Simultaneous Analysis of 27 Antidiabetic Drugs in Whole Blood by LC–MS/MS

Significance of the topic

Antidiabetic drugs cover multiple pharmacological classes (biguanides, sulfonylureas, DPP‑4 inhibitors, SGLT2 inhibitors, glinides, thiazolidinediones) and are widely prescribed. Overdose, polypharmacy and non‑medical use (e.g., weight‑loss misuse) can cause severe hypoglycemia and other adverse events that are relevant in clinical, forensic and toxicological investigations. Rapid, sensitive and multiplexed methods for detecting and quantifying a broad panel of antidiabetic agents in whole blood are therefore important for emergency toxicology, post‑mortem analysis and monitoring of illicit use.

Objectives and overview of the study

This work develops and validates a single‑run LC–MS/MS method to simultaneously quantify 27 antidiabetic drugs in human whole blood. The method aims to deliver high sensitivity (detection at ~0.1 ng/mL), broad dynamic range, good accuracy and precision, and compatibility with an existing forensic LC/MS/MS workflow to allow parallel screening of other drug classes.

Methodology

• Sample preparation: A Micro Volume QuEChERS kit was used. To prepare analytical samples, 100 µL whole blood was mixed with 200 µL water, 300 µL acetonitrile and 20 µL internal standard (diazepam‑d5, 0.2 µg/mL in MeOH). After mixing and centrifugation, the acetonitrile supernatant was evaporated under N2 and the residue reconstituted in 100 µL of 20% methanol for injection.
• Chromatography: Kinetex XB‑C18 column (100 mm × 2.1 mm, 2.6 µm). Column temperature 40 °C, injection volume 1 µL. Binary mobile phases: 10 mM ammonium formate + 0.1% formic acid in water (A) and in methanol (B). Flow rate 0.3 mL/min. The gradient program refers to the LC/MS/MS Forensic Toxicology Database workflow used by the authors.
• Mass spectrometry: Triple quadrupole LCMS‑8050RX with Corespray ESI operated in positive ion mode using scheduled MRM. MRM transitions and collision energies were optimized per compound; typical precursor ions were [M+H]+ or [M+NH4]+ depending on analyte.
• Calibration and validation: Calibration standards were prepared in whole‑blood equivalents at 0.1, 0.5, 1, 5, 10, 50, 100 and 500 ng/mL. Linearity, sensitivity, accuracy and repeatability were evaluated. Accuracy and repeatability assessed at 20 ng/mL (n = 5).

Used Instrumentation

• Shimadzu Nexera X3 UHPLC system.
• Shimadzu LCMS‑8050RX triple quadrupole mass spectrometer (Corespray ESI).
• Column: Phenomenex Kinetex XB‑C18 (100 × 2.1 mm, 2.6 µm, P/N 00D4496‑AN).
• Micro Volume QuEChERS kit for extraction (P/N: S225‑37870‑91).
• Internal standard: diazepam‑d5 (0.2 µg/mL in methanol).
• MS operating conditions (representative): nebulizing gas 3 L/min, drying gas 10 L/min, heating gas 10 L/min, DL temp 250 °C, heat block 400 °C, interface 300 °C.

Main results and discussion

• Analytes: The panel comprised 27 antidiabetic compounds spanning biguanides, DPP‑4 inhibitors, SGLT2 inhibitors, sulfonylureas, glinides and thiazolidinediones (metformin, imeglimin, buformin, vildagliptin, anagliptin, alogliptin, saxagliptin, trelagliptin, omarigliptin, sitagliptin, teneligliptin, linagliptin, chlorpropamide, pioglitazone, acetohexamide, empagliflozin, gliclazide, tofogliflozin, ipragliflozin, luseogliflozin, dapagliflozin, glibenclamide, mitiglinide, canagliflozin, glimepiride, nateglinide, repaglinide).
• Sensitivity: All 27 compounds produced detectable MRM peaks at 0.1 ng/mL in whole blood. Representative MRM chromatograms at 0.1 ng/mL were obtained for each analyte.
• Linearity: Excellent linearity was reported across compound‑specific calibration ranges. Most analytes showed R or R2 values above 0.995 (R values reported; R2 similarly high), with lower upper limits for a subset (several compounds calibrated up to 100 or 200 ng/mL; 15 compounds up to 500 ng/mL).
• Accuracy and precision: At 20 ng/mL (n = 5) accuracy for all analytes fell within 80–120%. Repeatability (%RSD) was ≤ 7.9% across the panel, indicating robust precision for routine quantification in whole blood.
• Chromatographic separation: Retention times spanned ~0.78 min (metformin) to ~8.01 min (repaglinide), enabling complete separation within a short analytical window and compatibility with scheduled MRM for multiplexed detection.

Benefits and practical applications of the method

• Single‑run quantification of a broad antidiabetic panel reduces instrument time and increases throughput compared with multiple targeted assays.
• High sensitivity (~0.1 ng/mL) and wide dynamic ranges suit both low‑level exposure detection and therapeutic/overdose quantification.
• Compatibility with an existing LC/MS/MS Forensic Toxicology Database facilitates integration into forensic and clinical toxicology workflows and parallel analysis of other drug classes.
• Simple, small‑volume sample prep (100 µL whole blood) using Micro Volume QuEChERS supports limited‑volume or post‑mortem samples and streamlines processing in high‑throughput laboratories.

Future trends and potential uses

• Panel expansion: Inclusion of emerging antidiabetic agents, metabolites and relevant adulterants to cover evolving misuse patterns.
• Stable isotope standards: Use of isotope‑labeled internal standards for each analyte would further improve quantitative accuracy and matrix compensation across diverse blood matrices.
• Automation and sample tracking: Integrating robotic extraction and automated data processing would increase throughput for routine forensic labs.
• Hybrid strategies: Combining targeted triple quadrupole quantification with high‑resolution LC–MS screening could provide both sensitive quant and untargeted suspect screening in a two‑tiered workflow.
• Alternative matrices: Method adaptation and validation for plasma, serum, oral fluid and dried blood spots would broaden clinical and field‑applicable testing scenarios.
• Regulatory implementation: Standardization and interlaboratory validation would support adoption for clinical toxicology, therapeutic drug monitoring and forensic casework.

Conclusion

The presented LC–MS/MS assay on the Nexera X3/LCMS‑8050RX platform demonstrates a practical, sensitive and precise approach to simultaneously quantify 27 antidiabetic drugs in 100 µL whole blood using a simple Micro Volume QuEChERS extraction. The method achieves detection at ~0.1 ng/mL, robust linearity and acceptable accuracy and precision, and is directly compatible with forensic toxicology workflows—supporting efficient casework, overdose investigations and monitoring of non‑medical use.

Reference

• Wasim Rauf Kadri, Khalid Raza, Comparison of Different Antidiabetic Medication Classes: Efficacy and Safety, European Journal of Cardiovascular Medicine, Vol. 15, Oct. 2025, p. 223–227.