Quantification of glucose in the plasma of healthy Chinese volunteers and a bioequivalence study using a surrogate matrix approach combined with UHPLC-MS/MS

Abstract

Acarbose works by competitively inhibiting α-glucosidase, delaying the breakdown of starch into glucose. Thus, it plays an important role in treating type 2 diabetes. Acarbose exhibits unique pharmacokinetic characteristics, and the United States of America (USA) Food and Drug Administration (FDA) has focused on pharmacodynamic parameters rather than pharmacokinetic ones in acarbose bioequivalence studies since issuing its 2009 draft guidance, which was formalized in 2017. A literature review on glucose measurement shows that glucose concentration data are primarily derived from serum matrices. In the 2022 draft guidance, the FDA reaffirmed that plasma glucose concentration is the most suitable pharmacodynamic endpoint for acarbose. This study employed a surrogate matrix method combined with ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) to measure plasma glucose levels. Because glucose is an endogenous substance, directly measuring its concentration in biological matrices does not effectively reflect the impact of acarbose on blood glucose levels. This study used Phosphate Buffered Saline (PBS) as the surrogate matrix to determine glucose concentrations and accurately achieve precise glucose quantification. Moreover, glucose and fructose are structural isomers that are difficult to quickly separate chromatographically, which can affect detection efficiency and introduce interference. Therefore, we aim to develop a rapid, simple, and accurate method for quantifying glucose concentration in human plasma using a surrogate matrix approach combined with UHPLC-MS/MS. Plasma samples were processed using protein precipitation, with glucose-[U-13C6] as the internal standard. Chromatographic separation was performed using a Waters ACQUITY UPLC BEH amide column (100 × 2.1 mm, 1.7 μm), and gradient elution was carried out at a flow rate of 0.4 mL min⁻¹ with a mobile phase of 0.05% aqueous ammonia and 0.05% ammonia in acetonitrile. Electrospray ionization (ESI) in negative ion multiple reaction monitoring (MRM) mode was used for detection, with a total analysis time of 6 min. Glucose exhibited good linearity in the concentration range of 2.00 to 20.00 mmol L⁻¹ (r² = 0.9980), with a precision and accuracy within and between batches of less than 10%. The method was fully validated to ensure data accuracy. This method was successfully applied to a bioequivalence study of acarbose tablets in healthy Chinese subjects in the fasting state, providing valuable data for evaluating the consistency of acarbose formulations.