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

Importance of the topic

A robust and rapid analytical method for measuring diclofenac levels in human plasma is vital for diverse applications including clinical pharmacokinetic studies, therapeutic drug monitoring, and safety evaluations. Diclofenac, a commonly prescribed NSAID, exhibits low nanogram-per-milliliter concentrations in plasma and coexists with complex biological matrices. Therefore, developing a workflow that combines efficient sample cleanup, high chromatographic speed, and sensitive detection is essential for reliable data and streamlined laboratory operations.

Objectives and Overview

This study presents a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed to quantify diclofenac in human plasma between 1 and 1000 ng/mL. Key objectives included:

• Implementing Thermo Scientific SOLA solid phase extraction (SPE) cartridges for fast and reproducible sample preparation.
• Employing an Accucore RP-MS core-shell UHPLC column for sub-two-minute separation.
• Establishing mass spectrometric transitions for diclofenac and its deuterated internal standard (diclofenac-d4) to achieve precise quantification.

Methodology and Instrumentation

The assay workflow consisted of the following steps:

• Sample pretreatment: 200 µL of human plasma spiked with 10 µL of diclofenac standard and diclofenac-d4 internal standard; acidified with 200 µL of 70% formic acid.
• SPE cleanup: Conditioning of SOLA 10 mg cartridges with methanol and water; sample loading; wash with 90:10 water/methanol; elution with acetonitrile; nitrogen evaporation at 40 °C; reconstitution in 100 µL of 50:50 water/acetonitrile.
• Chromatography: Thermo Scientific Accela 600 UHPLC; Accucore RP-MS column (2.6 µm, 50 × 2.1 mm); mobile phases A (water + 0.1% acetic acid) and B (acetonitrile + 0.1% acetic acid); linear gradient from 50% to 100% B over 1 minute; flow rate 0.6 mL/min; column temperature 40 °C; injection volume 20 µL.
• Mass spectrometry: Thermo Scientific TSQ Vantage with heated electrospray ionization in negative mode; optimized parameters included spray voltage 3000 V, vaporizer 300 °C, capillary 210 °C, collision gas pressure 1.5 mTorr; monitored transitions m/z 250.1→178.0 and 214.0 for diclofenac, and m/z 254.1→217.0 for diclofenac-d4.

Key Results and Discussion

The method delivered a linear calibration curve across 1 to 1000 ng/mL with an R2 of 0.999. The limit of quantification (LOQ) at 1 ng/mL showed a clear and well-defined peak shape. Quality control samples at 15 ng/mL demonstrated precision below 4.4% CV (n=6). Recovery experiments at the same concentration yielded an average extraction recovery of 85.8%, indicating efficient SPE cleanup. Accuracy across the dynamic range remained within ±17% of nominal values.

Benefits and Practical Applications

This LC-MS/MS method offers:

• High throughput: Rapid 1-minute chromatographic cycle accelerates sample processing.
• Robust cleanup: SOLA SPE cartridges reduce matrix effects and minimize phospholipid contamination.
• Sensitivity: LOQ of 1 ng/mL supports low-level quantification required in pharmacokinetic and toxicological studies.
• Reproducibility: Core-shell Accucore technology ensures consistent peak shape and low backpressure.

Future Trends and Opportunities

Potential developments include:

• Automation: Integration of SPE and UHPLC steps into robotic platforms for fully automated workflows.
• Miniaturization: Adoption of microflow LC-MS systems to decrease solvent consumption and further accelerate analysis.
• Multiplex assays: Expansion to multi-analyte panels for simultaneous quantification of NSAIDs and metabolites.
• Advanced ion sources: Evaluation of novel ionization techniques (e.g., sub-ambient pressure ionization) to enhance sensitivity and reduce matrix effects.

Conclusion

The described LC-MS/MS approach combining SOLA SPE and Accucore RP-MS chromatography achieves a fast, sensitive, and precise measurement of diclofenac in human plasma. With an LOQ of 1 ng/mL, recovery above 85%, and precision below 5% CV, the method is well suited for bioanalytical laboratories focused on pharmacokinetics, clinical research, and quality control.

References

• No external references were cited in the original document.