Quantitation of verapamil and norverapamil in postmortem and clinical samples using liquid-liquid extraction, solid phase extraction, and HPLC

Verapamil is a calcium channel blocking drug often prescribed to treat hypertension. The increase in verapamil prescriptions has led directly to an increase in overdoses, both intentional and accidental. The aim of this study was to develop a simple, fast and sensitive HPLC method with fluorescence detection for the quantitation of verapamil and its major metabolite norverapamil in postmortem samples. This study compared liquid-liquid and solid phase (C-2 silica) extraction techniques for quantitation of verapamil and norverapamil in postmortem blood, liver and kidney. The method was also applied to a blood sample collected in the emergency room from a patient suffering from a verapamil overdose. The experiment established a five-point standard curve that crossed both therapeutic and fatal concentrations of verapamil and norverapamil (100 to 3,000 ng mL(-1) for blood and 100 to 3,000 ng g(-1) for tissues). The standard curves were linear over the range of verapamil and norverapamil concentrations assayed and had correlation coefficients greater than 0.9784 +/- 0.0198 for both liquid-liquid and solid phase extractions. The limit of quantitation for verapamil and norverapamil in all specimens studied was established to be 100 ng mL(-1) and 100 ng g(-1). Intra-day variability was determined using 3-6 replicates of controls (2,500 ng mL(-1) of blood or 2,500 ng g(-1) of tissue homogenate) prepared in naive blood and tissues and analyzed on a single day. Inter-day variability was determined over a four day period analyzing replicates of controls. Control solutions prepared in naive blood, kidney and liver homogenates in the fatal concentration levels fell within the acceptable precision and accuracy range. Furthermore, liquid-liquid extraction is preferable to solid phase extraction for quantitation of verapamil and norverapamil in tissues because it is simpler, more versatile, more precise, more accurate and less expensive.