Gas Chromatography-negative Ion Chemical Ionization Mass Spectrometry Research Articles

Obesity Is Associated with Increased F2-Isoprostanes and IL-6 in Black Women

Obesity affects over 40% of the adult population and is a major risk factor for morbidity and mortality due to cardiovascular disease. Black women have one of the highest prevalences of obesity, insulin resistance, hypertension, and cardiovascular events in the US. We previously found that free radical-mediated lipid peroxidation contributes to IL-6 production in dendritic cells leading to inflammation and hypertension. Thus, we hypothesized that F2-isoprostanes (F2-IsoPs), products and biomarkers of endogenous lipid peroxidation, contribute to increased inflammation and IL-6 production among obese Black women. We studied a total of 88 obese Black women of age 42.0 ± 9.8 years, weight 102 ± 16 kg, and body mass index (BMI) 37.68 ± 5.08. Systolic and diastolic blood pressure were 124 ± 14/76.2 ± 9.9 mmHg, heart rate was 68.31 ± 10.26 beats/min, and fasting insulin was 15.0 ± 8.7 uU/mL. Plasma F2-IsoPs were measured using gas chromatography/negative ion chemical ionization mass spectrometry (GC/NICI-MS). Plasma cytokines, including IL-6, IL-8, IL-10, IL-1β, TNF-a, and C-reactive proteins were measured using multiplex Luminex technology. Anthropometric measurements were performed using dual-energy X-ray absorptiometry. Using Pearson’s correlation analysis, we found that BMI was positively correlated with plasma F2-IsoPs, while inversely correlated with insulin sensitivity in obese Black women. Further, F2-IsoPs were positively correlated with inflammatory marker IL-6 levels while negatively correlated with anti-inflammatory marker IL-10. In addition, we found that plasma F2-IsoPs levels were significantly associated with reduced insulin sensitivity. These results suggest that F2-IsoPs may be associated with obesity-induced cardiovascular risk in Black women by increasing the production of inflammatory cytokine IL-6 and decreasing the production of anti-inflammatory IL-10.

Abstract P076: Obesity Is Associated With Increased F2-isoprostanes And Il-6 In Black Women.

Obesity affects over 40% of the adult population and is a major risk factor for morbidity and mortality due to cardiovascular disease. Black women have one of the highest prevalence of obesity in the United States and approximately 80% are overweight or obese with increased insulin resistance, hypertension, and cardiovascular events. We previously found that isolevuglandins formed in the F2-isoprostane pathway of free radical–mediated lipid peroxidation contributes to IL-6 production in dendritic cells leading to inflammation and hypertension. Thus, we hypothesized that F2-isoprostanes contribute to increased inflammation and IL-6 production among obese Black women. We recruited 88 Black women of age 42.0±9.8 years, weight 102±16 kg, body mass index (BMI) 37.68±5.08, systolic blood pressure 124±14 mmHg, diastolic blood pressure 76.2±9.9 mmHg, and insulin 15.0±8.7 uU/ml. We measured plasma F2-isoprostane levels using gas chromatography/negative ion chemical ionization mass spectrometry (GC/NICI-MS) at the Eicosanoid Core at Vanderbilt University Medical Center. We also measured plasma cytokines including TNF-α 68±174, IL-1β 135±372, IL-6 1615±2769, IL-8 3536±1901, and IL-10 324±633 pg/mL. Using Pearson’s correlation analysis, we found that increased BMI correlated with increased plasma F2-isoprostane levels (Fig. 1A, R=0.23, p=0.037). Interestingly among all the inflammatory cytokines, elevated IL-6 correlated with increased F2-isoprostanes (Fig. 1B, R= 0.44, p=.0025). These results suggest that F2-isoprostanes may contribute to the obesity-induced cardiovascular risk in Black women by increasing production of inflammatory cytokine IL-6.

Assessment of urinary 15-F2 -isoprostanes in dogs with urothelial carcinoma of the urinary bladder and other lower urinary tract diseases.

BackgroundThe 15‐F2‐isoprostanes are by‐products of oxidative stress and are increased in the urine of people with lower urinary tract diseases (LUTD), especially urinary neoplasia. Urothelial carcinoma (UC) is the most common urinary neoplasm in dogs. Earlier detection of UC by noninvasive means could lead to improved outcomes. Urinary 15‐F2‐isoprostanes potentially could provide this means, but have not been evaluated in dogs with UC.ObjectiveThe objective of this study was to measure urinary 15‐F2‐isoprostanes in dogs with UC and dogs with other LUTD.AnimalsOne hundred seventeen dogs: 46 dogs with UC, 30 dogs with LUTD, and 25 control dogs.MethodsAny dog that was presented with dysuria was eligible for inclusion. Diagnosis of UC was confirmed histologically. Urinalysis was performed in each case, and 15‐F2‐isoprostanes quantified by gas chromatography‐negative ion chemical ionization‐mass spectrometry (GC‐NICI‐MS) and normalized to urinary creatinine concentration.ResultsDogs with urinary diseases (UC + LUTD) had higher median urinary 15‐F2‐isoprostanes when compared to control dogs (5.92 ng/mg [range, 0.46‐31.03] vs 3.73 [range, 1.8‐7.98]; P = .02). Urinary 15‐F2‐isoprostanes were similar in dogs with UC (5.33 ng/mg [range, 0.46‐31.03]) compared to dogs with LUTD (6.29 ng/mg [range, 0.54‐18.93]; P = .47) and control dogs (P = .06). Dogs with UC had higher qualitative measures of proteinuria (P = .004), hematuria (P = .01), and epithelial cells on urinalysis (P = .002) compared to the other groups.Conclusions and Clinical ImportanceUrinary F2‐isoprostanes are not useful for the detection of UC in dogs. Future research could evaluate urinary 15‐F2‐isoprostanes as a marker of inflammation in disease progression and prognosis.

Investigation of NG-hydroxy-l-arginine interference in the quantitative determination of nitrite and nitrate in human plasma and urine by GC-NICI-MS

NG-Hydroxy-l-arginine (NOHA) is the intermediate product of the conversion of l-arginine to l-citrulline and nitric oxide (NO) by nitric oxide synthase (NOS). NO is further oxidized to nitrite and nitrate which circulate in the blood and are excreted in the urine. Nitrite and nitrate may therefore serve as surrogates of NO synthesis. NOHA has been reported to occur in various cells and in blood of animals and humans. The concentration of nitrite in the circulation is comparable to the concentration of NOHA in plasma and serum of humans and laboratory animals. NOHA is a relatively unstable compound and the interaction of its NG-hydroxy group with redox active species or during sample treatment such as derivatization in the heat may yield N-containing compounds including nitrite and nitrate. In theory, NOHA may interfere with the analysis of nitrite and nitrate. In the present study, we investigated a possible interference of synthetic NOHA (0–400 μM) with the gas chromatography-negative ion chemical ionization–mass spectrometry (GC-NICI-MS) method of analysis of circulating and urinary nitrite and nitrate involving derivatization with pentafluorobenzyl (PFB) bromide in aqueous acetone at 50 °C for 5 min (nitrite) or for 60 min (nitrite and nitrate). Our results show that NOHA does not interfere with the measurement of nitrite and nitrate in human plasma and urine by this method at concentrations up to 400 μM.

Quantification of nitrated-polycyclic aromatic hydrocarbons in atmospheric aerosol samples with in-injection port thermal desorption-gas chromatography/ negative chemical ionization mass spectrometry method

In-injection port thermal desorption (TD) was extended in determination of particulate phase nitrated-polycyclic aromatic hydrocarbons (N-PAHs) on filter samples coupled with a gas chromatography/negative ion chemical ionization-mass spectrometry (GC/NCI-MS). The experimental and analytical parameters were optimized with standard testing and ambient samples. Fourteen of the most abundant N-PAHs in ambient airs were included for their quantification. A desorption temperature of 280 °C for 10 min was sufficient to transfer entire target compounds from injection port to the analytical system. Good linearity (R2 >0.995) on the calibrations for each analyte was achieved. The limit of detections (LODs) ranged from 46.5 to 152.3 pg per filter sample, which were 4–66% better than solvent extraction (SE) approach. Method precision, repeatability and reproducibility, determined by replicate analyses of calibration standards and ambient samples, were less than 6% for target compounds. Validation of the method were conducted with two batches of a total of 30 ambient aerosol-loaded filters using our TD and the traditional SE approaches. Reasonably good agreement (R2 = 0.98) by the two methods was demonstrated for most of N-PAHs, except for 9-nitrophenanthrene and 6-nitrobenzo(a)pyrene. The in-injection port TD can improve laboratory efficiency and reduce solvent-based costs for the measurement of N-PAHs.

Determination of 16 polychlorinated biphenyls in fish oil by gas chromatography-negative ion chemical ionization-mass spectrometry

An analytical method for the simultaneous determination of 16 polychlorinated biphenyls (PCBs) in fish oil was developed. PCBs were extracted from fish oil with n-hexane, purified by sulfuric acid and determined by using gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NCI-MS) in selected ion-monitoring (SIM) mode. A good linear relationship (r > 0.99) was observed with the PCBs concentrations from 0.01 µg/L to 10 µg/L, and the limits of quantification (LOQ, S/N = 10) were between 3 pg/g and 67 pg/g for different kinds of PCBs. The average recoveries ranged from 62.3% to 121.8% with the relative standard deviations ( RSDs, n = 3) smaller than 12%. Compared with the traditional pre-treatment of multiple material solid phase extraction, this new method is simple, rapid and less organic solvent usage. Meanwhile the method has good selectivity and sensitivity, and it is suitable for the determination of multiple trace PCBs in fish oil.

Analytical Variables Affecting Analysis of F2-Isoprostanes and F4-Neuroprostanes in Human Cerebrospinal Fluid by Gas Chromatography/Mass Spectrometry

F2-isoprostanes (F2-IsoPs) are a gold marker of lipid peroxidation in vivo, whereas F4-neuroprostanes (F4-NPs) measured in cerebrospinal fluid (CSF) or brain tissue selectively indicate neuronal oxidative damage. Gas chromatography/negative-ion chemical-ionization mass spectrometry (GC/NICI-MS) is the most sensitive and robust method for quantifying these compounds, which is essential for CSF samples because abundance of these compounds in CSF is very low. The present study revealed potential interferences on the analysis of F2-IsoPs and F4-NPs in CSF by GC/NICI-MS due to the use of improper analytical methods that have been employed in the literature. First, simultaneous quantification of F2-IsoPs and F4-NPs in CSF samples processed for F4-NPs analysis could cause poor chromatographic separation and falsely higher F2-IsoPs values for CSF samples with high levels of F2-IsoPs and F4-NPs. Second, retention of unknown substances in GC columns from CSF samples during F4-NPs analysis and from plasma samples during F2-IsoPs analysis might interfere with F4-NPs analysis of subsequent runs, which could be solved by holding columns at a high temperature for a period of time after data acquisition. Therefore, these special issues should be taken into consideration when performing analysis of F2-IsoPs and F4-NPs in CSF to avoid misleading results.

Isoprostanes and isofurans as non-traditional risk factors for cardiovascular disease among Canadian Inuit

Objectives: The aim of the present study was to investigate the potential importance of oxidative stress, measured by isoprostanes-related compounds, as non-traditional risk factor for cardiovascular disease. We planned to examine the relationship between concentrations of plasma F2-isoprostanes (F2-IsoPs), isofurans (IsoFs), measures of obesity and various cardiometabolic risk factors. Materials and methods: Cross-sectional study using a sub-sample from the population of a survey conducted in the summer and fall 2007 and 2008 by Canadian Coastguard Ship Amundsen in 36 Canadian Arctic Inuit communities. Subjects included a subset (n = 233) of a total study population (n = 2595) with a mean age 42.56 ± 15.39 years and body mass index 27.78 ± 5.65 kg/m2. Plasma levels of F2-IsoPs and IsoFs was determined by gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) method; and their relationships to waist circumference (WC), blood pressure C reactive proteins (CRP), blood lipids and fasting glucose were assessed by multivariate analyses. Results: Plasma F2-IsoPs correlated positively with CRP (r =.132, P =.048) and systolic blood pressure (SBP) (r =.157, P =.024) after adjustment for age, sex and body mass index. IsoFs correlated with WC (r =.190, P =.005) and SBP (r =.137, P =.048). F2-IsoPs were not found elevated in smokers (P =.034), whereas IsoFs were decreased in smokers (P =.001). WC, SBP and sex were found to be major correlates of oxidative stress in Canadian Inuit. Conclusions: Plasma measures of F2-IsoPs and IsoFs increase with increased obesity and associated cardiometabolic risk factors, including CRP and blood pressure. Simultaneous measurement of IsoFs provides an advantageous mechanistic insight into oxidative stress not captured by F2-IsoPs alone.

A rapid and sensitive analytical method for the determination of 14 pyrethroids in water samples

A simple, efficient and environmentally friendly analytical methodology is proposed for extracting and preconcentrating pyrethroids from water samples prior to gas chromatography-negative ion chemical ionization mass spectrometry (GC-NCI-MS) analysis. Fourteen pyrethroids were selected for this work: bifenthrin, cyfluthrin, λ-cyhalothrin, cypermethrin, deltamethrin, esfenvalerate, fenvalerate, fenpropathrin, τ-fluvalinate, permethrin, phenothrin, resmethrin, tetramethrin and tralomethrin. The method is based on ultrasound-assisted emulsification-extraction (UAEE) of a water-immiscible solvent in an aqueous medium. Chloroform was used as extraction solvent in the UAEE technique. Target analytes were quantitatively extracted achieving an enrichment factor of 200 when 20 mL aliquot of pure water spiked with pyrethroid standards was extracted. The method was also evaluated with tap water and river water samples. Method detection limits (MDLs) ranged from 0.03 to 35.8 ng L −1 with RSDs values ≤3–25% ( n = 5). The coefficients of estimation of the calibration curves obtained following the proposed methodology were ≥0.998. Recovery values were in the range of 45–106%, showing satisfactory robustness of the method for analyzing pyrethroids in water samples. The proposed methodology was applied for the analysis of river water samples. Cypermethrin was detected at concentration levels ranging from 4.94 to 30.5 ng L −1.

9016 Nutritional assessment on hospitalised lung cancer patients

A high-performance liquid chromatography (HPLC) and gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICI-MS) method was developed for the analysis of the combined antitumor drug S-1 (tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate[ and active metabolite 5-fluorouracil in human plasma and urine. Tegafur was fractionated from biological fluids by extraction with dichloromethane and analyzed by HPLC. 5-Fluorouracil and 5-chloro-2,4-dihydroxypyridine were extracted with ethyl acetate from the residual layer after extraction of tegafur, and converted to pentafluorobenzyl (PFB) derivatives. Potassium oxonate was cleaned up with an anion-exchange column (Bond Elut NH2). The extracted potassium oxonate was degraded to 5-azauracil and converted to PFB derivatives. The PFB derivatives were analyzed by GC-NICI-MS. A stable isotope was employed as the internal standard in the GC-NICI-MS analysis. The limits of quantitation of tegafur, 5-fluorouracil, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in plasma were 10, 1, 2 and 1 ng/ml, respectively. The reproducibility of the analytical method according to the statistical coefficients is ∼10%. The accuracy of the method is good; that is, the relative error is <10%. The methods were applied to pharmacokinetic studies of S-1 in patients.

MEASURING TOXICITY, INCORPORATION, AND REPAIR OF 5-HYDROXYMETHYL-2′-DEOXYURIDINE IN CELLS.

5-Hydroxymethyl-2′-deoxyuridine (HmdU) is among the most common endogenous oxidative DNA lesions and is repaired by more than one DNA glycosylase in human cells. This DNA-repair pathway may be exploited for use in cancer chemotherapy. Exogenous delivery of HmdU to cells in culture leads to its incorporation into the DNA and subsequent repair, resulting in death by apoptosis if the HmdU load is too heavy for the cell. Both incorporation into the DNA and repair of HmdU out of the DNA are necessary for cell death to occur. To measure the incorporation into and subsequent repair of HmdU out of the DNA of cells in culture, we have developed a highly sensitive and specific method using stable isotope dilution and gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) for measuring HmdU levels found in DNA. Using the osteosarcoma cell line U2OS as a model system, we treat cells with HmdU, harvest cells, and isolate DNA. Hydrolysis of the DNA with formic acid releases the free bases, and we convert the base 5-hydroxymethyluracil (HmU) into its methyl ester, 5-methoxymethyluracil, subsequently derivatizing with 3,5-bis(trifluoromethyl)benzyl bromide to yield a stable compound. When analyzing trace levels of HmdU deoxynucleoside standard, this derivatization process yields a detection limit of approximately 500 attomoles (5 × 10−16 mol) on column. We demonstrate in U2OS cells that toxicity of exogenously delivered HmdU is proportional to its incorporation into the DNA, and we show that repair of HmdU out of the DNA is occurring. Application of our method in cell lines with varying sensitivities to HmdU treatment will allow us to gain further insight into the mechanism of and resistance to this potential chemotherapeutic agent.

Determining15N enrichment of dissolved organic nitrogen in environmental waters by gas chromatography/negative-ion chemical ionization mass spectrometry

To minimize the required sample volume and simplify the overall procedure for determining 15N enrichment of dissolved organic nitrogen (DON) in environmental waters, we employed derivatization of NO3− with pentafluorobenzyl bromide and gas chromatography/negative-ion chemical ionization mass spectrometry (GC-NICI-MS). The pretreatment processes to remove inorganic nitrogen were optimized to ensure recovery of labile DON molecules, and the derivatization procedure for NO2− and NO3− was optimized for environmental samples. The required sample volume is 10 mL or less and the detection limit is about 0.015 µmol 15N L−1, or 0.3 atomic percent excess of 15N for water samples containing 5 µmol DON L−1. This method can also be applied to 15N enrichment analysis of particulate organic nitrogen. We applied the developed method to measure DON production rate by the hermatypic coral Porites cylindrica during incubation with 15N-labeled NO3−.

Effect of cigarette smoking on urinary 3,N4-ethenocytosine levels measured by gas chromatography/mass spectrometry.

Etheno DNA adducts are DNA damages derived from exogenous carcinogens as well as endogenous lipid peroxidation and oxidative stress. Elevated levels of etheno DNA adducts were found in cancer-prone tissues and blood samples, suggesting that these promutagenic lesions correlate with risk of cancers. We previously reported the detection of 3,N4-ethenocytosine (epsilon Cyt) in the urine samples of two smokers using the isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) assay (Chen et al., 2001, Chem. Res. Toxicol. 14, 1612-1619). Since smokers are found to have elevated levels of lipid peroxidation and oxidative stress, we examined the association between urinary epsilon Cyt levels with cigarette smoking. Among the 23 samples analyzed, the average concentration of urinary epsilon Cyt in smokers was significantly higher than that of nonsmokers, 2.65 +/- 4.0 versus 0.61 +/- 0.90 ng/kg/g creatinine (p= 0.03). Albeit the number of subjects is limited, the results indicate that the measurement of epsilon Cyt in human urine may provide a useful noninvasive biomarker for oxidative DNA damage and cancer chemoprevention studies.

Determination of decabromodiphenyl ether in sediments using selective pressurized liquid extraction followed by GC-NCI-MS.

A method based on selective pressurized liquid extraction (SPLE) followed by gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NCI-MS) has been evaluated for analysis of decabromodiphenyl ether (PBDE-209) in sediment samples. Instrumental operating conditions such as source temperature and system pressure were optimized in the NCI-MS system, giving an instrumental detection limit of 2 pg. The limit of determination of the entire SPLE-GC-NCI-MS procedure was around 50 pg g(-1) dry weight (dw), with repeatability of replicates between 4 and 21% relative standard deviation. Application of the method to 13 different river and marine sediment samples collected in Spain revealed that levels of decabromodiphenyl ether ranged between 2 and 132 ng g(-1) dry weight.

Enantioselective trace analysis of amphetamine in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry.

A method for the quantitative enantioselective analysis of amphetamine in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry (GC/NICI-MS) is presented. Five-fold deuterated analogues of both enantiomers were used as internal standard. Plasma sample preparation was performed by a rapid liquid-liquid extraction using n-hexane. Derivatization with (S)-(-)-N-(heptafluorobutyryl)prolyl chloride was accomplished directly in the n-hexane extract to avoid loss of amphetamine during sample concentration. The method was validated in the expected concentration range of 0.006 for a pharmacokinetic study. Calibration curves were linear within a range 0.006-50 ng/mL plasma. Precision and accuracy were acceptable over the entire calibration range. Baseline separation of the enantiomers was easily achieved on a 15-m nonchiral apolar column. The method is simple and robust, and has been applied to the batch analysis of amphetamine enantiomers.

Detection and quantification of 5-chlorocytosine in DNA by stable isotope dilution and gas chromatography/negative ion chemical ionization/mass spectrometry.

Hypochlorous acid (HOCl) is generated from activated phagocytes during infections and inflammation. One of the major products of HOCl reaction with DNA was 5-chlorocytosine (5Cl-Cyt). In this report, a gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) assay with stable isotope dilution was developed for detection and quantification of 5Cl-Cyt in DNA. During hydrolysis of DNA, 5Cl-Cyt undergoes spontaneous deamination quantitatively forming 5-chlorouracil (5Cl-Ura). The stable isotope of 5Cl-Ura with six mass units higher than the normal 5Cl-Ura was synthesized and used as internal standard of the assay. The adduct-enriched fraction of DNA hydrolysate was derivatized with pentafluorobenzyl bromide before GC/NICI/MS analysis with selected ion monitoring at [M - 181](-) fragments of bispentafluorobenzylated 5Cl-Ura and its isotope analogue. The limit of detection was 20 amol (S/N = 8) of bispentafluorobenzylated 5Cl-Ura injected on column with selective ion monitoring mode and the limit of quantification for the entire assay was 14 fmol of 5Cl-Cyt. Analysis of hypochlorous acid-treated calf thymus DNA by both GC/NICI/MS and HPLC/UV detection provided similar adduct levels and thus verified this new GC/NICI/MS assay. Using this highly specific and ultrasensitive GC/NICI/MS method, the levels of 5Cl-Cyt in untreated calf thymus DNA and human placental DNA were determined as 0.6 and 6.6 adducts per 10(7) normal cytosine, respectively. Peroxynitrite also contributed to 5Cl-Cyt formation in DNA. Level of 5Cl-Cyt in DNA treated with peroxynitrite in the presence of chloride was higher than that without addition of chloride. Thus, quantification of 5Cl-Cyt in DNA by this isotope dilution GC/NICI/MS assay may facilitate research on the role of DNA chlorination in carcinogenesis and in cancer development.

Methods for the determination of deoxynivalenol and other trichothecenes in foods.

Trichothecene mycotoxins are secondary metabolites of Fusarium moulds that routinely infect cereal crops. Processing can reduce, but not eliminate, trichothecenes from cereal-based foods, and the potential presence of the trichothecenes in cereal foods poses a significant health risk to consumers. Deoxynivalenol (vomitoxin, DON) is the most common of the trichothecenes detected in cereal crops and is subject to government regulation in many countries. Sensitive (ng/g) methods for the detection of trichothecenes in cereal grains and food products are needed to protect consumers, to provide data for dietary exposure estimates, and to support research into the control of moulds and subsequent toxin production. Laboratories require simple, rugged and reliable methods for routine testing, with unequivocal identification of suspect mycotoxins. A method employing gas chromatography-negative ion chemical ionisation/mass spectrometry (GC-NICI/MS) has been developed and used for the routine determination of eight of the most significant trichothecenes in a variety of commodities. This chapter discusses GC, liquid chromatographic (LC) and supercritical fluid chromatographic methods that are currently used for the analysis of trichothecene mycotoxins.

Analysis of 3,N(4)-ethenocytosine in DNA and in human urine by isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry.

The promutagenic etheno DNA adducts have been detected in tissue DNA of rodents and humans from various exogenous and endogenous sources. While other etheno DNA adducts have been detected and quantified by isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS), similar analysis for 3,N(4)-ethenocytosine (epsilonCyt) has not been available. In this report, a GC/NICI/MS assay was developed for detection and quantification of epsilonCyt in DNA and in human urine samples. The stable isotope of epsilonCyt with 7 mass units higher than the normal epsilonCyt was synthesized and used as internal standard of the assay. The adduct-enriched fraction of DNA hydrolysate was derivatized with pentafluorobenzyl bromide before GC/NICI/MS analysis with selective ion monitoring at [M - 181](-) fragments of pentafluorobenzylated epsilonCyt and its isotope analogue. One femtogram (S/N > 40) of pentafluorobenzylated epsilonCyt was detected when injected on column with selective ion monitoring mode. The limit of quantification for the entire assay was 7.4 fmol of epsilonCyt, which was approximately one thousand times lower than that of the HPLC/fluorescence assay for the nucleoside 3,N(4)-etheno-2'-deoxycytidine in DNA. Analysis of chloroacetaldehyde-treated calf thymus DNA by both GC/NICI/MS and HPLC/fluorescence methods provided similar adduct levels and thus verified the assay. This GC/NICI/MS method was used for analysis of epsilonCyt in two smokers' urine samples and the average level of epsilonCyt was 101 +/- 17 pg/mL/g of creatinine. Thus, quantification of epsilonCyt in DNA and in urine by this highly specific and ultrasensitive isotope dilution GC/NICI/MS assay may facilitate research on the role of epsilonCyt in carcinogenesis and in cancer development.

Determination of estrogens in river water by gas chromatography–negative-ion chemical-ionization mass spectrometry

A method for the determination of estrogens (17α-estradiol, 17β-estradiol, estrone, ethynyl estradiol, and estriol) as pentafluorobenzyl-trimethylsilyl (PFB–TMS) derivatives by gas chromatography–mass spectrometry (GC–MS) with negative-ion chemical-ionization (NICI) is described. The NICI of all the derivatives produced an intense [M–PFB] − ion as the base peak. The reagent gas (methane) flow-rate and the ion source temperature were determined to be 2.0 ml/min and 240°C, respectively, for the optimized NICI-selected ion monitoring (SIM) conditions. The sensitivities of the PFB–TMS derivatives in the NICI mode were 8.0–130 times higher than those of the PFB–TMS derivatives in electron ionization (EI) mode, and 12–25 times higher than those of all the TMS derivatives in the EI mode. This method was applied to the analysis of estrogens in river water using a solid-phase extraction as the sample preparation. The recoveries of the target chemicals from a river-water sample spiked with standards at 2 ng/l level were 85.8–126.5% (RSD, 6.2–13.0%). The methodical detection limits ranged from 0.10 to 0.28 ng/l.

Detection and quantification of 1,N(6)-ethenoadenine in human placental DNA by mass spectrometry.

Exocyclic DNA adducts have been reported to derive from various exogenous as well as endogenous sources, such as lipid peroxidation. Among them, 1,N(6)-ethenoadenine (epsilonAde) has previously been detected in tissue DNA of untreated rodents and humans by an immunoaffinity/(32)P-postlabeling method. This study reports detection and quantification of the endogenous epsilonAde adduct in the same human placental DNA by three independent assays, namely, GC/MS, LC/MS, and HPLC/fluorescence. Using a recently reported gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) method [Chen, H.-J. C., et al. (1998) Chem. Res. Toxicol. 11, 1474], the level of epsilonAde in human placental DNA from a commercial source was found to be 2.3 adducts per 10(6) Ade bases. To confirm these findings, a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method was developed for epsilondAdo. With this LC/MS assay, epsilondAdo was detected at the level of 2.5 adducts per 10(6) dAdo nucleosides in the same human placental DNA. The stable isotopes of epsilonAde and epsilondAdo were added as internal standards in both GC/MS and LC/ESI/MS/MS assays, respectively, and thus provided high specificity, reproducibility, and accurate quantification. The relatively high levels of epsilonAde in this human placental DNA detected by mass spectrometry were further verified by HPLC/fluorescence analysis. The GC/MS method was validated by the HPLC/fluorescence assay using calf thymus DNA treated with chloroacetaldehyde or by the LC/MS method with 2, 3-epoxy-4-hydroxynonanal-modified calf thymus DNA. The epsilonAde level in human placental DNA freshly isolated in the presence of an antioxidant was similar to that in DNA from the commercial source. Since epsilonAde is a potential mutagenic lesion, analysis of epsilonAde by the specific and sensitive GC/NICI/MS method may provide a useful biomarker in cancer risk assessment.