Early pregnancy metabolites predict gestational diabetes mellitus: implications for fetal programming.

BACKGROUND: Aberrant fetal programming in gestational diabetes mellitus seems to increase the risk of obesity, type 2 diabetes, and cardiovascular disease. The inability to accurately identify gestational diabetes mellitus in the first trimester of pregnancy has thwarted ascertaining whether early therapeutic interventions reduce the predisposition to these prevalent medical disorders. OBJECTIVE: A metabolomics study was conducted to determine whether advanced analytical methods could identify accurate predictors of gestational diabetes mellitus in early pregnancy. STUDY DESIGN: This nested observational case-control study was composed of 92 gravidas (46 in the gestational diabetes mellitus group and 46 in the control group) in early pregnancy, who were matched by maternal age, body mass index, and gestational age at urine collection. Gestational diabetes mellitus was diagnosed according to community standards. A comprehensive metabolomics platform measured 626 endogenous metabolites in randomly collected urine. Consensus multivariate criteria or the most important by 1 method identified low-molecular weight metabolites independently associated with gestational diabetes mellitus, and a classification tree selected a subset most predictive of gestational diabetes mellitus. RESULTS: Urine for both groups was collected at a mean gestational age of 12 weeks (range, 6-19 weeks' gestation). Consensus multivariate analysis identified 11 metabolites independently linked to gestational diabetes mellitus. Classification tree analysis selected a 7-metabolite subset that predicted gestational diabetes mellitus with an accuracy of 96.7%, independent of maternal age, body mass index, and time of urine collection. CONCLUSION: Validation of this high-accuracy model by a larger study is now needed to support future studies to determine whether therapeutic interventions in the first trimester of pregnancy for gestational diabetes mellitus reduce short- and long-term morbidity.

Absorption and metabolism of isothiocyanates formed from broccoli glucosinolates: effects of BMI and daily consumption in a randomised clinical trial.

Sulphoraphane originates from glucoraphanin in broccoli and is associated with anti-cancer effects. A preclinical study suggested that daily consumption of broccoli may increase the production of sulphoraphane and sulphoraphane metabolites available for absorption. The objective of this study was to determine whether daily broccoli consumption alters the absorption and metabolism of isothiocyanates derived from broccoli glucosinolates. We conducted a randomised cross-over human study (n 18) balanced for BMI and glutathione S-transferase µ 1 (GSTM1) genotype in which subjects consumed a control diet with no broccoli (NB) for 16 d or the same diet with 200 g of cooked broccoli and 20 g of raw daikon radish daily for 15 d (daily broccoli, DB) and 100 g of broccoli and 10 g of daikon radish on day 16. On day 17, all subjects consumed a meal of 200 g of broccoli and 20 g of daikon radish. Plasma and urine were collected for 24 h and analysed for sulphoraphane and metabolites of sulphoraphane and erucin by triple quadrupole tandem MS. For subjects with BMI >26 kg/m2 (median), plasma AUC and urinary excretion rates of total metabolites were higher on the NB diet than on the DB diet, whereas for subjects with BMI <26 kg/m2, plasma AUC and urinary excretion rates were higher on the DB diet than on the NB diet. Daily consumption of broccoli interacted with BMI but not GSTM1 genotype to affect plasma concentrations and urinary excretion of glucosinolate-derived compounds believed to confer protection against cancer. This trial was registered as NCT02346812.

Detection of rare species of volatile organic selenium metabolites in male golden hamster urine.

Selenium has been considered as an essential trace element in mammals and its intake comes mainly from food. Mammals can metabolize both inorganic and organic species, and urinary excretion is the primary elimination route of selenium. Selenosugars and trimethylselenonium ion have been identified as major urinary metabolites. Other metabolites have been reported, but they were detected in some studies and not in others. Still, a large portion of the ingested selenium eliminated from the body is unknown. Volatile selenium species may account for a certain portion of the unknown species since they can easily be lost during sample analyses. While we analyzed male golden hamster urine in search of potential volatile pheromone(s), four volatile selenium compounds were detected. They were dimethyl selenenylsulfide, dimethyl diselenide, dimethyl bis(thio)selenide, and dimethyl selenodisulfide. When the urine samples were aged and dried for 48 h, dimethyl selenodisulfide tended to increase, while others decreased. The increase might be due to the formation of dimethyl selenodisulfide via reaction of dimethyl diselenide and dimethyl trisulfide whose concentration increased as urine aged. To our knowledge, dimethyl bis(thio)selenide and dimethyl selenodisulfide have never been demonstrated in urine. It remains to be determined whether these species are common metabolites in other animals or hamster-specific.

Urinary Excretion Profiles of 2,5-Dimethoxy-4-alkylthiophenethylamine Analogs in Rats.

The urinary metabolic profiles of three hallucinogenic 2,5-dimethoxy-4-alkylthiophenethylamine analogs: 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4), and 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7), were investigated in rats. For each drug, four male Sprague-Dawley rats were orally administered 10 mg/kg of 2C-T-2, 2C-T-4, or 2C-T-7, and urine was collected 0-24 and 24-48 h after administration. The urine samples were processed by liquid-liquid extraction, and the extracts were analyzed by liquid chromatography/mass spectrometry to quantify the metabolites. The metabolic patterns of these drugs were different: for 2C-T-7, the principal metabolite was the ß-hydroxylated-N-acetylated-sulfoxide, whereas for 2C-T-2 and 2C-T-4 the major metabolites were the N-acetylated-sulfoxide and S-methylated-N-acetylated-sulfoxide, respectively.

Bioavailability study of arsenic and mercury in traditional Chinese medicines (TCM) using an animal model after a single dose exposure.

Traditional Chinese medicines (TCM) are increasingly being used as alternative medicines in many countries, and this has caused concern because of adverse health effects from toxic metal bioavailability such as mercury (Hg) and arsenic (As). The aim of this study was to investigate the bioavailability of As and Hg from TCM after a single exposure dose using an animal model of female Sprague-Dawley rats. The rats were divided into 6 groups which included four groups treated with sodium arsenite (NaAsO2), arsenic sulfide (As2S3), mercuric chloride (HgCl2), mercuric sulfide (HgS), and two groups treated with TCM containing high Hg or As (Liu Shen Wan: As 7.7-9.1% and Hg 1.4-5.0%; Niuhang Jie du Pian: As 6.2-7.9% and Hg <0.001%). The samples of urine, faeces, kidney and liver were collected for analysis and histological assay. The results indicated that relatively low levels of As and Hg from these TCM were retained in liver and kidney tissues. The levels of As in these tissues after TCM treatment were consistent with the levels from the As sulphide treated group. With the exception of the mercuric chloride treated group, the levels of Hg in urine from other groups were very low, and high levels of As and Hg from TCM were excreted in faeces. The study showed poor bioavailability of As and Hg from TCM as indicated by low relative bioavailability of As (0.60-1.10%) and Hg (<0.001%). Histopathological examination of rat kidney and liver tissues did not show toxic effects from TCM.

Gold-nanoparticle extraction and reversed-electrode-polarity stacking mode combined to enhance capillary electrophoresis sensitivity for conjugated nucleosides and oligonucleotides containing thioether linkers.

We present a capillary electrophoresis method for determining two different C8-conjugated deoxyadenosines, and for oligonucleotides containing them, in which a psoralen or an acridine molecule is bonded to the base via a short alkyl chain containing sulfur ethers at both ends. The sensitivity of the micellar electrokinetic chromatography (MEKC) method was increased by using two preconcentration techniques, micro solid-phase extraction (µSPE) followed by reversed-electrode-polarity stacking mode (REPSM). Variables that affect the efficiency of the extraction in µSPE and preconcentration by REPSM, including the type and volume of extraction nanoparticle, concentration, and injection time, were investigated. Under the optimum conditions, enrichment factors obtained were in the range 360-400. The limits of detection (LODs) at a signal-to-noise ratio of 3 ranged from 2 to 5 nmol L(-1). The relative recoveries of labelled adenosines from water samples were 95-103%. The proposed method provided high enrichment factors and good precision and accuracy with a short analysis time. On the basis of the advantages of simplicity, high selectivity, high sensitivity, and good reproducibility, the proposed method may have great potential for biochemical applications.

Simultaneous determination of rabeprazole and its two active metabolites in human urine by liquid chromatography with tandem mass spectrometry and its application in a urinary excretion study.

A simple and rapid liquid chromatography with tandem mass spectrometry method has been developed and validated for the determination of rabeprazole and its two active metabolites, rabeprazole thioether and desmethyl rabeprazole thioether, in human urine using donepezil as the internal standard. The sample preparation procedure involved a simple dilution of urine sample with methanol (1:3, v/v). The chromatographic separation was achieved on a Hedera ODS-2 C18 column using a mixture of methanol/10 mmol/L ammonium acetate solution (containing 0.05% formic acid; 55:45, v/v) as the mobile phase. The method was validated over the concentration ranges of 0.15-100 ng/mL for rabeprazole, 0.30-400 ng/mL for rabeprazole thioether, and 0.05-100 ng/mL for desmethyl rabeprazole thioether. The established method was highly sensitive with a lower limit of quantification of 0.15 ng/mL for rabeprazole, 0.30 ng/mL for rabeprazole thioether, and 0.05 ng/mL for desmethyl rabeprazole thioether. The intra- and interbatch precision was <4.5% for the low, medium, and high quality control samples of all the analytes. The recovery of the analytes was in the range 95.4-99.0%. The method was successfully applied to a urinary excretion profiles after intravenous infusion administration of 20 mg rabeprazole sodium in healthy volunteers.

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples.

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-L-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.

A robust high-throughput sample preparation and liquid chromatography/tandem mass spectrometry method for the quantitation of ß-lyase metabolites of sulfur mustard as 1,1'-sulfonylbis-[2-(methylthio)ethane] in human urine.

RATIONALE: Sulfur mustard (HD) is a major chemical warfare agent threat to humans. Since World War I, several incidents of human exposure to sulfur mustard have been reported. In order to assist health professionals during an exposure event and support biological monitoring, a rapid analytical method is required to measure the exposure of humans to HD. METHOD: The ß-lyase metabolites of HD, 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) and 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) were reduced to the single biomarker, 1,1'-sulfonylbis-[2-(methylthio)ethane] (SBMTE), using titanium(III) chloride. High-throughput sample preparation was performed on a Tecan Freedom EVO liquid handler and analysis was performed by electrospray ionization liquid chromatography and tandem mass spectrometry (LC/MS/MS) in the multiple-reaction monitoring mode. RESULTS: Each analytical run consisted of a matrix blank, calibration standards (0.1-100 ng/mL), low quality controls (QCs), 2.5 ng/mL, and high QCs, 25.0 ng/mL, of SBMTE in human urine. The method was validated with 20 analytical runs performed by four analysts. The mean calculated concentrations of the low and high QCs were 2.52 and 25.5 ng/mL with relative standard deviations of 3.6% and 2.3%, respectively. CONCLUSION: This semi-automated method has few manual transfer steps, thus minimizing common manual errors and saving time. Therefore, this method would be very helpful to responding laboratories in a large-scale exposure event related to HD.

Discovery of safety biomarkers for realgar in rat urine using UFLC-IT-TOF/MS and 1H NMR based metabolomics.

As an arsenical, realgar (As4S4) is known as a poison and paradoxically as a therapeutic agent. However, a complete understanding of the precise biochemical alterations accompanying the toxicity and therapy effects of realgar is lacking. Using a combined ultrafast liquid chromatography (UFLC) coupled with ion trap time-of-flight mass spectrometry (IT-TOF/MS) and (1)H NMR spectroscopy based metabolomics approach, we were able to delineate significantly altered metabolites in the urine samples of realgar-treated rats. The platform stability of the liquid chromatography LC/MS and NMR techniques was systematically investigated, and the data processing method was carefully optimized. Our results indicate significant perturbations in amino acid metabolism, citric acid cycle, choline metabolism, and porphyrin metabolism. Thirty-six metabolites were proposed as potential safety biomarkers related to disturbances caused by realgar, and glycine and serine are expected to serve as the central contacts in the metabolic pathways related to realgar-induced disturbance. The LC/MS and NMR based metabolomics approach established provided a systematic and holistic view of the biochemical effects of realgar on rats, and might be employed to investigate other drugs or xenobiotics in the future.

Massive human ingestion of orpiment (arsenic trisulfide).

BACKGROUND: Because the toxicity of arsenic is well known, arsenic-containing compounds have frequently been ingested for suicidal purposes. We report a case of attempted suicide by massive ingestion of arsenic trisulfide, an arsenic mineral of low solubility, which resulted in minimal symptoms. CASE REPORT: An asymptomatic 57-year-old man presented to an Emergency Department 13h after his reported ingestion of approximately 84g of arsenic contained in a mineral specimen of orpiment (arsenic trisulfide) that had been crushed and mixed with an alcoholic beverage and food. His only symptom before presentation was nausea. Physical examination was unremarkable, and diagnostic tests included a normal electrolyte panel, a normal serum lactate, and a normal electrocardiogram. An abdominal radiograph revealed hyper-dense material scattered throughout the large intestine. As per the recommendations of the regional poison center, the patient was managed with whole bowel irrigation with a polyethylene glycol solution, maintenance intravenous hydration, and observation on a telemetry unit. Chelation was not performed. A spot urine specimen collected 12h after admission contained 1490µg of total arsenic per liter (background range<50µg per liter). The patient remained asymptomatic throughout his hospital course. Follow-up studies revealed a diminution in both intra-abdominal radiopacities and urine arsenic concentration. X-ray diffraction analysis of the specimen confirmed its identity as arsenic trisulfide. CONCLUSIONS: Our experience demonstrates that massive ingestion of a poorly soluble inorganic arsenic compound can be successfully managed with gastrointestinal decontamination alone without chelation, provided that the patient remains asymptomatic during close clinical monitoring.

Effect of simultaneous exposure to benzene and ethanol on urinary thioether excretion.

The toxicity of benzene is not an issue of the past, especially in developing countries. Bone marrow toxicity is demonstrated among workers. In this study, the effect of simultaneous exposure to benzene and ethanol on benzene metabolism in mice was investigated by measuring the excretion of thioethers in urine. Urinary thioether excretion significantly decreased in the mice receiving both benzene and ethanol compared with the animals receiving benzene only. The assay of determining thioethers in urine samples in this study is a simple and low-cost method, thus suitable for routine use, especially in developing countries, not only for benzene, but also for other alkilating agents, which can be found during occupational exposure. Our results suggest that further research is needed to elucidate the mechanisms of decreased urinary excretion of thioether after simultaneous exposure to benzene and ethanol.

The use of high-performance liquid chromatography with diode array detector for the determination of sulfide ions in human urine samples using pyrylium salts.

Hydrogen sulfide is a toxic gas involved in the regulation of some essential biological processes. A novel, precise, accurate and rapid method based on high-performance liquid chromatography with diode array detection for the determination of sulfide ions in human urine sample is proposed. The method involves the derivatization of sulfide with pyrylium salts - (2,4,6-triphenylpyrylium hydrogensulfate(VI) (L1) and 4-[p-(N,N-dimethylamino)phenyl]-2,6-diphenylpyrylium chlorate(VII) (LN1). The separation occurs on InfinityLab Poroshell 120 EC C18 column using acetonitrile and phosphate buffer as a mobile phase. The detectors utilized a wavelength of 371 or 580 nm. The calibration curves were linear in the range of 2-150 µmol L-1 and 1-50 µmol L-1 for L1 and LN1 derivatives, respectively. The samples were found to be stable from sample collection to final analysis. The method was successfully applied to samples from apparently healthy volunteers.

Evidence of sulfur mustard exposure in victims of chemical terrorism by detection of urinary ß-lyase metabolites.

Background: Sulfur mustard (SM) is a vesicant chemical warfare agent. Ocular, dermal, and respiratory systems are the primary targets of SM exposure. The aims of this study were to perform a quantitative analysis of ß-lyase metabolites of SM as 1,1'-sulfonylbis[2-(methylthio) ethane] (SBMTE) in urine samples of chemical casualties and to investigate the relationship between the measured SBMTE levels and the severity of characteristic symptoms of SM poisoning.Methods: A bioanalytical method which is based on titanium (III) chloride reduction of ß-lyase metabolites was employed to analyze urine samples of individuals (n = 13, collected 30 h after SM exposure) using gas chromatography-tandem mass spectrometry.Results: Various levels of SBMTE were measured in urine samples of seven individuals, confirming SM exposure for each. There was a correlation observed between measured levels of SBMTE in human urine samples and severity of clinical findings including ocular, respiratory, and cutaneous lesions of SM.Discussion: In combination with clinical examination, measurement of SBMTE levels in human urine could be used as a prognostic factor for clinical outcomes in victims of SM exposure. This bioanalytical verification is also important for the documentation of alleged use of SM.Conclusion: SBMTE is an unambiguous biomarker of potential SM poisoning as it does not exist in urine samples of an unexposed population. Quantitation of urinary SBMTE concentrations in victims of SM could be used in order to enable improved interpretation of clinical findings.

Occurrence of S-methyl thioesters in urines of humans after they have eaten asparagus.

Gas chromatography-mass spectrometry was used to determine the odor-causing agent (or agents) present in the urines of humans after they have eaten asparagus. S-Methyl thioacrylate and S-methyl 3-(methylthio)thiopropionate were identified from methylene chloride extracts of such urines and appear to be the odor-causing compounds. Methanethiol, the previously reported odor-causing agent, was not detected in these methylene chloride extracts.

Relative oral bioavailability of arsenic from contaminated soils measured in the cynomolgus monkey.

A number of studies have found that gastrointestinal absorption of arsenic from soil is limited, indicating that a relative oral bioavailability (RBA) adjustment is warranted when calculating risks from exposure to arsenic-contaminated soil. However, few studies of arsenic bioavailability from soil have been conducted in animal models with phylogenetic similarity to humans, such as nonhuman primates. We report here the results of a study in which the RBA of arsenic in soil from a variety of types of contaminated sites was measured in male cynomolgus monkeys. A single oral dose of each contaminated soil was administered to five adult male cynomolgus monkeys by gavage, and the extent of oral absorption was evaluated through measurement of arsenic recovery in urine and feces. Urinary recovery of arsenic following doses of contaminated soil was compared with urinary recovery following oral administration of sodium arsenate in water in order to determine the RBA of each soil. RBA of arsenic in 14 soil samples from 12 different sites ranged from 0.05 to 0.31 (5-31%), with most RBA values in the 0.1-0.2 (10-20%) range. The RBA values were found to be inversely related to the amount of arsenic present with iron sulfate. No other significant correlations were observed between RBA and arsenic mineralogic phases in the test soils. The lack of clear relationships between arsenic mineralogy and RBA measured in vivo suggests that gastrointestinal absorption of arsenic from soil may be more complex than originally thought, and subject to factors other than simple dissolution behavior.

Utility of urinary biomarkers in oral cancer.

OBJECTIVE: Oral cancer is the leading malignancy in India, with tobacco playing a major role in the etiology. The aim of the present study was to quantify nitrate+nitrite (NO2+NO3) in tobacco products as well as to study tobacco exposure related biomarkers in controls, patients with oral precancers (OPC) and oral cancer patients. MATERIALS & METHODS: Healthy individuals (n=90) were grouped into without habit of tobacco (NHT, n=30) and healthy individuals with habit of tobacco (WHT, n=60). Oral cancer patients with a tobacco habit were classified into abstinence (n=62) and non-abstinence (n=64) groups according to status at the study time. Urinary nicotine and cotinine levels were analyzed by modified high-pressure liquid chromatography (HPLC) using a UV detector. Levels of NO2+NO3 in tobacco and urine, and urinary thioether levels were estimated by spectrophotometry. RESULTS: NO2+NO3 levels in different types of tobacco product ranged between 0.13 to 3.39 mg/g. The Odds Ratio (OR) analysis indicated positive associations of both smoking and chewing habits of tobacco with high risk of development of oral cancer. Urinary nicotine, cotinine and NO2+NO3 levels were significantly elevated in WHT, patients with OPC and oral cancer patients as compared with the NHT group. This was also the case for urinary thioether levels. Levels of urinary nicotine and cotinine were also higher in the non-abstinence group with oral cancers. CONCLUSION: The results confirmed that tobacco chewing and smoking habits are prominent risk factors for development of oral cancer in the western part of India (Gujarat). Urinary nicotine, cotinine, NO2+NO3 and thioether levels can be helpful for screening programs for oral cancer.

Carrot-derived carbon dots modified with polyethyleneimine and nile blue for ratiometric two-photon fluorescence turn-on sensing of sulfide anion in biological fluids.

In this article, a facile and green synthesis of carbon dots (CDs) was developed by using natural carrot as new carbon source. After direct hydrothermal carbonization for 5h at 180°C, CDs were prepared facilely. Then, CDs were conjugated with polyethyleneimine (PEI) and Nile Blue (NB) chloride to produce CDs/PEI/NB nanocomposites under electrostatic interactions. Upon excitation at 800nm, two-photon fluorescence (TPF) of the nanocomposites was observed, with TPF peaks of CDs at 415nm and NB at 675nm. The addition of Cu2+ could lead to TPF quenching of CDs via inner filter effect, but hardly any impacted on TPF of NB. Afterward, the added S2- combined with Cu2+ to form stable species that caused the separation of Cu2+ from CDs surface and the TPF recovery of CDs, with negligible effects on TPF of NB. Herein, a new CDs-based ratiometric TPF turn-on probe of S2- was developed and showed a good linear relationship (R2 =0.9933) between ratiometric TPF intensity (I415/I675) and S2- concentration (0.1-8.0µM), with a low detection limit of 0.06µM. This probe was highly selective and sensitive toward S2- over potential interferences in real biological fluids, with high detection recoveries.

Biological monitoring screening of patients provided antineoplastic drugs including adriamycin, cyclophosphamide, 5-fluorouracil, methotrexate, and vincristine.

The aim of the present study was to establish screening biomarkers of exposure to antineoplastic drugs administered to 11 patients undergoing cancer chemotherapy. Among the anticancer drugs administered were cyclophosphamide (all), Adriamycin (5 of 11), methotrexate (3 of 11), 5-fluorouracil (4 of 11), vincristine (3 of 11), megestrol acetate (1 of 11), and procarbazine (1 of 11). The noninvasive urinary parameters investigated were thioethers, D-glucaric acid, elements, and forward and reverse mutagenesis using bacterial bioassays. The data were analyzed in terms of the observed concentrations and those corrected for personal baseline. Personal baseline correction for parameters with significant nonexposure baseline levels was essential. While glucaric acid and thioethers were increased by the drug treatments, the correlations with baseline-uncorrected data showing an inverse relationship proved spurious, because saturation of the detoxification systems occurred at the high doses administered. Glucaric acid was also influenced by methotrexate and vincristine. Thioether content was affected by cyclophosphamide only. The forward mutagenesis assay was directly correlated to cyclophosphamide dose but the reverse assay was not, in the presence or absence of rat S9 fraction. The forward assay was not sensitive to the effects of smoking. Relative to controls, the elements changed by cyclophosphamide were K, S, and P. Those affected by Adriamycin were Ca, Mg, and Na; 5-fluorouracil affected Ca, Mg, Na, and C; methotrexate changed P and S. The forward mutagenesis assay and D-glucaric acid concentrations were the screening biomarkers best suited to monitoring for extent of exposure to these antineoplastic drugs.

Rapid detection and identification of N-acetyl-L-cysteine thioethers using constant neutral loss and theoretical multiple reaction monitoring combined with enhanced product-ion scans on a linear ion trap mass spectrometer.

A sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method based on the combination of constant neutral loss scans (CNL) with product ion scans was developed on a linear ion trap. The method is applicable for the detection and identification of analytes with identical chemical substructures (such as conjugates of xenobiotics formed in biological systems) which give common CNLs. A specific CNL was observed for thioethers of N-acetyl-L-cysteine (mercapturic acids, MA) by LC-MS/MS. MS and HPLC parameters were optimized with 16 MAs available as reference compounds. All of these provided a CNL of 129 Da in the negative-ion mode. To assess sensitivity, a multiple reaction monitoring (MRM) mode with 251 theoretical transitions using the CNL of 129 Da combined with a product ion scan (IDA thMRM) was compared with CNL combined with a product ion scan (IDA CNL). An information-dependent acquisition (IDA) uses a survey scan such as MRM (multiple reaction monitoring) to generate "informations" and starting a second acquisition experiment such as a product ion scan using these "informations." Th-MRM means calculated transitions and not transitions generated from an available standard in the tuning mode. The product ion spectra provide additional information on the chemical structure of the unknown analytes. All MA standards were spiked in low concentrations to rat urines and were detected with both methods with LODs ranging from 60 pmol/mL to 1.63 nmol/mL with IDA thMRM. The expected product ion spectra were observed in urine. Application of this screening method to biological samples indicated the presence of a number of MAs in urine of unexposed rats, and resulted in the identification of 1,4-dihydroxynonene mercapturic acid as one of these MAs by negative and positive product ion spectra. These results show that the developed methods have a high potential to serve as both a prescreen to detect unknown MAs and to identify these analytes in complex matrix.