Simultaneous analysis of antihyperglycemic small molecule drugs and peptide drugs by means of dual liquid chromatography high-resolution mass spectrometry.

OBJECTIVES: The study aimed to evaluate dual liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) for the simultaneous analysis of small and large molecule drugs by development and application of a validated bioanalytical method. METHODS: The oral antihyperglycemic drugs (OAD) dapagliflozin, empagliflozin, glibenclamide, glimepiride, metformin, pioglitazone, repaglinide, saxagliptin, sitagliptin, and vildagliptin, as well as the antihyperglycemic peptides exenatide, human insulin, insulin aspart, insulin degludec, insulin detemir, insulin glargine, insulin glulisine, insulin lispro, and semaglutide were included in the analytical procedure. Analytes were extracted using a combination of protein precipitation and solid-phase extraction. Two identical reversed-phase columns were used for separation followed by Orbitrap high-resolution mass spectrometry. The whole procedure was validated according to international recommendations. RESULTS: Different MS parameters had to be used for the two analyte groups, but dual LC separation allowed elution of all analytes within 12 min using the same column type. The analytical procedure was accurate and precise for most of the compounds except for exenatide, semaglutide, and insulin glargine, which were included qualitatively in the method. Analysis of proof-of-concept samples revealed OAD concentrations mostly within their therapeutic range, insulins could be detected in five cases but at concentrations below the lower limit of quantification except for one case. CONCLUSIONS: Dual LC in combination with HRMS was shown to be a suitable platform to analyze small and large molecules in parallel and the current method allowed the determination of a total of 19 antihyperglycemic drugs in blood plasma within 12 min.