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 compounds.

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 ClH using H 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 ClH, 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. This value can be significantly improved (to 0.002 mg L) via on-line sample pre-concentration using a trapping chromatographic column and a time-programmable 10 ports/2 positions micro valve.