[Determination of nine markers of tobacco exposure in urine by supported liquid extraction-liquid chromatography-mass spectrometry]

Abstract

Objective: To establish a liquid chromatography-mass spectrometry method for the simultaneous determination of nine tobacco exposure biomarkers in human urine, including: nicotine, cotinine, trans-3'-hydroxycotinine, nicotine nitrogen oxide, cotinine nitrogen oxide, nornicotinine, norcotinine, anatabine, and anabasine.

Methods: Urine samples were hydrolyzed by adding β-glucuronidase/arylsulfatase(1000 units/sample) and shaking at 37 ℃ overnight in the dark. Deuterium-labeled internal standards were added, and the samples were extracted using a 96-well supported liquid extraction plate. The plate was eluted with 1.8 mL of isopropanol-dichloromethane solution(5∶95, V/V) and dried under a stream of nitrogen. The residues were reconstituted in pure water for analysis. A Gemini® 3 μm NX-C18 110A(2.0 mm×150 mm, 1.9 μm) column was used for separation, with gradient elution using 0.1%(V/V) ammonia solution and methanol as the mobile phase. The method utilized liquid chromatography-tandem mass spectrometry(LC-MS/MS) with a heated electrospray ionization source, employing parallel reaction monitoring and positive ion mode scanning.

Results: The method exhibited correlation coefficients(r) for the standard curves of the nine tobacco exposure markers ranging from 0.9984 to 0.9999. The detection limits were between 0.02 and 1.40 ng/mL, and the quantitation limits were between 0.06 and 4.60 ng/mL. The matrix effect ranged from 85.9% to 109%. The spiked recoveries ranged from 83.5% to 115%, and the relative standard deviations ranged from 1.0% to 16.3%(n=6).

Conclusion: This method is characterized by high sensitivity, precision, and accuracy, making it suitable for the determination of the nine tobacco exposure markers in the urine of both non-smoking and smoking individuals.

Keywords: exposure marker; liquid chromatography-mass spectrometry; supported liquid extraction; tobacco; urine.