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.