Comparison of Liquid Chromatography Mass Spectrometry and Enzyme-Linked Immunosorbent Assay Methods to Measure Salivary Cotinine Levels in Ill Children

Abstract

Objective: Cotinine is the preferred biomarker to validate levels of tobacco smoke exposure (TSE) in children. Compared to enzyme-linked immunosorbent assay methods (ELISA) for quantifying cotinine in saliva, the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) has higher sensitivity and specificity to measure very low levels of TSE. We sought to compare LC-MS/MS and ELISA measures of cotinine in saliva samples from children overall and the associations of these measures with demographics and TSE patterns. Method: Participants were nonsmoking children (N = 218; age mean (SD) = 6.1 (5.1) years) presenting to a pediatric emergency department. Saliva samples were analyzed for cotinine using both LC-MS/MS and ELISA. Limit of quantitation (LOQ) for LC-MS/MS and ELISA was 0.1 ng/mL and 0.15 ng/mL, respectively. Results: Intraclass correlations (ICC) across methods = 0.884 and was consistent in sex and age subgroups. The geometric mean (GeoM) of LC-MS/MS = 4.1 (range: < LOQ to 382 ng/mL; 3% < LOQ) which was lower (p < 0.0001) than the ELISA GeoM = 5.7 (range: < LOQ to 364 ng/mL; 5% < LOQ). Similar associations of cotinine concentrations with age (β < −0.10, p < 0.0001), demographic characteristics (e.g., income), and number of cigarettes smoked by caregiver (β > 0.07, p < 0.0001) were found regardless of cotinine detection method; however, cotinine associations with sex and race/ethnicity were only found to be significant in models using LC-MS/MS-derived cotinine. Conclusions: Utilizing LC-MS/MS-based cotinine, associations of cotinine with sex and race/ethnicity of child were revealed that were not detectable using ELISA-based cotinine, demonstrating the benefits of utilizing the more sensitive LC-MS/MS assay for cotinine measurement when detecting low levels of TSE in children.includes exposure from both secondhand smoke (SHS) and thirdhand smoke (THS), measurement needs to be reliable, highly sensitive and specific so that even low levels of THS exposure can be detected [1,7]. Nicotine is predominantly derived from combustible and noncombustible tobacco/nicotine products. Over 70% of nicotine is metabolized to cotinine by the liver enzyme CYP2A6 [8]. Cotinine is nicotine's major proximate metabolite with a mean half-life of 16 h; some studies have reported higher median half-life levels of up to 28.3 h in infants and older children [1,[9][10][11]. In combination with cotinine, the other nicotine metabolites are nicotine, trans-3'-hydroxy cotinine (3HC), cotinine glucuronide, nicotine glucuronide, and 3HC glucuronide, which in combination, estimates the approximate daily intake of nicotine [1,12]. Cotinine concentrations are relatively stable and can be detected in the blood, saliva, urine, hair, and nails of children who are exposed to SHS and THS [1]. Thus, cotinine is the most commonly reported measure to indicate children's recent exposure to tobacco smoke. Since the concentration of cotinine in blood and saliva are highly correlated, saliva is often used to assess TSE in children since the collect...

A Parental Smoking Cessation Intervention in the Pediatric Emergency Setting: A Randomized Trial

“…Saliva was tested for cotinine, a measure of recent TSE [36] using enzyme-linked immunosorbent assay (ELISA) techniques by Salimetrics LLC [23] (78.2% of T0 and all T1 and T2 samples; level of detection (LOD) = 0.15 ng/mL) or by liquid chromatography tandem mass spectrometry (LC-MS/MS) with isotope dilution (21.9% of T0 and none of the T1 and T2 samples; LOD = 0.1 ng/mL) [37]. A prior publication indicates that ELISA is a cost-effective alternative to LC-MS/MS for detecting TSE to classify children into highly exposed versus not exposed to tobacco smoke [38]. Cotinine levels less than LOD were replaced with LOD/ √ 2.…”

The Associations of Trans-3′-Hydroxy Cotinine, Cotinine, and the Nicotine Metabolite Ratio in Pediatric Patients with Tobacco Smoke Exposure

“…Consistent with our hypothesis, we observed higher levels of COT and 3HC + COT sums, but lower 3HC/COT ratio levels in children who were non-Hispanic Black compared to children who were non-Hispanic White race/ethnicity, independent of other child sociodemographic and TSE patterns. Regarding COT, other prior research has also found that COT levels are higher in children who are of Black race and who are younger [2,7,32]. These higher COT levels may be due to slower metabolism secondary to genetic polymorphisms in the CYP2A6 gene in these child groups [4,[11][12][13].…”

“…Cotinine (COT) is a nicotine metabolite and it is a commonly used TSE biomarker [4]. Prior research indicates that up to 31% of children who live with smokers who seek care in emergency settings have high biochemically confirmed TSE levels that are equivalent to that of active smokers [5][6][7][8]. COT is measurable in the urine, saliva, blood, hair, and nails of exposed children who live with smokers and nonsmokers [3], and it has a half-life of approximately 16 h [5,9]; thus, COT levels reflect recent TSE.…”

Hand Nicotine and Cotinine In Children Exposed to Cigars: A Pilot Study

“…8 Research indicates that all children of cigarette smokers, even those whose parents enforce smoking bans, have detectable hand nicotine and cotinine with higher levels in younger children and those exposed to more cigarettes. 5,8,[10][11][12][13] Whereas the nicotine concentration in cigars compared to cigarettes is similar, the total tobacco content in cigars is higher than cigarettes, and the smoke emissions and TSE patterns from cigars and cigarettes may vary. 14 TSE biomarker levels differ by cigar type and use patterns and the use of other tobacco products (eg, dual-or poly-use with cigars, cigarettes, and/or marijuana).…”