Determination of three beta-blockers in biofluids and solid tissues by liquid chromatography-electrospray-mass...

A LC-MS method using clenbuterol as internal standard was developed and validated for three b-blockers (BB) (atenolol, metoprolol, and propranolol) in rabbit postmortem matrices: heart, lung, kidney, liver, brain, blood, vitreous humor, gastric liquid, and urine. The BB were extracted from 2.0 mL of biofluids or 200 mg of solid tissues (after grinding and homogenization) by liquid-liquid extraction using Extrelut columns. Chromatographic separation involved a Nucleosil C18 (150 mm x 1-mm i.d., 5 microm) column together with a gradient of acetonitrile in 2mM, pH 3 ammonium formate. The compounds were ionized in the ionspray source of the atmospheric pressure mass spectrometer and fragmented by in-source collisions. The fragment ions were detected in the positive selected ion monitoring mode, targeting one quantitation and two confirmation ions per compound. The extraction recovery ranged between 10 and 40%, depending on the matrices. The limits of quantitation were 50 ng/g in tissues, 50 microg/L in blood and urine, and 10 microg/L in vitreous humor. Indeed, as preliminary results in one rabbit administered 5 mg/kg of each BB showed that BBs were more concentrated in some postmortem organs, validation was performed in the relevant concentration area in these particular tissues. The technique was found to be linear between 50 ng/g and 5000 ng/g for heart and liver, between 50 microg/L and 5000 microg/L for urine extracts, between 1000 ng/g and 50 000 ng/g for lung and kidney, and between 500 microg/L and 5000 microg/L for gastric content. A quadratic equation best fitted the calibration curve in blood between 50 microg/L and 5000 microg/L, as well as in brain between 50 ng/g and 40,000 ng/g. The correlation coefficients were all higher than 0.997. Intra- and interassay precision and accuracy fulfilled the international requirements. This simple and sensitive assay was applied to the determination of three BB in the biofluids and tissues of a rabbit as part of a preliminary step of a postmortem redistribution study and is also suitable for the routine determination of BB in forensic investigations.