Simultaneous determination of nicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine in human oral fluid using solid phase extraction and gas chromatography-mass spectrometry

Nicotine is rapidly and extensively metabolized in humans. We present an analytical method to simultaneously quantify nicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine in human oral fluid. Solid phase extraction (SPE) and GC/MS/EI with selected ion monitoring (SIM) were utilized. Linearity ranged from 5 to 1000 ng/mL of oral fluid; correlation coefficients for calibration curves were >0.99. Recoveries were 90-115% nicotine, 76-117% cotinine, 88-101% norcotinine, and 67-77% trans-3'-hydroxycotinine. Intra-assay precision and accuracy ranged from 1.6 to 5.7% and 1.6 to 17.8%, respectively. Inter-assay precision and accuracy ranged from 4.3 to 10.2% and 0 to 12.8%, respectively. Suitable precision and accuracy were achieved for the simultaneous determination of nicotine and three metabolites in the oral fluid of smokers. This assay is applicable to pharmacokinetic studies of nicotine, cotinine, and trans-3'-hydroxycotinine from tobacco smokers and can be utilized for routine monitoring of tobacco smoke exposure. 3-Hydroxycotinine requires additional investigation to determine its usefulness as a biomarker for tobacco smoke exposure.     

Changes in the salivary cotinine cut-offs to discriminate smokers and non-smokers before and after Spanish smoke-free legislation

IntroductionHigh levels of cotinine in non-smokers indicate passive exposure to tobacco smoke. This study aims to evaluate variations in salivary cotinine cut-offs to discriminate smokers and non-smokers before and after the implementation of smoke-free legislation (Law 28/2005 and Law 42/2010) in a sample of the adult population of Barcelona, Spain.MethodsThis longitudinal study analyzes salivary cotinine samples and self-reported information from a representative sample (n = 676) of the adult population from Barcelona before and after the approval of smoke-free legislation. We calculated the receiver operating characteristic (ROC) curves, to obtain optimal cotinine cut-off points to discriminate between smokers and non-smokers overall, by sex and age, and their corresponding sensitivity, specificity, and area under the curve. We used linear mixed-effects models, with individuals as random effects, to model the percentage change of cotinine concentration before and after the implementation of both laws.ResultsThe mean salivary cotinine concentration was significantly lower post-2010 law (−85.8%, p < 0.001). The ROC curves determined that the optimal cotinine cut-off points for discriminating non-smokers and smokers were 10.8 ng/mL (pre-2005 law) and 5.6 ng/mL (post-2010 law), with a post-2010 law sensitivity of 92.6%, specificity of 98.4%, and an area under the curve of 97.0%. The post-2010 law cotinine cut-off points were 5.6 ng/mL for males and 1.9 ng/mL for females.ConclusionThe implementation of Spanish smoke-free legislation was effective in reducing secondhand smoke exposure and, therefore, also in reducing the cut-off point for salivary cotinine concentration. This value should be used to better assess tobacco smoke exposure in this population.     

DNA adducts in human placenta exposed to ambient environment and passive cigarette smoke during pregnancy

BACKGROUND: The risk of human diseases and abnormal development under the relatively reduced toxic environmental exposure conditions of passive cigarette smoke and urban pollution is emerging as significant. To assess the genotoxic potential of such exposure, we analyzed the DNA adducts of polynuclear aromatic hydrocarbons (PAH), a proven marker of genotoxicity, in human placental DNA samples of pregnancies monitored for passive cigarette smoke exposure. METHODS: Maternal exposure to active and passive cigarette smoke was evaluated by verbal disclosure and urinary nicotine and cotinine measurements. PAH-DNA adducts were assayed by ELISA using a polyclonal antibody against benzo[alpha]pyrene-diol-epoxide-DNA in placental DNA. Birth weights of infants were recorded in these monitored pregnancies. RESULTS: Urinary nicotine and cotinine values were reduced in the passive smoke-exposed group compared to smokers and similar to those in the nonsmoker ambient exposure group. PAH-DNA and nicotine/cotinine values were not correlated with birth weight of the infant. PAH-DNA adducts were present in approximately 25% of samples exposed to passive cigarette smoke and ambient environment. CONCLUSIONS: The study has revealed that a subpopulation of humans is predisposed to accumulating PAH adducts independent of high levels of PAH sources (e.g., maternal cigarette smoke exposure). Because DNA adducts promote genomic changes, it is likely that this subpopulation is susceptible to diverse changes in the genome that may influence human development.     

Quantification of cotinine in dried blood spots as a biomarker of exposure to tobacco smoke

Objective: We present an ultra-sensitive, minimally-invasive method for quantifying cotinine in dried blood spot (DBS) samples as a biomarker of exposure to tobacco smoke that can be collected using a simple heel or finger prick to obtain blood samples.

Methods: Cotinine levels were measured in matched plasma and reconstituted DBS samples from smokers and nonsmokers to evaluate assay parameters. In addition, we applied this new method to finger-prick DBS samples that were collected from infants, children and young adults ages 1–21 to estimate exposure to tobacco smoke. Partitioning of cotinine across red blood cells and haematocrit effects were investigated.

Results: Cotinine levels measured in matched plasma and reconstituted DBS samples from smokers and nonsmokers were found to be highly correlated (R²=0.94), with 100% sensitivity and 94% specificity to differentiate reported smokers from nonsmokers. With this method, the LOQ is <0.25?ng/mL using a single 3.2?mm punch of a DBS, and haematocrit effects are negligible.

Conclusions: This sensitive, high-throughput and minimally-invasive method for quantifying cotinine in DBS samples provides a simple and cost effective means for estimating exposure to tobacco smoke in population based studies, and has particular advantages in studies involving infants and children.     

Estimation of urinary cotinine cut-off points distinguishing non-smokers, passive and active smokers

An objective assessment of exposure to tobacco smoke may be accomplished by means of examining particular biomarkers in body fluids. The most common biomarker of tobacco smoke exposure is urinary, or serum, cotinine. In order to distinguish non-smokers from passive smokers and passive smokers from active smokers, it is necessary to estimate cotinine cut-off points. The objective of this article was to apply statistical distribution of urinary cotinine concentration to estimate cut-off points distinguishing the three above-mentioned groups. The examined group consisted of 327 volunteers (187 women and 140 men) who were ethnically homogenous inhabitants of the same urban agglomeration (Sosnowiec, Poland). The values which enabled differentiation of the examined population into groups and subgroups were as follows: 50 µg l^-1 (differentiation of non-smokers from passive smokers), 170 µg l^-1 (to divide the group of passive smokers into two subgroups: minimally and highly exposed to environmental tobacco smoke), 550 µg l^-1 (differentiation of passive smokers from active smokers), and 2100 µg l^-1 (to divide group of active smokers into two subgroups: minimally and highly exposed to tobacco smoke). The results suggest that statistical distribution of urinary cotinine concentration is useful for estimating urinary cotinine cut-off points and for assessing the smoking status of persons exposed to tobacco smoke.     

Passive smoking,salivary cotinine concentrations,and middle ear effusion in 7 year old children.

OBJECTIVE--To assess the contribution of passive exposure to tobacco smoke to the development of middle ear underpressure and effusion. DESIGN--Cross sectional observational study. SETTING--One third of the primary schools in Edinburgh. SUBJECTS--892 Children aged 6 1/2 to 7 1/2 were examined, and satisfactory tympanograms were obtained in 872. Results of assay of salivary cotinine concentrations were available for 770 children, and satisfactory tympanograms were available for 736 of these. END POINT--Correlation of the prevalence of middle ear underpressure and effusion with concentrations of the marker of nicotine, cotinine, in the saliva of the children. MEASUREMENTS AND MAIN RESULTS--Middle ear pressure and compliance were measured in both ears by impedance tympanometry. Salivary cotinine concentrations were assayed by gas-liquid chromatography. Cotinine concentrations increased with the number of smokers in the household. Girls had higher concentrations than boys, and children living in rented housing had higher concentrations than those living in housing owned by their parents. There was a trend towards more abnormal tympanometric findings with increasing cotinine concentration, the odds ratio for a doubling of the cotinine concentration being 1.14 (95% confidence interval 1.03 to 1.27). After adjustment for the sex of the child and housing tenure the odds ratio for a doubling of the cotinine concentration was 1.13 (1.00 to 1.28). CONCLUSIONS--The results of this study are consistent with those of case-control studies of children attending for an operation to relieve middle ear effusion. They indicate that the disease should be added to the list of recognised hazards associated with passive smoking. About one third of the cases of middle ear effusion in this study were statistically attributable to exposure to tobacco smoke.     

Determination of the nicotine metabolites cotinine and trans-3'-hydroxycotinine in biologic fluids of smokers and non-smokers using liquid chromatography-tandem mass spectrometry: biomarkers for tobacco smoke exposure and for phenotyping cytochrome P450 2A6 activity

The nicotine metabolite cotinine is widely used to assess the extent of tobacco use in smokers, and secondhand smoke exposure in non-smokers. The ratio of another nicotine metabolite, trans-3'-hydroxycotinine, to cotinine in biofluids is highly correlated with the rate of nicotine metabolism, which is catalyzed mainly by cytochrome P450 2A6 (CYP2A6). Consequently, this nicotine metabolite ratio is being used to phenotype individuals for CYP2A6 activity and to individualize pharmacotherapies for tobacco addiction. In this paper we describe a highly sensitive liquid chromatography-tandem mass spectrometry method for determination of the nicotine metabolites cotinine and trans-3'-hydroxycotinine in human plasma, urine, and saliva. Lower limits of quantitation range from 0.02 to 0.1ng/mL. The extraction procedure is straightforward and suitable for large-scale studies. The method has been applied to several thousand biofluid samples for pharmacogenetic studies and for studies of exposure to low levels of secondhand smoke. Concentrations of both metabolites in urine of non-smokers with different levels of secondhand smoke exposure are presented.     

Cotinine levels and self-reported smoking status in patients attending a bronchoscopy clinic.

The reliability of self-reported smoking behaviour can vary and may result in bias if errors in misclassification vary with outcome. We examined whether self-report was an accurate measure of current smoking status in patients with malignant or non-malignant respiratory disease. Smoking behaviour was assessed by self-report and by analysis of whole blood for cotinine, a biomarker of exposure to cigarette smoke, in 166 patients attending a bronchoscopy clinic. Cotinine levels ranged from 2.5 to >400 ng ml(-1) blood and were higher in self-reported current smokers (173+/-123 ng ml(-1)) than in never smokers (3.7+/-8.7 ng ml(-1)) or ex-smokers (20.5+/-49.0 ng ml(-1)). Cotinine levels in self-reported current smokers increased with the numbers of cigarettes smoked (p=0.06), and levels in smokers and ex-smokers decreased with the reported length of time since the last cigarette (p=0.001). Using a cotinine level of 20 ng ml(-1) and self-report as the gold standard, the sensitivity and specificity for defining current smoking status were 90.2% and 82.4%, respectively. Out of a total of 125 self-reported current non-smokers, 23 (18.4%) had cotinine levels greater than 20 ng ml(-1). Smoking prevalence was significantly underestimated by self-report (24.7%) when compared with that defined using blood cotinine levels (36.1%: p<0.001). Misclassification of current smoking status was particularly high in ex-smokers, in patients without malignant respiratory disease, in men, and in those below the median age. Such differential misclassification may result in bias in studies examining associations between current smoking habits and disease risk.     

Estimation of urinary cotinine cut-off points distinguishing non-smokers,passive and active smokers

Abstract

An objective assessment of exposure to tobacco smoke may be accomplished by means of examining particular biomarkers in body fluids. The most common biomarker of tobacco smoke exposure is urinary, or serum, cotinine. In order to distinguish non-smokers from passive smokers and passive smokers from active smokers, it is necessary to estimate cotinine cut-off points. The objective of this article was to apply statistical distribution of urinary cotinine concentration to estimate cut-off points distinguishing the three above-mentioned groups. The examined group consisted of 327 volunteers (187 women and 140 men) who were ethnically homogenous inhabitants of the same urban agglomeration (Sosnowiec, Poland). The values which enabled differentiation of the examined population into groups and subgroups were as follows: 50 µg l^?1 (differentiation of non-smokers from passive smokers), 170 µg l^?1 (to divide the group of passive smokers into two subgroups: minimally and highly exposed to environmental tobacco smoke), 550 µg l^?1 (differentiation of passive smokers from active smokers), and 2100 µg l^?1 (to divide group of active smokers into two subgroups: minimally and highly exposed to tobacco smoke). The results suggest that statistical distribution of urinary cotinine concentration is useful for estimating urinary cotinine cut-off points and for assessing the smoking status of persons exposed to tobacco smoke.     

Cotinine levels and self-reported smoking status in patients attending a bronchoscopy clinic

The reliability of self-reported smoking behaviour can vary and may result in bias if errors in misclassification vary with outcome. We examined whether self-report was an accurate measure of current smoking status in patients with malignant or non-malignant respiratory disease. Smoking behaviour was assessed by self-report and by analysis of whole blood for cotinine, a biomarker of exposure to cigarette smoke, in 166 patients attending a bronchoscopy clinic. Cotinine levels ranged from 2.5 to &gt;400 ng ml^?1 blood and were higher in self-reported current smokers (173±123 ng ml^?1) than in never smokers (3.7±8.7 ng ml^?1) or ex-smokers (20.5±49.0 ng ml^?1). Cotinine levels in self-reported current smokers increased with the numbers of cigarettes smoked (p=0.06), and levels in smokers and ex-smokers decreased with the reported length of time since the last cigarette (p=0.001). Using a cotinine level of 20 ng ml^?1 and self-report as the gold standard, the sensitivity and specificity for defining current smoking status were 90.2% and 82.4%, respectively. Out of a total of 125 self-reported current non-smokers, 23 (18.4%) had cotinine levels greater than 20 ng ml^?1. Smoking prevalence was significantly underestimated by self-report (24.7%) when compared with that defined using blood cotinine levels (36.1%: p&lt;0.001). Misclassification of current smoking status was particularly high in ex-smokers, in patients without malignant respiratory disease, in men, and in those below the median age. Such differential misclassification may result in bias in studies examining associations between current smoking habits and disease risk.     

Gas chromatographic analysis of nicotine and cotinine in hair

Non-invasive validation of cigarette- or cigar-smoking behaviour is necessary for large population studies. Urine or saliva samples can be used for confirmation of recent nicotine intake by analysis of cotinine, the major metabolite of nicotine. However, this test is not suitable for validation of survey data, since the quantification of cotinine in saliva only reflects nicotine exposure during the preceding week. To validate information on tobacco use, we investigated hair samples for quantifying nicotine and cotinine by gas chromatography—mass spectrometry. Hair (about 50–100 mg) was incubated in 1 M sodium hydroxide at 100°C for 10 min. After cooling, samples were extracted by diethyl ether, using ketamine as an internal standard. Drugs were separated on a 12-m BP-5 capillary column, and detected using selected-ion monitoring (m/z 84, 98 and 180 for nicotine, cotinine and ketamine, respectively). Hair from non-smokers and smokers contained nicotine and cotinine. Although it is difficult to determine an absolute cut-off concentration, more than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from non-smokers. Some applications of this technique are developed to determine the status of passive smokers, the gestational exposure in babies and the pattern of an individual's nicotine use by cutting strands of hair into sections of one-month intervals.     

Simultaneous estimation of nicotine and cotinine levels in biological fluids using high-resolution capillary-column gas chromatography combined with solid phase extraction work-up

A rapid and sensitive capillary gas-chromatographic method with nitrogen-sensitive detection is reported for the simultaneous analysis of nicotine and cotinine levels occurring in the plasma, saliva, and urine of regular tobacco smokers. The proposed assay has a linear output, has satisfactory accuracy over the range of concentrations of both amines encountered in active smokers, and has also been successful in the analysis of the urine samples of passive smokers. Its lower limit of sensitivity is 0.2 ng of nicotine and 0.5 ng of cotinine per ml of plasma or saliva or per 100 l of urine.The beneficial characteristics of the presented method were achieved by the combination of solid phase extraction of 0.1–1.0 ml of fluid specimens, capillary column gas chromatography with splitless injection and nitrogen sensitive detection, and the use of separate, structurally analogous compounds as internal standards for nicotine. The suitability of the assay is shown by plasma concentration-time curves of nicotine and cotinine in a steady smoker during a 24 hours period.     

Simultaneous determination of nicotine and eight nicotine metabolites in urine of smokers using liquid chromatography-tandem mass spectrometry

A method based on liquid chromatography tandem mass spectrometry (LC-MSMS) applying atmospheric pressure chemical ionisation (APCI) in the positive ion mode was developed for the direct determination of nicotine, cotinine, trans-3'-hydroxycotinine, their corresponding glucuronide conjugates as well as cotinine-N-oxide, norcotinine, and nicotine-N'-oxide in the urine of smokers. The assay involves filtration of crude urine, fast liquid chromatography on a reversed-phase column and mass-specific detection using MSMS transitions. Deuterium-labeled nicotine, cotinine, and trans-3'-hydroxycotinine were used as internal standards. Glucuronides used as reference material were either chemically (cotinine-N-glucuronide) or enzymatically synthesized (nicotine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide). Precision for the major nicotine analytes at levels observable in urine of smokers was better than 10%. Accuracy expressed in recovery rates in urine matrix for nicotine, cotinine, trans-3'-hydroxycotinine, and cotinine-N-glucuronide ranged from 87 to 113%. Quantitative results for the three glucuronides in urine samples of 15 smokers were compared to an indirect method in which the aglycons were determined with gas chromatography and nitrogen-selective detection (GC-NPD) before and after enzymatic splitting of the conjugates. Good agreement was found for cotinine-N-glucuronide (coefficient of variation, CV: 9%) and trans-3'-hydroxycotinine-O-glucuronide (CV: 20%), whereas the accordance between both methods was moderate for nicotine-N-glucuronide (CV: 33%). The described LC-MSMS method allows the simultaneous determination of nicotine and eight of its major metabolites in urine of smokers with good precision and accuracy. Since the method requires a minimum of sample clean-up and a very short time for chromatography (3 min), it is suitable for determining the nicotine dose in large-scale human biomonitoring studies.     

The effects of nicotine, cotinine and anabasine on rat adrenal 11 beta-hydroxylase and 21-hydroxylase

The effects of nicotine, cotinine and anabasine on rat adrenal steroidogenesis were examined by spectral and enzymatic techniques. The addition of nicotine, cotinine or anabasine to preparations of rat adrenal mitochondria produced type II cytochrome P-450 difference spectra. The addition of nicotine or anabasine, but not cotinine, to rat adrenal microsomes yielded type II cytochrome P-450 difference spectra. Nicotine and anabasine competitively inhibited rat adrenal mitochondrial 11 beta-hydroxylase and microsomal 21-hydroxylase. Cotinine competitively inhibited mitochondrial 11 beta-hydroxylase, but did not inhibit microsomal 21-hydroxylase. The apparent enzymatic inhibition constants for cotinine, nicotine, anabasine and metyrapone inhibition of the mitochondrial 11 beta-hydroxylase were 32, 96, 120 and 74 microM respectively. These studies suggest that components of cigarette smoke may alter patterns of adrenal steroidogenesis.     

A Rat Model of Cigarette Smoke Abuse Liability

We sought to develop a rat model of cigarette smoke exposure (CSE) that created cotinine serum levels comparable to those of smokers and induced conditioned place preference (CPP) suggestive of cigarette smoke abuse liability. Rats were exposed to sidestream cigarette smoke delivered semicontinuously for 2 periods of 20 (group S20), 40 (group S40), or 60 (group S60) min daily for 12 wk. Serum cotinine concentration in blood samples was determined at 1 and 20 h after CSE. A biased (black versus white chamber) CPP paradigm was used. In the high CSE group (group S60), serum cotinine at 1 h (250 to 300 ng/mL) was comparable to average cotinine levels reported for addicted smokers (around 300 ng/mL). Cotinine levels at 20 h after CSE were higher than the smoker–nonsmoker cut-off value (greater than 14 ng/mL) in all smoking groups, with the S60 group having the highest levels. All rats preferred the black chamber to the white chamber during the preexposure CPP test. The time spent in the white chamber was increased compared with 0-wk values in group S40 at 8 wk, group S60 at 4 and 8 wk, and the control group at 4 and 8 wk but not at 12 wk; however, the shift in CPP was significantly higher at 8 wk in group S60 compared with other groups. In conclusion, interrupted 2-h daily CSE for 8 wk induced serum cotinine levels in rats comparable to those of smokers and induced CPP suggestive of cigarette smoke abuse liability.Abbreviations: CPP, conditioned place preference; CSE, cigarette smoke exposureThe devastating consequences of smoking on health have been studied extensively in numerous clinical and animal studies over time. This chronic habit leads to dependence on tobacco smoke, with nicotine, a main active ingredient of tobacco products, being recognized as the basic addictive substance.³²The known health benefits of smoking cessation motivate smokers to quit tobacco use. However, unaided efforts usually are unsuccessful, resulting in smoking relapse. The fight against nicotine addiction may be undermined by potential weight gain after smoking cessation, potentially discouraging those attempting to quit smoking and contributing to relapse. During the past few years, research has been focused on 2 main areas of interest toward this direction: understanding the underlying biologic mechanisms related to nicotine addiction to effectively design therapeutic strategies to support those who wish to quit smoking and investigating the hormonal and molecular mechanisms responsible for weight gain after smoking cessation.So far, animal models used to study the consequences of smoking cessation involved the administration of nicotine as a sole agent until addiction was achieved.²³ However, nicotine-administration models do not completely represent the toxic and addictive effects of cigarette smoke, given that smoke contains more than 4000 chemicals whose actions or coactions have not been thoroughly evaluated yet.¹ Cigarette smoke exposure (CSE) animal models have been used in studies investigating the metabolic changes conferred by smoking^10-12 but not in those after its cessation. In toxicity studies, animals are exposed to tobacco smoke for various periods, which depend on the side effect under investigation.^18,25,27 Smoke exposure timetables usually do not involve weekends for practical reasons, and addiction of animals to tobacco smoke is not assessed in current models.In our opinion, an ideal animal model of cigarette smoke abuse liability suitable for the study of smoking cessation resembles the clinical situation in terms of chronic daily inhalation of cigarette smoke sufficient to attain blood nicotine levels comparable to those of smokers and in cessation of the CSE period after achieving tobacco smoke abuse liability. In the present project, we sought to establish such a model in rats by defining the daily timetable of CSE to induce serum levels of cotinine, nicotine''s major proximate metabolite, comparable to those of smokers and by determining the minimum total CSE period required to induce abuse liability to cigarette smoke. We assessed the CSE period by using a biased conditioned place preference (CPP) paradigm.⁸     

Determination of urinary and salivary cotinine using gas and liquid chromatography and enzyme-linked immunosorbent assay

The objective of this study was to compare cotinine concentrations in urine and saliva using gas chromatography (GC), high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). Ninety-four subjects were selected (27 smokers and 67 non-smokers) and interviewed using questionnaire. Of the non-smokers, 39 had been exposed to environmental tobacco smoke (ETS) and 28 had not been exposed to ETS. Cotinine levels among smokers were highest using all three measurements, followed by ETS exposed subjects and non-smokers. Cotinine levels in urine, using HPLC, correlated significantly with levels measured using ELISA (r=0.92) and GC-nitrogen-phosphorus detection (NPD) (r=0.92). Salivary cotinine levels measured using ELISA did not correlate significantly with either HPLC (r=0.37) or GC-NPD (r=0.33) measurements. Multiple regression models were used to adjust for age, gender, drug use and health status, and it was found that cotinine levels in urine and saliva were significantly correlated with smoking pack-year. The authors conclude that urinary cotinine concentration is a more accurate biomarker for ETS than salivary cotinine concentration.     

Analysis of [3',3'-d(2)]-nicotine and [3',3'-d(2)]-cotinine by capillary liquid chromatography-electrospray tandem mass spectrometry

A selective and sensitive LC/MS/MS assay was developed for the quantification of d(2)-nicotine and d(2)-cotinine in plasma of current and past smokers administered d(2)-nicotine. After solid phase extraction and liquid-liquid extraction, HPLC separation was achieved on a capillary hydrophilic interaction chromatography phase column. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated for d(2)-nicotine (0.03-6.0 ng/ml plasma) and d(2)-cotinine (0.15-25 ng/ml plasma). The lower limits of quantitation were 0.15 ng/ml and 0.25 ng/ml for d(2)-nicotine and d(2)-cotinine, respectively. The coefficient of variation was 3.7% for d(2)-nicotine and 2.5% for d(2)-cotinine. The method was applied to two ongoing studies of d(2)-nicotine metabolism in prior and current smokers. Preliminary analysis of a subset of subjects from these studies detected a significantly lower rate of nicotine conversion to cotinine by past smokers compared to current smokers.     

Deterioration of semen quality and sperm-DNA integrity as influenced by cigarette smoking in fertile and infertile human male smokers—A prospective study

In modernized lifestyle smoking is one of the trendy, psychological, and socioeconomic scenarios of young adolescents mainly in the age of the reproductive stage. Based on a number of cigarettes smoked, age, and duration of the smoke, the study aims to search for the profound effects of smoking and its impact on semen parameters, sperm-DNA integrity, and fragmentation of sperm DNA with cotinine and apoptotic caspase-3 marker in the seminal plasma of fertile and infertile smokers. To determine oxidative damage by 8-hydroxy deoxyguanosine (8-OHdG) from isolated sperm DNA (steps: reactive oxygen species washing by nitro blue tetrazolium (NBT), sperm lysis, salt digestion, ethanol washing, and finally with high-performance liquid chromatography analysis). Level of DNA fragmentation (percentage) in native and intact DNA, the activity of caspase-3 in infertile smokers will be compared with the control group of nonsmokers. Also, the sperm viability was visualized by eosin-nigrosin and aniline blue staining. Cotinine is one of the best markers of smoking. The cotinine level (2224.24 ± 1.19 *** ng/mL), when abundant it negative correlates with morphology and rapid motility in infertile smokers than nonsmokers. Gel preprogram measured the sperm integrity and was found to be less in smokers than nonsmokers. The spermatic oxidative marker 8-OHdG was high and gave an R ² value of 0.9104 with morphology and 0.9007 for rapid motility of infertile sperm, respectively. Infertile smoking subjects (<10 cigarettes/day) had significant changes increase in sperm fragmentation, caspase-3, and cotinine while negative impact with motility, morphology, and pH of semen compared with fertile, infertile nonsmoking subjects.     

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 mM 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 C₁₈ 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.     

A novel validated procedure for the determination of nicotine,eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry

A novel validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-β-d-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1′-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-β-d-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC–MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R²) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged between 52–88% in plasma and 51–118% in urine. The limits of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL, respectively, with the exception of cotinine-N-β-d-glucuronide, which was 50 ng/mL. Intra-day and inter-day imprecision were ≤14% and ≤17%, respectively. Matrix effect (%) was sufficiently minimized to ≤19% for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze–thaw cycles, 24 h at room temperature, 24 h in the refrigerator (4 °C) and 1 week in the freezer (?20 °C). Reconstituted plasma and urine extracts were stable for at least 72 h storage in the liquid chromatography autosampler at 4 °C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1′-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrollment) and on the pharmacokinetic study day.