Concurrent Determination of Second-Generation Antidepressants in Plasma by Using Gas Chromatography with Nitrogen–Phosphorus Detection

Abstract

Antidepressant (ADP) drugs are among the most commonly involved compounds in voluntary intoxications because of their large prescription to people with major depression and their benefical effect in some associated psychiatric disorders (1). The second-generation compounds have clear advantages in comparison with tricyclic ADPs, but numerous studies report the frequent occurrence of interaction when new ADPs are associated with tricyclics, antipsychotics, or anxiolytics (2)(3). Methods of analysis in biological fluids include gas chromatography (GC), GC–mass spectrometry (MS), HPLC, and immunoassays (4). Chromatographic methods usually appear to be complex and unable to provide chemical identification under screening conditions. We present in this study a simple and rapid GC procedure with a nitrogen–phosphorus detector (NPD) for the simultaneous determination of seven frequently requested new ADPs in plasma. As far as we know, no previous report has been published on the concurrent determination of ADPs by using a common extraction procedure and GC analysis. This assay was developed for the analysis of amoxapine, dothiepin, fluoxetine, fluvoxamine, medifoxamine, mianserin, and viloxazine. The reagents, apparatus (Varian-Star 3400 CX coupled to a Fisons-Chromcard for Windows Software), and procedure for the separation and measurement of the analytes have been previously described, according to the method for fluoxetine (5). Briefly, the patient’s alkalinized specimen (1 mL of a 20% ammonia solution mixed with an equal volume of serum), with added protriptyline as internal standard (IS) (15 μL of a 10 mg/L working solution in ethanol), is extracted in a one-step procedure with hexane:dichloromethane:isoamyl alcohol (57:42:1 by vol). The residue is dissolved in 50 μL of ethanol and directly chromatographed on a 25 m × 0.32 mm (i.d.) OV1 capillary column (25-μm-thick film) with helium as the carrier gas. The method gives a linear response to at least 2000 μg/L. The limits of detection ranged between 0.2 and 2.0 μg/L and the limit of quantification was established as between 0.5 and 5.0 μg/L (n = 5). Intraassay and between-day CVs were ≤10% (n = 10). The mean overall absolute recoveries (n = 5) ranged from 65% (medifoxamine) to 97% (fluoxetine). Plasma from drug-free patients did not exhibit significant peaks in the 30-min retention range. Calibrators and controls were prepared with drug-free plasma of healthy volunteers. We studied 57 compounds that can potentially interfere in plasma. A complete list, with retention times (tRs), is given in Table 1 . ADP metabolites may also be detected in plasma patients. So whenever possible, we tested the metabolites of the various analytes. No interference came from endogenous plasma constituents eluting during the complete analysis set. The potential for interference was judged by the presence of an interfering peak for coextracted drugs, the retention time of the peak, and peak height in relation to the drug’s plasma concentration. Many psychoactive drugs were tested on the OV1 capillary column because they could be associated with ADPs. Of all the drugs tested, only trimipramine coadministration could modify the results, since its metabolite, the desmethyltrimipramine, has the same tR as the IS (18.33 min). If a peak appeared at tR 18.16 min, it is highly probable that a tricyclic ADP (trimipramine or imipramine) was administered. No quantification is available in the case of trimipramine coadministration. Therefore only GC-MS appeared to be able to provide unequivocal chemical identification under screening conditions for toxicological samples. Fours drugs also showed peaks that would partially overlap within ±0.1 min tR of analyte peaks: cocaine and amitriptyline with mianserin, codeine with dothiepin, and desipramine with protriptyline. The GC-NPD method was applied to plasma samples from depressed patients, chronically treated with ADPs, who had intentionally ingested high doses of their treatment. In the case of a patient overdosed with dothiepin, our method allowed a successful resolution of dothiepin (tR 19.28 min) with its two major metabolites, the northiaden (tR 19.37 min) and the dothiepin sulfoxide (tR 19.69 min), in spite of close tRs. The main advantages of this technique are based on: 1) Use of NPD detector: The introduction of new generations of NPD detectors allows signals of high stability during long-term operation, and the nitrogen-containing structure of ADPs is compatible with a high sensitivity for NPD detection. The method’s measurement range (0.5–2000 μg/L for amoxapine and dothiepin and 5–4000 μg/L for fluvoxamine) makes it possible to avoid the potential need for sample dilution in cases of intoxication, and it also has a low detection limit (≤2 μg/L). 2) Simplicity of the procedure: The simple addition of an ammonia solution before the initial introduction of plasma and other reagents for extraction minimizes adsorption of ADPs on glass and also makes the biological material slightly alkaline (pH >10) for the extraction. Protriptyline was chosen as IS because this compound either has been withdrawn from the market in some countries, or is not as widely used as tricyclic ADPs. The decrease in the proportion of hexane (57%), the addition of a more polar solvent, dichloromethane (42%), and isoamyl alcohol (1%) to break up emulsions was shown to be preferable for recovery of tricyclic ADPs and fluoxetine, as we previously described in recent reports (5)(6). Overall recoveries ranged between 65% and 97%. 3) Good resolution: The benefit of capillary GC essentially resides in a high resolution, allowing for peak resolution of peaks with tRs within >0.1 min of each other. 4) Powerful detection tool for ADPs: Automated immunoassays are unable to detect ADPs unrelated to the imipramine structure (4). Chromatographic procedures are the most viable techniques used to identify and determine these drugs. The benefits of monitoring ADPs have been much discussed, but in many cases, the information obtained has proved useful for the management of psychiatric patients (7). In conclusion, the present assay is one of the most sensitive and specific assays for second-generation ADPs. Its ability to measure several new ADPs simultaneously makes it particularly useful for toxicological emergency and for managing psychiatric patients treated with ADPs. Lab. de Pharmacol. et Analyses Toxicol., Centre Hospitalier Universitaire de Grenoble, BP 217, F-38043 Grenoble Cedex 9, France Retention times of coextracted drugs in ADP GC-NPD assay. Assay was developed for concurrent analysis of these drugs. Interference occurs above high concentrations. Interference occurs at any concentration. Retention times of coextracted drugs in ADP GC-NPD assay. Assay was developed for concurrent analysis of these drugs. Interference occurs above high concentrations. Interference occurs at any concentration. We are grateful to Anphar-Rolland, Eli-Lilly, Lederle, Minden, Organon, Solvay-Pharma, and Zeneca companies for providing analytical calibrators.