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.     

Hair analysis of nicotine and cotinine for evaluating tobacco smoke exposure by liquid chromatography-mass spectrometry

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the determination of nicotine and cotinine in human hair was established. In the procedure, a hair sample (10 mg) was washed with dichloromethane and digested in 2.5 M sodium hydroxide. The digest was extracted with dichloromethane and then 25 mM hydrochloric acid in methanol was added to the extract, to prevent loss of analytes. The solution was evaporated and redissolved in the mobile phase, methanol/10 mM ammonium acetate (30/70, v/v). A 20 microL aliquot of redissolved solution was subjected to analysis. Nicotine and cotinine in human hair were quantified by using deuterated analytes as internal standards. The quantification limits were 8 microg/L for nicotine and 0.9 microg/L for cotinine. The proposed method was applied to measure the concentrations of nicotine and cotinine in hair of smokers and non-smokers to evaluate their self-reported smoking and exposure to environmental tobacco smoke. In both cases, the method provided good selectivity, accuracy and precision.     

A new quantitative thermospray LC-MS method for nicotine and its metabolites in biological fluids

A rapid thermospray liquid chromatography-mass spectrometry (TSP LC-MS) method is described for the simultaneous determination of nicotine and 17 of its metabolites. Chemical ionization of nicotine and its metabolites separated by reversed-phase HPLC is achieved by postcolumn addition of ammonium acetate buffer with the filament of the ion source turned off. Quantification is accomplished by selectively monitoring the unique protonated molecular ion of each metabolite. Trideuterated cotinine serves as an internal standard. Linear responses for cotinine, demethylcotinine, and trans-3'-hydroxycotinine were observed over a concentration range of 20-8000 ng/mL, and 80-8000 ng/ml for nicotine and nicotine-1'-N-oxide. Of the 17 metabolites examined, only nicotine, cotinine, demethylcotinine, and trans-3'-hydroxycotinine were detected in smokers' urine.     

亲水作用色谱-串联质谱测定尿液中尼古丁和可替宁

建立了测定人尿液中尼古丁和可替宁含量的亲水作用色谱-串联质谱(HILIC-MS/MS)方法。尿样加入尼古丁-d4和可替宁-d3同位素内标后,用水稀释10倍,经过滤后的滤液由超高效液相色谱-串联质谱(UPLC-MS/MS)进行分离分析。采用ACQUITY UPLC~BEH HILIC色谱柱(50 mm×3.0 mm,1.7μm),以甲醇和体积分数为0.1%的氨水为流动相,流速为0.2 mL/min,在电喷雾电离源正离子模式下测定尿液中尼古丁和可替宁的含量,用标准曲线法定量。尼古丁和可替宁在1.0~1 000μg/L范围内线性关系良好,相关系数分别为0.994 9和0.995 8;检出限分别为0.082μg/L和0.077μg/L;定量限分别为0.27μg/L和0.26μg/L;加标回收率分别为90.4%~103.5%和93.0%~104.6%;相对标准偏差分别为4.80%~6.21%和4.22%~7.15%。应用所建立的方法测定了200份尿样,结果表明,吸烟人群尿中尼古丁含量为26.68~854.30μg/L,可替宁含量为36.66~1 191.18μg/L(n=86,M_(nicotine)=76.00μg/L,M_(nicotine)=83.52μg/L,M为中位数);非吸烟人群尿中尼古丁含量为5.08~69.66μg/L,可替宁含量为3.16~28.21μg/L(n=114,Mnicotine=7.53μg/L,M_(nicotine)=3.79μg/L)。该方法快速灵敏,操作简单,适用于尿样中尼古丁和可替宁的批量测定,能满足烟草暴露评价的需要。     

Simultaneous determination of cotinine and trans‐3‐hydroxycotinine in urine by automated solid‐phase extraction using gas chromatography–mass spectrometry

A gas chromatography–mass spectrometry method was developed and validated for the simultaneous automated solid‐phase extraction and quantification of cotinine and trans‐3‐hydroxycotinine in human urine. Good linearity was observed over the concentration ranges studied (R² > 0.99). The limit of quantification was 10 ng/mL for both analytes. The limits of detection were 0.06 ng/mL for cotinine (COT) and 0.02 ng/mL for trans‐3‐hydroxycotinine (OH‐COT). Accuracy for COT ranged from 0.98 to 5.28% and the precision ranged from 1.24 to 8.78%. Accuracy for OH‐COT ranged from ?2.66 to 3.72% and the precision ranged from 3.15 to 7.07%. Mean recoveries for cotinine and trans‐3‐hydroxycotinine ranged from 77.7 to 89.1%, and from 75.4 to 90.2%, respectively. This analytical method for the simultaneous measurement of cotinine and trans‐3‐hydroxycotinine in urine will be used to monitor tobacco smoking in pregnant women and will permit the usefulness of trans‐3‐hydroxycotinine as a specific biomarker of tobacco exposure to be determined. © 2014 The Authors. Biomedical Chromatography published by John Wiley & Sons Ltd.     

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.     

Determination of the nicotine metabolite trans-3'-hydroxycotinine in urine of smokers using gas chromatography with nitrogen-selective detection or selected ion monitoring.

A gas chromatographic method for the determination of the nicotine metabolite trans-3'-hydroxycotinine is described. The method involves conversion of the metabolite to the tert.-butyldimethylsilyl derivative, chromatography on a fused-silica capillary column, and determination using nitrogen-phosphorus detection or electron ionization mass spectrometry with selected ion monitoring. A structural analogue, trans-3-hydroxy-1-methyl-5-(2-pyridyl)pyrrolidin-2-one (trans-3'-hydroxy-ortho-cotinine), was used as an internal standard. Using selected ion monitoring, good precision and accuracy were obtained for determination of trans-3'-hydroxycotinine in urine over the concentration range 10-10,000 ng/ml. There was a good correlation between concentrations determined by selected ion monitoring and by nitrogen-phosphorus detection in urine of smokers, although low concentrations determined using nitrogen-phosphorus detection tended to be somewhat higher, suggesting some interference from urinary constituents. Concentrations and 24-h excretion of trans-3'-hydroxycotinine in the urine of 22 cigarette smokers are reported and compared to concentrations and excretion of nicotine, cotinine, nicotine 1'-N-oxide, nornicotine, and cotinine N-oxide.     

A simple and sensitive assay for the quantitative analysis of paclitaxel and metabolites in human plasma using liquid chromatography/tandem mass spectrometry

A sensitive and specific LC-MS/MS assay for the determination of paclitaxel and its 3'p- and 6-alpha-hydroxy metabolites is presented. A 200 microL plasma aliquot was spiked with a 13C6-labeled paclitaxel internal standard and extracted with 1.0 mL tert-butylmethylether. Dried extracts were reconstituted in 0.1 M ammonium acetate-acetonitrile (1:1, v/v) and 25 microL volumes were injected onto the HPLC system. Separation was performed on a 150 x 2.1 mm C18 column using an alkaline eluent (10 mm ammonium hydroxide-methanol, 30:70, v/v). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range for paclitaxel from 0.25 to 1000 ng/mL and metabolites from 0.25 to 100 ng/mL using 200 microL human plasma samples. Validation results demonstrate that paclitaxel and metabolite concentrations can be accurately and precisely quantified in human plasma. This assay is now used to support clinical pharmacologic studies with paclitaxel.     

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.     

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.     

Simultaneous determination of cotinine and trans-3'-hydroxycotinine in human serum by high-performance liquid chromatography.

A high-performance liquid chromatographic method with ultraviolet photometric detection has been developed for the quantitation of cotinine and trans-3'-hydroxycotinine in human serum. A solid-phase extraction procedure was performed for the analytes and the internal standard, N-ethylnorcotinine, before chromatography. The use of a 30-cm reversed-phase column and a mobile phase of water-methanol-0.1 M sodium acetate-acetonitrile (67:24.5:6.5:2, v/v), pH 4.3, prevented the co-elution of caffeine with cotinine. The limit of quantitation observed with this method was 5 ng/ml for both cotinine and trans-3'-hydroxycotinine. The present method proved useful for the determination of serum levels of these metabolites, correlating with nicotine daily intake.     

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 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).     

High-performance liquid chromatographic determination of pyridostigmine bromide, nicotine, and their metabolites in rat plasma and urine

This study reports on the development of a rapid and simple method for the determination of the antinerve agent drug pyridostigmine bromide (3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) (PB), its metabolite N-methyl-3-hydroxypyridinium bromide, nicotine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidine), and its metabolites nornicotine (2-(3-pyridyl)pyrrolidine) and cotinine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidone) in rat plasma and urine. The compounds are extracted and eluted by methanol and acetonitrile using C18 Sep-Pak cartridges and separated using high-performance liquid chromatography by a gradient of methanol, acetonitrile, and water (pH 3.2) at a flow rate of 0.8 mL/min in a period of 14 min. UV detection was at 260 nm for nicotine and its metabolites and at 280 nm for PB and its metabolite. The limits of detection ranged between 20 and 70 ng/mL, and the limits of quantitation were 50-100 ng/mL. The average percent recovery of five spiked plasma samples were 85.7 +/- 7.3%, 80.4 +/- 5.8%, 78.9 +/- 5.4%, 76.7 +/- 6.4%, and 79.7 +/- 5.7% and for urine were 85.9 +/- 5.9%, 75.5 +/- 6.9%, 82.6 +/- 7.9%, 73.6 +/- 5.9%, and 77.7 +/- 6.3% for nicotine, nornicotine, cotinine, PB, and N-methyl-3-hydroxypyridinium bromide, respectively. The calibration curves for standard solutions of the compounds of peak areas and concentration are linear for a range between 100 and 1,000 ng/mL. This method is applied in order to analyze the previously mentioned chemicals and metabolites following their oral administration in rats.     

Application of HPLC-QQQ-MS/MS and New RP-HPLC-DAD System Utilizing the Chaotropic Effect for Determination of Nicotine and Its Major Metabolites Cotinine,and trans-3′-Hydroxycotinine in Human Plasma Samples

The routine techniques currently applied for the determination of nicotine and its major metabolites, cotinine, and trans-3′-hydroxycotinine, in biological fluids, include spectrophotometric, immunoassays, and chromatographic techniques. The aim of this study was to develop, and compare two new chromatographic methods high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS), and RP-HPLC enriched with chaotropic additives, which would allow reliable confirmation of tobacco smoke exposure in toxicological and epidemiological studies. The concentrations of analytes were determined in human plasma as the sample matrix. The methods were compared in terms of the linearity, accuracy, repeatability, detection and quantification limits (LOD and LOQ), and recovery. The obtained validation parameters met the ICH requirements for both proposed procedures. However, the limits of detection (LOD) were much better for HPLC-QQQ-MS/MS (0.07 ng mL⁻¹ for trans-3′-hydroxcotinine; 0.02 ng mL⁻¹ for cotinine; 0.04 ng mL⁻¹ for nicotine) in comparison to the RP-HPLC-DAD enriched with chaotropic additives (1.47 ng mL⁻¹ for trans-3′-hydroxcotinine; 1.59 ng mL⁻¹ for cotinine; 1.50 ng mL⁻¹ for nicotine). The extraction efficiency (%) was concentration-dependent and ranged between 96.66% and 99.39% for RP-HPLC-DAD and 76.8% to 96.4% for HPLC-QQQ-MS/MS. The usefulness of the elaborated analytical methods was checked on the example of the analysis of a blood sample taken from a tobacco smoker. The nicotine, cotinine, and trans-3′-hydroxycotinine contents in the smoker’s plasma quantified by the RP-HPLC-DAD method differed from the values measured by the HPLC-QQQ-MS/MS. However, the relative errors of measurements were smaller than 10% (6.80%, 6.72%, 2.04% respectively).     

烟草与尿样中的代谢标记物

卷烟烟气进入人体后会对人体的代谢物产生影响,目前研究较多的是稠环芳烃类、烟草特有亚硝胺类和尼古丁的代谢物。文章分别介绍了稠环芳烃类代谢物1-羟基芘,烟草特有亚硝胺类代谢物NNAL和NNAL-Gluc,尼古丁的代谢物可天宁,对这些代谢物的检测方法、体内的含量水平、各种条件的影响进行了综述。     

Simultaneous determination of morphine‐6‐d‐glucuronide,morphine‐3‐d‐glucuronide and morphine in human plasma and urine by ultra‐performance liquid chromatography–tandem mass spectrometry: Application to M6G injection pharmacokinetic study

A robust ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the determination of morphine‐6‐d ‐glucuronide (M6G), morphine‐3‐d ‐glucuronide (M3G) and morphine (MOR) in human plasma and urine has been developed and validated. The analytes of interest were extracted from plasma by protein precipitation. The urine sample was prepared by dilution. Both plasma and urine samples were chromatographed on an Acquity UPLC HSS T₃ column using gradient elution. Detection was performed on a Xevo TQ‐S tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. Matrix interferences were not observed at the retention time of the analytes and internal standard, naloxone‐D5. The lower limits of quantitation of plasma and urine were 2/0.5/0.5 and 20/4/2 ng/mL for M6G/M3G/MOR, respectively. Calibration curves were linear over the concentration ranges of 2–2000/0.5–500/0.5–500 and 20–20,000/4–4000/2–2000 ng/mL for M6G/M3G/MOR in plasma and urine samples, respectively. The precision was <7.14% and the accuracy was within 85–115%. Furthermore, stability of the analytes at various conditions, dilution integrity, extraction recovery and matrix effect were assessed. Finally, this quantitative method was successfully applied to the pharmacokinetic study of M6G injection in Chinese noncancer pain patients.     

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).     

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.     

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.