Plasma and Urine Levamisole in Clinical Samples Containing Benzoylecgonine: Absence of Aminorex.

Aminorex has been reported as a metabolite of levamisole in man, but data on the aminorex concentrations in clinical samples are scant. We thus measured levamisole, aminorex and benzoylecgonine in urine, and levamisole and aminorex in plasma using achiral liquid chromatography-high resolution mass spectrometry. Centrifuged urine (50 µL) was diluted with LC eluent containing internal standard (benzoylecgonine-D3, 25 µg/L) (450 µL). For plasma, sample (200 µL) and Tris solution (2 mol/L, pH 10.6, 100 µL) were added to a 60.5 × 7.5 mm i.d. glass test tube. Internal standard solution (ketamine-D4, 200 µg/L) (10 µL) was added and the tube contents vortex-mixed (5 s). Butyl acetate:butanol (9 + 1, v/v; 200 µL) was added and after vortex-mixing (30 s) and centrifugation (13,680 × g, 4 min), the extract was evaporated to dryness and reconstituted in 10 mmol/L aqueous ammonium formate containing 0.1% (v/v) formic acid (150 µL). Prepared samples and extracts (100 µL) were analyzed using an AccucoreTM Phenyl-Hexyl column (2.6 mm a.p.s., 100 × 2.1 mm i.d.) maintained at 40°C. MS detection was in positive mode using heated electrospray ionization (ThermoFisher Q-ExactiveTM). Intra- and inter-assay accuracy and precision were ±20%, and ≤11%, respectively, for all analytes in both matrices. Lower limits of quantitation were 0.1 and 1 µg/L (all analytes) in plasma and urine, respectively. Of 100 consecutive urine samples submitted for drugs of abuse screening containing benzoylecgonine, levamisole was detected in 72 (median 565, range 4-72,970 µg/L). Levamisole was also measured in eight plasma samples (median 10.6, range 0.9-64.1 µg/L). A number of metabolites of levamisole (4-hydroxylevamisole, levamisole sulfoxide, levamisole glucuronide, and hydroxylevamisole glucuronide) were tentatively identified in urine. Neither aminorex, nor any of its reported metabolites were detected in any sample.

Postmortem biochemistry: Current applications.

The results of biochemical analyses in specimens obtained postmortem may aid death investigation when diabetic and alcoholic ketoacidosis is suspected, when death may have been the result of drowning, anaphylaxis, or involved a prolonged stress response such as hypothermia, and in the diagnosis of disease processes such as inflammation, early myocardial infarction, or sepsis. There is often cross-over with different disciplines, in particular with clinical and forensic toxicology, since some endogenous substances such as sodium chloride, potassium chloride, and insulin can be used as poisons. The interpretation of results is often complicated because of the likelihood of postmortem change in analyte concentration or activity, and proper interpretation must take into account all the available evidence. The unpredictability of postmortem changes means that use of biochemical measurements in time of death estimation has little value. The use of vitreous humour is beneficial for many analytes as the eye is in a physically protected environment, this medium may be less affected by autolysis or microbial metabolism than blood, and the assays can be performed with due precaution using standard clinical chemistry analysers. However, interpretation of results may not be straightforward because (i) defined reference ranges in life are often lacking, (ii) there is a dearth of knowledge regarding, for example, the speed of equilibration of many analytes between blood, vitreous humour, and other fluids that may be sampled, and (iii) the effects of post-mortem change are difficult to quantify because of the lack of control data. A major limitation is that postmortem vitreous glucose measurements are of no help in diagnosing antemortem hypoglycaemia.