and applied to study the pharmacokinetics of fixed-dose formulations of MET and SGLT-2 inhibitors in healthy subjects.

A high-throughput LC-MS/MS method for determination of flunarizine in human plasma: Pharmacokinetic study with different doses.

A high-throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of flunarizine in human plasma. Liquid-liquid extraction under acidic conditions was used to extract flunarizine and flunarizine-d8 from 100 µL human plasma. The mean extraction recovery obtained for flunarizine was 98.85% without compromising the sensitivity of the method. The chromatographic separation was performed on Hypersil Gold C18 (50 × 2.1 mm, 3 µm) column using methanol-10 mm ammonium formate, pH 3.0 (90:10, v/v) as the mobile phase. A tandem mass spectrometer (API-5500) equipped with an electrospray ionization source in the positive ion mode was used for detection of flunarizine. Multiple reaction monitoring was selected for quantitation using the transitions, m/z 405.2 → 203.2 for flunarizine and m/z 413.1 → 203.2 for flunarizine-d8. The validated concentration range was established from 0.10 to 100 ng/mL. The accuracy (96.1-103.1%), intra-batch and inter-batch precision (CV ≤ 5.2%) were satisfactory and the drug was stable in human plasma under all tested conditions. The method was used to evaluate the pharmacokinetics of 5 and 10 mg flunarizine tablet formulation in 24 healthy subjects. The pharmacokinetic parameters Cmax and AUC were dose-proportional.