Development and validation of a novel quantification approach for gradient elution reversed phase high-performance liquid chromatography coupled to tandem ICP-mass spectrometry (RP-HPLC-ICP-MS/MS) and its application to diclofenac and its related compound

•Interference-free determination of Cl in organic matrix using ICP-MS/MS.•Investigation of the effects of organic solvents on the ICP-MS/MS signal of Cl.•Development of a novel quantification approach for gradient RP-HPLC-ICP-MS/MS.•Mathematical correction for the adverse effect of gradient elution.•Metabolite profiling of diclofenac and validation in human plasma matrix.

A novel quantification approach, compensating for the effect of gradient elution on the instrumental response during reversed phase high-performance liquid chromatography - inductively coupled plasma - tandem mass spectrometry analysis, has been developed and validated. As a proof of concept, diclofenac and its related compounds, including its major metabolite i.e. 4′-hydroxy-diclofenac, have been quantitatively determined in human plasma matrix. An inductively coupled plasma - tandem mass spectrometer has been applied for the interference-free determination of Cl as 35ClH2+ using H2 as a reaction gas in the collision/reaction cell. The effect of the eluent composition on the instrumental response for Cl has been thoroughly investigated for the most common organic solvents in reversed phase high-performance liquid chromatography, i.e. methanol and acetonitrile. A proper mathematical function describing the effect of the eluent composition on the sensitivity for Cl, monitored as 35ClH2+, permitted adequate correction for the otherwise detrimental effect of gradient elution for both solvents. Validation using synthetically degraded diclofenac samples spiked with its major metabolite, 4′-hydroxy-diclofenac, demonstrated appropriate accuracy (recovery for 4′-hydroxy-diclofenac between 95 and 105%) and >90% and >80% recovery for Cl using acetonitrile and methanol, respectively. When applied to spiked human plasma samples (the most important matrix in drug metabolism studies), a satisfactory accuracy (recovery of 92-98%) and precision (<4% RSD) were established for both 4′-hydroxy-diclofenac and diclofenac. The limit of quantification for Cl (as diclofenac) using the novel method was 0.05 mg L−1. This value can be significantly improved (to 0.002 mg L−1) via on-line sample pre-concentration using a trapping chromatographic column and a time-programmable 10 ports/2 positions micro valve.

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