Repetitive injection field-amplified sample stacking for cationic compounds determination.

The development of a field-amplified sample stacking technique is presented. Sensitivity enhancement in this technique was obtained by repetitive injections of a sample followed by steps of sample matrix removal through the application of counter-pressure. Under optimized conditions the background electrolyte (BGE) was composed of 80 mM H3PO4 while the sample matrix contained 0.5mM H3PO4 and 30% (v/v) methanol. The elaborated method enabled a 4-fold effective injection of the sample (53 s, 0.5 psi). Each injection was followed by a focusing step during which the application of a voltage (2 kV) and counter-pressure (-1 psi) was performed for 0.65 min. The method was developed for the determination of six psychiatric drugs (opipramol, hydroxyzine, promazine, amitriptyline, fluoxetine, and thioridazine). The elaborated method was applied for analysis of human urine samples after a simple liquid-liquid extraction procedure. The detection limits obtained were in the range of 2.23-6.21 ng/mL.

Urine sample preparation of tricyclic antidepressants by means of a supported liquid membrane technique for high-performance liquid chromatographic analysis.

Supported liquid membrane (SLM) technique for sample work-up and enrichment was used for determination of tricyclic antidepressant drugs in urine by high-performance liquid chromatography (HPLC) with UV detection. The studied antidepressant drugs were amitriptyline, opipramol, noxiptyline and additionally diethazine was used as possible internal standard. Alkaline phosphoric buffer with urine sample, as the donor solution, was passed over the liquid membrane into which investigated substances were extracted. On the other side of the membrane, analyzed compounds were trapped due to creating non-extractable form in acidic acceptor solution. Enriched and cleaned up drugs were then injected into a HPLC system with ultraviolet detection to analyze of their concentration in acceptor solution. Optimum extraction efficiency was determined by changing acceptor and donor solutions pH, application of different flow rates of donor solution and by using different solvents in the membrane. Also, donor solution volume, extraction time and concentration of analytes were varied to check the linearity of extraction process. The highest extraction efficiency: 43% for opipramol, 56% for noxiptyline, 43% for amitriptyline and 42% for diethazine (R.S.D. values were <6% and n=3) was achieved when 0.05 M phosphate buffer pH 4.0 and 9.5 were used as donor and acceptor solutions, respectively, n-undecane with 5% tri-n-octylphosphine oxide (TOPO) was used as liquid membrane. Limit of quantification (LOQ) for tricyclic antidepressants after enrichment of 100ml of urine sample was about 1 ng/ml.

Identification and determination of opipramol metabolites in plasma and urine.

In six cases of suspected opipramol overdose, commercially available immunoassays for tricyclic antidepressants (TCA) EMIT tox serum Assay and ADxR serum TCA Assay indicated arbitrarily high or toxic TCA concentrations. However, opipramol concentrations determined by high-performance liquid chromatography (HPLC) analysis were in the high-normal or low-toxic range. This finding prompted us to study opipramol metabolism by mass spectral techniques and to determine the cross-reactivity of opipramol and its metabolites in immunoassays. Three previously unknown metabolites (I, II, V) included an oxidation product of the hydroxyethyl moiety to an acetic acid group at the piperazine side chain (1), a decarboxylation product of the latter metabolite (II), and opipramol-N-oxide (V). In addition, two previously reported metabolites were identified, which included a deshydroxyethyl metabolite (III) and dibenzazepine (IV). One of the major metabolites of opipramol is the acetic acid metabolite (I), which may exceed the opipramol plasma concentration immensely and contribute to an arbitrarily high concentration in commercially available immunoassays. The cross-reactivities of the metabolite (I) were determined to be 64 and 66% with EMIT and ADx, respectively.