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The ultra-trace determination of nicotine and its 4 major metabolites (cotinine, nornicotine, norcotinine and anabasine) from rabbit plasma was achieved by a newly developed solid phase microextraction-liquid chromatography-tandem mass spectrometry method. Extraction of the target analytes was performed with hydrophilic/lipophilic balance-polyacrylonitrile SPME fibers. Dual fiber extraction was necessary to guarantee improved recovery at parts-per-trillion levels. Liquid chromatographic analysis was achieved in a 6-min run using a C18 (1.9 µm C18, 50 mm x 2.1 mm) column with a mobile phase flow rate of 0.4 mL/min. Tandem mass spectrometry was used for detection and quantification in positive electrospray ionization (ESI+) mode for all the targeted analytes. Two stable isotope-labeled internal standards were used for signal correction and accurate quantification. The mass spectrometer with laminar flow ion flux transport, guaranteed improved signal stability, minimal contamination of the ion guide and reproducibility into the first quadrupole analyzer. The method was validated in line with the Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The results met the acceptance criteria as proposed by the FDA: accuracy was tested at 0.35, 10 and 75 µg L  -   1 and ranged between 98.3-112.2% for nicotine, 94.1-101.9% for cotinine, 94.7-107.0% for nornicotine, 81.1-107.2% for norcotinine and 94.3-115.2% for anabasine, with precision up to 14.2%. Stability tests indicated that all the targeted analytes were stable in the desorption solution for at least 1 week. LOQs ranged from 0.05 to 1 µg L -1 . The method was successfully applied to analyze plasma samples obtained from rabbits following transdermal application of a smoking cessation formulation loaded with solid lipid nanoparticles containing a nicotine-stearic acid conjugate., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

A simple, effective, convenient and environmentally friendly methodology using high throughput bar adsorptive microextraction (HT-BAμE) with microliquid desorption in combination with large volume injection-gas chromatography-mass spectrometry operating in the selected-ion monitoring acquisition mode (LVI-GC-MS(SIM)) was applied for the determination of nicotine and cotinine in urine samples. Under optimized experimental conditions, the developed methodology allowed for linear dynamic ranges between 20.0 and 2000.0 μg L- 1 with determination coefficients of 0.9991 and 0.9992, as well as average recovery yields of 61.7-67.5% and 53.9-57.8% for nicotine and cotine, respectively. The developed methodology was applied to monitor urine samples from 86 volunteers having different smoking habits, where nicotine and cotinine were quantified in the range from 23.6 to 2612.6 μg L -1 . The target compounds were extracted in a HT-BAμE apparatus, which allows for simultaneous microextraction and subsequent back-extraction of up to 100 samples. This is a major improvement over other microextraction techniques. The data from the proposed methodology were satisfactory and in line with current green analytical chemistry guidelines, and proved to be an effective sample preparation alternative with substantial potential for high throughput bioanalysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: Fetal exposure to tobacco constituents is a risk factor for negative birth outcomes. We aimed to determine the relationships between nicotine and cotinine concentrations in amniotic fluid and maternal saliva., Methods: As part of a therapeutic trial, 42 pregnant smokers agreed to sample amniotic fluid (8 samples from amniocentesis, 34 at birth). Their smoking characteristics were collected along with the newborns' birth outcomes., Results: The median concentrations [IQR] in amniotic fluid and saliva were 11 [7-31] and 38 [7-174] μg/L for nicotine and 72 [22-123] μg/L and 55 [17-109] μg/L for cotinine, respectively. Multivariate models showed that saliva cotinine concentration predicted amniotic fluid nicotine and cotinine concentrations (R 2  =   0.398, p < 0.0001 and R 2  =   0.708, p < 0.0001 respectively). Amniotic fluid nicotine or cotinine concentration was not associated with birth weight. In multivariate analysis, the time elapsed since the last cigarette was the only variable associated with increased birth weight (R 2  =   0.237, p = 0.002)., Conclusions: Maternal saliva sampling for the determination of cotinine concentration is of interest to monitor fetal exposure to nicotine of any origin. Nevertheless, the time elapsed since the last cigarette was a better predictor of birth weight than the biomarkers' concentrations in amniotic fluid or maternal saliva.

A straightforward, high-throughput method was developed and fully validated for the simultaneous determination of the specific tobacco biomarkers nicotine and its main metabolite cotinine in a wide dynamic range and supporting the most common human biological matrices (urine, oral fluid and hair). Sample preparation was performed inside the very HPLC injection vials by pipetting 0.5 mL of the liquid samples, deuterated internal standards in alkaline solution and dichloromethane as extraction solvent. Solid samples (i.e. around 10 mg hair) were first submitted to alkaline digestion in the HPLC vials and processed accordingly. The organic phase (reached through the upper aqueous layer) was directly injected without further treatment. Instrumental analysis was performed using hydrophilic interaction (HILIC) ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Total chromatographic time was 2 min. The method covers a wide dynamic range making it fit-for-purpose for the analysis of samples covering entire populations, irrespective of the level of exposure or tobacco use. Calibration curves (r (2) > 0.995) covered the range 1-2000 ng/mL (or 0.05-100 ng/mg hair) for nicotine and 0.1-2000 ng/mL (or 0.005-100 ng/mg hair) for cotinine. Within-run and between-run precision and accuracy were typically below 10 %, and always below 20 % at the lower limit of quantification. The method was successfully applied to the analysis of samples from different projects involving multiple matrices.

Smoking is considered to be one of the main risk factors for cancer and other diseases and is the second leading cause of death worldwide. As the anti-tobacco legislation implemented in Europe has reduced secondhand smoke exposure levels, analytical methods must be adapted to these new levels. Recent research has demonstrated that cotinine is the best overall discriminator when biomarkers are used to determine whether a person has ongoing exposure to tobacco smoke. This work proposes a sensitive, simple and low-cost method based on solid-phase extraction and liquid chromatography with diode array detection for the assessment of tobacco smoke exposure by cotinine determination in urine. The analytical procedure is simple and fast (20 min) when compared to other similar methods existing in the literature, and it is cheaper than the mass spectrometry techniques usually used to quantify levels in nonsmokers. We obtained a quantification limit of 12.30 μg/L and a recovery of over 90%. The linearity ranges used were 12-250 and 250-4000 μg/L. The method was successfully used to determine cotinine in urine samples collected from different volunteers and is clearly an alternative routine method that allows active and passive smokers to be distinguished., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: The aim of the study was an objective assessment of active and passive exposure to cigarette smoke among patients after percutaneous transluminal coronary angioplasty (PTCA). A questionnaire was answered by 24 patients (18 men and 6 women) aged 48 to 76 (av. 61.2) years. Cotinine (metabolite of nicotine) level was measured in blood, urine and saliva of the patients as objective verification of the questionnaire answers., Conclusions: 1. Among the PTCA patients 41.6% of them were active smokers. 2. 30% of smokers concealed the fact of active smoking. 3. We assume that smoking habit among the patients should be verified by measurement of cotinine in serum, saliva and urine. 4. The main motivations for smoking cessation were myocardial infarction, PTCA and hospitalizations. 5. All patients were aware of the harmful effects of smoking and were able to identify the main risks.

Among available methods to estimate the exposure to tobacco smoke, cotinine, a major metabolite of nicotine is considered the most accurate marker. The aim of this study was to evaluate environmental tobacco smoke exposure in pregnancy and postpartum period. The cohort study was conducted in 2004 and 2006 in public maternity units in Lodz, Poland. The study population consisted of women between 32-36 weeks of pregnancy who have quit smoking within 2 months before pregnancy or no later than three months prior to participation in the study. Women were interviewed twice: during pregnancy and three months after delivery. Self-reported non-smoking status was verified using saliva cotinine level. Cotinine level in saliva sample was analyzed using Liquid Chromatography with Tandem Mass Spectrometry. We included into the analysis 62 women who, based on self-reported smoking status and saliva cotinine level were classified as non-smokers. There were no statistically significant differences in mean saliva cotinine level measured in pregnancy and postpartum period. Pregnant women who smoked more cigarettes per day before quitting smoking had significantly higher cotinine level comparing to women who smoked < or = 10 cigarettes per day (p = 0.03). Saliva cotinine level was significantly higher among women exposed to environmental tobacco smoke at home compared to non-exposed (p = 0.02).

In Poland, tobacco smoking by women in a procreational age as well as the pregnant women is a common phenomenon. The aim of the conducted research was to assess the usefulness of cotinine markers in different biological materials--mother and newborn's urine, cord blood se. rum, placenta--as a biomarker of tobacco smoking by delivering women, and dependence between these biomarkers and the newborn's health state. 218 pregnant women (117 smokers and 101 non-smokers), who were checked in at the Perinatology and Gynecology Clinic of the University of Medical Sciences in Poznan, took part in the first stage of the research (period between the twelfth and sixteenth week of pregnancy) carried out between years 2004-2006. In the second stage, 201 pairs of women (89 smokers and 112 non-smokers) and their newborns were checked after the women came to hospital to deliver. The research that was conducted showed that both cotinine in the urine of delivering women and in the urine of newborns as well as in the cord blood serum may be used as a biomarker of exposure of a foetus to tobacco smoke. For practical reasons, it must be assumed that the delivering women urine should be the material from choice. The research did not indicate the usefulness of the determination of cotinine in the placenta, in order to assess the exposure of the foetus to the components of tobacco smoke. On the other hand, again it confirmed the influence of tobacco smoking on the newborn's birth parameters, a correlation between the birth weight, body length and cotinine concentration in the urine of a mother makes it possible to predict the lowering of the antropometric parameters of the newborn as a result of tobacco smoking by pregnant women.

Kind of the main nicotine metabolites, occurrence frequency of trans-3'-hydroxycotinine (HK) and cotinine (K) in mother and foetus serum, amniotic fluid and urine of chosen group of children (10 - 12 years old) determined by planar chromatography with densitometry as well as ratio of (HK)/(K) has been compared. The obtained data allowed to state, that the main nicotine metabolite concentrations (HK and K) were higher in foetus serum in comparison to mother serum. In the case of occurrence both of them ratio of (HK)/(K) was close to 1 in 20% of examined body fluid samples, but it was above this value in significant majority cases. It can be a prove that trans-3'-hydroxycotinine was dominating metabolite of nicotine in these samples and statistically significant more often occur in foetus serum, in the case when cotinine was determined in mother serum.

Trifluoroacetic acid (TFA) is a commonly used additive in HPLC and LC-MS analysis of basic compounds. It is also routinely added to aqueous-organic mobile phases utilized in the hydrophilic interaction chromatography-electrospray tandem mass spectrometry (HILIC-ESI/MS/MS) technique used in our laboratories for bioanalysis. However, TFA is known to suppress the ESI signals of analytes due to its ability to form gas-phase ion pairs with positively-charged analyte ions. The most common method to overcome this problem involves the post-column addition of a mixture of propionic acid and isopropanol. However the post-column addition setup requires additional pumps and is not desirable for continuous analysis of large amounts of samples. In this paper we present a simple yet very effective means of minimizing the negative effect of TFA in bioanalysis by direct addition of 0.5% acetic acid or 1% propionic acid to mobile phases containing either 0.025 or 0.05% TFA. A factor of two- to five-fold signal enhancement was achieved for eight basic compounds studied. Furthermore, chromatography integrity was maintained even with the addition of acetic acid and propionic acid to existing TFA mobile phases. This method has been successfully applied to the HILIC-ESI/MS/MS high-throughput analysis of extracted biological samples to support pre-clinical and clinical studies.

Background: Environmental tobacco smoke (ETS) is an important risk factor. The aim of this study is to evaluate effects of the counseling method "Smoke-free children" that focuses on protection of infants., Methods: The counseling method, "Smoke-free children", has been developed and implemented at Swedish child health centers. The counseling method's point of departure is based upon a client-centered approach. Saliva cotinine samples from the mothers were collected when the child was 1-4 weeks and 3 months of age. Interviews regarding mothers' smoking habits and self-reported maternal smoking were also carried out., Results: Forty-one mothers participated in the study, 26 in the intervention group and 15 in the control group. Cotinine was collected from 22 subjects in the intervention and 8 in the control group. Before the intervention, the mean cotinine level was 185 ng/mL in the intervention group and 245 ng/mL in the control group. After the intervention, cotinine levels were reduced in the intervention group (165 ng/mL) and increased in the control group (346 ng/mL). Yet, after the intervention, the mothers themselves reported more smoking in the intervention group than in the control group. Only weak correlations were found between self-reported smoking and cotinine., Conclusions: The statistical analysis supports the view that a client-centered intervention, aimed at increasing self-efficacy, exerts a positive effect on maternal smoking in the prevention of infant exposure to ETS, when applied in a routine clinical setting.

Analysis of nicotine and cotinine in human hair can provide information on nicotine intake and exposure to environmental tobacco smoke over a long period of time. Nonetheless, to better assess the usefulness of hair analysis to determine smoking habits or exposures, all procedures have to be standardized. Various solvents were tested as washing solvents to eliminate external contamination from nicotine. Dichloromethane was found effective when used for two washes prior to the extraction. Basic and acid digestion of hair followed by solid phase extraction with Extrelut-3 glass column using dichloromethane:isopropyl alcohol (9:1) as eluting mixture both gave good recoveries of nicotine and cotinine, when compared with extractions reported in the literature. The extraction method was free from substances, which could interfere in the chromatographic analysis. Furthermore, the addition of methanolic HCl to the eluting mixture prevented the loss of nicotine during the evaporation step before chromatography. Chromatography was performed using a reversed-phase column and a U.V. detection at 254 nm. Furthermore, hair treatments (dyes, permanent wave, hydrogen peroxide) caused a major decrease in the nicotine content in hair, and a smaller effect on cotinine levels. However, the effect of various treatments was not reproducible. Several attempts to produce reference materials were carried out. Nicotine and cotinine standard solutions at different concentrations were added to blank hair soaked in dimethylsulfoxide, methanol and water.

The health consequences of exposure to environmental tobacco smoke (ETS) are well documented. Although nonsmokers are generally aware of the health risks of ETS exposure, the majority of nonsmokers are regularly exposed. The most common source of exposure is the workplace. Restrictive workplace smoking policies are being used as a primary means of reducing ETS exposure. However, few studies have focused on the relation between workplace policy and ETS exposure. We performed two studies which examined the relationship between smoking policy, self-reported ETS exposure, and salivary cotinine concentrations. Study I, a pilot study, focused on a workplace-based sample of 106 volunteers; Study 2 examined exposure among 881 nonsmokers in workplace settings. In both studies, more restrictive workplace smoking policies were associated with a lower proportion of nonsmoking volunteers with detectable salivary cotinine. In Study 2, the larger study, the only other variable found to be significantly related to cotinine detection was the presence of smokers in the home. These results suggest that restrictive workplace smoking policies may reduce employees' overall ETS exposure.

A method for the synthesis of (3'R,5'S-trans-3'-hydroxycotinine, a major metabolite of nicotine in humans, is described. The method involves deprotonation of (S)-cotinine with lithium diisopropylamide (LDA) followed by oxidation with the transition metal peroxide oxodiperoxymolybdenum(pyridine)(hexamethylphosphoric triamide) (MoOPH) to give an 80:20 mixture of trans-/cis-3'-hydroxycotinine. The pure (greater than 98%) trans isomer is obtained by conversion to the solid hexanoate ester, recrystallization, and cleavage of the ester by heating with n-butylamine. GC-MS analysis of urine extracts from several smokers indicated that in humans metabolic 3'-hydroxycotinine is 95-98% trans.

The S-(-)- and R-(+)-nicotine isomers were administered subcutaneously via Alzet osmotic pumps to male Hartley guinea-pigs (n = 5 with each isomer) over a 23-day period. Estimated dosage rate throughout the experiment was 0.6 mg-1. Urine samples were collected over this time and the levels of urinary oxidative and N-methylated nicotine metabolites were measured by cation-exchange HPLC analysis. S-(-)-Nicotine formed only oxidative metabolites, whereas the R-(+)-isomer formed both oxidative and N-methylated metabolites. 3'-Hydroxycotinine and nicotine-1'-oxide were major metabolites of both enantiomers; cotinine and nornicotine were only minor metabolites. The major N-methylated metabolite of R-(+)-nicotine was N-methylnicotinium ion; N-methylcotininium ion and N-methylnornicotinium ion were also identified as metabolites of this nicotine isomer. Total N-methylated quaternary ammonium metabolites accounted for 15 to 20% of the administered dose of R-(+)-nicotine. An interesting enantioselective reduction in the percent of oxidative urinary metabolites formed S-(-)-nicotine was observed over 23 days. This may indicate the enantioselective induction of an uncharacterized metabolic pathway for this nicotine isomer.