LC-MS/MS Method for the Determination of Diclofenac in Human Plasma

Importance of the Topic

Accurate quantification of diclofenac in human plasma is essential for pharmacokinetic studies, therapeutic drug monitoring, and safety assessments. High-throughput bioanalytical methods with low limits of quantification support clinical trials, drug–drug interaction studies and quality control in pharmaceutical research.

Study Objectives and Overview

This work aimed to develop a rapid, sensitive and reproducible LC–MS/MS assay for diclofenac in human plasma. Key goals included minimizing sample preparation time, achieving a wide dynamic range (1–1000 ng/mL) and ensuring robust chromatographic performance within a one-minute cycle.

Methodology

Sample pretreatment used solid-phase extraction on Thermo Scientific SOLA cartridges. Human plasma (200 µL) was spiked with diclofenac and an isotopically labeled internal standard (diclofenac-d4), acidified with formic acid and loaded onto conditioned SPE media. After washing, analytes were eluted with acetonitrile, evaporated under nitrogen, and reconstituted in 50:50 water/acetonitrile.

Chromatographic separation employed a reversed-phase Accucore RP-MS column (2.6 µm, 50×2.1 mm) with a 50% to 100% acetonitrile gradient (both phases containing 0.1% acetic acid) over one minute at 0.6 mL/min and 40 °C. A 20 µL injection volume and rapid needle washes ensured minimal carryover.

Used Instrumentation

• Thermo Scientific Accela 600 UHPLC system with Accucore RP-MS column (50×2.1 mm, 2.6 µm)
• Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer equipped with a heated electrospray ionization (HESI) source
• Thermo Scientific SOLA 10 mg/1 mL SPE cartridges and 96-well plates
• Thermo Scientific UltraVap sample evaporator and 96-well plate vacuum manifold
• LCQuan software for data processing

Results and Discussion

The calibration curve for diclofenac was linear from 1 to 1000 ng/mL with r2=0.999. Accuracy across eight calibration levels remained within ±17% at the lowest concentration and within ±1–2% at higher levels. The limit of quantification at 1 ng/mL produced clean, well-shaped peaks with negligible tailing. Six replicate quality control samples at 15 ng/mL showed precision below 4.4% CV. Recovery experiments yielded an average extraction efficiency of 85.8%.

Benefits and Practical Applications

• High throughput: one-minute chromatographic cycle accelerates sample analysis in clinical and bioanalytical labs.
• Low solvent consumption and simplified SPE workflow reduce operational costs.
• Robust performance ensures reproducibility and confidence in pharmacokinetic and toxicology studies.

Future Trends and Potential Applications

Emerging directions include multiplexing assays for simultaneous NSAID profiling, integration of on-line SPE for full automation, miniaturized workflows for microsampling, and coupling with high-resolution mass spectrometry for untargeted metabolite identification.

Conclusion

The described LC–MS/MS method combining SOLA SPE and Accucore RP-MS UHPLC provides a fast, sensitive and reproducible approach for quantifying diclofenac in human plasma. Achieving an LOQ of 1 ng/mL, recovery >86% and precision <5% CV, this assay meets the demands of high-throughput bioanalysis and clinical research.