A Novel Ultra‐Performance Liquid Chromatography‐Electrospray Ionisation‐Tandem Mass Spectrometry Method for Quantification of Imeglimin in Human Plasma: Application to a Bioequivalence Study

ABSTRACT The pharmaceutical market has recently witnessed the advent of a novel tetrahydrotriazene molecule belonging to the new pharmacological class “Glimins”, named Imeglimin (IMG). It has been approved in Japan as a safe and highly effective oral diabetic drug for type II diabetic patients. It enhances the function of β‐cells in the human pancreas and increases insulin sensitivity. Hence, this work was directed to provide a fast, sensitive and highly reliable bioanalytical method for its quantification in human plasma. What makes this work greatly distinctive is first: adopting an ultra‐performance liquid chromatography (UPLC) with protein precipitation as a fast and straightforward sample preparation protocol with the highest extraction recovery. Secondly, utilisation of the stable isotope‐labelled molecule, IMG‐d6, rather than a structurally similar analogue, to avoid the interference of any co‐administered drugs in human plasma. Samples were extracted with acetonitrile and then chromatographically separated using an Acquity UPLC BEH HILIC column of 1.7 µm particle size along with a mobile phase solution composed of 70:30 (v/v) acetonitrile and 10 mM ammonium formate buffer acidified with 0.1% formic acid. Upon adjusting the flow rate to 0.3 mL/min, IMG was eluted at 1.3 min with a total run time of only 2.0 min. Mass quantification was performed in positive electrospray ionisation operated in multiple reaction monitoring mode. IMG was quantified at m/z of 156.05 → 112.91 transition pairs while IMG‐d6, at m/z 162.11 → 119.04. The proposed UPLC‐tandem mass spectrometry method was thoroughly validated as per Food and Drug Administration principles over a linearity range of 10.0–3000.0 ng/mL and successfully applied for IMG quantification in human plasma samples. The scope of the work was extended to encompass real analysis of collected human blood samples, calculation of IMG pharmacokinetics parameters and conductance of a bioequivalence study between the IMG generic product versus brand one.