Determination of tricyclic antidepressants in human urine samples by the three-step sample pretreatment followed by HPLC-UV analysis: an efficient analytical method for further pharmacokinetic and forensic studies

In this work, an efficient sample pretreatment method has been developed by combining salt induced-homogenous liquid-liquid extraction, dispersive solid phase extraction, and dispersive liquid-liquid microextraction based on the solidification of floating organic droplet for the extraction of some widely used tricyclic antidepressant (TCA) drugs (nortriptyline, amitriptyline, desipramine, clomipramine, and imipramine) in human urine samples before their determination by high performance liquid chromatography-ultraviolet detection. In brief, the target analytes are first isolated from urine samples into acetonitrile (ACN) separated by adding a salt. Then the obtained ACN phase is treated with a mixture of appropriate sorbents to remove interferences. Afterward, the purified ACN is mixed with menthol as an extractant and rapidly injected into alkaline HPLC-grade water as a preconcentration step. Next, the obtained solution is placed in an ice bath and menthol collects on top of the solution after solidification. The solidified drop is then withdrawn and injected into separation system after dissolving in 10 µL ACN. Under the optimum experimental conditions, extraction recoveries and enrichment factors of the selected drugs ranged from 69-84 % and 345-420, respectively. The limits of detection and quantification were obtained at the ranges of 0.22-0.31, and 0.71-1.1 µg L(-1), respectively. The relative standard deviations of the proposed method were ≤ 6 % for intra- (n=6) and inter-day (n=4) precisions at a concentration of 10 µg L(-1) (each drug). Finally, the suggested approach was applied to determine of TCA drugs in different patients' urine samples. The method could be applied in further TCAs pharmacokinetic and forensic studies.