Application of an assay for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine for the assessment of tobacco-related harm

Background: The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is formed from nicotine and related compounds during tobacco curing and is classified as a human carcinogen. Its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), is thought to be useful in the assessment of cigarette smoking harm minimisation strategies.

Methodology: Urine samples were collected from 24 current Caucasian smokers participating in a smoking cessation study; before and four weeks after a quit attempt. Samples were spiked with NNAL-d3 internal standard, extracted with ethyl acetate using liquid–liquid extraction, reconstituted with deionised water and analysed using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). Both free (unconjugated) and total NNAL was measured, with totals determined by cleavage of NNAL-glucuronide using standard enzymatic hydrolysis methods.

Results: Free NNAL levels (193.5 ± 158.2 pg/mL [mean ± standard deviation]) measured in urine samples obtained at baseline (during ad lib smoking) were indicative of biological exposure to NNK. Free NNAL levels were significantly reduced four weeks after the quit attempt (64.5 ± 77.6 pg/mL; p < 0.002). Assay performance met acceptance criteria with mean recovery of 59 ± 23%, intra-day accuracy was 3.7%, 3.7 and 3.6% and precision was 11.3%, 5.1% and 5.3% at 5 pg/mL, 20 pg/mL and 100 pg/mL levels respectively. Enzymatic hydrolysis of NNAL-glucuronide proved unreliable with complete loss of NNAL aglycone in some subject samples following the hydrolysis procedure.

Conclusion: Liquid-liquid extraction with UPLC–MS/MS is a convenient approach to measure low levels of NNAL in urine as a marker of biological NNK exposure, which can be used to assess the effectiveness of smoking reduction harm minimisation strategies. Enzymatic hydrolysis of NNAL-glucuronide and measuring the aglycone is not recommended.