Automated Quantification of Doxylamine and Diphenhydramine in Human Plasma using on-line extraction-HPLC-DAD (TOX.I.S.)

Aim: The purpose of this study was to develop a screening method for the identification of weak basic, neutral and weak acidic substances in human plasma using on-line extractionHPLC-DAD (TOX.I.S. - toxicological identification system). Beside the identification of xenobiotics, the method was validated for the automatic quantification of two compounds in human plasma: doxylamine (DA) and diphenhydramine (DPH). In the context of STA routine analysis more than 450 intoxication cases were analyzed by the described method. Two intoxication cases are presented. Methods: The quantification of DA and DPH in human plasma was based on a methanolic extraction of plasma (0.2 ml) and basic (pH = 9) automated on-line extraction (Strata-X, 25 µm, 20x2 mm) followed by on-line extraction followed by HPLC-DAD detection (TOX.I.S). Analytical separation was carried out on Gemini NX (150x4.6 mm, 3µm) using gradient elution. The mobile phase consisted of 0.05M potassium dihydrogen phosphate buffer (pH=2.3) and acetonitrile/water (90/10, v/v). Peak identification, retention time (RT) and relative retention time (RRT) were carried out by chromatographic and spectral data comparison with a library containing >750 UV-spectra of weak basic, weak acidic and neutral compounds. Criteria for peak identification were a 95% agreement between the measured and the library spectrum (similarity !0.995) and a maximum difference in RRTs of ± 5%. The validation procedure was performed according to the GTFCh guidelines. Results and Discussion: A new method for the identification of xenobiotics in human plasma was successfully developed, integrated and validated using TOX.I.S. The tested model compounds for the automated quantification yielded following results: the calibration range of DA and DPH was linear from 0.25-5.0 mg/L (r 2 = 0.999). The LLOQs (0.15 mg/L for DPH and 0.25 mg/L for DA) were low enough for the detection of intoxications with theses substances. In intoxication case 1, DPH was detected in toxic concentration (3.24 mg/L); Dinordiphenhydramine and nordiphenhydramine were determined qualitatively. In case 2, sub-therapeutic concentrations of DPH (0.29 mg/L) and of DA (<0.25 mg/L) were found. Moreover, amitriptyline (0.084 mg/L) and nortriptyline (0.158 mg/L) and other metabolites of amitriptyline were quantitated in the same analytical run. Concentrations of amitriptyline and of nortriptyline were in therapeutic range. Conclusion: In TOX.I.S., an additional method for the automated identification of a broad range of weak basic, weak acidic and neutral compounds in human plasma was integrated and successfully validated. A highly automated online-SPE-HPLC-DAD method was capable of identifying (and automated quantification of doxylamine and diphenhydramine) xenobiotics in human plasma including sample preparation, data evaluation and comprehensive reporting.