Development and validation of an HPLC method for the determination of six 1,4-benzodiazepines in pharmaceuticals and human biological fluids

A sensitive, fast, and accurate reversed phased High Performance Liquid Chromatographic method, coupled with UV/DAD set at 240nm has been developed for the separation and quantification of six benzodiazepines (alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam) in pharmaceutical and biological matrices after solid phase extraction. The separation was achieved within 10min. on a Kromasill (C(8)-5 mu m, 250 x 4 mm, 100) analytical column using a mobile phase consisting of CH(3)OH:0.05M, CH(3)COONH(4):CH(3)CN delivered under a gradient program at a constant flow rate of 1.0mL/min. Colchicine (4ng/mu L) was used as internal standard. The drug/internal standard peak area ratios were linked in quadratic relationships to its concentrations giving a linearity range of 0.20-15.00ng/mu L for each BDZ, except BRZ that of which was 0.1-18.0 ng/mu L with correlation coefficients greater than 0.993. The precision and accuracy were evaluated at three concentrations. Validation was also performed in terms of selectivity, extraction recovery, stability, and robustness. Recoveries from pharmaceutical/biological samples were between 88-113% and intra-inter day RSD were 0.5-12%. The method was also applied to a real urine sample from a patient under treatment with alprazolam.