Journal of Chromatography B: Biomedical Sciences and Applications
Short communicationImproved highly sensitive method for determination of nicotine and cotinine in human plasma by high-performance liquid chromatography
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.
References (23)
- et al.
J. Chromatogr.
(1998) - et al.
J. Chromatogr.
(1996) - et al.
J. Chromatogr.
(1979) - et al.
J. Chromatogr.
(1985) J. Chromatogr.
(1987)- et al.
J. Chromatogr.
(1996) - et al.
J. Chromatogr.
(1988) - et al.
J. Pharm. Biomed. Anal.
(1999) - et al.
J. Chromatogr.
(1997) - et al.
J. Chromatogr.
(1982)
Drug Metab. Rev.
Cited by (66)
Nano-TiO<inf>2</inf> modified carbon paste sensor for electrochemical nicotine detection using anionic surfactant
2016, Biosensors and BioelectronicsCitation Excerpt :Many methods for NIC determination in the human body or in tobacco samples have been used like HPLC technique with various detectors such as electrochemical (HPLC–EC) (Mahoney and Al-Delaimy, 2001) and ultraviolet detectors (HPLC-UV) (Page-Sharp et al., 2003), and high-sensitivity gas chromatography mass spectrometry (GC–MS) (Shin et al., 2002). HPLC technique still normally employed in clinical laboratories for accurate sensing (Maurer, 1998; Nakajima et al., 2000; Papadoyannis et al., 2002) with disadvantage of time-consumption, very sophisticated, require preliminary extraction and purification of nicotine from the sample matrix and involves many steps. Electrochemical sensing methods have a great interest among researchers because of its fast response, relatively highly sensitive, simple, low cost, reliability and could be miniaturized (Yang et al., 2004; Suffredini et al., 2005; Lin et al. 2008; Sims et al., 2010; Švorc et al., 2014).
Cigarette smoking substantially alters plasma microRNA profiles in healthy subjects
2013, Toxicology and Applied PharmacologyCitation Excerpt :Principal component analysis (PCA) was performed to visualize the difference between groups of the expression profiles of miRNAs that exceeded the cutoff value. Plasma concentrations of nicotine and cotinine were measured as described previously (Nakajima et al., 2000) with slight modifications. The plasma sample (0.5 ml) was alkalinized with 25 μl of 10 M NaOH.
Rapid and sensitive determination of nicotine in formulations and biological fluid using micellar liquid chromatography with electrochemical detection
2010, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesCitation Excerpt :The importance of the determination of nicotine is evident from the number of scientific works carried out for the detection of nicotine in formulation [6] and products i.e. cigarette butt [7], chewing gum [8], pharmaceuticals [9], nasal powder [10], dietary nicotine [11], fertilizer [12] as well as nicotine and its metabolite in various biological samples like plasma [13–16], urine [14], hair [17,18], saliva [11], meconium [19], cutaneous nicotine [20] and milk [21] leaving apart experimental animals and animal products [22–24]. Analytical techniques for the determination of nicotine in these samples includes enzyme linked immunoassay and radio immunoassay [25,26], gas chromatography [9], gas chromatography–mass spectrometry [10,11,24], high performance liquid chromatography (HPLC) coupled with ultraviolet detector [6,12,14,15,18,20–24], diode array detection [18], electrochemical detector [17] and mass spectrometry [7,11,13,16,19]. In the biological samples, using a simple UV detection mode one may encounter other interfering substances.
An inhalation chamber model for controlled studies of tobacco smoke toxicity in rodents
2010, Archivos de BronconeumologiaSimultaneous and sensitive measurement of nicotine, cotinine, trans-3′-hydroxycotinine and norcotinine in human plasma by liquid chromatography-tandem mass spectrometry
2009, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesDetection of nicotine based on molecularly imprinted TiO<inf>2</inf>-modified electrodes
2009, Analytica Chimica ActaCitation Excerpt :In some cases, MIPs are immobilized in a bulk polymer [18,19]. Although innumerable methods have been developed for detecting NIC in the human body and tobacco samples, the HPLC technique is still normally employed in clinical laboratories for accurate sensing [20–22]. However, this method is time-consuming and involves a number of steps.