Chromatography Negative Ion Chemical Ionization Research Articles

Development of simple analytical methods of polychlorinated biphenyls in human serum by gas chromatography negative ion chemical ionization quadrupole mass spectrometry

In this study, we developed a highly sensitive, robust method for determining 12 congeners of two to ten chlorinated polychlorinated biphenyls (PCBs) in serum samples using gas chromatography (GC)–mass spectrometry (MS) operating in selected ion monitoring mode (SIM: m/z 35) with negative ion chemical ionization (NICI), and the results were compared with those from GC coupled with high-resolution MS (HRMS) with electron impact (EI). The recovery rates of the surrogate PCB congeners were 97.2%–112% (coefficient of variation: 5.3%–8.5%), and the method detection limits for PCBs in each matrix obtained by GC–NICI–quadrupole mass spectrometry (qMS) were 1.9–20 pg g−1 wet wt. The analytical values of the target compounds in the samples analyzed by GC–NICI–qMS and GC–EI–HRMS were comparable (Passing–Bablok regression: R = 0.888–0.967), and the analytical values obtained via GC–NICI–qMS were almost comparable with those of the certified serum samples from National Institute of Standards and Technology (NIST: SRM1957), indicating that GC–NICI–qMS is suitable for the analysis of tetra- to hepta-chlorinated PCBs in serum samples.

Bis-pentafluorobenzyl derivatives of N-acetyl-l-methionine and N-acetyl-l-selenomethionine for the quantitative determination in human plasma by gas chromatography–negative ion chemical ionisation mass spectrometry

Targeted anti-cancer combination therapy with infusion of N-acetyl-l-methionine (NALM) and N-acetyl-l-selenomethionine (NASeLM) shows promising results in cancer treatment. Selenium has been recognised as a valuable additive in cancer therapeutics due to its ability to minimise side effects of chemotherapy and its role in cancer prevention and therapy. Due to the promising results of this new therapeutic approach evaluation of pharmacokinetic data for NALM and NASeLM is of ultimate importance. We have therefore elaborated a method for the quantitative measurement of these compounds in human plasma based on GC–negative ion chemical ionisation-MS. The derivatisation sequence elaborated can be regarded as a novel strategy for the chemical modification of delicate sulphur- and selenium-containing compounds, and underlines the enhanced reactivity of selenium-analogues of sulphur-containing amino acids. The target compounds were extracted from plasma with ethyl acetate and converted to the S/Se-pentafluorobenzyl-homocysteine pentafluorobenzyl ester derivative. Reaction conditions were optimised for derivative yield. Calibration graphs were established in the range of 2.938–481.105ng/0.5mL plasma (NALM) and 0.233–59.543ng/0.5mL plasma (NASeLM). Accuracy, precision and stability data were elaborated. The method was applied to pharmacokinetic profiling of the compounds after infusion into human volunteers.

Improved gas chromatography–negative ion chemical ionization tandem mass spectrometric method for determination of 11-nor-Δ 9-tetrahydrocannabinol-9-carboxylic acid in hair using mechanical pulverization and bead-assisted liquid–liquid extraction

A gas chromatography–negative ion chemical ionization tandem mass spectrometric (GC–NCI-MS/MS) method was developed and validated for the determination of 11-nor-Δ 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in human hair. After decontamination, hair samples were weighed (25 mg), mechanically pulverized with a bead mill, and incubated in 0.7 mL of 1.0 M sodium hydroxide at 95 °C for 30 min. Bead-assisted liquid–liquid extraction was performed with n-hexane:ethyl acetate (9:1, v/v), a method developed in our laboratory. The extract was evaporated to dryness, derivatized with pentafluoropropanol and pentafluoropropionic anhydride, and analyzed by GC–MS/MS in the negative ion chemical ionization mode using methane as the reagent gas. The linear ranges were 0.05–10.0 pg/mg for THC-COOH with the coefficient of determination ( r 2 = 0.9976). The intra-day and inter-day precisions were within 1.7 and 13.8%, respectively. The intra-day and inter-day accuracies were −4.8 to 10.0% and −3.9 to 3.8%, respectively. The limit of detection and quantification were 0.015 and 0.05 pg/mg, respectively. The recoveries were in the range of 79.4–87.1%. The results indicate that the proposed method is simple, rapid, accurate, and precise for determination of THC-COOH in hair. The method identified THC-COOH in hair specimens from suspected marijuana abusers.

Contribution of nitrated polycyclic aromatic hydrocarbons to the mutagenicity of ultrafine particles in the roadside atmosphere

This is the first report of the quantification of nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) in ultrafine particles in the roadside atmosphere and their contribution to the direct-acting mutagenicity of ultrafine particles. The detailed size distributions of six nitro-PAHs (2-nitrofluoranthene, 1-nitropyrene, 6-nitrobenzo[a]pyrene, 1,3-dinitropyrene, 1,6-dinitropyrene, and 1,8-dinitropyrene) were measured by highly sensitive gas chromatography–negative ion chemical ionization tandem mass spectrometry. Direct-acting mutagenicity of size-fractionated particulate matter (PM) was determined by the Ames test using Salmonella typhimurium strains TA98 and YG1024. The amounts of nitro-PAHs per unit mass of ultrafine particles (<0.12 μm) were significantly higher than those of accumulation mode particles (0.12–2.1 μm) and of coarse particles (>2.1 μm). Therefore, more than 20% of each nitro-PAH, with the exception of 2-nitrofluoranthene, was observed in the ultrafine particle fraction, although the contribution of ultrafine particles to the total PM mass in the roadside atmosphere was only 2.3%. Also, in both tester strains TA98 and YG1024, the mutagenicity per unit mass of ultrafine particles was significantly higher than those of accumulation mode particles or coarse particles. The contributions of 2-nitrofluoranthene, 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene, and 1,8-dinitropyrene to the direct-acting mutagenicity of ultrafine particles were 0.56, 1.5, 0.57, 2.2, and 9.2%, respectively, in the TA98 strain, and 0.54, 1.1, 0.71, 5.0, and 17%, respectively, in the YG1024 strain, while the contribution of 6-nitrobenzo[a]pyrene was less than 0.01% in both strains. 1,8-Dinitropyrene was the largest contributor to the mutagenicity not only of ultrafine particles but also of accumulation mode particles in both strains. Only five nitro-PAHs accounted for as much as 14 and 24% of the direct-acting mutagenicity of ultrafine particles in the roadside atmosphere in the TA98 strain and the YG1024 strain, respectively. This result indicated that nitro-PAHs, especially 1,8-dinitropyrene, were important contributors to the high direct-acting mutagenicity of ultrafine particles in the roadside atmosphere.

Gas chromatography negative ion chemical ionization mass spectrometry: Application to the detection of alkyl nitrates and halocarbons in the atmosphere

Alkyl nitrates and very short-lived halocarbon species are important atmospheric trace gas species that are present in the low to sub parts per trillion concentration range. This presents an analytical challenge for their detection and quantification that requires instrumentation with high sensitivity and selectivity. In this paper, we present a new in situ gas chromatograph negative ion chemical ionization mass spectrometer (GC/NICI–MS) coupled to a non-cryogen sample pre-concentration system. This instrument, with detection limits of <0.01 ppt, is capable of detecting and quantifying a large suite of alkyl nitrate and halocarbon species with high sensitivity and precision. The effects of ion source temperature and reagent gas pressure on the ionization efficiency of the NICI mode are investigated and the results are used to optimize the sensitivity. The NICI mode is compared to the more frequently used electron impact (EI) ionization and the enhancements in sensitivity are presented for all the calibrated compounds.

Free 3-nitrotyrosine in exhaled breath condensates of children fails as a marker for oxidative stress in stable cystic fibrosis and asthma

3-Nitrotyrosine (3-NT) is considered as a marker of oxidative stress, which occurs during inflammation. Since 3-NT levels in exhaled breath condensate (EBC) are very low, we applied a specific and sensitive gas chromatography–negative ion chemical ionization–mass spectrometry (GC–NICI–MS) method and high performance liquid chromatography (HPLC) with electrochemical detection for the analysis of free 3-NT in EBC. A total of 42 children (aged 5–17 years) were enrolled in this study, including children with asthma ( n = 12), cystic fibrosis ( n = 12), and healthy controls ( n = 18). Additionally, 14 healthy non-smoking adults (aged 18–59 years) were included. An EcoScreen system was used for the collection of EBC samples. Free 3-NT levels in EBC ranged from 0.54–6.8 nM. Median (interquartile range) concentrations (nM) were similar in all groups: 1.46 (0.97–2.49) in healthy adults, 2.51 (1.22–3.51) in healthy children, 1.46 (0.88–2.02) in children with asthma, and 1.97 (1.37–2.35) in CF children, respectively ( p = 0.24, Kruskall–Walis test). No difference was found between the children with airway disease and age-matched healthy controls. In healthy subjects, there was no effect of age on 3-NT concentrations. HPLC analyses provided similar concentration ranges for EBC 3-NT when compared with GC–NICI–MS. Our study has clearly demonstrated that free 3-NT in EBC fails as a marker for oxidative stress in children with stable CF and asthma.

Reduction of brain lipid peroxidation by CSF drainage in Alzheimer's disease patients.

Alzheimer's disease (AD) is associated with an increase in cerebrospinal fluid (CSF) levels of the isoprostane 8,12-iso-iPF2alpha-VI, a specific marker of in vivo lipid peroxidation. Poor cerebral clearance of end products of oxidative reactions via CSF circulation may contribute to and sustain ongoing stress. CSF drainage via a low-flow ventriculoperitoneal (VP) shunt may improve removal of these products, reducing oxidative stress. We quantified this biomarker in patients with AD undergoing to VP shunt placement at baseline and after one-year period. CSF sampling occurred at baseline and quarterly visits for one year. Levels of this isoprostane were determined simultaneously at the end of the study by gas chromatography negative ion chemical ionization mass spectrometry. Over one-year, CSF 8,12-iso-iPF2alpha-VI levels consistently decreased versus baseline (51% of initial level), while CSF protein, glucose, cell count and IgG concentrations remained within normal limits. This finding supports the hypothesis that improving CSF drainage enhances extra-cellular clearance of end products of oxidative reactions and lowers brain lipid peroxidation.

Determination of perfluorocarboxylic acids in aqueous matrices by ion-pair solid-phase microextraction–in-port derivatization–gas chromatography–negative ion chemical ionization mass spectrometry

A rapid, selective and simple analytical procedure using tetrabutylammonium as ion pair in conjunction with solid-phase microextraction followed by in-port derivatization–GC–negative ion chemical ionization mass spectrometry was developed. The procedure allows an accurate determination of perfluoroalkylcarboxylic acids in aqueous samples at ng L −1 levels (i.e. method detection limit 20 ng L −1 for perfluorodecanoic acid) improving previous GC methods in terms of analysis time and sensitivity. Ammonia as reagent gas in the negative ion chemical ionization mass spectrometry increased the sensitivity at least 3-fold compared to methane for perfluorocarboxylic acid butyl esters. The developed procedure was successfully applied to effluents from wastewater treatment plants (i.e. 0.05–8.2 μg L −1) and harbor seawaters.

Quantitation of estrogens in ground water and swine lagoon samples using solid-phase extraction, pentafluorobenzyl/trimethylsilyl derivatizations and gas chromatography–negative ion chemical ionization tandem mass spectrometry

A method was developed for the confirmed identification and quantitation of 17β-estradiol, estrone, 17α-ethynylestradiol and 16α-hydroxy-17β-estradiol (estriol) in ground water and swine lagoon samples. Centrifuged and filtered samples were extracted using solid-phase extraction (SPE), and extracts were derivatized using pentafluorobenzyl bromide (PFBBR) and N-trimethylsilylimidazole (TMSI). Analysis was done using negative ion chemical ionization (NICI) gas chromatography–mass spectrometry–mass spectrometry (GC–MS–MS). Deuterated analogs of each of the estrogens were used as isotope dilution standards (IDS) and were added to the samples before extraction. A limit of quantitation of 1 ng/l in ground water was obtained using 500 ml of ground water sample, 1.0 ml of extract volume and the lowest calibration standard of 0.5 pg/μl. For a 25 ml swine lagoon sample, the limit of quantitation was 40 ng/l. The average recovery of the four estrogens spiked into 500 ml of distilled water and ground water samples ( n=16) at 2 ng/l was 103% (S.D. 14%). For 25 ml of swine lagoon samples spiked at 500, 1000 and 10,000 ng/l, the average recovery for the four estrogens was 103% (S.D. 15%). The method detection limits (MDLs) of the four estrogens spiked at 2 ng/l in a 500 ml of ground water sample ranged from 0.2 to 0.6 ng/l. In swine lagoon samples from three different types of swine operations, estrone was found at levels up to 25,000 ng/l, followed by estriol and estradiol up to levels at 10,000 and 3000 ng/l, respectively. It was found that pretreatment of swine lagoon samples with formaldehyde was necessary to prevent conversion of estradiol to estrone.

Improved sample preparation for the quantitative analysis of paroxetine in human plasma by stable isotope dilution negative ion chemical ionisation gas chromatography–mass spectrometry

An improved sample work-up and derivatisation procedure for the quantitative determination of paroxetine in human plasma by gas chromatography–negative ion chemical ionisation mass spectrometry is presented. Solvent extraction from plasma samples at alkaline pH was combined with derivatisation to the pentafluorobenzyl carbamate derivative in one step and subsequently analysed without any further purification. Thus, lengthy and time-consuming solvent evaporation steps are avoided to assure high-throughput analysis. Complete validation data are presented. The method is rugged, rapid and robust and has been applied to the batch analysis of paroxetine during pharmacokinetic profiling of the drug.

Analysis of a stable halogenated derivative of muramic acid by gas chromatography–negative ion chemical ionization tandem mass spectrometry

Muramic acid (Mur) is present in the cell wall of Eubacteria and serves as a chemical marker for the trace detection of bacteria and bacterial cell wall debris in complex matrices. There have been numerous studies using a variety of derivatives of Mur, particularly in combination with gas chromatography–tandem mass spectrometry (GC–MS–MS) where the detection limit has been steadily lowered. A stable, halogenated derivative, the pentafluorobenzyl oxime (PFBO) acetate of Mur, has been developed by others and successfully used for GC with electron-capture detection. The current report is the first use of this derivative for GC–MS–MS analysis of Mur, or indeed any other carbohydrate, using negative ion chemical ionization (NICI) with GC–MS–MS. Mur was readily detected in settled surface dust (166 ng/mg), as well as dust collected from indoor air (1.4–5.9 ng/mg). Analyses of Mur as a PFBO acetate by GC–NICI-MS–MS or as alditol acetates by electron impact GC–electron impact ionization MS–MS serve as complementary approaches for trace detection in complex matrices.

Validation of a method for the detection and confirmation of nitroimidazoles and corresponding hydroxy metabolites in turkey and swine muscle by means of gas chromatography–negative ion chemical ionization mass spectrometry

The results of a validation study of a GC–NCI–MS method for the quantitative determination of 5-nitroimidazoles {1,2-Dimethyl-5-nitroimidazole (dimetridazole, DMZ), 1-methyl-2-[(carbamoyloxy)methyl]-5-nitroimidazole (ronidazole, RNZ), 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole, MNZ) and 2-isopropyl-1-methyl-5-nitroimidazole (ipronidazole, IPZ)} including the hydroxy metabolites of these agents {2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI), 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole (MNZOH), and 1-methyl-2-(2′-hydroxyisopropyl)-5-nitroimidazole (IPZOH)} in turkey and swine muscle are presented. The validation was carried out according to the requirements of the draft for the revision of Commission Decision 93/256/EC, which is expected to be adopted by the European Commission in due course. The determination of the method’s performance parameters revealed decision limits (CC α ) between 0.65 and 2.8 μg/kg for DMZ, RNZ/HMMNI, MNZ and MNZOH. Confirmatory analyses according to the requirements of the forthcoming EC decision are possible for all analytes except for IPZ and IPZOH where already the decision limits (CC α ) were higher (5.2 μg/kg) than for the above-mentioned nitroimidazoles. The within-laboratory reproducibility and the mean recovery were in an acceptable range for all analytes.

Quantitative analysis of morphine in human plasma by gas chromatography–negative ion chemical ionization mass spectrometry

A sensitive and specific method for the quantitative determination of morphine in human plasma is presented. Morphine was extracted from plasma by solvent extraction with ethyl acetate and derivatized to its heptafluorobutyrate derivative. The derivatives were measured by gas chromatography–negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostic useful fragment ion at m/z 637 is obtained at high relative abundance. Deuterated morphine was used as an internal standard. Calibration graphs were linear within a range of 0.78 ng/ml and 50 ng/ml. Inter-assay precision was 2.3% (2.85 ng/ml) and 1.4% (14.25 ng/ml), intra-assay variability was found to be 1.5% (3.71 ng/ml) and 0.5% (14.54 ng/ml). Accuracy showed deviations of −9.3% (2.85 ng/ml) and −4.2% (14.25 ng/ml). The method is rugged and robust and has been applied to the batch analysis of morphine during pharmacokinetic profiling of the drug.

Testing of the anabolic stanozolol in human hair by gas chromatography–negative ion chemical ionization mass spectrometry

A sensitive, specific and reproducible method for the quantitative determination of stanozolol in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride and the sonication in methanol of 100 mg of powdered hair for 2 h. After elimination of the solvent, the hair sample was solubilized in 1 ml 1 M NaOH, 15 min at 95°C, in the presence of 10 ng stanozolol-d 3 used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase (Isolute C 18) and a liquid–liquid (pentane) extraction. After evaporation of the final organic phase, the dry extract was derivatized using 40 μl MBHFA–TMSI (1000:20, v/v), incubated for 5 min at 80°C, followed by 10 μl of MBHFBA, incubated for 30 min at 80°C. The derivatized extract was analyzed by a Hewlett-Packard GC–MS system with a 5989 B Engine operating in the negative chemical ionization mode of detection. Linearity of the detector response was observed for stanozolol concentrations ranging from 5 to 200 pg/mg with a correlation coefficient of 0.998. The assay was capable of detecting 2 pg of stanozolol per mg of hair when approximately 100 mg hair material was processed, with a quantification limit set at 5 pg/mg. Intra-day precision was 5.9% at 50 pg/mg and 7.8% at 25 pg/mg with extraction recoveries of 79.8 and 75.1%, respectively. The analysis of a 3-cm long hair strand, obtained from a young bodybuilder (27 year old) assuming to be a regular user of Winstrol (stanozolol, 2 mg), revealed the presence of stanozolol at the concentration of 15 pg/mg.

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE–Hb) and serum albumin (BPDE–SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m−3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography–negative ion chemical ionization–mass spectrometry. BPDE–Hb adducts were detected in 16% of the population and BPDE–SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE–Hb and BPDE–SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE–Hb and BPDE–SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced theresults to some degree.

Biomonitoring of heterocyclic aromatic amine metabolites in human urine

Human exposure to heterocyclic aromatic amines such as MeIQx (2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline) may be monitored by measuring the levels of the heterocyclic aromatic amine in urine. In order to investigate the contribution of N-oxidation to the metabolism of MeIQx in vivo, we developed a biomonitoring procedure for the analysis and quantification of the N 2-glucuronide conjugate of 2-hydroxyamino-3,8-dimethylimidazo[4,5- f]quinoxaline in human urine. Subjects ( n=66) in the dietary study ingested a uniform diet of cooked meat containing known amounts of MeIQx, and urine was collected after consumption of the test meal. A method based on solid-phase extraction and immunoaffinity separation was used to isolate N 2-(β-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo[4,5- f]quinoxaline and its stable isotope-labeled internal standard from urine. The isolated conjugate was converted to the deaminated product 2-hydroxy-3,8-dimethylimidazo[4,5- f]quinoxaline by treatment with acetic acid under moderate heating. 2-Hydroxy-3,8-dimethylimidazo[4,5- f]quinoxaline and the [ 2H 3]methyl analog were derivatized to form the corresponding 3,5- bis(trifluoromethyl)benzyl ether derivatives and quantified by capillary gas chromatography–negative ion chemical ionization mass spectrometry employing selected ion monitoring procedures. The amounts of N 2-(β-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo[4,5- f]quinoxaline recovered in urine collected 0–12 h after the test meal accounted for 2.2–17.1% of the ingested dose, with a median value of 9.5%. The variability in the proportion of the dose excreted among the subjects may be reflective of several factors, including interindividual variation in the enzymic activity of CYP1A2 and/or conjugation reactions of the N-hydroxylamine metabolite with N-glucuronosyltransferase(s).

Gas chromatography–negative ion chemical ionization mass spectrometry as a powerful tool for the detection of mercapturic acids and DNA and protein adducts as biomarkers of exposure to halogenated olefins

The studies on metabolism of halogenated olefins presented here outline the advantages of modern mass spectrometry. The perchloroethene (PER) metabolite N-acetyl- S-(trichlorovinyl)- l-cysteine ( N-ac-TCVC) is an important biomarker for the glutathione dependent biotransformation of PER. In urine of rats and humans exposed to PER, N-ac-TCVC was quantified as methyl ester after BF 3–MeOH derivatization by gas chromatography with chemical ionization and negative ion detection mass spectrometry (GC–NCI-MS). The detection limit was 10 fmol/μl injected solution using [ 2H 3] N-ac-TCVC methyl ester as the stable isotope internal standard. Cleavage of S-(trichlorovinyl)- l-cysteine by β-lyase enzymes results in an electrophilic and highly reactive thioketene which reacts with nucleophilic groups in DNA and proteins. Protein adduct formation was shown in kidney mitochondria by identification of dichloroacetylated lysine after derivatization with 1,1,3,3-tetrafluoro-1,3-dichloroacetone by GC–NCI-MS. In addition, chlorothioketene was generated in organic solvents and reacted with cytosine to give N 4-chlorothioacetyl cytosine. After derivatization with pentafluorobenzyl bromide this compound exhibited good gas chromatographic properties and was detectable with a limit of detection of 50 fmol/injected volume. The detection of chemically induced protein modifications in the target organ of toxic metabolite formation and the study of DNA modifications with chemically generated metabolites provide important information on organ toxicity and possible tumorigenicity of halogenated olefins.

Phosphorylation of cytosolic phospholipase A2 by IL-3 is associated with increased free arachidonic acid generation and leukotriene C4 release in human basophils

Background: Human basophils secrete leukotriene C4 (LTC4) in response to various stimuli, and a short treatment with IL-3 enhances LTC4 release, although IL-3 alone does not induce LTC4 release. However, the mechanism of this priming effect of IL-3 for LTC4 generation remains unknown in human basophils. Objective: This study was designed to explore the mechanisms by which short treatments with IL-3 enhance stimulated secretion of LTC4, with a focus on the activation of cytosolic phospholipase A2 (cPLA2). Methods: The phosphorylation state of cPLA2 in human basophils was examined by its shift in electrophoretic mobility as detected by Western blotting. Free arachidonic acid (AA) and LTC4 were measured by gas chromatography–negative ion chemical ionization mass spectrometry and LTC4-specific RIA, respectively. Result: Human basophils expressed cPLA2. IL-3, as well as the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, caused a shift in the electrophoretic mobility of cPLA2, which indicated phosphorylation of cPLA2 and therefore its activation. Ionomycin at a concentration of 0.1 μg/mL was used to induce a modest elevation of cytosolic calcium response ([Ca 2+] I), no apparent cPLA2 phosphorylation, and little free AA and LTC4 generation. Pretreatment with IL-3 (1 to 10 ng/mL) markedly enhanced ionomycin (0.1 μg/mL)-mediated AA and LTC4 generation. The concentration dependence of cPLA2 phosphorylation by IL-3 and its effects on free AA and LTC4 generation were similar. The selective PKC inhibitors, bis-indolylmaleimide II and Ro-31-8220 inhibited the phorbol 12-myristate 13-acetate–mediated cPLA2 electrophoretic mobility shift, but not the IL-3–mediated shift, suggesting that the IL-3 effect is PKC independent. Both the anaphylatoxin split product of the C component C5 (C5a) and f-Met-Leu-Phe induced PKC-independent cPLA2 phosphorylation with a similar time course most notable for the absence of observable changes in cPLA2 phosphorylation before 30 seconds. These results suggested an explanation for the absence of free AA generation by C5a. When [Ca 2+] I was elevated in response to C5a, there was no phosphorylation of cPLA2, and by the time cPLA2 became phosphorylated, [Ca 2+] I had returned to resting levels. Treatment with IL-3 preconditioned the cPLA2 by causing its phosphorylation so that the transient [Ca 2+] I response, which followed stimulation by C5a, could induce the generation of free AA and LTC4. Conclusion: Taken together, these results suggest that the effect of IL-3 for free AA generation and LTC4 release might be due to induction of cPLA2 phosphorylation. The studies demonstrated a need for synchronous cPLA2 phosphorylation and elevations in [Ca 2+] I. (J Allergy Clin Immunol 1998;102:512-20.)

Trace analysis of tributyltin and triphenyltin compounds in sea water by gas chromatography–negative ion chemical ionization mass spectrometry

An analytical gas chromatography–mass spectrometry (GC–MS) method using negative ion chemical ionization (NICI) has been investigated for the determination of trace tributyltin (TBT) and triphenyltin (TPhT) compounds in sea water. TBT and TPhT were extracted from samples as chloride under the acidic condition of HCl. Doping of the GC system with a dilute HBr–methanolic solution resulted in direct detection of the chlorides of TBT, TPhT and tripentyltin (TPenT, internal standard). As the result of HBr doping, a sharp peak of the respective organotin bromides appeared: during GC analysis, halogen exchange from the chloride to the bromide occurred. NICI-MS was highly selective and sensitive for the detection of TBT, TPhT and TPenT bromides. In the selected ion monitoring mode of NICI-MS, the minimum detectable amounts defined as the signal equal to three times the standard deviation (3 σ) of the baseline noise were 20 and 25 pg ml −1 for TBT and TPhT, respectively. These amounts are approximately 250–400 times better than those in electron impact mode. The combination of GC using an apolar capillary column doped with a dilute HBr–methanolic solution and NICI-MS made it possible to determine TBT and TPhT at less than the ng l −1 level in sea water.

Quantitation of 1-Stearoyl-2-arachidonoyl-sn-3-glycerol in Human Basophils via Gas Chromatography–Negative Ion Chemical Ionization Mass Spectrometry

Investigation of the role of diacylglycerol molecular species in signal transduction in human basophils has been impeded by the lack of an assay method with adequate sensitivity and selectivity. Conversion of 1-stearoyl-2-arachidonoyl-sn-3-glycerol to the pentafluorobenzoyl ester conveys electron-capture properties to the diacylglycerol. The electron-capture derivative of the diacylglycerol is amenable to gas chromatographic analysis and undergoes limited fragmentation under negative ion mass spectrometric conditions with generation of an intense molecular anion atm/z838. Monitoringm/z838 for detection of 1-stearoyl-2-arachidonoyl-sn-3-glycerol andm/z841 for detection of 1-trideuterostearoyl-3-arachidonoyl-sn-2-glycerol employed as the internal standard provides the analytical basis for GC–MS quantitation of the endogenous diacylglycerol in human basophils. The assay displays excellent reproducibility over a wide range of concentrations with variations ≤10%. The GC–MS assay is highly selective and exquisitely sensitive with a detection limit of ≤0.20 pg (∼30 fmol) for endogenous 1-stearoyl-2-arachidonoyl-sn-3-glycerol per injection. Approximately 400 fmol of the diacylglycerol were extracted from 105stimulated human basophils.