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 C~8~ analytical column (250×4 mm, 5 μm) was used for the separation, with a mobile phase consisting of 0.05 M CH~3~COONH~4~ and CH~3~CN (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.