Development of a fluorescence polarization immunoassay for lorazepam quantification

Lorazepam concentrations have been quantified in biological fluids using gas chromatography and high-performance liquid chromatography (HPLC). However, these methods are too time consuming and labor intensive for most nonresearch laboratories to offer. A fluorescence polarization immunoassay (FPIA) method for quantification of lorazepam was developed and validated using a reverse-phase HPLC method as the reference method. The FPIA method involves a single liquid-liquid extraction of 100 microliters of either serum or plasma, then direct analysis using the TDxFLx (Abbott Diagnostics, North Chicago, IL). FPIA calibrations were linear between 50 and 800 ng/ml using five calibrators prepared in human serum. Within-run precision (n = 10) for three serum controls (75, 300, and 600 ng/ml) resulted in coefficients of variation (CVs) of 5.7%, 5.4%, and 4.2%, respectively. Between-day precision studies for the three serum controls were 7.0%, 8.9% and 4.9%, respectively (n = 5). The analytical recovery of the three serum controls was 104.9%, 97.3% and 98.3%, respectively. There was an excellent linear correlation between the FPIA and HPLC determinations of 43 patient specimens (r = 0.990, slope = 0.961, intercept = 16.3). No interferences were found from many commonly prescribed nonbenzodiazepine drugs; however, other benzodiazepines that were tested will cross-react with this procedure and give inaccurate results. Therefore, this method should not be used in patients who are receiving other benzodiazepines in addition to lorazepam. The FPIA method described herein can be adapted to reliably measure lorazepam concentrations in serum or plasma.