Rapid and sensitive high-performance liquid chromatographic determination of nicotine and cotinine in nonsmoker human and rat urines

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Abstract

A simple reversed-phase high-performance liquid chromatographic method with paired-ion and UV detection has been developed for the rapid quantification of urinary nicotine and cotinine. A one-step solid–liquid extraction on Extrelut® was used. Separation from endogenous substances was achieved with a decreasing flow-rate. With 20 ml of urine for extraction, the limit of quantification was 0.5 ng/ml for cotinine and 5 ng/ml for nicotine; linearity was obtained from 50 to 5000 ng/ml. The intra- and inter-day coefficients of variation were less than 9% for cotinine and 30% for nicotine. Average recoveries for cotinine were 92–100% and 47–86% for nicotine. The present method was applied to the urine analysis of smokers, nonsmoker children, and experimental animals.

Introduction

The determination of nicotine and one of its major metabolites, cotinine, in biological fluids has aroused particular interest during the last decades. Indeed these biochemical markers have been used to estimate active smoking behaviour, to validate abstinence smoking, to evaluate exposure to environmental tobacco smoke (ETS) [1]and its effects on health, and to supplement information for epidemiological studies.

Many analytical methods have been described for the analysis of nicotine and cotinine: gas chromatography (GC) 2, 3on capillary or packed columns with a nitrogen-sensitive detector [4]or an alkali flame ionization detector, but the low long-term stability of this latter detector limits its routine application. GC coupled with a mass spectrometer [5], a highly specific gas detector, is very expensive for routine analysis. High-performance liquid chromatography (HPLC) is the preferred technique 6, 7, 8, 9, 10, 11, sometimes involving precolumn derivatization with diethylthiobarbituric acid [12], but these are not suitable for routine assays because the coloured complexes are unstable. Enzyme-linked immunosorbent assay (ELISA) [13]and radio-immunoassay (RIA) have also been described 14, 15but the former lack sensitivity and the latter is very expensive.

Progress in analytical methodology has improved the understanding of nicotine metabolism [1]. Metabolites such as trans-3′-hydroxycotinine and glucuronide conjugates of nicotine, cotinine, and trans-3′-hydroxycotinine have been studied 16, 17, 18, 19, 20, 21, 22, 23but unconjugated trans-3′-hydroxycotinine is difficult to quantify in urine since its hydrophilicity hinders extraction [18], and otherwise the assay of glucuronide metabolites requires longer pretreatment. All these methods include liquid–liquid or solid–liquid extractions, and all the procedures reported are fairly complicated, time consuming, and not suitable for application to large numbers of samples with low concentrations.

In this paper, a simple, fast and sensitive reversed-phase ion-paired liquid chromatographic procedure using ultra-violet detection was developed with a single-step solid-phase extraction. The aim of this study was to evaluate this procedure in routine and simultaneous analysis of nicotine and cotinine in urine of smokers, nonsmokers exposed to ETS, and animals, in order to evaluate the pharmacological effects of this tobacco alkaloid.

Section snippets

Reagents and standards

Nicotine and cotinine were purchased commercially from Sigma (St. Louis, MO, USA). Methanol, acetonitrile, chloroform, 2-phenylimidazole, citric acid, disodium hydrogenphosphate dodecahydrate, triethylamine and sodium octanesulphonate were from Aldrich (Strasbourg, France). All solvents were analytical grade.

Stock solutions of nicotine, cotinine and 2-phenylimidazole, as internal standard (I.S.), were prepared separately for each compound at a concentration of 1 mg/ml in methanol. These stock

Results

Fig. 1 shows typical results obtained with a blank nonsmoker's urine and the same blank urine spiked with nicotine, cotinine and I.S. solutions; and a low exposure urine sample from a child.

Discussion

The number of cigarettes smoked by parents inside and outside the house is a very subjective indicator of passive smoking in children, because smoking status is difficult to establish, and several other sources could introduce a bias, e.g., visitors at home, public transport, public buildings. Thus it was necessary to validate a method of evaluation of such exposure, one suitable for the processing of large numbers of samples for routine application and also for experimental animal models.

The

Acknowledgements

We express our gratitude to Pr. B. Bruguerolle, for providing the experimental animals.

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