The determination of cotinine in serum, urine and saliva is widely used as a quantitative marker of cigarette smoking/smoke intake, on account of its sensitivity and specificity [Pojer et al., Clinical Chemistry, 1984, 30, 1377-1380], particularly for research on smoking-related diseases [Hill et al., Journal of Chronic Diseases, 1983, 36, 439-449], and health risks associated with passive smoking [Jarvis et al., Thorax, 1983, 38, 829-833]. Cotinine is more sensitive than nicotine since it has a longer plasma half-life, in the region of ˜11-37 hours, compared with ˜30 minutes for nicotine.
Cotinine level monitoring can be used to provide an objective quantitative assessment of smoking status, which is a useful addition to self-reported smoking information (such as number of cigarettes smoked per day), which can be unreliable, partly due to differences in inhalation as a result of individual smoking techniques [Vogt et al., Preventive Medicine, 1979, 8, 23-33; Pettiti et al., American Journal of Public Health, 1981, 71, 308-311; Hall et al., Clinical Phamacology & Therapeutics, 1984, 35, 810-814; Lewis et al., Biomarkers, 2003, 8, 218-228; Britton et al., Journal of Obstetric, Gynecologic, & Neonatal Nursing, 2004, 33, 306-311; Studts et al., Cancer Epidemiology Biomarkers & Prevention, 2006, 15, 1825-1828; Gorber et al., Nicotine & Tobacco Research, 2009, 11, 12-24]. Cotinine is often referred to as the major nicotine metabolite, however some investigations have shown it to account for only ˜2-30% of total nicotine metabolites in urine, and total colorimetric assay of numerous nicotine breakdown products to give equivalent cotinine values ˜8 times higher than radioimmunoassay (RIA) of cotinine only levels in the same samples [Barlow et al., Clinica Chimica Acta, 1987, 165, 45-52]. Thus cotinine, although being a significant nicotine metabolite, is just one of many metabolites in the metabolic degradation sequence of nicotine (FIG. 1), and nicotine metabolite ratios can be used for prediction of cigarette consumption [Benowitz et al., Nicotine & Tobacco Research, 2003, 5, 621-624].
Methods for cotinine analysis include colorimetric methods, gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography, and radioimmunoassay (RIA). The GC and HPLC methods are not suited to either large-scale studies (such as epidemiological studies) or routine assessment of smoking status since the equipment is expensive, requires skilled staff for reliable reproducible operation and the methods are time consuming. The RIA methods are more amenable to general use but the reagents are not widely available.
Colorimetric methods are initially based upon a chemical reaction similar to that originally described by König [Journal für Praktische Chemie, 1904, 70, 19-56 & Journal für Praktische Chemie, 1904, 69, 105-137]. An example of the chemistry used in such an assay is shown in FIG. 2 using Meldrum's acid as an example.
These colorimetric methods may be used as stand-alone assays or as pre- or post-column derivatisation methods for HPLC. Chromophore-generating reagents may include barbituric acid (BA), 1,3-diethyl-2-thiobarbituric acid (DETBA), and Meldrum's acid (MA).
Cotinine equivalent measurements may use cyanide and a chromophore-generating reagent (e.g. BA, MA, DETBA) for determination of pyridine derivatives (specifically nicotine metabolites). BA is known for use with some pyridine derivatives for such colorimetric determinations of cyanide. DETBA and MA have been incorporated into near-patient/point-of-care (poc) tests for nicotine metabolites in urine or saliva to assess smoking habit [Cope et al., Annals of Clinical Biochemistry, 2000, 37, 666-673], provide biochemical feedback to improve smoking cessation interventions [Cope, Smoking Cessation: Theory, Interventions and Prevention (Landow, J. E. (ed.)), Nova Science Publishers, Inc., New York, N.Y., USA, 2008, pp. 373-383], and quantify exposure to environmental tobacco smoke [Cope et al., Annals of Clinical Biochemistry, 2000, 37, 795-796].
We now provide an improved colorimetric nicotine-metabolite (such as cotinine) assay using a pyrazolone as chromophore-generating agent. Also provided is a method of detecting and/or quantitatively measuring nicotine-metabolite concentration (such as cotinine concentration) in sample (such as a human body fluid sample) and methods which relate this concentration to smoking habit. Nicotine-metabolite assay kits also form part of the present disclosure as are near-patient/point-of-care (poc) tests for assessment of smoking habit.