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Improved highly sensitive method for determination of nicotine and cotinine in human plasma by high-performance liquid chromatography

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Abstract

A highly sensitive and reliable method for the determination of nicotine and its metabolite cotinine in human plasma by high-performance liquid chromatography was developed. Nicotine and cotinine were extracted from alkalinized plasma with dichloromethane and the volatility of nicotine was prevented by the addition of conc. HCl to the organic solvent during evaporation. The sensitivity of quantification at 260 nm absorption was improved by using a noise-base clean Uni-3 to 0.2 ng/ml nicotine and 1.0 ng/ml cotinine. The method was validated over linear ranges of 0.2–25.0 ng/ml for nicotine and 1.0–80.0 ng/ml for cotinine. The intra-day precision and accuracy were ≤15.9% relative standard variation (RSD) and 89.9–103.5% for nicotine and ≤8.0% RSD and 98.7–103.0% for cotinine. The inter-day precision and accuracy were ≤17.0% RSD and 94.2–100.9% for nicotine and ≤8.2% RSD and 98.0–105.1% for cotinine.

Introduction

Nicotine taken up from cigarette smoking is primarily metabolized to cotinine in human liver [1]. Previously, we clarified that the enzyme responsible for the metabolism of nicotine to cotinine is cytochrome P4502A6 (CYP2A6) [2]. Recently, it has been reported that there is a whole deletion allele of the CYP2A6 gene and that the metabolic capacities in subjects whose CYP2A6 gene was deleted were poor [3], [4], [5]. Therefore, it is suspected that smokers with the deleted CYP2A6 gene would have a high nicotine plasma concentration and/or negligible cotinine plasma concentration. Thus, the determination of nicotine and cotinine in human plasma or urine is of particular interest to investigators studying the pharmacokinetics of nicotine and cotinine.

Various methods for the determination of nicotine and cotinine in biological specimens have been reported in the literature including radioimmunoassay [6], [7], high-performance liquid chromatography (HPLC) [8], [9], [10], gas chromatography (GC) using electron-capture detection, flame ionization detection, mass spectrometry (MS) [11], [12], [13], [14], and liquid chromatography (LC)–MS [15], [16]. In this study, we established and evaluated a highly sensitive HPLC procedure for nicotine and cotinine with modifications of the mobile phases, columns, extraction methods, and using a noise-base clean Uni-3.

Section snippets

Materials

Nicotine and cotinine were purchased from Sigma (St. Louis, MO, USA). Acetanilide was purchased from Wako (Osaka, Japan). All other chemicals and solvents were of the highest grade commercially available.

Extraction procedure

For the determination of the nicotine concentration, the plasma sample (1 ml) was alkalinized by 50 μl of 10 M NaOH. After the addition of 10 ng of acetanilide as an internal standard, the mixture was extracted with 4 ml of dichloromethane by shaking for 10 min. After centrifugation at 1000 g

Assay characteristics

Fig. 1A shows a representative chromatogram of nicotine and acetanilide. Fig. 1B and C represent chromatograms of extracts from human plasma samples before and after smoking, respectively. Fig. 1D shows a representative chromatogram of cotinine. Fig. 1E and F represent chromatograms of extracts from human plasma sample before and after smoking, respectively. None of these chromatograms showed any interfering peaks. For cotinine assay, an appropriate internal standard could not be found.

Conclusions

A highly sensitive and reliable HPLC method for the determination of nicotine and cotinine was developed and validated. The sensitivity for nicotine and cotinine was high enough for pharmacokinetic studies after the smoking of one cigarette or the chewing of one piece of nicotine gum. This method can be used in the processing and quantification of a large series of plasma samples.

Acknowledgements

This study was supported by an SRF Grant for Biomedical Research. We acknowledge Mr. Brent Bell for reviewing the manuscript.

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