Simultaneous chiral separation and determination of carvedilol and 5'-hydroxyphenyl carvedilol enantiomers from human urine by high performance liquid chromatography coupled with fluorescent detection

A sensitive and specific high performance liquid chromatography coupled with fluorescent detection (HPLC-FL) and tandem mass spectrometry detection (HPLC-MS/MS) methods for separation and determination of carvedilol (CAR) enantiomers and 5'-hydroxyphenyl carvedilol (5'-HCAR) enantiomers has been developed and validated. The analysed compounds were extracted from human urine by solid phase extraction. Good enantioseparation of the studied enantiomers was achieved on CHIRALCELA (R) OD-RH column using 0.05% trifluoroacetic acid and 0.05% diethylamine in water and acetonitrile in a gradient elution. The mass spectrometric data were acquired using the multiple reaction monitoring mode by positive electrospray ionisation. The method was validated over the concentration range from 25.0 ng mL(-1) to 200 ng mL(-1) for the analysed compounds. The limit of quantification varied from 14.2 ng mL(-1) to 24.2 ng mL(-1). Both the repeatability and inter-day precisions were below 10.0%, and the accuracy varied from -13.2% to 3.77%. The extraction recoveries ranged from 79.2% to 108%. The present paper reports the method for the simultaneous determination of CAR enantiomers and their metabolite enantiomers (5'-HCAR) in human urine samples. This newly developed method was successfully used to analyse the aforementioned analytes in human urine samples obtained from patients suffering from cardiovascular disease.