HPLC separation and simultaneous determination of tricyclic antidepressant drugs and their main metabolites in human plasma

In the last twenty years, several new antidepressant drugs have been introduced in therapy; however, the traditional tricyclic antidepressants (TCAs), which act by inhibiting the uptake of norepinephrine and serotonin, are still widely used by psychiatrists. TCAs are very effective against major depression. However, TCAs can cause several, potentially dangerous side effects, such as cardiovascular effects. Furthermore, the therapeutic window is quite narrow and even modest overdosing can cause severe toxic effects. Thus, in the last few years the practice of therapeutic drug monitoring (TDM) has been receiving increasing attention as a way of optimising therapy effectiveness and to increase patient compliance. Most TCAs have very long half-lives and generate active metabolites, which can complicate the TDM. Futhermore, the high structural similarity makes it quite difficult to discriminate between the different TCAs. To obtain reliable monitoring data, a new HPLC method has been developed, which allows the simultaneous determination of the plasma levels of seven TCAs (dibenzepine, amoxapine, protriptyline, imipramine, amitriptyline, maprotiline and clomipramine) and seven of their main active metabolites (8-hydroxyamoxapine, 8-hydroxyclomipramine, N-desmethylmaprotiline, nortriptyline, dinorclomipramine, amitriptyline N-oxide and norclomipramine) in a single chromatographic run. The method is based on the use of a Phenomenex C8 reversed-phase column as the stationary phase and a mixture of acetonitrile and a phosphate buffer as the mobile phase. Using this system, all fourteen analytes and the internal standard loxapine are baseline separated within 16 minutes. Spectrophotometric detection is carried out at 220 nm. The plasma sample pre-treatment is performed on C2 cartridges, using only 250 µL of plasma. The analytes are eluted with methanol and, when low levels are suspected, concentrated twice with respect to the original amount. This solid phase extraction (SPE) procedure eliminates all endogenous interference, while obtaining very satisfactory extraction yields for the analytes. In fact, extraction yield results range from 80% for protriptyline to 99% for amitriptyline. The preliminary results from its application to real samples from depressed patients are very encouraging, thus the method is promising for the TDM of TCAs in human plasma.