Determination of urinary and salivary cotinine using gas and liquid chromatography and enzyme-linked immunosorbent assay

Abstract

The objective of this study was to compare cotinine concentrations in urine and saliva using gas chromatography (GC), high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). Ninety-four subjects were selected (27 smokers and 67 non-smokers) and interviewed using questionnaire. Of the non-smokers, 39 had been exposed to environmental tobacco smoke (ETS) and 28 had not been exposed to ETS. Cotinine levels among smokers were highest using all three measurements, followed by ETS exposed subjects and non-smokers. Cotinine levels in urine, using HPLC, correlated significantly with levels measured using ELISA (r=0.92) and GC–nitrogen–phosphorus detection (NPD) (r=0.92). Salivary cotinine levels measured using ELISA did not correlate significantly with either HPLC (r=0.37) or GC–NPD (r=0.33) measurements. Multiple regression models were used to adjust for age, gender, drug use and health status, and it was found that cotinine levels in urine and saliva were significantly correlated with smoking pack-year. The authors conclude that urinary cotinine concentration is a more accurate biomarker for ETS than salivary cotinine concentration.

Introduction

Cotinine is the major proximate metabolite of nicotine and has been widely used as a biomarker of environmental tobacco smoke (ETS) exposure. Cotinine levels in plasma, urine and saliva of non-smokers have been used in the assessment of ETS exposure and risk of ETS-related lung cancer [1]. Another biomarker for ETS exposure is COHb (blood carboxyhemoglobin) but this best represents acute exposure and cannot show daily variations in ETS exposure. Thiocynate has been used as a biomarker for ETS exposure, however it displays a lack of specificity and sensitivity [2]. CO, thiocynate and plasma nicotine concentrations have been shown to be unrelated to ETS exposure. Cotinine levels provide the best biomarker for exposure to passive smoke [3]. Of all the biomarkers for ETS exposure, nicotine and cotinine have been shown to be the most specific and most sensitive, however, the former has a short (6 h) half-life. The quantitative analysis of cotinine in physiological fluids can be achieved using gas chromatography with nitrogen–phosphorus detection (GC–NPD), radioimmunoassay (RIA), high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) [4]. Monoclonal antibodies were used to develop non-isotopic and RIA for quantitative determination of cotinine levels and results showed a strong correlation with values obtained by RIA or by GC [5]. ELISA gives a reliable quantitative measure of cotinine as an indicator of active and passive exposure to tobacco smoke [6]. GC–NPD is well known for such sensitive and simultaneous measurements of both nicotine and cotinine using a well maintained capillary column [7]. However, HPLC values for nicotine and cotinine in urine samples from passive smokers compare quite well with those of the more sensitive and simpler GC method [8]. Salivary cotinine levels over 0.4 ng/ml corresponded to an increased risk of lung cancer and heart disease due to ETS exposure by 1/1000 and 1/100, respectively [9]. There are many factors which could affect the condition of saliva which makes it difficult to collect standard specimens of saliva to accurately represent ETS exposure. Also, factors such as diet, time and duration of smoking can affect salivary cotinine. There are few studies in the literature which have compared salivary and urinary cotinine using different analytical methods. In 1997, the Taiwan government introduced the Tobacco Control Act which aims to reduce tobacco consumption and thereby reduce the population’s ETS exposure. There is no available data in Taiwan to investigate the relative reliability of biomarkers of ETS exposure using physiological fluids, such as serum, urine and saliva. Urine and saliva have been more widely investigated since they can be obtained non-invasively. The objective of this study was to compare the cotinine concentrations in urine and saliva using GC, HPLC and ELISA.