Validation of SPE-HPLC determination of 1,4-benzodiazepines and metabolites in blood plasma, urine, and saliva.

A simple, sensitive, selective, and reproducible RP-HPLC method with DAD detection at 240 nm was developed for the determination of six 1,4-benzodiazepines: bromazepam (BRZ), clonazepam (CLZ), diazepam (DZP), flunitrazepam (FNZ), lorazepam (LRZ), alprazolam (APZ); and two metabolites: alpha-hydroxyalprazolam (HALZ) and alpha-hydroxytriazolam (HTZL) in human plasma, urine, and saliva, using colchicine as internal standard, after SPE using Nexus Varian cartridges. Separation was performed on a Kromasil C(8) (250 mm x 5 mm, 5 microm) analytical column with a gradient mobile phase containing methanol, ACN and 0.05 M ammonium acetate. Linearity was held within the range 0.3-20.0 ng/microL, with coefficients of determination (r(2)) better than 0.997. The within- and between-day assay RSD at 2, 4, 8 ng/microL ranged from 0.03 to 4.7% and 0.5 to 7.0%, respectively in standards, from 1.3 to 7.9% and 3.3 to 7.3%, respectively in plasma, from 2.1 to 6.0% and 2.1 to 7.8%, respectively in urine and at 0.5, 1.0, 2.0 ng/microL ranged from 2.22 to 5.8% and 2.2 to 8.1%, respectively, in saliva. The mean relative recoveries were 96.3-108.6, 96.0-108.2, 94.3-107.1, 97.0-107.0% in within-day assay and 96.8-107.7, 94.6-107.6, 93.2-105.8, 96.0-108.6 in between-day assay for standard, plasma, urine, and saliva, respectively. The LOD and LOQ were 0.02-0.47 and 0.07-1.57 ng/microL, respectively.