LC–MS assay for quantitative determination of cardio glycoside in human blood samples

A method is described for liquid chromatography–mass spectrometry analysis of the cardio glycosides digoxin and digitoxin in biological samples. The method was optimized for use in the forensic field and, therefore, comprises the determination from whole blood and tissue samples. Sample cleanup by solid phase extraction (SPE) on a functionalized polymeric phase was sufficient to limit matrix suppression to <10% for all analytes. Chromatographic separation was achieved using an RP-8 column. Detection of the cardio glycosides was performed with electrospray ionization in the positive mode. The system was run in single ion monitoring mode, measuring the sodium adducts (M + Na)⁺ of the analyte and of the internal standard, respectively. The method was fully validated for the analysis of blood samples and was also successfully applied in forensic cases. The method was accurate and precise over a linear concentration range up to 50 ng/g blood. Lower limit of quantitation was 0.2 ng/g for digoxin and 2 ng/g for digitoxin, respectively. As deuterated analyte was used as internal standard, we also present a new microwave-enhanced method for the fast preparation of the labelled analyte within 20 min.