Liquid Chromatography− and Capillary Electrophoresis−Mass Spectrometry in Glucuronide Analysis by

Phase II metabolites, or conjugates, are highly polar, non-volatile and thermolabile compounds that need to be analysed in biological matrices such as urine, bile and plasma. Liquid chromatography (LC) and capillary electrophoresis (CE) are proven techniques for the analysis of compounds of this kind, but the standard detectors in LC and CE lack sensitivity and especially specificity. Coupling of LC or CE to mass spectrometry (MS) provides a powerful tool for qualitative and quantitative analysis of drug conjugates. Atmospheric pressure ionisation (API) methods produce soft ionisation of the compounds and are well suited for the analysis of thermolabile secondary metabolites. The mass spectrometric and tandem mass spectrometric (MS/MS) behaviour of nitrocatecholtype glucuronides was systematically studied using atmospheric pressure chemical ionisation (APCI) and electrospray ionisation (ESI). The effect of operational parameters on the fragmentation of the compounds and the MS and MS/MS spectra is described. The applicability of APCI and ESI and a new atmospheric pressure photoionisation (APPI) method for the analysis of several phase II metabolites in biological matrices was evaluated. With the help of enzymatically synthesised standard compounds, three direct methods relying on liquid chromatography−tandem mass spectrometry (LC−MS/MS) and capillary electrophoresis−tandem mass spectrometry (CE−MS/MS) were developed for the quantitative analysis of nitrocatechol glucuronides. ESI in negative ion mode giving deprotonated molecules as base peaks in the spectra was found to be the most suitable for the analysis of acidic glucuronides. Conditions in the ion source were optimised to produce high abundance of deprotonated glucuronide as parent ion, to be fragmented by collision induced dissociation to the product ions that were analysed. All methods were based on highly specific selected reaction monitoring. The applicability of the methods was demonstrated in the analysis of intact glucuronides in urine and plasma. Urine samples were purified before analysis by simple solid phase extraction (SPE). For plasma samples, on-line purification using column-switching technique and restricted access material (RAM) column was more effective. All methods were shown to be repeatable and sensitive, the limits of quantitation being few femtomoles of glucuronide to MS.