Determination of muscarine in human urine by electrospray liquid chromatographic–mass spectrometric

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2011-09, Vol.879 (25), p.2549-2553
• Main Authors: Merová, Barbora, Ondra, Peter, Staňková, Marie, Soural, Miroslav, Stříbrný, Jan, Hebká, Lenka, Lemr, Karel
• Format: Article
• Language: English
• Subjects: acetonitrile; Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography; Chromatography, Reverse-Phase - methods; Determination; Extraction; Fundamental and applied biological sciences. Psychology; General pharmacology; Human; Human urine; Humans; Intoxication; Linearity; Liquid chromatography; Liquids; Male; Mass spectrometry; Medical sciences; microscopy; Middle Aged; Muscarine; Muscarine - urine; Pharmacology. Drug treatments; poisoning; Reproducibility of Results; screening; Sensitivity and Specificity; solid phase extraction; Spectrometry, Mass, Electrospray Ionization - methods; Spectroscopy; spores; Urine
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2011.07.009

A liquid chromatography–mass spectrometry based method for determination of muscarine in human urine was developed and validated. The method involved a solid phase extraction of muscarine from urine using Strata X-CW column. Separation of muscarine was achieved within 16.0 min on a reversed phase Gemini C18 analytical column (150 mm × 2.0 mm i.d., 5 μm) with a mobile phase consisted of aqueous 8 mmol/L heptafluorobutyric acid and acetonitrile in a gradient mode. Mass spectrometric detection was performed at m/ z 174 and m/ z 216 for muscarine and acetylmuscarine (internal standard), respectively. The linearity was satisfactory with a coefficient of determination ( R 2) 0.9993 at concentration range from 0.3 ng/mL to 2.0 μg/mL, LOD and LOQ for muscarine was 0.09 ng/mL and 0.3 ng/mL, respectively. The found out recoveries of muscarine were 96% or 95% for concentration 0.3 ng/mL and 0.2 μg/mL or 2.0 μg/mL, respectively. The precision in the intra-assay-study varied from 0.48% to 1.39% and in the inter-assay-study from 2.39% to 5.49%. The accuracy ranged from −3.3% to −6%. The validation results demonstrated that the method fulfilled satisfactory requirements for precision and accuracy across the calibration curve. The applicability of the method has been demonstrated by analyzing clinical urine samples. The method offers the fast objective identification of intoxication by muscarine and can become a routine screening alternative to more difficult microscopic examination of spores in the gastric content in clinical practice.