DEVELOPMENT OF A FLUORESCENCE POLARIZATION IMMUNOASSAY FOR LORAZEPAM QUANTIFICATION

Lorazepam concentrations have been quantified in biological fluids usi ng gas chromatography and high-performance liquid chromatography (HPLC ). However, these methods are too time consuming and labor intensive f or most nonresearch laboratories to offer. A fluorescence polarization immunoassay (FPIA) method for quantification of lorazepam was develop ed and validated using a reverse-phase HPLC method as the reference me thod. The FPIA method involves a single liquid-liquid extraction of 10 0 mu l of either serum or plasma, then direct analysis using the TDxFL x (Abbott Diagnostics, North Chicago, IL). FPIA calibrations were line ar between 50 and 800 ng/ml using five calibrators prepared in human s erum. 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 thr ee 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 man y commonly prescribed nonbenzodiazepine drugs; however, other benzodia zepines that were tested will cross-react with this procedure and give inaccurate results. Therefore, this method should not be used in pati ents who are receiving other benzodiazepines in addition to lorazepam. The FPIA method described herein can be adapted to reliably measure l orazepam concentrations in serum or plasma.