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 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).     

Simultaneous determination of thirteen plant alkaloids in a human specimen by SPE and HPLC

A new screening method for the simultaneous determination of thirteen plant alkaloids (aconitine, anabasine, atropine, brucine, colchicine, cotinine, cytisine, harmine, ibogaine, nicotine, scopolamine, strychnine, yohimbine) in a human specimen was developed based on solid-phase extraction and reversed-phase liquid chromatography with photodiode array detection. The validated method enables selective identification as well as accurate and sensitive quantification. The analysis of forensic and clinical samples emphasizes the applicability for intoxications and drug abuse, as well as for compliance control.     

Determination of nicotine and cotinine in rat plasma by liquid chromatography-tandem mass spectrometry

A method was developed for the efficient determination of nicotine and cotinine in rat plasma samples originating from nicotine exposure studies. Nicotine and cotinine were extracted from plasma samples with dichloromethane and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of hydrochloric acid to the organic solvent during evaporation. The samples were analysed using liquid chromatography with triple quadrupole mass spectrometry. For quantification, the deuterated internal standards were added and the most intensive MS-MS ion of the analyte and internal standards were monitored. For confirmatory analysis, two specific MS-MS ions, viz. m/z 132 and 106 for nicotine and m/z 80 and 98 for cotinine, were monitored and the ratios between the ions were calculated and compared with those of standards. The ratios have to be within the tolerances of the EU criteria. The limit of identification of the developed method was 1 microg/l. The repeatability ranged from 5 to 12% (R.S.D.) for nicotine and from 3 to 5% for cotinine at the concentration level of 1-60 microg/l (n = 4).     

Analyses of tobacco alkaloids by cation-selective exhaustive injection sweeping microemulsion electrokinetic chromatography

In this study, an on-line concentration method which coupled cation-selective exhaustive injection (CSEI) sweeping technology with microemulsion electrokinetic chromatography (MEEKC) was used to detect and analyze several tobacco alkaloids (nornicotine, anabasine, anatabine, nicotine, myosmine and cotinine) that are commonly found in various tobacco products. First, the effects of microemulsion compositions (oil, cosurfactant and solution pH) were examined in order to optimize the alkaloid separations in conventional MEEKC. The pH value and the injection length of basic plug were found to be the predominant influences on the alkaloid stacking. This optimal CSEI sweeping MEEKC method provided approximately 180- to 540-fold increase in detection sensitivity in terms of peak height without any loss in separation efficiency when compared to normal MEEKC separation. Furthermore, this proposed CSEI sweeping MEEKC method was applied successfully for the detection of the minor alkaloids nornicotine, anabasine and anatabine in tobacco products.     

Simultaneous determination of plasma nicotine and cotinine by UHPLC–MS/MS in C57BL/6 mice and its application in a pharmacokinetic study

Plasma concentrations of nicotine and its active metabolite cotinine are highly correlated with its biological effects. A UHPLC–MS/MS method was developed, validated and applied for nicotine and cotinine analysis in mice plasma. Chromatographic separation was achieved on a BEH HILIC column using acetonitrile (0.1% formic acid) and 10 mm ammonium formate as mobile phase. The gradient elution was performed at 0.4 mL/min with a run time of 3.6 min. The quantitative ion transition was m/z 163.1 > 130.0 for nicotine, m/z 177.1 > 80.0 for cotinine and m/z 167.1 > 134.0 for nicotine‐D₄ (internal standard, IS). For both nicotine and cotinine, the calibration range was 5–500 ng/mL with 5 ng/mL as the lower limit of quantitation, and the intra‐ and inter‐day bias and imprecision were ?4.61–12.00% and <11.12%. The IS normalized recovery was 90.62–98.95% for nicotine and 89.18–101.53% for cotinine, and the IS normalized matrix factor was 106.00–116.44% for nicotine and 100.34–109.85% for cotinine. Both nicotine and cotinine were stable under conventional storage conditions. The validated method has been applied to a pharmacokinetic study in mice to calculate the pharmacokinetic parameters for both analytes.     

Bestimmung kleiner Mengen Nicotin und Cotinin in tierischem Gewebe

The method permits the specific determination of nicotine and cotinine in the μg range in 2 g of tissue. Nicotine or cotinine are extracted from tissue homogenates with ether or chloroform and are separated from accompanying substances by thin-layer chromatography. The determination is performed spectrophotometrically by utilization of König's reaction. The recovery for both compounds is 90–95%. The procedure is especially suitable for investigating questions in connection with the reduction of nicotine by animal tissue.     

A simple,fast, and sensitive method for the measurement of serum nicotine,cotinine, and nornicotine by LC–MS/MS

The measurement of nicotine and its metabolites has been used to monitor tobacco use. A high‐sensitivity method (<1 ng/mL) is necessary for the measurement in serum or plasma to differentiate nonsmokers from passive smokers. Here, we report a novel LC–MS/MS method to quantify nicotine, cotinine, and nornicotine in serum with high sensitivity. Sample preparation involved only protein precipitation, followed by online turbulent flow extraction and analysis on a porous graphitic carbon column in alkaline conditions. The chromatography time was 4 min. No significant matrix effects or interference were observed. The lower limit of quantification was 0.36, 0.32, and 0.38 ng/mL for nicotine, cotinine, and nornicotine, respectively, while accuracy was 91.6–117.1%. No carryover was observed up to a concentration of 48 , 550, and 48 ng/mL for nicotine, cotinine, and nornicotine, respectively. Total CV was <6.5%. The measurement of nicotine and cotinine was compared with an independent LC–MS/MS method and concordant results were obtained. In conclusion, this new method was simple, fast, sensitive, and accurate. It was validated to measure nicotine, cotinine, and nornicotine in serum for monitoring tobacco use.     

Application of response surface methodology to vortex‐assisted dispersive liquid–liquid extraction for the determination of nicotine and cotinine in urine by gas chromatography–tandem mass spectrometry

A method of vortex‐assisted dispersive liquid–liquid extraction coupled with gas chromatography and tandem mass spectrometry for the determination of nicotine and cotinine in urine was developed. Response surface methodology was applied to obtain the optimum extraction conditions. In this method, Plackett–Burman design was utilized to evaluate the impact of five selected factors on pretreatment procedure. Then, three main factors were optimized using a Box–Behnken design. The optimized method showed good linearities at 1–2000 μg/L with correlation coefficients of 0.9998 for nicotine and 0.9986 for cotinine. Recovery was 91.4–106 and 91.7–108% for nicotine and cotinine, respectively. The intraday relative standard derivations of determination were 1.47–4.06% for nicotine and 0.41–3.16% for cotinine, and interday relative standard derivations were 3.03–6.70% for nicotine and 1.64–6.38% for cotinine. The method detection limits for nicotine and cotinine were 0.33 and 0.34 μg/L, respectively. A total of 87 urine samples from smokers and nonsmokers were tested with the proposed method. Urinary nicotine and cotinine were 23.0–6.67 × 10³ and 18.4–4.17 × 10³ μg/(g·cr) for smokers and 1.31–286 and 1.39–131 μg/(g·cr) for nonsmokers, respectively. The method is sensitive, suitable and reliable for the determination of nicotine and cotinine in urine and meets the requirements for evaluating short‐term tobacco exposure.     

A rapid LC-MS-MS method for the determination of nicotine and cotinine in serum and saliva samples from smokers: validation and comparison with a radioimmunoassay method

The development and validation of a rapid liquid chromatography (LC)-tandem mass spectrometry (MS-MS) method for determination of nicotine and cotinine in smokers' serum is described. The method is based on solid-phase extraction in a 96-well plate format and requires only 100 microL of serum. Using normal-phase chromatography, both analytes elute in less than 1 min, which permits high sample throughput applications. The calibrated range is 2-100 ng/mL nicotine and 20-1,000 ng/mL cotinine. For known samples, recovery is 95-116% for nicotine and 93-94% for cotinine. The method is extended to rat serum and human saliva (cotinine only) using partial validation techniques. When compared with an existing radioimmunoassay method in our laboratory, the LC-MS-MS method gives improved accuracy, precision, and sample throughput.     

Determination of Cotinine, 3′-Hydroxycotinine,and Their Glucuronides in Urine by Ultra-high Performance Liquid Chromatography

The measurement of the primary nicotine metabolites, cotinine and trans-3′-hydroxycotinine, is a useful biomarker of nicotine exposure and metabolism genetics for smoking cessation research. Herein is described an ultra-high performance liquid chromatography–tandem mass spectrometry method for the determination of these primary nicotine metabolites in urine. Urine samples were diluted one hundred-fold with water and introduced into an ultra-high performance liquid chromatography triple quadrupole mass spectrometer using positive ion electrospray ionization with multiple reaction monitoring. Levels of urinary nicotine metabolites: cotinine, trans-3′-hydroxycotinine, and their respective glucuronides were determined directly using deuterated internal standards and compared with indirect determination by enzymatic hydrolysis. The assay was applied to a community sample of smokers’ urine (n = 280). The assay demonstrated satisfactory performance (relative standard deviation of 1.6–6.5 percent at the 1000 nanograms per milliliter level and >98 percent recovery) suitable for application to smoking studies with a run time less than five minutes. The mean (min-max) levels of cotinine and cotinine-glucuronide were 968 (31-3831) and 976 (9-5607) nanograms per milliliter. The mean (min-max) levels of trans-3′-hydroxycotinine and trans-3′-hydroxycotinine-glucuronide were 3529 (13-21337) and 722 (0-4633) nanograms per milliliter. Direct determination of glucuronide metabolites was superior to indirect measurement using enzymatic hydrolysis, where there was evidence of loss of metabolites during sample preparation. A sensitive and selective ultra-high performance liquid chromatography–tandem mass spectrometry assay was successfully developed for the determination of cotinine, trans-3′-hydroxycotinine, and their glucuronides in urine. The rapid and simple sample preparation makes this assay suitable for high throughput studies involving nicotine metabolism phenotype for both cytochrome P450 2A6 and uridine 5′-diphospho-glucuronosyltransferase, smoking prevalence, and cessation studies.     

Optimization study for the reversed-phase ion-pair liquid chromatographic determination of nicotine in commercial tobacco products.

The availability of published methods for the determination of nicotine in commercial tobacco products based on state-of-the-art chromatographic methods is limited. Nicotine is a diprotic base with pKa's of 3.12 (pyridine ring) and 8.02 (pyrrolidine ring). Other monoprotic and diprotic bases are also present in commercial tobacco including anatabine, nornicotine, anabasine, and cotinine. In this paper, the chromatography of nicotine and the minor tobacco alkaloids under reversed-phase ion-pairing conditions is thoroughly studied. The results of this study are used to understand the retention mechanisms of the tobacco alkaloids, to examine their observed elution order with respect to fundamental analyte properties (size, functionality, and acid-base strength), and to select optimum chromatographic conditions for the determination of nicotine in commercial tobacco products.     

Characterisation of nicotine and related compounds using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their detection by liquid chromatography/electrospray ionisation mass spectrometry

Electrospray ionisation ion trap mass spectrometry (ESI-MS(n)) has been used to study the fragmentation patterns of nicotine and nine of its related compounds. From this study certain characteristic fragmentations are apparent with generally the pyrrolidine or piperidine ring being subject to chemical modifications. The structures of the product ions proposed for the ESI-MS(n) study have been supported by results from electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Compounds with pyrrolidine and piperidine rings that possess an unsubstituted N atom have been shown to lose NH(3) at the MS(2) stage. Those compounds with N-methyl groups lose CH(3)NH(2) at the MS(2) stage. The loss of NH(3) or CH(3)NH(2) leaves the corresponding rings opened and this is followed by ring closure at the pyridine-2 carbon atom. Mono-N-oxides fragment in a similar way but the di-N-oxide can also fragment by cleavage of the bond between the pyridine and pyrrolidine rings. Cotinine also can undergo cleavage of this bond between the rings.This data therefore provides useful information on how substituents and the nature of the non-pyridine ring can affect the fragmentation patterns of nicotine and its related compounds. This information can be used in the characterisation of these compounds by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) which results in the separation of nicotine and its related compounds with limits of detection (LODs) ranging from 15 to 105 ng/mL. The use of LC/ESI-MS to study nicotine-containing samples resulted in the simultaneous and unambiguous identification of seven of the compounds discussed in this paper: cotinine identified at retention time 12.5 min (with its [M+H](+) ion at m/z 177), nornicotine 16.0 min (m/z 149), anatabine 18.0 min (m/z 161), myosmine 18.5 min (m/z 147), anabasine 20.4 min (m/z 163), nicotine 22.2 min (m/z 163), and nicotyrine 31.4 min (m/z 159). For quality control of nicotine replacement therapy products, these nicotine impurities can be readily identified and determined at levels up to 0.3% for single impurities and up to 1.0% for total impurities.     

On-line molecularly imprinted solid-phase extraction for the selective spectrophotometric determination of nicotine in the urine of smokers

This work describes an on-line molecularly imprinted solid-phase extraction (MISPE) method for spectrophotometric determination of nicotine in urine samples of smokers. This method is based on manganese (VII) to manganese (VI) reduction in an alkaline medium, promoted by nicotine. Two wash solutions (1:4 (v/v) acetonitrile:sodium hydroxide - pH 11.4, and nitric acid - pH 2.5) were employed to circumvent interferences. Aqueous solutions containing nicotine plus different possible concomitants (cotinine, anabasine, norcotinine and caffeine) were tested individually. The analytical calibration curve was prepared in urine samples collected from non-smokers and spiked with nicotine standard from 1.1 to 60 μmol L⁻¹ (r² > 0.998). The limit of quantification and the analytical frequency were 1.1 μmol L⁻¹ and 11 h⁻¹, respectively. The precision, evaluated using 3, 10 and 30 μmol L⁻¹ nicotine in urine, was 10, 10 and 4% (intra-day precision) and 12, 13 and 5% (inter-day precision), respectively. Accuracy was checked through high performance liquid chromatography and the results did not present significant differences at the 95% confidence level according to the Student's t-test.     

Liquid chromatography/electrospray ionization tandem mass spectrometry assay for determination of nicotine and metabolites, caffeine and arecoline in breast milk

A procedure based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) is described for the determination of nicotine and its principal metabolites cotinine, trans-3-hydroxycotinine and cotinine-N-oxide, caffeine and arecoline in breast milk, using N-ethylnorcotinine as internal standard. Liquid/liquid extraction with chloroform/isopropanol (95:5, v/v) was used for nicotine, cotinine, trans-3-hydroxycotinine, cotinine-N-oxide and caffeine under neutral conditions and for arecoline under basic conditions. Chromatography was performed on a C(8) reversed-phase column using a gradient of 50 mM ammonium formate, pH 5.0, and acetonitrile as a mobile phase at a flow rate of 0.5 mL/min. Separated analytes were determined by electrospray ionization tandem mass spectrometry in the positive ion mode using multiple reaction monitoring. Limits of quantification were 5 microg/L for nicotine, cotinine, trans-3-hydroxycotinine, cotinine-N-oxide and caffeine, and 50 microg/L for arecoline using 1 mL human milk per assay. Calibration curves were linear over the calibration ranges for all the substances under investigation, with a minimum r(2) > 0.998. At three concentrations spanning the linear dynamic range of the assay, mean recoveries from breast milk ranged between 71.8 and 77.4% for different analytes. This method was applied to the analysis of analytes in human milk to assess substance exposure in breast-fed infants in relation to eventual clinical outcomes. This LC/MS/MS assay provides adequate sensitivity and performance characteristics for the simultaneous quantification of biomarkers of three of the drugs most commonly used worldwide (tobacco, caffeine and areca nut).     

Medical swab touch spray‐mass spectrometry for newborn screening of nicotine and cotinine in meconium

Newborn screening is one of public health concerns designed to screen infants shortly after birth. Prenatal exposure to tobacco smoke such as nicotine has been reported to affect babies. Levels of nicotine and cotinine in meconium were widely used to evaluate the tobacco exposure of foetuses during pregnancy in a polluted environment. In this study, medical swabs were applied by using touch spray‐mass spectrometry (TS‐MS) to collect meconium from newborn infants for detection of nicotine and cotinine. Parameters such as choice of spray solvents, solvent volume and collision energy for screening of nicotine and cotinine were optimized. The limits of detection, reproducibility and matrix effect for analysis of meconium were also investigated. In this study, the levels of nicotine and cotinine in 54 puerpera volunteers were screened by TS‐MS and were validated by using traditional liquid chromatography‐mass spectrometry. These results showed that medical swab TS‐MS would be useful for newborn screening of nicotine and cotinine in meconium with high reproducibility, speed, sensitivity and specificity. The use of disposable medical swabs involves no sample preparation and no chromatographic separation, significantly reducing the cost and time required for screening a large number of clinical sample. Copyright © 2016 John Wiley & Sons, Ltd.     

Application of liquid separation techniques to the determination of the main urinary nicotine metabolites

A rapid procedure for the analysis of the main nicotine metabolites (cotinine, trans-3′-hydroxycotinine) in urine has been worked out. The procedure includes isolation of nicotine and its metabolites from urine by means solid–liquid extraction technique using resin Amberlite XAD-2 and then quantitation by the use of thin-layer chromatography with densitometry (in reflection mode). GC–MS was applied to confirm the results obtained by TLC. The procedure was applied to the analysis of cotinine concentrations in urine samples taken from children living in Upper Silesia region (Poland). Among 444 investigated children we did not find cotinine almost in 60% but in 15% of this population, there were children who could have been exposed to cigarette smoke.     

Improved gas chromatographic method for the determination of nicotine and cotinine in biologic fluids

Improved methods have been developed for the determination of nicotine and its major metabolite, cotinine, in blood, plasma, and urine samples. These methods utilize gas chromatography with alkali flame ionization (nitrogen--phosphorus) detection and structural analogs of nicotine and cotinine as internal standards.     

Review of HPLC–MS methods for the analysis of nicotine and its active metabolite cotinine in various biological matrices

In recent years, tobacco smoking is a risk factor for a series of diseases, including cardiovascular diseases, cerebrovascular diseases, and cancers. Nicotine, the primary component of tobacco smoke, is mainly transformed to its active metabolite cotinine, which is often used as a biomarker for tobacco exposure for its higher blood concentration and longer residence time than nicotine. Various analytical methods have been developed for the determination of nicotine and cotinine in biological matrices. This article reviewed the HPLC–MS based methods for nicotine and/or cotinine analysis in various biological matrices. The sample preparation, mass and chromatographic conditions, and method validation results of these methods have been summarized and analyzed. The sample was mainly pretreated by protein precipitation and/or extraction. Separation was achieved using methanol and/or acetonitrile:water (with or without ammonium acetate) on C18 columns and acetonitrile:water (with formic acid, ammonium acetate/formate) on HILIC columns. Nicotine-d3, nicotine-d4, and cotinine-d3 were commonly used internal standards (ISs). Other non-deuterated ISs such as ritonavir, N-ethylnorcotinine, and milrinone were also used. For both nicotine and cotinine, the calibration range was 0.005–35,000 ng/mL, the matrix effect was 75.96–126.8%, and the recovery was 53–124.5%. The two analytes were stable at room temperature for 1–10 days, at −80°C for up to 6 months, and after three to six freeze–thaw cycles. Comedications did not affect nicotine and cotinine analyses.     

A simple, sensitive, and rapid method for the determination of cotinine in urine by high-performance liquid chromatography with UV detection

Cotinine levels in biological fluids are a reliable indicator of the presence of nicotine. In this paper, a simple and sensitive high-performance liquid chromatography (HPLC) procedure for the determination of cotinine in urine following liquid-liquid extraction with dichloromethane in an alkaline medium is described. Calibration curves show linearity over the 50 to 3000 ng/mL range with low intra- and interday variability as well as good selectivity and specificity. No solid-phase extraction is performed because the liquid dichloromethane extraction step yields excellent results. This method is a good alternative for routine analysis of urinary cotinine in laboratories where gas chromatography or HPLC-mass spectrometry is not available.