Determination of Diclofenac and Its Related Compounds using RP-HPLCICP- QQQ

Importance of the topic

Quantitative profiling of drug molecules and their metabolites is critical in pharmaceutical development. Traditional compound-specific radiolabeling methods are accurate but time-consuming, costly, and involve handling of radioactive materials. A structure-independent detection strategy simplifies workflow, reduces hazards, and enables consistent quantification across diverse metabolites.

Aims and overview of the study

This study aims to develop and validate a reversed-phase HPLC method coupled with triple quadrupole ICP-MS (ICP-QQQ) to measure diclofenac and its related compounds via the chlorine heteroatom. The approach seeks to provide compound-independent response, compensate for gradient elution effects, and assess performance in human plasma matrix.

Methodology and instrumentation

• Sample preparation: Pooled human plasma spiked with diclofenac and 4'-hydroxydiclofenac; synthetically degraded diclofenac for mass balance.
• Chromatography: Agilent 1260 HPLC system with Waters XBridge BEH C18 column; gradient elution from 30 to 100% acetonitrile over 25 minutes; flow rate 1.0 mL/min; sample temperature 5°C; injection volume 50 μL (1500 μL with preconcentration).
• ICP-QQQ detection: Agilent 8800 with PFA nebulizer, platinum cones, hydrogen cell gas (3.5 mL/min) for mass-shift measurement of 35Cl to 37ClH2+, oxygen addition (20% in Ar at 0.2 L/min) to stabilize plasma, reversed phase gradient compensation via mathematical correction.

Main results and discussion

• Selectivity: Baseline separation of diclofenac, 4'-hydroxydiclofenac, and degradation products confirmed by retention times and mass-shift detection.
• Linearity and sensitivity: Linear response from 0.05 to 5.0 mg/L Cl (R2>0.99); LOQ of 0.05 mg/L Cl; preconcentration improved LOQ to 0.002 mg/L Cl (25-fold enhancement).
• Accuracy and precision: Recoveries between 90–100% in plasma; intraday and interday RSD below 4%.
• Matrix effects: Mass balance study in plasma yielded recoveries of 92–93%, with relative differences below 5% for most peaks, demonstrating matrix-independent quantification.

Benefits and practical applications

The HPLC-ICP-QQQ method offers a rapid, nonradioactive, and structure-independent approach to quantifying chlorine-containing drugs and metabolites. It eliminates the need for individual metabolite standards, reduces sample preparation complexity, and is directly applicable to biological matrices.

Future trends and opportunities

• Extension to other halogen- or nonmetal-containing pharmaceuticals.
• Integration with high-resolution separation techniques for detailed metabolite profiling.
• Automation of online preconcentration to enhance throughput.
• Application to clinical pharmacokinetics, environmental monitoring, and quality control.

Conclusion

The developed RP-HPLC-ICP-QQQ method reliably measures diclofenac and its related compounds in human plasma with high accuracy, precision, and sensitivity. The compound-independent chlorine detection simplifies quantification workflows and presents a versatile tool for pharmaceutical analysis.

References

1. Handbook of Hyphenated ICP-MS Applications, Agilent publication, 2012, 5989-9473EN
2. Klencsar B., Balcaen L., Cuyckens F., Lynen F., Vanhaecke F. Analytica Chimica Acta 974 (2017) 43–53
3. Sugiyama N. Agilent publication, 2013, 5991-2303EN