Simultaneous Quantification of Saxagliptin, 5-Hydroxy Saxagliptin, and Dapagliflozin in Human Plasma Using SPE and UPLC-MS/MS Analysis

Significance of the Topic

The growing incidence of type 2 diabetes has driven the need for precise bioanalytical methods to measure therapeutic agents and their metabolites in human plasma. Saxagliptin, a DPP-4 inhibitor, its active metabolite 5-hydroxy saxagliptin, and the SGLT2 inhibitor dapagliflozin represent key drugs whose simultaneous quantification supports pharmacokinetic profiling, drug development, and clinical monitoring.

Objectives and Study Overview

This study describes a streamlined protocol for concurrent extraction and quantification of saxagliptin, 5-hydroxy saxagliptin, and dapagliflozin in human plasma. The main goals were to achieve sub-nanogram per milliliter detection limits, ensure linear response across a broad concentration range, and deliver rapid analysis suited for high-throughput bioanalysis.

Methodology

• Preparation of calibration standards (0.146–300 ng/mL) and QC samples in human plasma without internal standard.
• Acidification with 2% formic acid followed by mixed-mode solid phase extraction using an Oasis MCX 96-well μElution plate.
• Chromatographic separation on an ACQUITY UPLC HSS C18 column (2.1×100 mm, 1.8 μm) at 40 °C, 0.4 mL/min flow, 5 min gradient (5–95% B).
• Detection by UPLC-MS/MS with multiple reaction monitoring: positive mode for saxagliptin (316.22>180.19) and 5-hydroxy saxagliptin (332.30>196.20), negative mode for dapagliflozin (467.22>329.15).

Used Instrumentation

• ACQUITY UPLC I-Class FTN System
• Xevo TQ-S micro Tandem Quadrupole Mass Spectrometer
• ACQUITY UPLC HSS C18 Column
• Oasis MCX 96-well μElution Plate

Main Results and Discussion

Analyte recoveries were 100% for saxagliptin, 71% for 5-hydroxy saxagliptin, and 59% for dapagliflozin. Limits of quantification were established at 0.15 ng/mL, 0.20 ng/mL, and 0.60 ng/mL, respectively. Calibration curves demonstrated linearity over each range with R2 > 0.99 (1/x2 weighting). Inter- and intra-day precision and accuracy met regulatory criteria, with QC accuracy between 94.6–104.8% and precision ≤ 7.8% RSD. Signal-to-noise ratios at LLOQ levels were excellent, confirming method sensitivity.

Benefits and Practical Applications

This approach offers fast sample processing in a μElution format, five-minute run times, and high analytical sensitivity, making it ideal for pharmacokinetic studies, bioequivalence trials, therapeutic drug monitoring, and routine clinical research.

Future Trends and Opportunities

• Integration of automated sample preparation and data processing for higher throughput.
• Expansion to multiplex assays covering additional antidiabetic compounds and metabolites.
• Miniaturization using microfluidic SPE and nanoflow LC-MS for reduced sample consumption.
• Application of high-resolution mass spectrometry for comprehensive metabolite profiling.
• Personalized medicine monitoring with real-time therapeutic drug analysis.

Conclusion

The described SPE-UPLC-MS/MS method provides a robust, sensitive, and rapid solution for simultaneous quantification of saxagliptin, 5-hydroxy saxagliptin, and dapagliflozin in human plasma. Its high recovery, low detection limits, and regulatory compliance make it well suited to support pharmaceutical research and clinical bioanalysis.

References

• Xu XS et al. Liquid Chromatography and tandem mass spectrometry method for the quantitative determination of saxagliptin and its major active metabolite in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;889-890:77–86.
• Shubrook J et al. Saxagliptin: A selective DPP-4 inhibitor for the treatment of Type 2 Diabetes Mellitus. Clin Med Insights Endocrinol Diabetes. 2011;4:1–12.
• FDA Guidance for Industry. Bioanalytical Method Validation, CDER.