MSACL 2019 EU Abstract

Simple and High-throughput LC-MS/MS Method for Simultaneous Measurement of Five Smoking-related Metabolites in Urine

Mi-Ryung Chun, Jongwon Oh, Min-Seung Park, and Soo-Youn Lee
Department of Laboratory Medicine and Genetics, Samsung Medical Center,

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Mi ryung Chun (Presenter) Samsung Medical Center

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of multiple nicotine metabolites can provide accurate information on smoking status and smoke exposure. However, current SPE or LLE for sample preparation is time consuming and labor intensive. Therefore, we focused on developing a simple and high-throughput method for measuring five smoking-related metabolites (nicotine, cotinine, 3-OH cotinine, nornicotine and anabasine) in population-based urine samples.

Methods: Each 30 µl of urine sample was deproteinized with 90 µL of acetonitrile and then further diluted by 120 µl of distilled water. Chromatographic separation used a Kinetex EVO C18 column (2.1mm×150mm, 5 µm; Phenomenex, CA, USA) and acetonitrile/30 mM ammonium bicarbonate (9/91, v/v) as mobile phase, at a flow rate of 0.40 mL/min. LC-MS/MS analysis was performed by a XEVO TQ-S tandem quadrupole MS equipped with an Acquity UPLC system (Waters, MA, USA). The transitions of MRM were m/z 163.2→ 84.2 for nicotine, m/z 177.2→ 98.1 for cotinine, m/z 193.2→ 86.2 for 3-OH cotinine, m/z 149.1→ 130.2 for nornicotine, m/z 163.2→ 134.2 for anabasine, m/z 167.3→ 134.2 for nicotine-d4, m/z 180.2→ 101.1 for cotinine-d3, m/z 196.2→ 134.2 for 3-OH cotinine-d3, m/z 153.2→ 134.2 for nornicotine-d4, and m/z 167.3→ 150.3 for anabasine-d4, respectively. The limit of quantification, selectivity, precision, accuracy, linearity, extraction recovery, matrix effect, and carry-over were evaluated according to CLSI guidelines. The validated method was used to analyze random urine samples from 268 healthy subjects.

Results:
The sample preparation time for a batch with 100 specimens was <1 hour. Chromatographic run time for each sample was 6.5 min. Our method showed excellent precision and accuracy on 4 concentrations of all the analytes; within-run CVs 2.9-9.4%, between-run CVs 1.5-9.8%, and bias <+/-10%, Linear dynamic ranges were 1.0–10,000 ng/mL for nicotine, nornicotine, and anabasine, 2.0–5,000 ng/mL for cotinine, and 5.0–15,000 ng/mL for 3-OH cotinine. The extraction recoveries ranged from 91.7% to 115.3%. No significant matrix effects or carry-over was observed. The urine concentrations of nicotine, cotinine, 3-OH cotinine, nornicotine and anabasine in non-smokers were <5.0 ng/mL, <8.7 ng/mL, <48.3 ng/mL, <1.4 ng/mL and <7.6 ng/mL, respectively.

Conclusion: Our LC-MS/MS method with simple sample preparation technique is sensitive and effective in screening of smoking status and smoke exposure in the general population.