Development of an HPLC method for the monitoring of tricyclic antidepressants in biofluids

Abstract

A simple, rapid and sensitive HPLC method was developed and validated for the determination of four tricyclic antidepressants (TCAs): amitriptyline, doxepin, clomipramine (CLO) and imipramine, in pharmaceutical formulations and biological fluids. A Kromasil C8 analytical column (250 × 4 ram, 5 μm) was used for the separation, with a mobile phase consisting of 0.05 M CH3COONH4 and CH3CN (45:55 v/v) delivered at 1.5 mL/min isocratically. Quantification was performed at 238 nm, with bromazepam (1.5 ng/μL) as the internal standard. The determination of TCAs in blood plasma was performed after protein precipitation. Urine analysis was performed by means of SPE using Lichrolut RP-18 Merck cartridges providing high absolute recoveries (>94%). Direct analysis of urine was also performed after two-fold dilution. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity. Repeatability (n = 5) and between-day precision (n = 5) revealed RSD <13%. Recoveries from biological samples ranged from 91.0 to 114.0%. The absolute detection limit of the method was calculated as 0.1-0.6 ng in blood plasma and 0.2-0.5 ng in extracted urine or 0.4-0.7 in diluted urine. The method was applied to real samples of plasma from a patient under CLO treatment. © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.