Development and validation of an HPLC method for the determination of benzodiazepines and tricyclic antidepressants in biological fluids after sequential SPE

A novel method for the simultaneous determination of six benzodiazepines (BZDs) and four tricyclic antidepressants (TCAs) in biological fluids by HPLC with UV detection at 240 nm has been developed. After a deproteinization step biological fluids were analyzed by direct injection. SPE on Nexus cartridges was also applied. Since two compounds, namely imipramine and diazepam, were coeluting, a sequential SPE protocol has been developed. BZDs were eluted by a mixture of methanol/ACN (1:1), followed by the elution of TCAs with methanol. Separation was performed on a Kromasil C 8 column (250×4 mm 2 id, 5 μm) using a mobile phase of 0.05 M CH 3 COONH 4 /ACN/methanol (initial composition 55:15:30 v/v/v) at a flow rate of 1.0 mL/min delivered by a gradient program within 15 min. Colchicine was used as the internal standard (4 ng/μL). The method was linear for all analytes up to 20 ng/μL, with coefficients of regression between 0.996 and 0.99996. LODs and LOQs were 0.08–1.17 and 0.28–3.91 ng/μL, respectively. Recovery was in the range of 92.8–108.7% for within‐day and 91.9–109.9% for between‐day assays, with RSD values lower than 10.0% for all matrices.