Sujeewa Piyankarage (Presenter)
Centers for Disease Control and Prevention (CDC)
Bio: Dr. Sujeewa Piyankarage is an Analytical Chemist working at the Tobacco and Volatiles Branch, National Center for Environmental Health, Centers for Disease Control and Prevention (CDC).
Authorship: Sujeewa C. Piyankarage, June Feng, Lanqing Wang
Tobacco and Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA
| Short Abstract Characterization of menthol and nicotine and its metabolites in human samples in large-scale epidemiological studies is important when determining the role of menthol in tobacco use. This method quantifies menthol-glucuronide, nicotine and its unconjugated metabolites, minor tobacco alkaloids, and smoking-cessation drugs in human urine. The automated sample preparation process includes cleaning urine with mixed-mode solid-phase extraction. Compounds separated with reversed-phase liquid chromatography are detected with polarity-switching tandem mass spectrometry. Separation of 14 different compounds is achieved in 4.3 minutes while a linear calibration range from 1-80,000 ng/mL is maintained for the analytes that showed a broad concentration range in urine. |
Long Abstract
Introduction
Menthol has been widely used in tobacco products as a flavor enhancer. Menthol may also numb the respiratory tract allowing deep inhalation of tobacco smoke. Effects of menthol in decreasing nicotine metabolism and in increasing an individual’s nicotine dependence are among its potential adverse health effects related to tobacco use. Measurement of urinary nicotine and its metabolites, minor tobacco alkaloids, and smoking-cessation drugs in human urine enables evaluating tobacco exposure and profiling nicotine metabolism, and assessing compliance with smoking cessation programs. Large scale studies quantifying menthol, tobacco alkaloids, nicotine metabolites require a high throughput and sensitive analytical method. The high throughput analytical method described here enables quantifying menthol glucuronide, nicotine, cotinine, trans-3’-hydroxycotinine, anabasine, anatabine, isonicoteine, myosmine, β-nicotyrine, arecoline, arecaidine, bupropion, cytisine, and verenicline.
Methods
Hamilton STARlet system is used for automated sample aliquoting where urine, internal standards, and formic acid (4%) are pipetted into 96-well plates. Aliquoted samples are extracted by automated Solid Phase Extraction (SPE) with a Hamilton [MPE]2 unit followed by nitrogen-drying and reconstitution. Fourteen analytes are separated with reversed-phase (Phenomenex Evo C18) liquid chromatography using a gradient elution (A: 20 mM ammonium acetate buffer, B: Acetonitrile) on a Shimadzu Nexera system. A Sciex 6500 mass spectrometer with Atmospheric Pressure Chemical Ionization (APCI) and Multiple Reaction Monitoring (MRM) is used for detecting menthol-glucuronide and other analytes in the negative and positive modes, respectively. The LC-MS/MS data are analyzed using the Indigo software.
Results
Mixed-mode SPE products from 4 different manufacturers were compared for analyte recoveries. We selected Waters Oasis MCX 96 well plates that showed over 90% recovery for the majority of the analytes. The automated sample preparation, including aliquoting, SPE, and reconstitution, allows 96 urine samples to be processed in under 3.5 hours. The automated process showed excellent precision (RDS< 5%) and good accuracy (100±15%) when tested with spiked and smoker urine samples. Seven reversed-phase LC columns were assessed. Separation of structurally and chemically diverse analytes was achieved with the Phenomenex Evo column (C18, 2.1 x 100 mm, 2.6 µm) within 4.3 minutes. The method maintained an overall linear calibration range from 1-80,000 ng/mL while retaining the required sensitivities to cover the significant variations in analyte concentrations in urine. Analyses of anonymous-smoker urine samples showed 100% detection rates for menthol glucuronide, nicotine, cotinine, trans-3’-hydroxycotinine, anatabine, and anabasine. The analytical process can accommodate determination of 14 analytes in 200 samples per day.
Conclusions & Discussion
The automated sample processing technique and the LC-MS/MS method demonstrated in this study enable high throughput analyses of menthol, nicotine and its metabolites, minor tobacco alkaloids, and cessation drugs in human urine.
Disclaimer:
“The findings and conclusions in this study are those of the authors and do not necessarily represent the official position of the U.S. Department of Health and Human Services, or the U.S. Centers for Disease Control and Prevention. Use of trade names and commercial sources is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, or the U.S. Centers for Disease Control and Prevention.”
References & Acknowledgements:
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