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.

The effect of intermittent dosing of Nicotiana glauca on teratogenesis in goats.

Sustained inhibition of fetal movement in livestock species, induced by several poisonous plants, can result in numerous skeletal-contracture malformations. Lupines are responsible for a condition in cattle referred to as "crooked calf syndrome" that occurs when pregnant cattle graze teratogenic lupines. Similar malformations are also seen in animals poisoned by Conium maculatum (coniine) and Nicotiana glauca (anabasine). A proposed management strategy to limit these types of birth defects includes utilizing an intermittent grazing schedule to allow short durations of grazing lupine-infested areas interrupted by movement to a lupine-free pasture. The objective of this study was to use a goat model to determine if an intermittent schedule of five continuous days on treatment followed by two days off treatment would be sufficient to decrease, or prevent, the incidence of anabasine-induced malformations. The data from this study suggest that, for N. glauca in goats, the intermittent grazing program of five days exposure with two days of non-exposure is insufficient to prevent significant skeletal malformations from occurring. However, this study did demonstrate an inverse relationship between the amount of serum anabasine in the dam and the extent of fetal movement.

Determination of nicotine, anabasine, and cotinine in urine and saliva samples using single-drop microextraction.

A simple, sensitive, and inexpensive singe-drop microextraction (SDME) followed by gas chromatography and flame-ionization detection (GC-FID) was developed for determination of nicotine, anabasine, and cotinine in human urine and saliva samples. The target compounds were extracted from alkaline aqueous sample solution into an organic acceptor drop suspended on the tip of a 25-µL GC microsyringe in the aqueous sample solution. This microsyringe was also used for direct injection after extraction. Under optimized experimental conditions, calibration plots were found to be linear in the range of 0.5-25.0, 0.5-65.0, and 0.5-45.0mgL(-1) for nicotine, anabasines and cotinine, respectively. The method detection limit values were in the range of 0.33-0.45mgL(-1). Intra-day and inter-day precisions for peak area ratios were in the range of 1.3-9.2% and 2.0-7.0%, respectively. The proposed procedure was successfully applied to the determination of analytes in spiked urine and saliva samples with satisfactory results. The mean relative recoveries of spiked water samples ranged over 71.2-111.0%, with relative standard deviations varying from 2.3% to 10.0%.

Quantitation of nicotine, its metabolites, and other related alkaloids in urine, serum, and plasma using LC-MS-MS.

We describe a method for the quantitative analysis of nicotine, cotinine, trans-3'-hydroxy cotinine, nornicotine, and anabasine in urine, serum, and plasma using liquid chromatography-tandem mass spectrometry. A mix of deuterium-labeled internal standards (IS) is added to a specimen aliquot. The aliquot is extracted using mixed-mode solid phase extraction and eluted into an autosampler vial for injection into an LC-MS-MS system. An Atlantis silica column is used for LC separation in hydrophilic interaction mode. Tandem mass spectrometry detection is performed in positive ion mode with electrospray ionization and two multiple reaction monitoring (MRM) transitions monitored for each analyte and IS.

Simultaneous analysis of nicotine, nicotine metabolites, and tobacco alkaloids in serum or urine by tandem mass spectrometry, with clinically relevant metabolic profiles.

BACKGROUND: Assessment of nicotine metabolism and disposition has become an integral part of nicotine dependency treatment programs. Serum nicotine concentrations or urine cotinine concentrations can be used to guide nicotine patch dose to achieve biological concentrations adequate to provide the patient with immediate relief from nicotine withdrawal symptoms, an important factor in nicotine withdrawal success. Absence of nicotine metabolites and anabasine can be used to document abstinence from tobacco products, an indicator of treatment success. METHODS: The procedure was designed to quantify nicotine, cotinine, trans-3'-hydroxycotinine, anabasine, and nornicotine in human serum or urine. The technique required simple extraction of the sample with quantification by HPLC-tandem mass spectrometry. RESULTS: The procedure for simultaneous analysis of nicotine, its metabolites, and tobacco alkaloids simultaneously quantified five different analytes. Test limit of quantification, linearity, imprecision, and accuracy were adequate for clinical evaluation of patients undergoing treatment for tobacco dependency. The test readily distinguished individuals who had no exposure to tobacco products from individuals who were either passively exposed or were abstinent past-tobacco users from those who were actively using a tobacco or nicotine product. CONCLUSIONS: Nicotine, cotinine, trans-3'-hydroxycotinine, nornicotine, and anabasine can be simultaneously and accurately quantified in either serum or urine by HPLC-tandem mass spectrometry with imprecision <10% at physiologic concentrations and limits of quantification ranging from 0.5 to 5 micro g/L. Knowledge of serum or urine concentrations of these analytes can be used to guide nicotine replacement therapy or to assess tobacco abstinence in nicotine dependency treatment. These measurements are now an integral part of the clinical treatment and management of patients who wish to overcome tobacco dependence.