Biocompatible SPME fibers for direct monitoring of nicotine and its metabolites at ultra trace concentration in rabbit plasma following the application of smoking cessation formulations.

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.

Simultaneous determination of urinary 31 metabolites of VOCs, 8-hydroxy-2'-deoxyguanosine, and trans-3'-hydroxycotinine by UPLC-MS/MS: 13C- and 15N-labeled isotoped internal standards are more effective on reduction of matrix effect.

Human beings are inevitably exposed to volatile organic compounds (VOCs) of anthropogenic emissions as they are ubiquitous atmospheric pollutants. Smoking is an important exposure route of VOCs for the general population. Health effects induced by VOC exposure raise more concerns as they are identified with carcinogenicity, genotoxicity, neurotoxicity, and reproductive toxicity. trans-3'-Hydroxycotinine (OH-Cot) is a urinary biomarker of smoking, and 8-hydroxy-2'-deoxyguanosine (8-OHDG) is a urinary biomarker of DNA oxidative damage. To develop a method for quantifying VOC exposure levels of the general population and assessing the health risks induced by VOCs from second-hand smoking, an effective, rapid, and high-throughput method for the simultaneous determination of 31 metabolites of VOCs, 8-OHDG, and OH-Cot using solid-phase extraction coupled with UPLC-MS/MS was developed and validated. Method precision and accuracy, extraction recoveries, matrix effects, and storage stabilities of most analytes met the criterion (80-120%). Extraction recoveries increased from 85.1 to 100% after adjustment by isotoped internal standards (ISs). Furthermore, 13C- and 15N-labeled ISs were more effective to reduce the influence of matrix effects on recoveries and precisions than the deuterated analogs (73.0-116% vs. 53.6-140%). This developed method was successfully applied to determine urine samples collected from children. Results showed that N-acetyl-S-(3,4-dihydrobutyl)-L-cysteine, 2,2'-thiodiacetic acid (TGA), and N-acetyl-S-(3-hydroxypropyl-1-methyl)-L-cysteine (HPMMA) were well correlated with 8-OHDG with coefficients higher than 0.82, indicating those VOCs might easily lead to DNA damage. In conclusion, our co-monitoring of metabolites of VOCs with 8-OHDG and OH-Cot in one method provides a robust analytical method, which not only suggests the potential adverse health effects induced by VOCs but also discriminates and evaluates the contribution of passive smoking in human VOC exposure. Graphical abstract.

Revised and extended serum cotinine cut-offs to classify smokers and non-smokers.

PURPOSE: To revise and extend the previously published serum cotinine cut offs to classify smokers and non-smokers for US adolescents and adults. MATERIALS AND METHODS: Cross-sectional data (N = 10171) from National Health and Nutrition Examination Survey for 2011-2014 were used to compute serum cotinine cut-offs to classify smokers and non-smokers for US adults aged ≥20 years and 2007-2014 (N = 4583) data were used to compute serum cotinine cut-offs for US adolescents aged 12-19 years. RESULTS: Specificities and sensitivities for the cut-offs among adults were ≥95% and ≥75% among adolescents. For adults, serum cotinine cut-offs in ng/mL to classify smokers from non-smokers were 3.3 for the total population, 4.13 for males, 2.99 for females, 4.03 for non-Hispanic whites, 8.85 for non-Hispanic blacks, 0.377 for Mexican Americans, 1.72 for other Hispanics and 1.41 for non-Hispanic Asians. For adolescents, serum cotinine cut-offs in ng/mL to classify smokers from non-smokers were 0.765 for the total population, 1.1 for males, 0.408 for females, 1.2 for non-Hispanic whites, 1.98 for non-Hispanic blacks, 0.215 for Mexican Americans and 0.321 for other Hispanics. CONCLUSIONS: Serum cotinine cut-offs to distinguish smokers from non-smokers for US adults and adolescents were developed.

A simple near-patient test for nicotine and its metabolites in urine to assess smoking habit.

We describe a disposable, near-patient urine test to monitor cigarette smoking. A plastic device contains the sealed dried reagents to measure nicotine and its metabolites, by a colorimetric assay. The device can be used to give a qualitative assessment of tobacco consumption, simply by observing a colour change. Alternatively, the test can be quantified by measuring the light absorbance with a simple colorimeter, and a concentration of nicotinic metabolites obtained with reference to a cotinine standard. A correction factor for the concentration of the urine sample, based on light absorbance, allows the result to be expressed as a ratio to urine concentration. This method correlates with reported daily cigarette consumption (r = 0.69, p < 0.0001) and compares favourably with cotinine, as measured by gas chromatography (r = 0.89, p < 0.0001). The method provides a simple-to-use, inexpensive way to monitoring tobacco consumption in extralaboratory situations.

Separation of pH, dilution, ionic strength and chemical matrix effects for biological monitoring of urines with the Microtox test using nicotine, cotinine and reference urines.

The aim was to investigate the factors influencing light emission from Photobacterium phosphoreum in the Microtox test to interpret bioassay results for urine. Four reference urines were assessed as reference materials for the bioassay. Nicotine and cotinine were investigated as urinary markers for tobacco exposure. The optimum luminescence conditions were: 1.85%-3.25% NaCl, 0.33-0.58 mol/L ionic strength, and pH 5.8-6.7. Low pH values and high concentration of toxic trace metals were important factors in this study. Unexpected toxicity for a Standard Reference Material was attributed to zinc contamination. Nicotine and cotinine together exhibited antagonistic effects in 2% saline but this could not be observed in the urines because of substantial urine toxicity. Thus practical urinary biological monitoring with the Microtox test necessitates excretion of metabolites and compounds that are much more toxic than the urine components. Also, separation of the effects of physical factors like pH, ionic strength and dilution is essential before chemical toxicity effects can be assigned. This is the first report of Microtox EC50 values for nicotine and cotinine. The results have application to environmental samples since analyses are often uncontrolled relative to pH, ionic strength and dilution.