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: α‐hydroxyalprazolam (HALZ) and α‐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 C₈ (250 mm×5 mm, 5 μm) 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/μL, with coefficients of determination (r²) better than 0.997. The within‐ and between‐day assay RSD at 2, 4, 8 ng/μL 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/μL 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/μL, respectively.