SIMULTANEOUS DETERMINATION OF 17 TOXIC ALKALOIDS IN HUMAN FLUIDS BY LIQUID CHROMATOGRAPHY COUPLED WITH ELECTROSPRAY IONIZATION TANDEM MASS SPECTROMETRY

Abstract

A sensitive and specific method was proposed and validated for the simultaneous determination of 17 poisonous alkaloids in human blood and gastric juice by liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). After mixing with boric acid buffer solution (pH 9), the samples were extracted with chloroform followed by chloroform-ether (2:1, v/v). The simultaneous qualitative and quantitative analysis of 17 alkaloids, including koumine, atropine, scopolamine, brucine, strychnine, aconitine, bulleyaconitine A, ephedrine, pilocarpine, lobeline, ergometrine, anabasine, oxymatrine, theophylline, colchicine, tetrahydropalmatine, and eserine, were performed by using ESI⁺ in multiple-reaction monitoring (MRM) mode. Under the optimized conditions, the calibration curves of the 17 compounds presented good linearity (r≧0.9952) in the ranges of 0.5–50 µg/L and 0.5–500 µg/L. The quantitation limits (LOQs) and detection limits (LODs) ranged from 0.5–1 and 0.1–0.5 µg/L, respectively. The average extraction recoveries were obtained in the range of 81.7–102.3%. The intra-day and inter-day relative standard deviations (RSDs) were less than 10% and 15%, respectively. The results indicated that the method was simple, rapid, selective, sensitive, and stable enough to examine toxic alkaloids and was suitable for poisoning diagnosis and forensic toxicology.

Keywords:

• blood
• gastric juice
• high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)
• simultaneous determination
• toxic alkaloids

ACKNOWLEDGMENT

This work was supported by the Scientific and Technology Plan Project from Guangdong province (No: 2008A030203010) and Scientific and Technology Plan Project from Yuexiu Region, Guangzhou (No: 2009-PT-005).

Notes

Each alkaloid standard solution at concentration of 1 mg/L was injected into LC-ESI-MS/MS system, to optimize fragment voltage for parent ion and collision energy for daughter ions. The ESI-MS/MS was set in the positive ion mode. MS conditions: dry gas, gas temperature 350°C, gas flow 10 L /min; nebulizer pressure: 40 psi; capillary voltage: 4000 V.

The spiked blank blood and gastric juice at concentration ranged from 0.5–500 µg/L were prepared and analyzed by the proposed method. The linearity calibration curves of 17 alkaloids were acquired. The regression equation was set Y = Ax + B, where Y and x were the values of the peak-area and concentrations for the alkaloids, respectively.

The calculated recoveries based on working standard solution prepared in method (R_M), and in blank blood and gastric juice (R_B). The RSDs were obtained based on the data calculated from working standard solution prepared in blank blood and gastric juice.