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

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.