Development and Validation of a Sensitive LC-MS/MS Method for the Simultaneous Analysis of Three-Tropane Alkaloids in Blood and Urine

Abstract

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous analysis of three tropane alkaloids in blood and urine. After 1 mL of a blood or urine sample was extracted using a liquid–liquid extraction method with ethyl acetate at pH 8 and homatropine as the internal standard, the tropane alkaloids were separated. An Allure PFP propyl column (50 mm × 2.1 mm, 5 µm) separated the tropane alkaloids using an acetonitrile-buffer solution (20 mmol/L ammonium acetate and 0.1% formic acid, pH 4) (70:30) as the mobile phase at a flow-rate of 0.2 mL/min in isocratic mode, with the LC-MS/MS in the positive ionization mode. For each compound, detection was related to two daughter ions (scopolamine: m/z 304.4 → 138.1 and 155.9; atropine: m/z 290.3 → 124.0 and 93.1; anisodamine: m/z 306.3 → 140.1 and 91.1; and homatropine: m/z 276.3 → 124.3 and 142.1). The tropane alkaloids exhibited excellent linearity in the range of 0.05–100 ng/mL in blood and 0.2–100 ng/mL in urine, with a limit of detection range from 0.02 to 0.05 ng/mL for biological materials. The extraction recoveries of atropine, scopolamine, and anisodamine were more than 53% in the blood and urine; the interday and intraday RSDs were less than 10%; the within-day and between-day accuracy were between −9.8% and +8.8%. The present method is simple and rapid, as shown by its application to a clinical case. This method is useful for routine analysis of tropane alkaloids in cases of suspected tropane alkaloid poisoning.