Quantification Of Nicotine Research Articles

Simultaneous quantification of nicotine and metabolites in rat brain by liquid chromatography–tandem mass spectrometry

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of nicotine (NIC), cotinine (COT), nornicotine (NNIC), norcotinine (NCOT), nicotine-N-β- d-glucuronide (NIC GLUC), cotinine-N-β- d-glucuronide (COT GLUC), nicotine-1′-oxide (NNO), cotinine-N-oxide (CNO), trans-3′-hydroxycotinine (3-HC), anabasine (AB) and anatabine (AT) was modified and validated for quantification of these selected analytes in rat brain tissue. This analytical method provides support for preclinical NIC pharmacokinetic and toxicological studies after controlled dosing protocols. After brain homogenization and solid-phase extraction, target analytes and corresponding deuterated internal standards were chromatographically separated on a Discovery ® HS F5 HPLC column with gradient elution and analyzed by LC–MS/MS in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. Method linearity was assessed and calibration curves were determined over the following ranges: 0.1–7.5 ng/mg for NIC, COT GLUC and AB; and 0.025–7.5 ng/mg for COT, NNIC, NCOT, NIC GLUC, NNO, CNO, 3-HC and AT ( R 2 ≥ 0.99 for all analytes). Extraction recoveries ranged from 64% to 115%, LC–MS/MS matrix effects were ≤21%, and overall process efficiency ranged from 57% to 93% at low and high quality control concentrations. Intra- and inter-assay imprecisions and accuracy for all analytes were ≤12.9% and ≥86%, respectively. The method was successfully applied to quantification of NIC and metabolites in the brain of post-natal day 90 rats that were sacrificed 2-h after a single 0.8 mg/kg s.c. administration of (−)NIC. In these tissues, striatal concentrations were 204.8 ± 49.4, 138.2 ± 14.2 and 36.1 ± 6.1 pg/mg of NIC, COT and NNIC, respectively. Concentrations of NIC, COT and NNIC in the remaining whole brain (RWhB) were 183.3 ± 68.0, 130.0 ± 14.1 and 46.7 ± 10.3 pg/mg, respectively. Quantification of these same analytes in plasma was also performed by a previously validated method. NIC, COT, NNIC, NCOT, NNO and CNO were detected in plasma with concentrations comparable to those reported in previous studies. However, and in contrast to brain tissues, COT concentrations in plasma were significantly higher than were those of NIC (194.6 ± 18.6 ng/mL versus 52.7 ± 12.9 ng/mL). Taken together, these results demonstrate that a sensitive and selective method has been developed for the determination of NIC biomarkers in rat brain.

Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Detection of Nicotine Biomarkers in Hair and an Evaluation of Wash Procedures for Removal of Environmental Nicotine

The aim of this exploratory study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the quantification of nicotine, eight nicotine metabolites, and two minor tobacco alkaloids in fortified analyte-free hair and subsequently apply this method to hair samples collected from active smokers. An additional aim of the study was to include an evaluation of different wash procedures for the effective removal of environmentally deposited nicotine from tobacco smoke. An apparatus was designed for the purpose of exposing analyte-free hair to environmental tobacco smoke in order to deposit nicotine onto the hair surface. A shampoo/water wash procedure was identified as the most effective means of removing nicotine. This wash procedure was utilized for a comparison of washed and unwashed heavy smoker hair samples. Analytes and corresponding deuterated internal standards were extracted using a cation-exchange solid-phase cartridge. LC-MS-MS was carried out using an Acquity™ UPLC(®) system (Waters) and a Quattro Premier XE™ triple quadrupole MS (Waters) operated in electrospray positive ionization mode, with multiple reaction monitoring data acquisition. The developed method was applied to hair samples collected from heavy smokers (n = 3) and low-level smokers (n = 3) collected through IRB-approved protocols. Nicotine, cotinine, and nornicotine were quantified in both the washed and unwashed hair samples collected from three heavy smokers, whereas 3-hydroxycotinine was quantified in only one unwashed sample and nicotine-1'-oxide in the washed and unwashed hair samples from two heavy smokers. In contrast, nicotine-1'-oxide was quantified in one of the three low-level smoker samples; nicotine was quantified in the other two low-level smoker samples. No other analytes were detected in the hair of the three low-level smokers.

A one-year monitoring of nicotine use in sport: Frontier between potential performance enhancement and addiction issues

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

Development and validation of a hydrophilic interaction chromatography–tandem mass spectrometry for quantification of nicotine and its metabolites in human maternal and cord sera

A selective and sensitive HILIC-MS/MS method for the simultaneous quantification of nicotine and its metabolites in human maternal and cord sera was developed and validated. After solid-phase extraction, LC separation was achieved on a hydrophilic interaction chromatography. The validated method is capable of selective identification as well as accurate and sensitive quantification. Analyte recovery ranged from 86.2 to 107.7% and intra- and inter-day assay precision were less than 15% relative standard deviation. This sensitive HILIC-MS/MS method can be used to determine nicotine and its metabolic profile in smokers. This validated method is useful for the determination of nicotine and its metabolites in human serum in future studies of the effects of nicotine exposure on neonatal outcome.

A liquid chromatography-mass spectrometry method for nicotine and cotinine; utility in screening tobacco exposure in patients taking amiodarone

A liquid chromatographic mass spectrometric (LC-MS) assay for the quantification of nicotine and cotinine in human specimens was developed. Human serum and urine (100 μL) were subjected to liquid-liquid extraction. For glucuronidated cotinine, serum was alkalinized and hydrolyzed before extraction. The dried samples were reconstituted and run using gradient flow reverse-phase liquid chromatography with MS detection. The ions utilized for quantification of nicotine, cotinine and milrinone (internal standard) were 162.8, 176.9 and 211.9 m/z, respectively. The mean recoveries were over 80% for cotinine and nicotine with excellent linearity between nominal concentrations and peak area ratios, over a wide concentration range. The percentage coefficient of variation and mean error of the inter- and intra-day validations were <15% for nicotine and cotinine. Analysis of serum from cardiac patients receiving amiodarone suggested that a number of patients were either active smokers or exposed to second-hand smoke. Significant concentrations of nicotine and cotinine were measured in the urine of a known smoking volunteer. The method was highly specific, sensitive and applicable as a tool in detecting and monitoring the passive exposure to tobacco smoke using small specimen volumes (0.1 mL).

Determination of nicotine and nicotine metabolites in urine by hydrophilic interaction chromatography–tandem mass spectrometry: Potential use of smokeless tobacco products by ice hockey players

Consumption of nicotine in the form of smokeless tobacco (snus, snuff, chewing tobacco) or nicotine-containing medication (gum, patch) may benefit sport practice. Indeed, use of snus seems to be a growing trend and investigating nicotine consumption amongst professional athletes is of major interest to sport authorities. Thus, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the detection and quantification of nicotine and its principal metabolites cotinine, trans-3-hydroxycotinine, nicotine- N′-oxide and cotinine- N-oxide in urine was developed. Sample preparation was performed by liquid–liquid extraction followed by hydrophilic interaction chromatography–tandem mass spectrometry (HILIC–MS/MS) operated in electrospray positive ionization (ESI) mode with selective reaction monitoring (SRM) data acquisition. The method was validated and calibration curves were linear over the selected concentration ranges of 10–10,000 ng/mL for nicotine, cotinine, trans-3-hydroxycotinine and 10–5000 ng/mL for nicotine- N′-oxide and cotinine- N-oxide, with calculated coefficients of determination ( R 2) greater than 0.95. The total extraction efficiency (%) was concentration dependent and ranged between 70.4 and 100.4%. The lower limit of quantification (LLOQ) for all analytes was 10 ng/mL. Repeatability and intermediate precision were ≤9.4 and ≤9.9%, respectively. In order to measure the prevalence of nicotine exposure during the 2009 Ice Hockey World Championships, 72 samples were collected and analyzed after the minimum of 3 months storage period and complete removal of identification means as required by the 2009 International Standards for Laboratories (ISL). Nicotine and/or metabolites were detected in every urine sample, while concentration measurements indicated an exposure within the last 3 days for eight specimens out of ten. Concentrations of nicotine, cotinine, trans-3-hydroxycotinine, nicotine- N′-oxide and cotinine- N-oxide were found to range between 11 and 19,750, 13 and 10,475, 10 and 8217, 11 and 3396, and 13 and 1640 ng/mL, respectively. When proposing conservative concentration limits for nicotine consumption prior and/or during the games (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine- N′-oxide and cotinine- N-oxide), about half of the hockey players were qualified as consumers. These findings significantly support the likelihood of extensive smokeless nicotine consumption. However, since such conclusions can only be hypothesized, the potential use of smokeless tobacco as a doping agent in ice hockey requires further investigation.

Real-Time UV Imaging of Nicotine Release from Transdermal Patch

This study was conducted to characterize UV imaging as a platform for performing in vitro release studies using Nicorette® nicotine patches as a model drug delivery system. The rate of nicotine release from 2mm diameter patch samples (Nicorette®) into 0.067M phosphate buffer, pH 7.40, was studied by UV imaging (Actipix SDI300 dissolution imaging system) at 254nm. The release rates were compared to those obtained using the paddle-over-disk method. Calibration curves were successfully established which allowed temporally and spatially resolved quantification of nicotine. Release profiles obtained from UV imaging were in qualitative agreement with results from the paddle-over-disk release method. Visualization as well as quantification of nicotine concentration gradients was achieved by UV imaging in real time. UV imaging has the potential to become an important technology platform for conducting in vitro drug release studies.

Quantification of nicotine in commercial brand cigarettes

The main objective of this experiment is to determine the amount of nicotine in commercial brand cigarettes by means of a nonaqueous acid-base titration. A simple glass device simulating a smoker is proposed, which allows the determination of the volatilized, filter retained, and inhaled portions. Students will readily see that the amount of nicotine/cigarette stated on the label (∼0.5-1.0 mg) refers indeed to the inhaled portion only, rather than to the total amount/cigarette (usually more than 10 mg). Even so, values for inhaled nicotine may be significantly higher than those reported for several brands. Students will also be able to make a critical evaluation of the true content of nicotine in the inhaled portion and confront it with the reported value for a given brand. In addition, the theoretical approach, supported by HPLC data, provides an excellent experience on nonaqueous acid-base volumetric analysis.

Rapid and sensitive determination of nicotine in formulations and biological fluid using micellar liquid chromatography with electrochemical detection

Nicotine can be determined in pharmaceuticals and biological fluids by micellar liquid chromatography (MLC) using a C18 column, a mobile phase containing sodium dodecyl sulphate 0.15 M–6% (v/v) pentanol–NaH 2PO 4 0.01 M (pH 6)–KCl 0.001 M, with electrochemical detection at 0.8 V. In the optimization step, the influence of the modifiers propanol, butanol and pentanol, and the voltage has been studied. With the proposed method the analysis time is below than 8 min, linearity better than 0.999, limits of detection and quantification (ng/ml) was 4 and 12 respectively, repeatability and intermediate precision below 1.8%, and all these parameters are adequate for the quantification of nicotine in chewing gum, dermal patches, tobacco and serum samples either by a pharmacologist, pathologist or toxicologist.

In vivo nicotine exposure in the zebra finch: A promising innovative animal model to use in neurodegenerative disorders related research

Nicotine improves cognitive enhancement and there are indications that neurodegenerative (age-related) cognitive disorders could be treated with nicotine-based drugs. The zebra finch is a well-recognized model to study cognitive functioning; hence this model could be used to study the effects of nicotine in neurodegenerative cognitive disorders. However, nicotine's in vivo physiological and behavioral effects have never been studied in the zebra finch. Here we present the first in vivo nicotine study in zebra finches. We evaluated the dose–response effects of nicotine on locomotor activity, song production, food intake and body weight. A liquid chromatography tandem mass spectrometry method was developed and validated for quantification of nicotine and cotinine in feces. The subcutaneous nicotine drug regiment (0.054–0.54 mg/kg) induced physiologically significant values of nicotine and cotinine. The mid (0.18 mg/kg) and high (0.54 mg/kg) dose of nicotine promoted the development and expression of a sensitized response of song production and locomotor activity. Food intake and body weight were not affected following nicotine exposure. In conclusion, the zebra finch can be used as an innovative animal model not only in nicotine-related research studying cognitive functioning, but also in studies examining nicotine dependence and addictive mechanisms.

Two UK suicides using nicotine extracted from tobacco employing instructions available on the Internet

Tobacco is one of the most easily accessible and commonly abused drugs world-wide. Nicotine, one of its principal constituents, can cause serious or fatal overdoses. Whilst the deliberate ingestion of this substance appears to be relatively rare, often the important signs of its consumption are not recognised, sometimes with fatal results. Here we describe two cases of intentional fatal ingestion of nicotine. The nicotine was extracted from tobacco using instructions available on the Internet. The first case involved a male aged 19 who died in 2008. The post-mortem blood and urine levels of nicotine were 5.5 mg/l and >80 mg/l respectively; the blood level is in line with the generally recognised fatal level of >5 mg/l. The levels of nicotine's main metabolite, cotinine, for this case were 2.5 mg/l and 7.9 mg/l for blood and urine respectively. A comparative case in 1999 involved a 32 year-old male. The level of post-mortem nicotine in his blood was 1.0 mg/l. These are believed to be the first UK suicides by nicotine using instructions from the Internet reported in the literature. Information that nicotine was the agent responsible only became apparent some time after death. There may be more deaths due to this cause that go unrecognised because quantification of nicotine and cotinine levels is not often conducted, due to the wide prevalence of smoking. It is important that all evidence at the scene of a sudden and unexplained death is carefully evaluated, including potential clues on PCs and lap-tops.

Optimization and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, cotinine, trans-3′-hydroxycotinine and norcotinine in human oral fluid

An analytical procedure was developed and validated for the simultaneous identification and quantification of nicotine, cotinine, trans-3'-hydroxycotinine, and norcotinine in 0.5 mL of human oral fluid collected with the Quantisal oral fluid collection device. Solid phase extraction and liquid chromatography-tandem mass spectrometry with multiple reaction monitoring were utilized. Endogenous and exogenous interferences were extensively evaluated. Limits of quantification were empirically identified by decreasing analyte concentrations. Linearity was from 1 to 2,000 ng/mL for nicotine and norcotinine, 0.5 to 2,000 ng/mL for trans-3'-hydroxycotinine, and 0.2 to 2,000 ng/mL for cotinine. Correlation coefficients for calibration curves were >0.99 and analytes quantified within +/-13% of target at all calibrator concentrations. Suitable analytical recovery (>91%) was achieved with extraction efficiencies >56% and matrix effects <29%. This assay will be applied to the quantification of nicotine and metabolites in oral fluid in a clinical study determining the most appropriate nicotine biomarker concentrations differentiating active, passive, and environmental nicotine exposure.

Simultaneous and sensitive measurement of nicotine, cotinine, trans-3′-hydroxycotinine and norcotinine in human plasma by liquid chromatography-tandem mass spectrometry

An LC-MS/MS method for the simultaneous quantification of nicotine, cotinine, trans-3′-hydroxycotinine and norcotinine in human plasma was developed and fully validated. Potential endogenous and exogenous interferences were extensively evaluated and limits of quantification were determined by decreasing analyte concentration. Analytical ranges were 1–500 ng/mL for nicotine and cotinine, 5–500 ng/mL for trans-3′-hydroxycotinine and norcotinine. Mean intra- and inter-assay analytical recoveries were between 101.9 and 116.8%, and intra- and inter-assay imprecision were less than 11% RSD for all analytes: parameters were evaluated at three different concentrations across the linear range of the assay. Extraction efficiency was ≥70% for all analytes. This validated method is useful for the determination of nicotine and metabolites in human plasma to support research on the role of nicotine biomarkers on neuronal systems mediating cognitive and affective processes and to differentiate active, passive and environmental exposure.

Validation of a GC–FID method for rapid quantification of nicotine in fermented extracts prepared from Nicotiana tabacum fresh leaves and studies of nicotine metabolites

A new GC–FID method, which allows rapid and reliable quantitation of nicotine in tobacco leaf extracts, was developed and validated. To avoid nicotine adsorption on the column, an amine-deactivated capillary column was used. The method developed was applied to study the degradation of nicotine in a fermented aqueous extract, and a loss of nearly 20% of nicotine over 12 months was observed. Careful inspection of GC–MS runs from concentrated samples of the same extract revealed the presence of nicotine metabolites such as nornicotine, anatabine, myosmine, 2,3′-bipyridyl, and 2-pyrrolidinone.

Simultaneous quantification of nicotine, opioids, cocaine, and metabolites in human fetal postmortem brain by liquid chromatography tandem mass spectrometry.

A validated method for simultaneous LCMSMS quantification of nicotine, cocaine, 6-acetylmorphine (6AM), codeine, and metabolites in 100 mg fetal human brain was developed and validated. After homogenization and solid-phase extraction, analytes were resolved on a Hydro-RP analytical column with gradient elution. Empirically determined linearity was from 5-5,000 pg/mg for cocaine and benzoylecgonine (BE), 25-5,000 pg/mg for cotinine, ecgonine methyl ester (EME) and 6AM, 50-5000 pg/mg for trans-3-hydroxycotinine (OH-cotinine) and codeine, and 250-5,000 pg/mg for nicotine. Potential endogenous and exogenous interferences were resolved. Intra- and inter-assay analytical recoveries were > or = 92%, intra- and inter-day and total assay imprecision were < or = 14% RSD and extraction efficiencies were > or = 67.2% with < or = 83% matrix effect. Method applicability was demonstrated with a postmortem fetal brain containing 40 pg/mg cotinine, 65 pg/mg OH-cotinine, 13 pg/mg cocaine, 34 pg/mg EME, and 525 pg/mg BE. This validated method is useful for determination of nicotine, opioid, and cocaine biomarkers in brain.

Quantification of nicotine, cotinine, trans-3′-hydroxycotinine, nornicotine and norcotinine in human meconium by liquid chromatography/tandem mass spectrometry

There are no analytical methods that simultaneously quantify nicotine, cotinine, trans-3′-hydroxycotinine, nornicotine and norcotinine in human meconium. Such a method could improve identification of in utero tobacco exposure, determine if maternal dose–meconium concentration relationships exist, and whether nicotine meconium concentrations predict neonatal outcomes. The first liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry method for simultaneous quantification of these analytes in meconium was developed and validated. Specimen preparation included homogenization, enzyme hydrolysis and solid phase extraction. The linear range was 1.25 or 5–500 ng/g. Method applicability was evaluated with meconium collected from an in utero tobacco exposed infant.

Determination of Nicotine and Cotinine in Human Serum for Evaluation of Tobacco Smoke Exposure by Hydrophilic Interaction Chromatography/Mass Spectrometry

Tobacco smoke contains more than 200 harmful compounds and 40 carcinogenic compounds. Tobacco smoke exposure is a significant risk of growth suppression for fetuses and infants. A highly sensitive and selective method for the determination of nicotine and cotinine in human serum samples, to evaluate the exposure amount of tobacco smoke, was developed. Solid-phase extraction was employed for sample preparation, and a measurement was performed by hydrophilic interaction chromatography with mass spectrometry (HILIC/MS). The limits of detection of nicotine and cotinine were 0.2 and 0.1 ng/mL, respectively. Moreover, the limits of quantification of nicotine and cotinine were 1.0 and 0.4 ng/mL, respectively. The average recoveries of nicotine and cotinine were over 90% (RSD<6%, n = 6). The proposed method was applied to measure the serum and umbilical cord serum of a pregnant woman. The detection rate of cotinine in the pregnant woman serum and umbilical cord serum were 51 to 14 and 11 to 1.

A validated method for the determination of nicotine, cotinine, trans‐3′‐hydroxycotinine, and norcotinine in human plasma using solid‐phase extraction and liquid chromatography‐atmospheric pressure chemical ionization‐mass spectrometry

A liquid chromatographic-mass spectrometric method for the simultaneous determination of nicotine, cotinine, trans-3'-hydroxycotinine, and norcotinine in human plasma was developed and validated. Analytes and deuterated internal standards were extracted from human plasma using solid-phase extraction and analyzed by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometric detection with selected ion monitoring (SIM). Limits of detection and quantification were 1.0 and 2.5 ng/ml, respectively, for all analytes. Linearity ranged from 2.5 to 500 ng/ml of human plasma using a weighting factor of 1/x; correlation coefficients for the calibration curves were > 0.99. Intra- and inter-assay precision and accuracy were < 15.0%. Recoveries were 108.2-110.8% nicotine, 95.8-108.7% cotinine, 90.5-99.5% trans-3'-hydroxycotinine, and 99.5-109.5% norcotinine. The method was also partially validated in bovine serum, owing to the difficulty of obtaining nicotine-free human plasma for the preparation of calibrators and quality control (QC) samples. This method proved to be robust and accurate for the quantification of nicotine, cotinine, trans-3'-hydroxycotinine, and norcotinine in human plasma collected in clinical studies of acute nicotine effects on brain activity and on the development of neonates of maternal smokers.

A SIMPLE AND RAPID METHOD FOR NICOTINE ASSAY BY HPLC FROM CUTANEOUS MICRODIALYSIS SAMPLES

A simple reversed-phase high-performance liquid chromatographic method with UV detection has been developed for the quantification of nicotine in cutaneous microdialysis studies. Nicotine was analyzed using 5 μm Lichrospher® 60 RP-18 Select B column (125 × 4 mm i.d.). The mobile phase used was water-methanol-0.1 M buffer acetate (pH = 4.5)–acetonitrile – acetic acid (74 : 3 : 20 : 2 : 1 v/v), adjusted to pH = 4.2 with diethylamine at a flow-rate of 0.7 ml/min. Detection was carried out at 254 nm at room temperature. The limit of quantification was 20 ng/ml; linearity was obtained from 50 to 5000 ng/ml. The intra- and inter assay coefficient of variation was less than 5%. The present method was applied to analysis of nicotine in in vivo cutaneous microdialysis samples.

Quantification of nicotine, chlorpyrifos and their metabolites in rat plasma and urine using high-performance liquid chromatography

This study describes a high-performance liquid chromatographic method for the separation and quantification of nicotine, its metabolites nornicotine and cotinine, the insecticide chlorpyrifos ( O, O-diethyl- O[3,5,6-trichloro-2-pyridinyl]phosphorothioate), and its metabolites chlorpyrifos-oxon ( O, O-diethyl- O[3,5,6-trichloro-2-pyridinyl]phosphate), and TCP (3,5,6-trichloro-2-pyridinol) in rat plasma and urine. The compounds were separated using gradient mobile phase of methanol, acetonitrile and water (pH 3.20) at a flow-rate of 0.8 ml/min in a period of 17 min, and gradient UV detection ranging between 260 and 280 nm. The retention times ranged from 3.4 to 16.7 min. The limits of detection were ranged between 20 and 150 ng/ml, while limits of quantitation were 50–200 ng/ml. Average percentage recovery of five spiked plasma samples were 84.7±8.3, 78.2±7.6, 80.1±7.6, 79.0±6.4, 74.0±7.4, 87.6±7.5, and from urine 85.1±5.2, 75.9±7.0, 82.1±6.1, 79.5±6.1, 71.3±7.4 and 81.3±6.9 for nicotine, nornicotine, cotinine chlorpyrifos, chlorpyrifos-oxon and TCP, respectively. Intra-day accuracy and precision for this method were ranged between 2.2–3.6 and 2.1–2.8%, respectively. The relationship between peak areas and concentration was linear over range between 200 and 2000 ng/ml. This method was applied to analyze the above chemicals and metabolites following combined oral administration in rats.