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 x 4 mm, 5 microm) 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/microL) 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.
HPLC is discussed as an essential tool in medicinal chemistry for the monitoring of tricyclic antidepressants in biofluids, providing clinicians with efficient fast and reliable methods to define individual optimum therapeutic concentrations in treatment of depressions. Additional information on mechanism of action, structure activity relationship and metabolism is provided.
This work describes the development of a dedicated, fully automated sequential-injection analysis (SIA) apparatus suitable for simultaneous electrogenerated chemiluminescence (ECL) and amperometric detection. The instrument is composed of a peristaltic pump, a multiposition selection valve, a home-made potentiostat, a thin-layer electrochemical/optical flow-through cell, and a light detector. Control of the experimental sequence and simultaneous data acquisition of the light and the current intensities were performed in LabVIEW6.1. The CL reagents and the sample were first aspirated as distinct zones into the holding coil of the analyser and, then, delivered to the cell; during their travel, the individual zones mixed and the ECL reaction was initiated as soon as the mixed regents reached the cell. The utility of the analyser was demonstrated for the detection of oxalate and
H
2
O
2
based on their ECL reactions with
Ru
(
bpy
)
3
2
and luminol, respectively.
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