GC–MS method for the simultaneous determination of β-blockers, flavonoids, isoflavones and their metabolites in human urine

Abstract

A sensitive and selective method based on gas chromatography hyphenated to mass spectrometry (GC–MS) for the screening of 23 different compounds including β-blockers, flavonoids, isoflavones and metabolites in human urine sample was developed and validated. The present paper reports, for the first time, the method for the simultaneous determination of β-blockers, isoflavones, flavonoids and metabolites in human urine samples. When flavonoids are ingested in combination with drugs that have a narrow therapeutic range, interactions between flavonoids and drugs should be investigated. Substances of interest were extracted from urine samples by solid-phase extraction (SPE) employing a mixture of tert-butyl methyl ether:methanol:formic acid (4.5:4.5:1; v/v/v) as a mobile phase and Oasis HLB (Waters) as a stationary phase. Before extraction, urine samples were incubated with β-glucuronidase/sulfatase in order to achieve enzymatic hydrolysis. Before GC–MS analysis the analytes had to be derivatized with N-methyl- N-(trimethylsilyl)trifluoroacetamide (MSTFA) into their trimethylsilyl derivatives by incubating for 60 min at 60 °C. Statistical central composite design and response surface analysis were used to optimize the derivatization reagent. These multivariate procedures were efficient in determining the optimal separation condition, using peak areas as responses. The calibration curves were indicative of high linearity ( r 2 ≥ 0.9992) in the range of interest for each analyte. LODs (S/N = 3) ranged between 0.6 and 9.7 ng/ml. Intra-day and inter-day precision (CV, %) was less than 4.96%, accuracy between 0.01 and 4.98% and recovery was found in the range from 70.20 to 99.55%. The developed method can be applied to the routine determination of examined compounds’ concentrations in human urine. Moreover the method is suitable for detecting pharmaceutical compounds containing β-blockers, isoflavones and flavonoids in urine after administration to humans.