Determination Of Cotinine Research Articles

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

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.

Determination of nicotine, cotinine and caffeine in meconium using high-performance liquid chromatography

A high-performance liquid chromatographic method with diode-array detection for the determination of nicotine and its metabolites, cotinine and caffeine, in meconium is described. This method is suitable to assess foetus exposure to tobacco smoke. The analytes were extracted by solid-phase extraction before chromatography. From among 30 meconium samples 11 were positive for cotinine (20–86 ng/g) and 27 for caffeine (10–45 ng/g). No nicotine was present in the samples because of its rapid metabolism into cotinine.

Development and validation of sensitive method for determination of serum cotinine in smokers and nonsmokers by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry

We describe a sensitive and specific method for measuring cotinine in serum by HPLC coupled to an atmospheric pressure chemical ionization tandem mass spectrometer. This method can analyze 100 samples/day on a routine basis, and its limit of detection of 50 ng/L makes it applicable to the analysis of samples from nonsmokers potentially exposed to environmental tobacco smoke. Analytical accuracy has been demonstrated from the analysis of NIST cotinine standards and from comparative analyses by both the current method and gas chromatography/high-resolution mass spectrometry. Precision has been examined through the repetitive analysis of a series of bench and blind QC materials. This method has been applied to the analysis of cotinine in serum samples collected as part of the Third National Health and Nutrition Examination Survey (NHANES III).

Detection of benzo[a]pyrene-DNA adducts in human placenta and umbilical cord blood.

Placenta constitutes a vital organ of exchange between mother and foetus. In addition to this favourable effect for foetal development, placenta indirectly may allow transfer of several maternal blood xenobiotics. Human placenta and umbilical cord blood are interesting models for investigating maternal environment and the metabolism, the bioactivation and the transfer of carcinogens such as polycyclic aromatic hydrocarbons. We used them to assess the effect of a woman's smoking on the foetus. Few studies cover this subject. In pregnant women who have continued to smoke, benzo[a]pyrene compound of cigarette smoke is metabolically activated to diol-epoxide derivative: benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide, ultimate carcinogen (BPDE-I). This derivative is covalently fixed on DNA and gives BPDE-I-DNA adducts. By a competitive immunoassay technique, we determined BDPE-I-DNA adducts in 20 samples of placenta and umbilical cord blood from women who smoked (n = 15) and who did not (n = 10). Tobacco consumption was checked by urinary cotinine determination. In the group of smokers levels of adducts were found in 13 specimens of placenta (from 10 to 60 fmol/50 micrograms of DNA) and 12 umbilical cord blood (from 10 to 22.15 fmol/50 micrograms of DNA) samples. These results indicate that a mother's tobacco consumption is linked to the accumulation of BPDE-I-DNA adducts in the placenta, which are seen in smaller quantities in the umbilical cord blood, probably because of the metabolic capacity of the placenta and the transfer of B[a]P from the mother to the foetus.

Smoking Cessation in Hospitalized Patients

Few research studies have evaluated the effectiveness of smoking interventions in hospitalized patients. This randomized controlled trial compared the efficacy of 2 smoking cessation programs in patients hospitalized in 4 community hospitals in a large health maintenance organization within the San Francisco Bay Area in California. Patients were randomly assigned to usual care (n = 990), nurse-mediated, behaviorally oriented inpatient counseling focused on relapse prevention with 1 postdischarge telephone contact (minimal intervention, n = 473), or the same inpatient counseling with 4 postdischarge telephone contacts (intensive intervention, n = 561). The main outcome measure, smoking cessation rate, was corroborated by plasma cotinine determination or family confirmation, 1 year after enrollment. At 1 year smoking cessation rates were 27%, 22%, and 20% for intensive intervention, minimal intervention, and usual care groups, respectively (P = .009 for intensive vs usual care). Subgroup analyses by diagnosis revealed that the odds of cessation among patients with cardiovascular disease or other internal medical conditions were greater among those receiving the intensive intervention than among their counterparts receiving usual care (odds ratios, 1.6 and 2.0, respectively). A multicomponent smoking cessation program consisting of physician advice; in-hospital, nurse-mediated counseling; and multiple postdischarge telephone contacts was effective in increasing smoking cessation rates among hospitalized smokers. Hospital-wide smoking cessation programs could substantially increase the effectiveness of hospital smoking bans.

Interference of Nicotine Metabolites in Cotinine Determination by RIA

Cotinine (COT), a major metabolite of nicotine (NIC), has been used as a biomarker in many studies of active and passive smoking (1)(2)(3)(4). Methods of analysis for COT in biological fluids include gas chromatography, gas chromatography–mass spectrometry, HPLC, HPLC–mass spectrometry, and immunoassays (5)(6)(7)(8)(9)(10). In particular, the RIA developed for COT and NIC by Langone and Van Vunakis (11) was the method reported by the International Agency for Research on Cancer (IARC) for determination of these analytes in several biological matrices (12)(13). In the description of the assay (11), no mention was made about cross-reactivity of anti-NIC and anti-COT antibodies with trans -3′-hydroxycotinine (THOC) and cotinine glucuronide (COT-G). Indeed, these two compounds were found to be the most abundant metabolites of NIC in urine of smokers (14). Other studies, however, have investigated the cross-reactivity of the anti-COT antiserum with THOC and other metabolites in an ELISA (15). The objective of this study was to investigate the cross-reactivity of the anti-COT and anti-NIC antisera with THOC and COT-G in the RIA (11)(12)(13). We also used urine samples from active and passive smokers to compare data from the RIA with data obtained by HPLC. The rabbit antiserum, (−)[3H]COT, and (−)[3H]NIC were …

Determination of nicotine and cotinine in human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical ionization interface

Here we report a sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method capable of quantifying nicotine down to 1 ng/ml and cotinine to 10 ng/ml from 1.0 ml of human plasma. The method was validated over linear ranges of 1.0–50.0 ng/ml for nicotine and 10.0–500.0 ng/ml for cotinine, using deuterated internal standards. Compounds were simply extracted from alkalinized human heparinized plasma with methylene chloride, reconstituted into a solution of acetonitrile, methanol and 10 m M ammonium acetate (53:32:15, v/v) after the organic phase was dried down, and analyzed on the LC-MS-MS, which is a PE Sciex API III system equipped with a Keystone BDS Hypersil C 18 column and atmospheric pressure chemical ionization (APCI) interface. The between-run precision and accuracy of the calibration standards were ≤6.42% relative standard deviation (R.S.D.) and ≤11.8%n relative error (R.E.) for both nicotine and cotinine. The between-run and within-run precision and accuracy of quality controls. (2.5, 15.0, 37.5 ng/ml for nicotine and 25.0, 150.0, 375.0 ng/ml for cotinine), were ≤6.34% R.S.D. and ≤7.62% R.E. for both analytes. Sample stabilities in chromatography, in processing and in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of nicotine and cotinine in human plasma.

Determination of volatile organic compounds and respirable suspended particulate matter in New Jersey and Pennsylvania homes and workplaces

One hundred-four self-reported nonsmoking married women participated in a home and workplace personal environmental tobacco smoke (ETS) exposure study for 33 volatile organic compounds (VOCs), total volatile organic compounds (TVOCs), respirable suspended particulate matter (RSP), and ETS-RSP. The women were selected and classified according to socioeconomic categories based on age (25–39 y and 40+ y), total annual household income (<$40K and >$40K), and reported ETS exposure status at home and at work (SH = smoking home, NSH = nonsmoking home, SW = smoking work, and NSW = nonsmoking work). Saliva samples were collected at the start and at the end of the study for cotinine determinations. Five participants (4.8% of the total), married to smokers and working in smoking workplaces, were excluded because they had average salivary cotinine concentrations greater than 10 ng/mL indicating that they were likely smokers. The background correction factor for cotinine (SH/NSH) or Z, indicated that total exposure was 4.8 times greater for those living with a smoker versus those not living with a smoker. Apportionment of TVOCs indicated that 3.4% of the TVOCs in the smoking homes and 0.8% of the TVOCs in the smoking workplaces were attributable to ETS. Apportionment of benzene and styrene indicated that 11.4% and 13.4%, respectively, were attributable to ETS in smoking homes; 11.5% and 6.2%, respectively, were attributable to ETS in smoking workplaces. RSP apportionment based on solanesol particulate matter (Sol-PM) indicated that 28.7% of the RSP in smoking homes and 22.7% of the RSP in smoking workplaces were attributable to ETS. RSP apportionment based on scopoletin particulate matter (Sco-PM) indicated that 12.9% of the RSP in smoking homes and 9.6% of the RSP in smoking workplaces were attributable to ETS. Median daily and weekly exposures to VOCs, TVOCs, and RSP were calculated from the concentrations determined and tended to follow the trend: SH > NSH > SW > NSW. The home/work exposure differential (SH/SW) indicated that ETS exposure was higher for living with a smoker than for working with a smoker by a factor of 3.7 for RSP and ETS-RSP and 2.4 for VOCs and TVOCs.

Simultaneous Determination of Nicotine and Cotinine in Untreated Human Urine by Micellar Liquid Chromatography

A simple, precise, accurate and sensitive method for the simultaneous determination of nicotine and cotinine by direct injection of human urine was developed and validated. This method utilizes nontoxic, nonflammable and relatively inexpensive sodium dodecylsulphate (SDS) as a mobile phase and eliminates the need for pretreating the urine. Optimum conditions for the separation of cotinine and nicotine, the column efficiency, and the elution of non-alkaloid components in urine were investigated. Nicotine and cotinine in untreated human urine were separated and quantitatively eluted using 0.20 M SDS and 3% 2-propanol at a pH of 4.60 and column temperature at 40 o C. Retention times for nicotine and cotinine were: 10. 85 and 9.48 minutes respectively, and all components in the urine sample were eluted within 15 minutes. This method may be suitable for use in hospitals, research centers, or in a physicina's office

Simultaneous determination of nicotine and cotinine in various human tissues using capillary gas chromatography/mass spectrometry.

A reliable and sensitive method for the simultaneous determination of nicotine and cotinine concentrations in various human tissues was developed using capillary gas chromatography/mass spectrometry. Nicotine and cotinine were extracted using a 3-step solvent extraction procedure and quinoline as an internal standard. Quantification was carried out by single ion monitoring using ions of m/z 133 for nicotine, m/z 176 for cotinine and m/z 129 for quinoline. The lower limit of detection was 5 ng/g for nicotine and 10 ng/g for cotinine, in each tissue sample. The calibration curves of various tissues were linear in the concentration range from 5-1,200 ng/g for nicotine and 10-1,500 ng/g for cotinine. The accuracy and precision of this method were examined using human tissues and the results were satisfactory. The distribution of nicotine and cotinine was measured in tissues from 10 human autopsies. Nicotine was detected in every tissue examined at a level seen in habitual smokers. The nicotine concentration was high in the liver, kidney, spleen and lung, and low in adipose tissue. The cotinine level was highest in the liver. The tissue/blood concentration ratios of nicotine and cotinine were most stable in skeletal muscle, where the level of these drugs was close to that in whole blood. Skeletal muscle is, therefore, considered to be the most suitable tissue sample for toxicological examination, when acquisition of blood samples is not feasible.

Plasma Cotinine: Stability in Smokers and Validation of Self-Reported Smoke Exposure in Nonsmokers

The stability of plasma cotinine was studied in smokers and the validity of a brief questionnaire on environmental tobacco smoke (ETS) exposure was assessed in nonsmokers. The correlation between two cotinine levels measured 14 weeks apart was 0.81 in smokers ( n = 148). A single measurement will classify 95% of all subjects within 30% of their habitual plasma cotinine level. Plasma cotinine was higher in ETS-exposed nonsmokers ( n = 50) than in ETS-unexposed nonsmokers ( n = 55; 1.6 ng/ml vs 0.6 ng/ml; P < 0.0001), but there was a substantial part of intersubject overlap. The sensitivity and specificity with respect to self-reported smoke exposure were 56 and 89%, respectively. This study shows that a single plasma cotinine determination gives a good impression of cotinine levels. It is dubious if a brief questionnaire about hours of passive smoke exposure in nonsmokers is valid to distinguish ETS-exposed from ETS-unexposed nonsmokers.

Comparison of a mechanized version of the ‘König’ reaction and a fluorescence polarization immunoassay for the determination of nicotine metabolites in urine

Smoking can be detected by the determination of cotinine in urine. We compared the performance of an automated modification of the ‘König’ reaction adapted to a centrifugal analyzer with an automated commercial fluorescence polarization immunoassay (TDX system). In the latter assay, cotinine, as the primary metabolite of nicotine, can be measured with high specificity. In contrast, the ‘König’ reaction also detects nicotine metabolites other than cotinine by a group colour reaction. Analysis speed of the ‘König’ reaction was about 66 samples/h with a detection limit 2 S.D. above the mean value of urine samples of non-smokers. Analysis speed of the TDX system was 41 samples/h. The coefficient of variation (C.V.) of both methods in smokers' urine was 8.6% (‘König’ reaction) vs 3.4% (TDX system) in the high range and 16.4% vs 9.5% in the low range. In a controlled, prospective study recruiting 86 cigarette-smoking volunteers, 83.7% were correctly classified as being smokers by both systems, 13.9% were classified as smokers by the ‘König’ reaction only and 2.4% were misclassified as non-smokers by both systems. Thus, the sensitivity of the ‘König’ reaction seems to be higher than in the TDX system (97.6% vs. 83.7%). Of 33 non-smoking individuals, 81.8% were correctly classified as non-smokers by both system, 18.2 were misclassified as smokers by the ‘König’ reaction and no person was misclassified by the fluorescence polarization immunoassay. Thus, the specificity of the TDX system seems to be higher than that of the ‘König’ reaction (100% vs. 81.8%). We conclude that both systems are applicable to detect individuals who smoke regularly by simple urine testing. The higher specificity of the TDX system is outweighted by the sensitivy of the ‘König’ reaction at much lower cost.

Simple selected ion monitoring capillary gas chromatographic—mass spectrometric method for the determination of cotinine in serum, urine and oral samples

A capillary gas chromatographic—mass spectrometric method using selected ion monitoring was developed for the analysis of cotinine in urine, serum and oral samples. The procedure requires 500 μl of an oral sample, 250 μl of a serum sample and 50 μl of urine and can detect 5 ng/ml cotinine in oral samples, 10 ng/ml in serum and 50 ng/ml in urine with good precision and accuracy. The method was used to determine the cotinine concentration in samples of all three fluids collected from a group of smokers and non-smokers.

A Double-blind Trial of a Nicotine Inhaler for Smoking Cessation

To evaluate the efficacy of a new nicotine inhaler system for smoking cessation. A 1-year, randomized, double-blind, placebo-controlled study. Medical outpatient clinic with physicians experienced in smoking cessation assistance. A total of 286 volunteers who smoked at least 10 cigarettes daily recruited through a local newspaper. Subjects were randomly allocated to nicotine inhalers (n = 145) or placebo (n = 141) to be used for 3 months followed by tapering for 3 months in the context of minimal levels of advice and support. Continuous smoking abstinence at weeks 6, 12, 24, and 52, verified by measurements of carbon monoxide in expired air. Continuous smoking abstinence was significantly higher for the active nicotine inhaler group compared with the placebo inhaler group. The respective success rates were 28% and 12% after 6 weeks, 21% and 9% after 12 weeks, 17% and 8% after 6 months, and 15% and 5% after 1 year (P = .02 to .001). The mean nicotine substitution based on cotinine determinations after 2 weeks was 43% (SD, 45%) of smoking levels. The treatment was well tolerated, and no serious adverse events were reported. In this setting the nicotine inhaler appeared safe to use and increased success rates of smoking cessation attempts.

Simultaneous determination of cotinine and creatinine by high performance liquid chromatography.

Simultaneous determination of cotinine and creatinine by high performance liquid chromatography.

Simultaneous determination of cotinine and trans-3′-hydroxycotinine in human serum by high-performance liquid chromatography

A high-performance liquid chromatographic method with ultraviolet photometric detection has been developed for the quantitation of cotinine and trans-3′-hydroxycotinine in human serum. A solid-phase extraction procedure was performed for the analytes and the internal standard, N-ethylnorcotinine, before chromatography. The use of a 30-cm reversed-phase column and a mobile phase of water—methanol—0.1 M sodium acetate—acetonitrile (67:24.5:6.5:2, v/v), pH 4.3, prevented the co-elution of caffeine with cotinine. The limit of quantitation observed with this method was 5 ng/ml for both cotinine and trans-3′-hydroxycotinine. The present method proved useful for the determination of serum levels of these metabolites, correlating with nicotine daily intake.

Elimination of caffeine interference in high-performance liquid chromatographic determination of cotinine in human plasma

A high-performance liquid chromatographic method with ultraviolet photometric detection for the determination of cotinine in human plasma was described. The use of a 30-cm reversed-phase column and of a mobile phase consisting of water—methanol—0.1 M sodium acetate—acetonitrile (72:21:5.6:1.4, v/v), pH 4.1, eliminated caffeine interference. A simplified solid-phase extraction procedure was also performed for plasma samples.

Liquid-chromatographic determination of nicotine and cotinine in urine from passive smokers: comparison with gas chromatography with a nitrogen-specific detector

We describe a simple, sensitive, and specific high-performance liquid-chromatographic method with ultraviolet detection (256 nm) for the simultaneous analysis of nicotine and cotinine in urine of passive smokers. The analytes are extracted and purified from the complex and impure matrix in two stages; first, by liquid-liquid extraction and followed by solid-phase extraction (C2 column). We used a "DB" C8 5-microns-particle column (25 x 0.46 cm) and a mobile phase of phosphate-citrate buffer and acetonitrile (91:9 by vol) containing 5 mL of triethylamine and 600 mg of heptanesulfonate per liter, adjusted to pH 4.4, to separate the compounds. Two internal standards (2-phenylimidazole and N-ethylnorcotinine) were used. The detection limit of the HPLC assay was less than 1 microgram/L for both analytes. The average interassay CV for nicotine was 7.6%, for cotinine 6.5%, in the concentration range 0-60 micrograms/L. The mean analytical recovery of nicotine with respect to the internal standard N-ethylnorcotinine was 102% and that for cotinine was 99%; the mean absolute recoveries of the compounds were as follows: nicotine 85%, cotinine 87%, and N-ethylnorcotinine 87%. To compare the HPLC assay results of 20 samples with the more-sensitive in-laboratory gas-chromatographic method with a nitrogen-phosphorus detector, we used both N-ethylnornicotine and N-ethylnorcotinine as internal standards. The mean correlation coefficient for nicotine values between the two methods was 0.934; for cotinine, 0.987.

Selected ion monitoring method for determination of nicotine, cotinine and deuterium-labeled analogs: Absence of an isotope effect in the clearance of (S)-nicotine-3′,3′-d2 in humans

A method for simultaneous determination of nicotine, its metabolite cotinine, and the stable isotope-labeled analogs nicotine-3',3'-d2 and cotinine-4',4'-d2 in human plasma has been developed. The method utilizes capillary column gas chromatography with detection by electron impact mass spectrometry and selected ion monitoring. Sensitivity is adequate for determination of nicotine and nicotine-d2 at concentrations as low as 1 ng ml-1, and cotinine and cotinine-d2 at concentrations as low as 10 ng ml-1 with good precision and accuracy. The method has been used to compare the elimination kinetics of (S)-nicotine-3',3'-d2 with natural nicotine in human subjects. Total clearance of nicotine-3',3'-d2 was virtually identical to the total clearance of natural nicotine, which validates the use of the deuterium-labeled analog in quantitative studies of nicotine metabolic disposition.

Highly Sensitive Immuno-Assays for the Determination of Cotinine in Serum and Saliva. Comparison Between RIA and an Avidin-Biotin ELISA

Two immuno-assay methods (RIA and ELISA) have been developed for the accurate and sensitive measurement of cotinine in human body fluids (serum, saliva). RIA uses [3H]cotinine as antigen and charcoal/dextran for separating cotinine-bound antibodies from the free derivative. Another technique (ELISA) was developed to avoid the use of radio-labelled compounds and to determine cotinine in large populations, including passive or non-smokers who usually present very low concentrations. The two techniques were analytically validated. The detection limit was similar (0.1 micrograms/l) and the precision was better than 10% for both techniques. Non-smoker values ranged from 0.1 to 17 micrograms/l by ELISA and 0.1 to 27.5 micrograms/l by RIA, whereas smoker values ranged from 50 to 1000 micrograms/l (ELISA) and from 70 to 800 micrograms/l (RIA). The comparative analysis of cotinine in 96 human sera revealed a good correlation between the two methods (r = 0.97) and a reliable discrimination between the populations of non-smokers and smokers. As usual, the ELISA is more rapid (4 h 30 min) than the RIA (longer than 48 h). ELISA is proposed for use in the epidemiological investigation of the human tobacco risk.