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.     

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.     

Optimization and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine,cotinine, <Emphasis Type="Italic">trans</Emphasis>-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.     

Occurrence,fate and determination of tobacco (nicotine) and alcohol (ethanol) residues in waste- and environmental waters

This review includes one hundred and two peer reviewed papers that focus on metabolic residues of the two most used licit drugs globally, nicotine (nicotine, cotinine, trans-3’-hydroxycotinine – HCOT) and alcohol (ethyl sulphate and ethyl glucuronide), in waste- and environmental waters. Sampling strategies and analytical methods are also summarised and discussed. Although grab sampling is the most widely applied method for collecting environmental samples (74% cases), wastewater samples are typically composite samples collected automatically at the wastewater treatment plants (66% cases). Sample preparation and analysis usually include solid-phase extraction (SPE) followed by reverse-phased liquid chromatography with tandem mass spectrometry detection (RP-LC-MS/MS) for nicotine residues. In contrast, alcohol residues are commonly determined via direct injection onto the LC-MS/MS using an ion-pair reagent to improve retention, leaving room for method improvement, e.g., introducing a suitable extraction procedure to achieve lower detection limits and quantification. In comparison to alcohol residues, more studies look into nicotine residues (85% of the studies). Concentration ranges for nicotine, cotinine, HCOT and ethyl sulphate were < 424,000, < 42,300, 50–52,000 and 500–33,000 ng/L in wastewater influents and 15–32,000, < 18,000, 15–1,552 and < 500 ng/L in effluents, while nicotine (12.6–947 ng/L) and cotinine (17–62 ng/L) were detected in reclaimed waters. Among environmental waters, the highest concentrations of nicotine residues were measured in surface waters (nicotine: < 9,340 ng/L, cotinine: < 6,582 ng/L and HCOT: 14–777 ng/L), while their concentrations in groundwater and drinking water were generally in the low ng/L range. This review also reveals the discrepancy between the number of studies in developed countries (90%) compared to developing countries and the need for more studies in the former, where most wastewater flows untreated into the environment.     

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.     

Simultaneous determination of atractylenolide II and atractylenolide III by liquid chromatography-tandem mass spectrometry in rat plasma and its application in a pharmacokinetic study after oral administration of Atractylodes Macrocephala Rhizoma extract

Atractylenolide II (AII) and atractylenolide III (AIII) are the major active components in Atractylodes Macrocephala Rhizoma (AMR). In this study, a sensitive, rapid and selective liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous determination of AII and AIII in rat plasma using loliolide as internal standard (IS). After protein precipitation with ethyl acetate, the analytes were injected into an LC‐MS/MS system for quantification. Chromatography was performed using a C₁₈ column, eluting with water and acetonitrile (45:55, v/v) at 0.2 mL/min. All analytes including IS were monitored under positive ionization conditions by multiple reaction monitoring with an electrospray ionization source. The validated method was successfully applied to the pharmacokinetic study of AII and AIII in rat plasma after oral administration of AMR extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine. Copyright © 2012 John Wiley & Sons, Ltd.     

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).     

Determination of abamectin, doramectin, emamectin, eprinomectin, ivermectin, and moxidectin in milk by liquid chromatography electrospray tandem mass specrometry

The avermectin and milbemycin families of compounds are derived from naturally occurring yeasts. They have proven to be potent preventatives against a variety of pests such as insects and parasites. Only eprinomectin and moxidectin are currently approved for use on lactating cattle with tolerances in milk of 12 microg/kg for eprinomectin and 40 microg/kg for moxidectin. Detection of misuse or inadvertent contamination in milk requires a sensitive and definitive analytical method. A method has been developed for the determination of 5 avermectins and 1 milbemycin in milk using a simple liquid-liquid extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Ivermectin (IVR), doramectin (DOR), abamectin (ABA), eprinomectin (EPR), emamectin (EMA), and moxidectin (MOX) were extracted from whole milk by partitioning into acetonitrile with a subsequent solvent exchange into methanol-water. Simultaneous confirmation and quantification were achieved with LC separation, positive electrospray ionization (ESI+), and MS/MS. The limits of detection ranged from 16 pg/g (ppt) for EMA to 1.7 microg/g (ppb) for MOX.     

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.     

Detection and validated quantification of toxic alkaloids in human blood plasma--comparison of LC-APCI-MS with LC-ESI-MS/MS

Poisonings with toxic plants may occur after abuse, intentional or accidental ingestion of plants. For diagnosis of such poisonings, multianalyte procedures were developed for detection and validated quantification of the toxic alkaloids aconitine, atropine, colchicine, coniine, cytisine, nicotine and its metabolite cotinine, physostigmine, and scopolamine in plasma using LC-APCI-MS and LC-ESI-MS/MS. After mixed-mode solid-phase extraction of 1 ml of plasma, the analytes were separated using a C8 base select separation column and gradient elution (acetonitrile/ammonium formate, pH 3.5). Calibration curves were used for quantification with cotinine-d(3), benzoylecgonine-d(3), and trimipramine-d(3) as internal standards. The method was validated according to international guidelines. Both assays were selective for the tested compounds. No instability was observed after repeated freezing and thawing or in processed samples. The assays were linear for coniine, cytisine, nicotine and its metabolite cotinine, from 50 to 1000 ng/ml using LC-APCI-MS and 1 to 1000 ng/ml using LC-ESI-MS/MS, respectively, and for aconitine, atropine, colchicine, physostigmine, and scopolamine from 5 to 100 ng/ml for LC-APCI-MS and 0.1 to 100 ng/ml for LC-ESI-MS/MS, respectively. Accuracy ranged from -38.6 to 14.0%, repeatability from 2.5 to 13.5%, and intermediate precision from 4.8 to 13.5% using LC-APCI-MS and from -38.3 to 8.3% for accuracy, from 3.5 to 13.8%, for repeatability, and from 4.3 to 14.7% for intermediate precision using LC-ESI-MS/MS. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments. With the exception of the greater sensitivity and higher identification power, LC-ESI-MS/MS had no major advantages over LC-APCI-MS. Both presented assays were applicable for sensitive detection of all studied analytes and for accurate and precise quantification, with the exception of the rather volatile nicotine. The applicability of the assays was demonstrated by analysis of plasma samples from suspected poisoning cases.     

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.     

Quantification of cyclophosphamide and its metabolites in urine using liquid chromatography/tandem mass spectrometry

A reliable and easy to use liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantification of urinary concentrations of cyclophosphamide (CP) and its main metabolites excreted in urine, i.e. N-dechloroethylcyclophosphamide (DCL-CP), 4-ketocyclophosphamide (4KetoCP), and carboxyphosphamide (CarboxyCP). Sample preparation consisted of dilution of urine with an aqueous solution of the internal standard D(4)-CP and methanol, and centrifugation. LC/MS/MS detection was performed using a triple-quadrupole mass spectrometer working in selected reaction monitoring mode. All analytes were quantified in a single run within 11.5 min. The limits of detection were 5 ng/mL for CP and 4KetoCP, 1 ng/mL for DCL-CP, and 30 ng/mL for CarboxyCP. Quantification ranges were adjusted to the expected concentrations in 24-h urine collections of patients treated with a polychemotherapy regimen (3-175 microg/mL for CP, 0.5-27 microg/mL for 4KetoCP and 0.17-9 microg/mL for CarboxyCP and DCL-CP, respectively). The method was validated according to international guidelines of the ICH and the FDA.     

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.     

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.     

Simultaneous determination of nicotine and its metabolite, cotinine, in rat blood and brain tissue using microdialysis coupled with liquid chromatography: pharmacokinetic application

To elucidate the disposition of nicotine in the brain is important because the neuropharmacological effects from nicotine exposure are centrally predominated. The aim of the present study was to develop a rapid and simple method for the simultaneous determination of unbound nicotine and its main metabolite, cotinine, in rat blood and brain tissue. We coupled a multiple sites microdialysis sampling technique with HPLC-UV system to characterize the pharmacokinetics of both nicotine and cotinine. Microdialysis probes were inserted into the jugular vein/right atrium and brain striatum of Sprague-Dawley rats, and nicotine (2 mg/kg, i.v.) was administered via the femoral vein. Dialysates were collected every 10 min and injected directly into a HPLC system. Both nicotine and cotinine were separated by a phenyl-hexyl column (150 mm x 4.6 mm) from dialysates within 12 min. The mobile phase consisted of an acetonitrile-methanol-20 mM monosodium phosphate buffer (55:45:900, v/v/v, pH adjusted to 5.1) with a flow-rate of 1 ml/min. The wavelength of the UV detector was set at 260 nm. The limit of quantification for nicotine and cotinine were 0.25 microg/ml and 0.05 microg/ml, respectively. Intra- and inter-day precision and accuracy of both measurements fell well within the predefined limits of acceptability. The blood and brain concentration-time profile of nicotine and cotinine suggests that nicotine is easily to get into the central nervous system and cotinine exhibits a long retention time and accumulates in blood.     

Simultaneous determination of seventeen glucocorticoids residues in milk and eggs by ultra-performance liquid chromatography/electrospray tandem mass spectrometry

A comprehensive analytical method has been developed and validated for the simultaneous determination of seventeen glucocorticoid residues in eggs and milk. The mass spectrometer parameters, the composition of the mobile phase and the sample preparation method were firstly optimized to obtain maximum sensitivity. The samples were deconjugated with beta-glucuronidase/arylsulfatase enzyme and concentrated using an Oasis HLB solid-phase extraction cartridge, followed by cleanup with a dual Sep-pak silica and aminopropyl cartridge. The analytes were quantified by ultra-performance liquid chromatography (using a C18 column)/electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) operating in the negative ion mode. The assay for the 17 glucocorticoids was linear over the range of 1-200 microg/L for milk and egg samples with a high correlation coefficient (>0.99). The limits of quantification (LOQs) for the target analytes were 0.04-1.27 microg/kg for the egg samples and 0.03-0.73 microg/kg for the milk samples. The average extraction recoveries of the glucocorticoids from eggs and milk at two concentration levels (spiked at 0.40 and 2.00 microg/kg) were 65.6-118.7% and 61.5-119.6%, respectively, with relative standard deviations between 1.8-17.0% and 2.4-18.4%, respectively. Because of its high sensitivity, good precision and specificity, the method was found to be suitable for trace analysis of synthetic and natural glucocorticoids in complex biosamples such as eggs and milk.     

Simultaneous Determination of Nicotine and Its Nine Metabolites in Human Urine by LC–MS–MS

A method based on liquid chromatography tandem mass spectrometry was developed for the direct determination of nicotine, cotinine, trans-3′-hydroxycotinine, their corresponding glucuronide conjugates as well as nornicotine, norcotinine, cotinine-N-oxide and nicotine-N′-oxide in the urine of smokers. The assay only involves centrifugation and filtration of diluted urine. The analysis was performed on a C18 reversed-phase column using a gradient of 10 mM ammonium acetate, pH 6.8, and methanol as mobile phase at a flow rate of 1 mL min^?1. Nicotine-methyl-d₃, Cotinine-methyl-d₃ and trans-3′-hydroxycotinine-methyl-d₃ were used as internal standards. Precisions (RSD) for all the analytes at three levels were between 2.1 and 17.0%. Recoveries for nicotine and nine nicotine metabolites ranged from 78.4 to 115.6%. The described method was suitable for determining the nicotine dose in large-scale human biomonitoring studies.     

Determination of three natural pesticides in processed fruit and vegetables using high-performance liquid chromatography/tandem mass spectrometry

This paper describes a method for the sensitive and selective determination of two macrocyclic lactones (abamectin and spinosad) and azadirachtin in apple purée, concentrated lemon juice, tomato purée and canned peas. The general sample extraction-partitioning method for our gas chromatography and liquid chromatography multiresidue methods has been used. The analytical procedure involves an extraction with acetone and liquid-liquid partitioning with ethyl acetate/cyclohexane combined in one step. The extracts are analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) without any further clean-up step. The pesticides are separated on a reversed-phase C12 column using a gradient elution. Thirteen simultaneous MS/MS transitions of precursor ions were monitored. Studies at fortification levels of 2.5-10 microg/kg and 25-100 microg/kg gave mean recoveries ranging from 70-100% for all compounds with satisfactory precision (relative standard deviation (RSD) from 3-20%). The excellent selectivity and sensitivity allows quantification and identification of low levels of pesticides in canned peas, tomato and apple purées (limits of quantitation (LOQs) 1-5 microg/kg) and in concentrated lemon juice (LOQs 2-10 microg/kg). The quantification of analytes was carried out using the most sensitive transition for every compound and by 'matrix-matched' standards calibration.     

Determination of cefuroxime in human serum or plasma by liquid chromatography with electrospray tandem mass spectrometry

Cefuroxime is a second-generation cephalosporin used against different kinds of bacterial infections. To be able to optimize the dosing it is necessary to characterize the pharmacokinetics of cefuroxime which requires a selective and sensitive analytical method for cefuroxime in plasma or serum. A new rapid liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) method, using cefotaxime as internal standard, was developed for analysis of cefuroxime in human serum. The work-up procedure consisted of protein precipitation with acetonitrile/cefotaxime, and after centrifugation the supernatant was dissolved in mobile phase. The sample was injected on a SB-CN column and the detection was performed using tandem mass spectrometry (MS/MS). The limit of quantification was determined to 0.025 microg/mL. The method was linear in the range 0.025-50 microg/mL with a coefficient of correlation >0.999. The limit of quantification and intra-day variability were found to be the same for plasma samples, which indicates that the method is valid for serum as well as plasma samples.     

Sensitive and rapid method for the determination of urinary cotinine in non-smokers: an application for studies assessing exposures to second hand smoke (SHS)

We describe a sensitive and rapid method to assay urinary cotinine levels among non-smokers using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) and its application in studies assessing exposures to second hand smoke (SHS). Cotinine was initially extracted from 1 ml of urine with methylene chloride by using a liquid-liquid extraction Chem Elut™ column. The extracted sample was further separated by using a BetaBasic C18 column (1 mm × 150 mm, 3 μm) with isocratic elution (60:40 acetonitrile and 5 mM ammonium acetate at pH 5), and then examined using a triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in multiple-reaction-monitoring (MRM) mode. The elution of cotinine from the LC column took approximately 2.3 min and the detection of cotinine by ESI/MS/MS provided a limit of detection (LOD) of 0.3 ng/ml. The ESI/MS/MS detection was able to easily distinguish between cotinine and nicotine. This method, validated using a cotinine concentration range from 0.8 to 102.4 ng/ml, was successfully applied in a cross-sectional study examining differences in levels and sources of second hand smoke (SHS) exposure among non-smokers. Self-reported measures of SHS exposure were significantly associated with urinary cotinine levels. This urinary cotinine assay using LC-ESI/MS/MS provides a robust, high throughput and very sensitive method for the evaluation of SHS exposure for use in epidemiologic and clinical research studies.