Research Article Received: 3 June 2008, Accepted: 19 June 2008 Published online 23 September 2008 in Wiley Interscience (www.interscience.wiley.com) DOI 10.1002/bmc.1110 Biomed. Chromatogr. 2009; 23: 273–279 Copyright © 2008 John Wiley & Sons, Ltd. 273 John Wiley & Sons, Ltd. Method validation for measurement of hair nicotine level in nonsmokers Method validation for measurement of hair nicotine level in nonsmokers Sung Roul Kim, a * Heather Wipfli, a Erika Avila-Tang, a Jonathan M. Samet a and Patrick N. Breysse b ABSTRACT: The development of strategies to address the growing worldwide burden of exposure to secondhand smoke (SHS) would be facilitated by sensitive and accurate methods for assessing SHS exposure. Hair provides a readily available matrix for assessing biomarkers of typical SHS exposure. We developed and applied an optimized analytical method using an isotope dilu- tion gas chromatography–mass spectrometry (GC/MS) for hair nicotine measurement. The utility of this optimized method is illustrated by presenting data on SHS exposure of women and children from 31 countries. Using this isotope dilution method with spiked samples (3.3 ng/mg), we found that the greatest hair nicotine extraction efficiency was obtained with a 60 min shaking time. In the field study (n = 2400), a positive association was evident between hair nicotine concentrations from nonsmokers and higher numbers of cigarettes smoked per day in a household. Copyright © 2008 John Wiley & Sons, Ltd. Keywords: nicotine; hair; secondhand smoke; children; gas chromatography–mass spectrometry Introduction In the presence of smokers, nonsmokers inhale secondhand smoke (SHS), the combination of side-stream smoke released from the cigarette’s burning and mainstream smoke exhaled by the active smoker (Guerin et al., 1992). Exposure to SHS is a worldwide public health problem (IARC, 1986; NRC, 1986; US DHHS, 1986; Warren et al., 2006; Wipfli et al., 2008). Evaluating the magnitude of SHS exposure and identifying its sources are fundamental steps needed to reduce exposure and to target prevention strategies. SHS exposure is assessed by diverse methods, including questionnaires (Al-Delaimy et al., 2000; Delfino et al., 1993; Gaffney et al., 2003; Klepeis, 1999), air nicotine monitoring (Hammond and Leaderer, 1987; Leaderer, 1990; Navas-Acien et al., 2006; Repace et al., 2006; Wipfli et al., 2008), and biomonitoring of nicotine or cotinine in such biofluids as serum, urine or saliva (Benowitz, 1999; Coultas et al., 1987; Seccareccia et al., 2003; Pirkle et al., 2006). The most commonly used biomarker for SHS exposure is nicotine or its metabolite, cotinine, in human body fluids such as urine, saliva and serum (Pichini et al., 2000; Simoni et al., 2006; Thaqi et al., 2005). The utility of nicotine or cotinine concentrations in body fluids is limited, however, by the relatively short half-lives of these biom- arkers. The half-life of nicotine in body fluids is approximately 2–3 h, and that of cotinine is 1–2 days (Benowitz, 1996; Jaakkola and Jaakkola, 1997). Therefore, levels of these markers in the body fluids are indicative of recent, rather than long-term, exposure. Longer-term biomarkers of SHS exposure have a potential utility for documenting usual patterns of exposure and for inves- tigating health risks of chronic exposure to SHS (IARC, 1986; NRC, 1986; US DHHS, 1986; Warren et al., 2006). Several studies indicate that hair nicotine concentration is a useful biomarker of longer term smoke exposure (Al-Delaimy et al., 2002; Eliopoulos et al., 1996; Nafstad et al., 1995; Pichini et al., 1997b). With a rate of hair growth of about 1.1 cm/month (Uematsu, 1993), a small amount of hair (2–3 cm) from the scalp can potentially represent expo- sure to SHS over 2–3 months. In addition, hair nicotine has shown better ability to discriminate exposure status when com- pared with urinary cotinine (Al-Delaimy et al., 2002; Nafstad et al., 1995). Al-delaimy et al. reported that hair nicotine concentrations in a sample of children were more strongly associated with cate- gories of smoking within a household (no smokers, smoking only outside the house, and smoking inside the house) than urinary cotinine concentrations (Al-Delaimy et al., 2002). Hair nicotine concentrations have been assessed using various chromatographic techniques, including high-performance liquid chromatography (HPLC) with an ultraviolet detector (Pichini et al., 1997a), an electrochemical detector (Mahoney and Al-Delaimy, 2001), and mass spectrometry (MS, MS/MS) (Chetiyanukornkul et al., 2004; Kronstrand et al., 2004; Pirkle et al., 2006); and gas chromatography (GC) with a nitrogen–phosphorus detector or mass spectrometry (Kintz, 1992; Zahlsen and Nilsen, 1994; Torano * Correspondence to: Sung Roul Kim, 615 N. Wolf Street W7010H, Baltimore, MD 21205, USA. E-mail: srkim@jhsph.edu a The Institute for Global Tobacco Control, Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA b Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA Abbreviations used: GC/MS, gas chromatography–mass spectrometry; LOD, limit of detection; SHS, secondhand smoke. Contract/grant sponsor: Flight Attendants Medical Research Institute; Con- tract/grant number: FAMRI 24066. Contract/grant sponsor: Johns Hopkins Center for Childhood Asthma in the Urban Environment; Contract/grant number: USEPA 0826724015, NIEHS ES09606. Contract/grant sponsor: Center in Urban Environmental Health; Contract/ grant number: ES03819. Contract/grant sponsor: Fogarty International Center; Contract/grant number: R01-HL-73699.