Development and validation of UHPLC-ESI-MS/MS method for the determination of selected cardiovascular drugs, polyphenols and their metabolites in human urine

Sylwia Magiera, Irena Baranowska, Jacek Kusa
Talanta 2012 January 30, 89: 47-56
A sensitive ultra-performance liquid chromatography tandem mass spectrometry method with electrospray ionisation (UHPLC-ESI-MS/MS) was developed for the simultaneous determination of 52 compounds: β-blockers, polyphenols (antioxidants) and their metabolites in mixture of standards and after addition the 52 standard solutions to human urine samples. The analyses of urine samples obtained from patients treated with β-blockers were also carried out. The separation of analytes was performed on a Hypersil GOLD™ column (100 mm × 2.1mm, 1.9 μm) using a gradient elution profile for 10 min and mobile phase consisting of 0.1% formic acid in water and acetonitrile. In these conditions, some of the tested compounds were not separated, but this was compensated by the use of MS/MS detection. The drugs, polyphenols and their metabolites were detected with a tandem mass spectrometer after being ionised positively or negatively (depending on the molecule) using an electrospray ionisation (ESI) source. The MS system was operated in the selected reaction monitoring (SRM) mode, where one quantitation and one confirmation transition was done for each analyte. The quantitative method was validated for selectivity, linearity, low limits of quantitation, accuracy, precision, recovery, matrix effect and analyte stability. The LLOQ varied from 0.01 to 0.40 ng mL(-1) for β-blockers and from 0.05 to 40.0 ng mL(-1) for polyphenols. The linear range was 0.08-1000 ng mL(-1) for the drugs and 0.10-2300 ng mL(-1) for the polyphenols. Intra-day and inter-day precision was less than 8%, and the accuracy ranged from -4.40 to 2.23% for all analytes. The average recoveries for all compounds analysed were better than 90%. The developed method can be successfully used to monitor cardiovascular drugs and their metabolites in urine samples of patients treated with β-blockers and can also be used to study the effect of polyphenols on the metabolism of drugs.

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