A new liquid chromatography/mass spectrometry method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine

4‐(Methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) is a carcinogenic nitrosamine produced upon curing tobacco. It is present in tobacco smoke and undergoes metabolism to 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL) in the lungs. NNAL undergoes further uridine diphosphate glucuronosyltransferase (UGT)‐mediated metabolism to give N‐ and O‐glucuronide metabolites, which together with free (non‐conjugated) NNAL are then excreted in the urine. The ability to conduct validated analyses of free and conjugated NNAL in human urine is important in order to assess inter‐individual differences in lung cancer risk from exposure to cigarette smoke. The use of stable isotope dilution (SID) methodology in combination with liquid chromatography/multiple reaction monitoring/mass spectrometry (LC/MRM‐MS) provides the highest bioanalytical specificity possible for such analyses. We describe a novel derivatization procedure, which results in the formation of a pre‐ionized N‐propyl‐NNAL derivative. The increased LC/MS sensitivity arising from this derivative then makes it possible to analyze free NNAL in only 0.25 mL urine. This substantial reduction in urine volume when compared with other methods that have been developed will help preserve the limited amounts of stored urine samples that are available from on‐going longitudinal biomarker studies. The new high sensitivity SID LC/MRM‐MS assay was employed to determine free and conjugated NNAL concentrations in urine samples from 60 individual disease‐free smokers. Effects of inter‐individual differences in urinary creatinine clearance on NNAL concentrations were then assessed and three metabolizer phenotypes were identified in the 60 subjects from the ratio of urinary NNAL glucuronides/free NNAL. Poor metabolizers (PMs, 14 subjects) with a ratio of NNAL glucuronides/free NNAL <2 (mean = 1.3), intermediate metabolizers (IMs, 36 subjects) with a ratio between 2 and 5 (mean = 3.4), and extensive metabolizers (EMs, 10 subjects) with a ratio >5 (mean = 11.1). Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid quantitation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in rat urine using ultra-fast liquid chromatography mass spectrometry (UFLC/MS/MS)

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a component of tobacco smoke and is rapidly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Limited information is available on the relative systemic exposures resulting from NNK administration via the oral, intraperitoneal injection, and inhalation routes. Moreover, there is a need for a rapid method for simultaneous quantitative analyses of NNK and NNAL in rat urine. We developed a method based on Ultra Fast Liquid Chromatography Mass Spectrometry (UFLC/MS/MS) for the extraction and analysis of the potent lung carcinogens NNK and NNAL. Following addition of synthetic labeled internal standards, urine was introduced to 96 well plate Evolute^® Express CX 30?mg solid phase extraction system. The eluates were dried under vacuum and reconstituted in mobile phase before injecting to the LC system. The use of UFLC allowed for a 7.1?min run time. The precision and accuracy of the samples was 1.2-6.6% relative standard deviation (%RSD) and 91-113% of the concentration added, respectively. The limits of detection for NNK and NNAL were 70 and 3.0?pg/mL, respectively. The selectivity and sensitivity of this method improves the ability to measure these compounds at low concentrations and greatly facilitate toxicological studies of the NNK and NNAL.     

Development, validation and transfer of a hydrophilic interaction liquid chromatography/tandem mass spectrometric method for the analysis of the tobacco-specific nitrosamine metabolite NNAL in human plasma at low picogram per milliliter concentrations

A highly sensitive bioanalytical method based on a simple liquid/liquid extraction and hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS) analysis has been developed, validated and transferred for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a tobacco-specific nitrosamine metabolite. Deuterated NNAL (NNAL-d(4)) was synthesized and used as the internal standard. This method can be used for the analysis of free and total NNAL (free NNAL plus NNAL-gluc) in K(3)-EDTA human plasma. Free NNAL and NNAL-d(4) are extracted from human plasma by liquid/liquid extraction. To analyze for total NNAL and the internal standard, a separate aliquot of the K(3)-EDTA human plasma is treated with beta-glucuronidase to deconjugate the NNAL-gluc; the total NNAL and internal standard are then extracted using liquid/liquid extraction. After drying down under nitrogen, the residue is reconstituted with acetonitrile and analyzed using positive ion electrospray and HILIC/MS/MS at a flow rate of 1.0 mL/min. The chromatographic run time is 1.0 min per injection, with retention time for both NNAL and NNAL-d(4) of 0.75 min with a capacity factor (k') of 2. The standard curve range for this assay is from 5.00-1000 pg/mL for both free and total NNAL, using a total plasma sample volume of 1.0 mL. The interday precision and accuracy of the quality control (QC) samples demonstrated <7.6% relative standard deviation (RSD) and <3.3% relative error (RE) for free NNAL. For total NNAL, the interday precision and accuracy of the QC samples demonstrated <11.7% RSD and <2.8% RE. Optimization of enzyme hydrolysis of NNAL-gluc is discussed in detail. The overall recoveries for free and total NNAL and IS were 68.2 and 71.5% (free) and 70.7 and 65.5% (total). No adverse matrix effects were noticed for this assay.     

Development, validation, and application of a liquid chromatography–tandem mass spectrometry method for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human hair

The tobacco-specific nitrosamine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a valuable biomarker for human exposure to the carcinogenic nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in tobacco and tobacco smoke. In this work, an efficient and sensitive method for the analysis of NNAL in human hair was developed and validated. The hair sample was extracted by NaOH solution digestion, purified by C(18) solid-phase extraction (SPE) and molecularly imprinted solid-phase extraction, further enriched by reverse-phase ultrasound-assisted dispersive liquid-liquid microextraction (USA-DLLME) into 1.0?% aqueous formic acid, and finally analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Good linearity was obtained in the range of 0.24-10.0?pg/mg hair with a correlation coefficient of 0.9982, when 150?mg hair was analyzed. The limit of detection and lower limit of quantification were 0.08 and 0.24?pg/mg hair, respectively. Accuracies determined from hair samples spiked with three different levels of NNAL ranged between 87.3 and 107.7?%. Intra- and inter-day relative standard deviations varied from 4.1 to 8.5?% and from 6.9 to 11.3?%, respectively. Under the optimized conditions, an enrichment factor of 20 was obtained. Finally, the developed method was applied for the analysis of NNAL in smokers' hair. The proposed sample preparation procedure combining selectivity of two-step SPE and enrichment of DLLME significantly improves the purification and enrichment of the analyte and should be useful to analyze NNAL in hair samples for cancer risk evaluation and cancer prevention in relation to exposure to the tobacco-specific carcinogen NNK.     

Simultaneous determination of NNK and its seven metabolites in rabbit blood by hydrophilic interaction liquid chromatography–tandem mass spectrometry

A hydrophilic interaction liquid chromatographic–tandem mass spectrometric (HILIC–MS–MS) method for investigation of the in vivo metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent carcinogen, in rabbit blood has been developed and validated. This method achieved excellent repeatability and accuracy. Recovery ranged from 76.9 to 116.3 % and precision (as RSD) between 0.53 and 6.52 %. Linearity was good for all compounds (R ²?>?0.9990) and the limit of detection (LOD) ranged from 0.016 to 0.082 ng mL^?1. Pharmacokinetic analysis indicated that NNK was rapidly eliminated in vivo in rabbit blood and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was the major metabolite. The hydroxy acid, keto acid, and NNAL-N-oxide were also important metabolites in rabbit blood. It is probable that α-methylene hydroxylation was the major pathway of α-hydroxylation of NNK and NNAL in the rabbit.

暴露于环境烟气中的大鼠尿样代谢物分析

4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁醇(NNAL)是烟草特有亚硝胺4-(甲基亚硝胺基)-1-(3-吡啶)-1-丁酮(NNK)在生物体内的一种代谢标记物,分析暴露于烟气中的生物体内NNAL的含量是研究卷烟烟气对生物体健康影响的有效手段.基于人体的个体差异性很大,本文以饲养的大鼠为研究对象,采用LC-MS/MS...     

Determination and excretion study of gestrinone in human urine by high performance liquid chromatography and gas chromatography/mass spectrometry

Gestrinone was studied by HPLC for screening and by GC/MS for confirmation. Three unknown peaks were found by HPLC which are probably the metabolites of gestrinone, and conjugated gestrinone in dosed human urine. The metabolites and gestrinone were excreted as the conjugated forms. The total amounts of metabolite 1 and conjugated gestrinone, recovered after 48 h, were 0.20 and 0.32 mg, respectively. When metabolite 1 was tested by LC/MS and LC/MS/MS, the parent ion was m/z 327, [MH](+), and fragment ions were seen at m/z 309 [MH - H(2)O](+), 291 [MH - 2H(2)O](+), 283, 263 and 239. The TMS-enol-TMS ether derivative of gestrinone has three peaks in the GC/MS chromatogram formed by tautomerism. The reproducibility of the derivatization method was stable and recoveries were over 87% when spiked into blank urine.     

Metabolic study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone to the enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in vitro in human bronchial epithelial cells using chiral capillary electrophoresis

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) with one chiral center at the carbinol is a major metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). As tobacco specific N-nitrosamines (TSNAs), NNK and NNAL are the most pulmonary carcinogens in tobacco products and smoke. In this paper, a chiral CE method modified with highly sulfated β-cyclodextrin (S-β-CD) was developed to investigate the stereoselective formation of NNAL from NNK in vitro in normal human bronchial epithelial (NHBE) cells. Combined with solid phase extraction (SPE) of the cell samples, NNK and NNAL enantiomers were baseline separated under the proposed CE conditions, with satisfactory recoveries (72.5-113% for NNK and (±)-NNAL) and low limits of detection (LOD, 2.5-3 μg/mL for NNK and (±)-NNAL). The cytotoxicity of NNK in NHBE cells was investigated through the cell counting kit (CCK) assay and proved to be highly dependent on the NNK's concentration. The metabolic results obtained from CE analysis demonstrated that NNK was preferentially metabolized to (+)-NNAL through carbonyl reduction. Meanwhile, the ratio of [(+)-NNAL]/[(-)-NNAL] was independent of NHBE cells' incubation time with NNK, but could be changed according to the original incubation concentration of NNK. This chiral CE method could be useful for the study on toxicology and metabolic transformations of related TSNAs.     

High-performance liquid chromatographic analysis of rosoxacin and its N-oxide metabolite in plasma and urine

A high-pressure liquid chromatographic method for the analysis of rosoxacin and its pyridyl N-oxide metabolite in plasma and urine extracts is described. A statistical evaluation of the assay data has shown acceptable accuracy and precision for 0.5 to 25 microgram of rosoxacin or the metabolite per ml of plasma and for 2.5 to 60 microgram/ml of either compound in urine. The minimum quantifiable level for rosoxacin was 0.13 microgram/ml in plasma and 0.64 microgram/ml in urine; for the metabolite in plasma and urine, the corresponding values were 0.21 and 0.60 microgram/ml, respectively. The method was applied to plasma and urine from three dogs medicated orally with 5 mg/kg of rosoxacin. The pharmacokinetic parameters calculated for rosoxacin were: plasma halflife, 1.9 h; plasma clearance, 65 ml/min; volume of distribution, 11.31. The average total urinary excretion of rosoxacin as free and conjugated rosoxacin and its free N-oxide was 7.7 +/- 0.2% over the 48-h collection period.     

Simultaneous determination of NNK and its metabolites in mouse tissue for evaluating the effects of chronic alcohol consumption on the metabolism of NNK in mouse liver and lung

A hydrophilic-interaction liquid chromatography–tandem mass spectrometry (HILIC–MS–MS) method was developed for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolites in mouse liver and lung. The limits of detection of all analytes were in the range 0.017–0.057 ng mL^?1, and recovery ranged from 88.4–119.8 % with intra and inter-day precision in the range 0.89–6.03 % and 1.01–6.97 %, respectively. This simple and accurate method was used to evaluate the effect of chronic alcohol consumption on NNK bioactivation in mouse tissue. Time-course curves for NNK and its metabolites were generated, and the areas under the curves (AUCs) were compared. It was found that target tissues of NNK carcinogenesis in C57BL/6 mice contained high levels of α-hydroxylation metabolites of NNK and its carbonyl reduction metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). The most pronounced effect of alcohol was to enhance α-hydroxylation of NNK in mouse lung and liver, which suggests that chronic alcohol consumption may increase the risk of carcinogenicity associated with NNK in mice.

UPLC‐MS/MS method for the determination of tobacco‐specific biomarker NNAL,tamoxifen and its main metabolites in rat plasma

Cigarette smoke is known to interact with tamoxifen‐metabolizing enzymes and transporters and potentially affect its treatment outcome. 4‐(N‐ nitrosomethylamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL) is an important metabolite of 4‐(methylnitro‐samino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) because it is frequently used as a biomarker to assess human smoke exposure. In order to study the potential pharmacokinetic interaction between cigarette smoke and tamoxifen in rats a UPLC‐MS/MS method for the simultaneous determination of NNAL and tamoxifen along with its metabolites in rat plasma has been developed and validated. Analytes were extracted with methanol and separated on a HSS T3 column by a gradient elution with the mobile phase consisting of acetonitrile and water. The lower limits of quantitation ranged from 0.05 to 0.62 ng/mL. Precisions showed RSD <15.8% and accuracy in the range 80.6–116.0%. Mean analyte recoveries ranged from 76.9 to 108.4%. The method was successfully applied to study the effects of cigarette smoke condensate (CSC), NNK and benzo(a)pyrene pre‐treatment on the pharmacokinetics of tamoxifen and its metabolites in rats. Significant effects of CSC, NNK, benzo(a)pyrene were observed on pharmacokinetics of tamoxifen and its metabolites. We also found that plasma NNAL levels are statistically significant correlated with plasma 4‐hydroxy‐tamoxifen and endoxifen.     

Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.     

Liquid chromatography/tandem mass spectrometric method for the simultaneous determination of tobacco-specific nitrosamine NNK and its five metabolites

A sensitive and robust high-performance liquid chromatography-electrospray ionization tandem mass spectrometry method to analyze 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its five metabolites in one passage was developed and validated. The method achieved excellent reproducibility and accuracy. Linearity was observed for all six compounds (R² = 0.999) with detection limits (S/N ≥ 3) ranging from 0.2 to 2.4 pg on column and 0.01-0.12 ng ml⁻¹ in samples injected. Average intra-day and inter-day variations (% R.S.D.) were 1.2 and 3.5%, respectively. A sample preparation method involving C8 and C18 solid phase extraction provided satisfactory recovery of the analytes in mouse urine. Each NNK metabolite was identified by its chromatographic retention time and specific fragmentation pattern. Since the carcinogenicity of NNK is related to its metabolism, the method described in this report should facilitate toxicological investigations into the carcinogenesis due to NNK exposure in the environment.     

Determination of free and conjugated cis-3,3,5-trimethylcyclohexanol in plasma and urine

A gas chromatographic procedure was developed to determine free and conjugated cis-3,3,5-trimethylcylcohexanol in plasma and urine. The sample is extracted with dichloromethane when free cis-3,3,5-trimethylcyclohexanol is determined, or with hexane after enzymatic hydrolysis, when conjugated cis-3,3,5-trimethylcyclohexanol is determined. An aliquot of the organic extract is injected into a stainless-steel column (packed with Carbowax 20M, 15% on Chromosorb W AW 100-120 mesh) and detected with a flame ionization detector. Extraction recovery from plasma and urine was almost 100% and the limit of quantification was fixed at 100 ng/ml plasma or urine. The procedure was evaluated in a pharmacokinetic study of cyclandelate and its metabolite cis-3,3,5-trimethylcyclohexanol.     

Pharmacokinetics and excretion study of bergenin and its phase II metabolite in rats by liquid chromatography tandem mass spectrometry

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for the determination of bergenin and its phase II metabolite in rat plasma, bile and urine has been developed. Biological samples were pretreated with protein precipitation extraction procedure and enzymatic hydrolysis method was used for converting glucuronide metabolite to its free form bergenin. Detection and quantitation were performed by MS/MS using electrospray ionization and multiple reaction monitoring. Negative electrospray ionization was employed as the ionization source. Sulfamethoxazole was used as the internal standard. The separation was performed on a reverse‐phase C₁₈ (250 × 4.6 mm, 5 μm) column with gradient elution consisting of methanol and 0.5% aqueous formic acid. The concentrations of bergenin in all biological samples were in accordance with the requirements of validation of the method. After oral administration of 12 mg/kg of the prototype drug, bergenin and its glucuronide metabolite were determined in plasma, bile and urine. Bergenin in bile was completely excreted in 24 h, and the main excreted amount of bergenin was 97.67% in the first 12 h. The drug recovery in bile within 24 h was 8.97%. In urine, the main excreted amount of bergenin was 95.69% in the first 24 h, and the drug recovery within 24 h was <22.34%. Total recovery of bergenin and its glucuronide metabolite was about 52.51% (20.31% in bile within 24 h, 32.20% in urine within 48 h). The validated method was successfully applied to pharmacokinetic and excretion studies of bergenin.     

On-line concentration and determination of tobacco-specific N-nitrosamines by cation-selective exhaustive injection-sweeping-micellar electrokinetic chromatography

In this paper, a micellar electrokinetic chromatography (MEKC) method combined with cation-selective exhaustive injection (CSEI) and sweeping was developed to separate and concentrate four tobacco-specific N-nitrosamines (TSNAs) including N′-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL). Experimental parameters affecting separation efficiency and enhancement factors were investigated in detail. Under the optimum MEKC condition, NAB, NNK, NNAL and iso-NNAL were baseline separated with high separation efficiencies and good peak shapes. Furthermore, with the preconcentration by CSEI-sweeping-MEKC, the sensitivity enhancement factors for NAB, NNK, NNAL and iso-NNAL in terms of peak areas ranged from 6.0 × 10³ to 1.5 × 10⁴, and the detection limits (LOD, S/N = 3) of four TSNAs were in the range of 0.004-0.016 μg/mL. In addition, this method had fairly good repeatability, and the RSDs of retention time and peak area were less than 1% and 5%, respectively. Finally, this method showed promising capabilities in the application of detecting and analyzing TSNAs in human urine samples.     

63Ni electron-capture gas chromatographic assay for buprenorphine and metabolites in human urine and feces

A 63Ni electron-capture gas chromatographic assay is described for buprenorphine, a potent narcotic agonist--antagonist. In addition, the assay is useful for the measurement of the metabolite norbuprenorphine and demethoxybuprenorphine, a rearrangement product resulting when buprenorphine is exposed to acid and heat. An extraction procedure was developed which optimized recovery of buprenorphine from biological samples and produced minimal background interferences and emulsion problems. Extract residues were derivatized with pentafluoropropionic anhydride and assayed by gas chromatography. Samples were analyzed with and without enzyme hydrolysis, thus providing a selective and sensitive assay for both free and conjugated buprenorphine, norbuprenorphine and demethoxybuprenorphine. The lower limits of detection following extraction of a 1-ml sample were ca. 10 ng/ml for buprenorphine and demethoxybuprenorphine and 5 ng/ml for norbuprenorphine. Application of the assay to human samples following a 40-mg oral dose of buprenorphine produced no evidence for the presence of demethoxybuprenorphine in urine or feces. Norbuprenorphine (free and conjugated) was present in urinary and fecal samples; buprenorphine (free and conjugated) was found in high amounts only in feces and in trace amounts in urine as conjugated buprenorphine. The urinary and fecal excretion pattern observed for a human subject following oral dosing of buprenorphine suggests enterohepatic circulation of buprenorphine.     

High-performance liquid chromatographic determination of the 1,4-diketo and 1,4-diketo-2,3-dihydroxy metabolites of lonapalene in rat urine

Individual high-performance liquid chromatographic (HPLC) methods have been developed for the determination of two major metabolites of lonapalene in rat urine. The highly unstable and polar 1,4-diketo-2,3-dihydroxy metabolite (II) is extracted from urine by two extraction columns (phenyl followed by silica), further purified by means of HPLC with a fully end-capped C18 HPLC column and quantified by an ultraviolet detector at 280 nm. Ascorbic acid is used as an antioxidant during extraction and overnight injection of II. Urine samples for total II (free plus conjugated) determination are incubated with arylsulfatase and beta-glucuronadase prior to extraction. The 1,4-diketo metabolite (III) is extracted from urine with a C18 extraction column, further purified with a C18 HPLC column, and quantified by an ultraviolet detector at 260 nm. The detection limit for both metabolites is 100 ng/ml of urine (signal-to-noise = 2.5). The methods were used to analyze urine samples from a long-term toxicology study of lonapalene in rats and to determine the linearity of dose-concentration relationships for both metabolites.     

New designer drug 4-iodo-2,5-dimethoxy-beta-phenethylamine (2C-I): studies on its metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric and capillary electrophoretic/mass spectrometric techniques

Studies are described on the metabolism and the toxicological analysis of the phenethylamine-derived designer drug 4-iodo-2,5-dimethoxy-beta-phenethylamine (2C-I) in rat urine using gas chromatographic/mass spectrometric (GC/MS) techniques, and for a particular question, using capillary electrophoretic/mass spectrometric (CE/MS) techniques. The identified metabolites indicated that 2C-I was metabolized on the one hand by O-demethylation in position 2 and 5, respectively, followed either by N-acetylation or by deamination with subsequent oxidation to the corresponding acid or reduction to the corresponding alcohol, respectively. The latter metabolite was hydroxylated in beta-position and further oxidized to the corresponding oxo metabolite. On the other hand, 2C-I was metabolized by deamination with subsequent oxidation to the corresponding acid or reduction to the corresponding alcohol, respectively. 2C-I and most of its metabolites were partially excreted in conjugated form. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-I in rat urine that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-I in human urine.     

SPE–HPLC–MS/MS method for the trace analysis of tobacco‐specific N‐nitrosamines and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol in rabbit plasma using tetraazacalix[2]arene[2]triazine‐modified silica as a sorbent

Tobacco‐specific N‐nitrosamines (TSNAs), including N′‐nitrosonornicotine, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone, N′‐nitrosoanatabine, and N′‐nitrosoanabasine, have been implicated as a source of carcinogenicity in tobacco and cigarette smoke. We present a rapid and effective method comprising SPE based on tetraazacalix[2]arene[2]triazine‐modified silica as sorbent and analysis with HPLC–MS/MS for the determination of TSNAs and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), a metabolite of 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone, in rabbit plasma. The linear dynamic ranges were 10–2000 pg/mL for NNAL and 4–2000 pg/mL for the four TSNAs with good correlation coefficients (>0.9965). The LODs were in the range of 0.9–3.7 pg/mL, and the LOQs were between 2.9 and 12.3 pg/mL. The accuracies of the method were also evaluated and found to be in the range of 90.1–113.3%. This method is promising to be applied to the preconcentration and determination of TSNAs and NNAL in smoke and human body fluids.