Development and validation of Empagliflozin and Linagliptin simultaneous estimation in rat plasma using freezing lipid precipitation and SCX-SPE assisted HPLC–MS/MS method and its application in pharmacokinetic studies

A quick and sensitive liquid chromatography-mass spectrometry technique was designed, improved, and validated for simultaneous determination of Empagliflozin (EPG) and Linagliptin (LNG) using Empagliflozin-d4 (EPG-d4) and linagliptin-d4 (LNG-d4) as internal standards (IS) in rat plasma. Target analytes and the IS were extracted using freezing lipid precipitation (FLP) and optimized using the strong cation exchange solid phase extraction (SCX-SPE) method to achieve the maximum sample clean-up. In particular, when combined with SPE clean-up, FLP can efficiently eliminate the plasma sample’s high lipid content. More than 84.14% of plasma lipids were rapidly removed during the FLP procedure, with minimal loss of EPG and LNG. We used LC-atmospheric chemical ionization (APCI)—mass spectrometry was employed to assess the efficiency of FLP in lipid removal. The SCX-SPE cartridges removed the remaining impurities from EPG and LNG, allowing for further purification. The samples were chromatographically separated using a Spherisorb RP/Cyano column by pumping a gradient mobile phase comprised of acetonitrile and 25 mM ammonium acetate buffer (pH 8.1) in positive ion mode at a flow rate of 0.8 mL/min. The selected reaction monitoring technique was performed using a Waters triple-stage quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source. The chromatographic separation was accomplished using a Waters Acquity® high-performance liquid chromatography (HPLC) system. Mass transition (m/z) of 451.15/71.12 for EPG, m/z 473.27/419.94 for LNG; m/z 455.19/71.12 for EPG-d4, and 477.27/423.94 for LNG-d4 was successfully achieved. This study successfully examined the concentration ranges of 25–1050 ng/mL for EPG and 0.35–15 ng/mL for LNG. The results showed that the linearity of EPG ranged from 25.14 to 985.26 ng/mL, while the linearity of LNG ranged from 0.59 to 14.86 ng/mL. The relative standard deviation (RSD) for both EPG and LNG, within and between days, were below 3.83%, indicating that they fall within acceptable limits. This novel approach demonstrated favourable outcomes in a pharmacokinetic study involving healthy rats, where EPG and LNG were co-administered. This study found that the co-administration of both drugs did not have a significant impact on their pharmacokinetic behavior, suggesting the absence of any drug-drug interactions.