Determination of a bound musk xylene metabolite in carp hemoglobin as a biomarker of exposure by gas chromatography-mass spectrometry using selected ion monitoring.

Musk xylene (MX) is widely used as a fragrance ingredient in commercial toiletries. Identification and quantitation of a bound 4-amino-MX (AMX) metabolite was carried out by gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). Detection of AMX occurred after the cysteine adducts in carp hemoglobin (Hb), derived from the nitroso metabolite, were released by alkaline hydrolysis. The released AMX metabolite was extracted into n-hexane. The extract was preconcentrated by evaporation and analyzed by GC-SIM-MS. The concentration of AMX metabolite was found to range from 6.0 to 30.6 ng/g in the carp Hb, collected from the Las Vegas Wash and Lake Mead, NV areas. The presence of an AMX metabolite in the carp Hb was confirmed when similar mass spectral features and the same retention time of the AMX metabolite were obtained for both standard AMX and carp Hb extract solutions. In the nonhydrolyzed and reagent blank extracts, the AMX metabolite was not detected.

Environmental screening of acidic compounds based on capillary zone electrophoresis/laser-induced fluorescence detection with identification by gas chromatography/mass spectrometry and gas chromatography/high-resolution mass spectrometry.

This paper describes the application of capillary zone electrophoresis/laser-induced fluorescence detection (CZE/LIF) to the discovery of acidic compounds in environmental matrixes or the screening of extracts for acidic components. Published studies indicate that coal-derived materials contain a significant fraction of acidic compounds relative to materials derived from petroleum and shales. Such compounds may be useful as marker compounds for site assessment and source apportionment issues, and their identification may be important in toxicological and other health issues. We used deep-UV light from the frequency-doubled Ar ion laser at 244 and 257 nm to study extracts of samples. The CZE/LIF technique possesses good sensitivity and therefore overcomes one of the limitations of CZE with UV detection. The present work depends on high pressure/temperature solvent extraction of polynuclear aromatic hydrocarbon (PNA)-contaminated soil, followed by separation using CZE. The anionic analytes were separated by using borate or phosphate buffer (pH 9.2-12.3) after a chemical class separation. Samples were also characterized by gas chromatography/mass spectrometry (GC/MS) using full scans at low resolution, and elemental compositions were determined unequivocally by GC/high-resolution MS (GC/HRMS) using mass peak profiling (MPP). The similarity of low-resolution electron ionization mass spectra for a standard, 1-hydroxypyrene, and for a series of compounds in a contaminated-soil extract suggested that several types of phenolic and hydroxy-PNAs were present, including hydroxylated derivatives of fluorenes, fluoranthenes, and pyrenes. GC/HRMS using MPP confirmed the elemental compositions of the hydroxyfluorenes and hydroxypyrenes (and presumably hydroxyfluoranthenes) as [C13H10O] and [C16H10O], respectively. A new version of the MPP software was written for the Finnigan-MAT 900S-Trap and was similar to that developed previously for the VG 250SE. Inclusion of a calibration ion in addition to a lock mass ion in the multiple-ion detection descriptor provided errors of <1 ppm for the 3 partial profiles of the analytes. A mass resolution of 31,000 was used to resolve the analyte signals from interferences evident in the full M+1 and M+2 profiles in the case of the hydroxyfluorenes. Derivatization was also performed to form the tert-butyldimethylsilyl derivatives of phenolic hydroxy groups as a further confirmation of structure.

Capillary electrophoresis and capillary electrochromatography of organic pollutants.

Capillary electrophoresis (CE) has a unique capability for separation of analytes of environmental concern, particularly those that are more polar and ionic, based on the complementary separation principle of electrophoresis. In the past few years, CE has been selectively used to analyze various classes of compounds having current or potential environmental relevance. This review outlines the current status of CE for the determination of environmental pollutants, based predominantly on research results published from the beginning of 1997 to early 1999. Covered are environmental pollutants of all types except pesticides and inorganics. Certain naturally produced toxins are also covered because of their significant impacts upon human health and the environment. CE methods, as with all methods, must be judged on their ability to provide approaches that are reliable, sensitive, selective, and rapid, while meeting "green chemistry" initiatives for pollution prevention. We also compare CE methods to benchmark environmental techniques involving gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and high performance liquid chromatography (HPLC).

Development of separation systems for polynuclear aromatic hydrocarbon environmental contaminants using micellar electrokinetic chromatography with molecular micelles and free zone electrophoresis.

Of four systems available from the literature, based on cyclodextrins, dioctylsulfosuccinate, bile salts, and molecular micelles consisting of oligomers of undecylenic acid, the most successful separation system in our hands is based on the molecular micelles, oligomers of sodium undecylenic acid (OSUA). We have employed organic additives of acetonitrile, acetone, and tetrahydrofuran in achieving separations of polyaromatic hydrocarbons (PNAs) using molecular micelles. Generally, successful separations are achieved with 20-40% composition as the organic additive in an 8 mM borate buffer. We separated 16 PNAs with 20% tetrahydrofuran in a system of 8 mM borate and 0.125 g/10 mL (ca. 6.25 mM) of OSUA. Typical extracts of environmental samples contain additional analytes besides the typical 16 target compounds. Among these are the nitrogen-containing aromatics that can act as cations under conditions of low pH and additional compounds that can act as anions under basic conditions in free-zone electrophoresis. These additional classes of analytes are separated by capillary zone electrophoresis/laser-induced fluorescence detection using a frequency-doubled laser operated at 257 nm.

Capillary electrophoresis/laser-induced fluorescence detection of fluorescein as a groundwater migration tracer.

Capillary electrophoresis (CE) has been applied to the determination of the groundwater migration tracer dye fluorescein based on laser-induced fluorescence (LIF) detection and compared to determinations obtained with traditional spectrofluorimetry. Detection limits of injected dye in the low parts per trillion (ppt) ranges have been accomplished with both CE/LIF based on the Ar ion laser and with a spectrofluorimeter. This approach was used for a real-world problem in determining groundwater migration between adjacent Resource Conservation and Recovery Act (RCRA) and Superfund sites by the Environmental Sciences Division in response to regional needs and as application of new analytical tools under development. Fluorescent dye was injected into source wells and then was determined in monitoring wells by extracting pads that adsorbed the dye or by directly determining the dye in the water using solid-phase extraction (SPE), a preconcentration technique. The approaches based on CE/LIF exhibits increased specificity over existing approaches due to the separation and unique migration time of the dye. Additional studies were aimed at achieving sub-ppt levels in the water using solid-phase extraction and field-amplified injection techniques.

A kinetic study of the methanolysis of the sulfonylureas bensulfuron methyl and sulfometuron methyl using capillary electrophoresis.

The instability of sulfonylureas in solution in methanol has led us to a kinetic study of methanolysis of two sulfonylureas using capillary electrophoresis. In a preliminary experiment, solutions of the seven compounds, bensulfuron methyl, sulfometuron methyl, nicosulfuron, chlorimuron ethyl, thifensulfuron methyl, metsulfuron methyl, and chlorsulfuron were prepared in methanol and in acetonitrile. After six weeks at room temperature the compounds dissolved in acetonitrile were quite stable as shown by electropherograms obtained with free zone capillary electrophoresis (CE). All seven of the compounds dissolved in methanol had undergone extensive degradation and in some cases the sulfonylurea could not be detected. Bensulfuron methyl and sulfometuron methyl were selected for further detailed study. Solutions of these two compounds in methanol were heated at 5, 40, 57, and 65 degrees C. No decomposition of either compound was observed in similar solutions of acetonitrile incubated at 57 degrees C. The rates of decomposition in methanolic solutions were estimated from the decrease in instrumental response representing the peaks of the two sulfonylureas. The methanolysis of both compounds proceeded with pseudo first-order kinetics as evidenced by the fact that semilogarithmic plots of the concentration of the substrate as a function of time were linear (correlation coefficients of 0.99). Rate constants and half-lives for the reactions at 40, 50, 57, and 65 degrees C were determined. These varied by factors of 18 to 22 depending upon the reaction temperature. The energies of activation for these reactions were estimated from the appropriate Arrhenius plots and found to be 26 kcal per mole for bensulfuron methyl and 24.5 kcal per mole for sulfometuron methyl. The kinetics of appearance of the products of methanolysis were directly related to and accurately predicted by the pseudo first-order rate constants observed for disappearance of the sulfonylureas. Kinetic study of the reaction mixture by negative and positive-ion mass spectrometry indicated that the products of methanolysis of sulfometuron methyl were 2-carboxymethyl (N-carboxymethyl)benzsulfonamide and 2-amino-4,6-di-methyl pyrimidine and suggested a general mechanism for methanolysis of each of the seven compounds studied.

Applications of capillary electrophoresis/laser-induced fluorescence detection to groundwater migration studies.

Capillary electrophoresis has been applied to the determination of groundwater migration based on laser-induced fluorescence (LIF) detection and traditional spectrofluorimetry. Detection limits of injected dye-fluorescent whitening agent (tinopal) in the low ppt ranges have been accomplished with both a spectrofluorometer and with CE/LIF based on the HeCd laser. The real-world problem was the determination of groundwater migration between adjacent Resource Conservation and Recovery Act (RCRA) and Superfund sites. Fluorescent dyes were injected into wells and were discovered in monitoring wells by extracting pads that adsorbed the dye. The methodology based on CE/LIF exhibits increased specificity over existing methodology due to the separation and unique migration time of the dye. Additional studies were aimed at achieving sub-ppt levels in the water directly using solid-phase extraction (SPE) and field-amplified injection techniques.

Capillary electrophoretic behavior of seven sulfonylureas.

The electrophoretic behavior of seven sulfonylureas (bensulfuron methyl, sulfometuron methyl, nicosulfuron [accent], chlorimuron ethyl, thifensulfuron methyl [harmony], metsulfuron methyl, and chlorsulfuron) was studied under capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) conditions. Mixtures of these compounds were separated with very high efficiencies (2 x 10(5) theoretical plates) in a running buffer consisting of 3 parts acetate buffer (25 mM, pH 5.0) and 1 part acetonitrile. In this buffer system, acetonitrile was shown to be superior to methanol, acetone, and ethanol as a nonpolar additive, but any of these solvents can be used to reduce electroosmotic flow (EOF) and to obtain adequate separation. On-column detection limits at 214 nM were of the order of 80-100 fM. Micellar agents such as sodium dodecyl sulfate (SDS) and sodium cholate (but not monosialoganglioside-Gm1 or starburst dendrimer, generation 2.5) improved separation in phosphate and borate buffers. Implications of these results for the development of methods to detect these compounds on matrices of environmental origin are discussed. In particular, the instability of these compounds in methanol is noted and degradation products are detected using free zone CE. The methanolysis products of sulfometuron are tentatively identified by tandem MS (negative ion conditions) as 2-amino-4,6-dimethylpyrimidine and 2-carboxymethylbenz(N-carboxymethyl)sulfonamide.

Modular methodology for determination of polychlorinated biphenyls in soil as Aroclors and individual congeners.

A comprehensive screening and confirmatory method was developed for monitoring polychlorinated biphenyls (PCBs), both as Aroclors and as individual congeners. This approach incorporates extraction, extract cleanup, and analysis modules designed to match cost, time, and data quality requirements. Soxhlet, sonication, supercritical fluid, and accelerated solvent extractions were evaluated. Carbon chromatographic cleanup procedures were used for separation of congeners on the basis of ortho substitutions, which permitted calculation of toxicity equivalents. Individual congener determinations, congener total histograms, and peak comparison techniques for Aroclor identification were elaborated by using high and low resolution mass spectrometric data. A screening procedure based on immunoassay using the Ohmicron PCB RaPID Assay kit gave results comparable to those obtained by gas chromatography with electron capture detection in the range 0.40-230 ppm, when the appropriate Aroclor calibrator was used.

Determination of elemental compositions from mass peak profiles of the molecular ion (m) and the m + 1 and m + 2 ions.

The relative abundances of M + 1 and M + 2 ions help to identify the elemental composition of the molecular ion (M). But scan speed, sensitivity, and resolution limitations of mass spectrometers have impeded determination of these abundances. Mass peak profiling from selected ion recording data (MPPSIRD) provided faster sampling and enhanced sensitivity, which permitted use of higher resolution. M + 2 profiles having only a few percent of the ion abundance of M were monitored at 20 000 resolution. The relative abundances, exact masses, and shapes of M, M + 1, and M + 2 mass peak profiles were determined. By applying five criteria based on these quantities, elemental compositions were determined even for ions too large (up to 766 Da) to be uniquely assigned from their exact mass and accuracy limits alone. A profile generation model (PGM) was written to predict these resolution-dependent quantities by considering all M + 1 and M + 2 ions for each candidate composition. The model also provided assurance that no other compositions were possible. Characterization of the M + 1 and M + 2 profiles by MPPSIRD and the PGM greatly expanded the practical ability of high-resolution mass spectrometry to determine elemental compositions.

Comparison of micellar electrokinetic chromatography (MEKC) with capillary gas chromatography in the separation of phenols, anilines and polynuclear aromatics potential field-screening applications of MEKC.

Capillary electrophoresis (CE) is known to be complementary to liquid chromatography, but comparison of CE with capillary gas chromatography (GC) for applicable analytes has not been extensive. Capillary GC has been the preeminent separation technique for environmental analysis, but CE has yet to be applied systematically to the determination of environmental analytes. We present data on separations of three classes of semivolatile analytes of interest to environmental analysis: phenols, anilines and polynuclear aromatic hydrocarbons (PNAs). Standard GC conditions were used to illustrate typical separations observed on 30-m and 40-m columns. Rapid analyses were addressed using a high-temperature 15-m column of thinner film. CE separations employed borate buffer with sodium cholate as the micellar agent in micellar electrokinetic chromatography (MEKC). The effects of organic additives were studied using methanol, acetone and tetrahydrofuran. gamma-Cyclodextrin was also used in MEKC to enhance the separation of polynuclear aromatic hydrocarbons and to examine its effects on separations of phenols and anilines. Short capillaries effected very rapid (< 3 min) compound-class characterization, an approach which has potential use in site characterization/remediation (field-screening) studies.

Collisional activation mass spectra of M-. ions of azo dyes containing 2-naphthol.

Collisionally activated decomposition mass spectra of M-. ions of azo dyes are presented. The compounds are of general structure Ar(1)--N = N--Ar(2), where Ar(1) is substituted phenyl and Ar(2) is 2-naphthol. Characteristic fragment ions observed include m/z 157, which corresponds to the 2-naphthol substituent with cleavage of the--N = N--bond represented as [Ar(2)--N]-.. Ion of general structure [Ar(1)--NH]- are also observed. Parent ion scans of m/z 157 provide a potential screening technique for 2-naphthol-containing axo dyes. Specific results are reported for the chloroform extract of FD&C Red #8, and capillary gas chromatographic introduction is compared with direct exposure probe introduction for the identification of dyes.

Electron impact mass spectrometry of BHT and its alteration products.

The electron impact (EI) mass spectra of 2,6-di-tert-butyl-4-methylphenol (BHT) and certain of its alteration products are described in detail. Accurate mass measurements confirm the elemental compositions of important fragment ions in the EI spectra. Collisionally activated mass spectra are also used to study fragmentation and suggest common ion structures. The reference spectra provide the basis for identifying various alteration products of BHT by capillary gas chromatography/mass spectrometry (GC/MS) without the necessity of isolating individual components. Application of GC/MS is made to three studies: (i) pyrolysis of hydroperoxy-BHT as a potential pathway to alteration products in food; (ii) GC/MS pyrolysis of hydroperoxy-BHT as a model study; and (iii) alteration of BHT in ethanol/water as a food-simulating solvent.

Reactions of antioxidants in foods.

The chemical transformations of BHT and BHA in the deep-fat frying of potatoes were studied. Approximately 80% of the BHT and its associated decomposition products was lost from the lard after six batches of french fries. This may have been due to steam distillation caused by the water boiled out of the potatoes. In contrast, 80% of the radiocarbon introduced as radiolabelled BHA was retained by the frying medium even after 12 batches of french fries. The concentration of intact BHA decreased to undetectable levels after four batches. Because of the complexity of the products formed in the heated fats, studies were undertaken on the thermolysis of a hydroperoxy derivative of BHT, 2,6-di-tert-butyl-4-hydroperoxy-4-methylcyclohexa-2,3-dienone++ + (HBHT). The product mixture, which was identified by mass spectrometry and gas chromatography-mass spectrometry, included BHT, 2,6-di-tert-butyl-4-hydroxy-4-methylcyclohexa-2,5-dienone and 3,5-di-tert-butyl-4-hydroxybenzaldehyde. Elemental compositions of other unidentified products were determined by high-resolution mass spectrometry.

Negative ion chemical ionization mass spectrometric method for confirmation of identity of aflatoxin B1: collaborative study.

An interlaboratory study of a negative ion chemical ionization mass spectrometric (MS) confirmation procedure for aflatoxin B1 was conducted in laboratories in the United States, England, and West Germany. Twelve partially purified, dry film extracts from naturally and artificially contaminated roasted peanuts, cottonseed, and ginger root containing varying quantities of aflatoxin B1 were distributed to the participating laboratories. The extracts required additional cleanup before MS analysis, using either an acidic alumina column and preparative thin layer chromatography (TLC) or a 2-dimensional TLC procedure. Recovery of purified aflatoxin B1 was influenced by the degree of recovery of sample from acid alumina and/or the TLC plate and incomplete elution of aflatoxin B1 from silica gel. Factors affecting MS confirmation included the purity and recovery of aflatoxin and MS instrument sensitivity. Aflatoxin B1 identity was confirmed in 19.5, 90.9, and 100% of samples containing less than 5, 5-10, and greater than 10 ng aflatoxin B1/g product, respectively, by solid probe introduction using full mass scans. The MS method has been adopted official first action.

Mass spectrometry and identification of sterols in vegetable oils as butyryl esters and relative quantitation by gas chromatography with flame ionization detection.

Electron ionization mass spectrometry (MS) of sterol butyrates is described. Fragmentation of common sterol butyrates is related to structure and is discussed in relation to the fragmentation of free sterols and of commonly used sterol derivatives. Derivatized samples of vegetable oils are introduced using a 10 m capillary gas chromatographic (GC) column for complete separation of the sterol butyrates. Quantitation of sterol butyrates in vegetable oils by packed column GC/flame ionization detection is based on percent relative area of peaks identified by MS. Results of analyses of sunflower, castor, rapeseed, and virgin olive oils, and other oils are presented. These techniques have been applied to the rapid screening of marketed olive oils for possible adulteration.

Rapid quantitation and confirmation of aflatoxins in corn and peanut butter, using a disposable silica gel column, thin layer chromatography, and gas chromatography/mass spectrometry.

A simple, rapid, and solvent-efficient method for determining aflatoxins in corn and peanut butter is described. Aflatoxins B1, B2, G1, and G2 were extracted from 50 g sample with 200 mL methanol-water (85 + 15). A portion of the extract was diluted with 10% NaCl solution to a final concentration of 50% methanol, and then defatted with hexane. The aflatoxins were partitioned into chloroform. The chloroform solution was evaporated, and the residue was placed on a 0.5 g disposable silica gel column. The column was washed with 3 mL each of hexane, ethyl ether, and methylene chloride. Aflatoxins were eluted with 6 mL chloroform-acetone (9 + 1). The solvent was removed by evaporation on a steam bath, and the aflatoxins were determined using thin layer chromatography (TLC) with silica gel plates and a chloroform-acetone (9 + 1) developing solvent. Overall average recovery of aflatoxin B1 from corn was 82%, and the limit of determination was 2 ng/g. For mass spectrometric (MS) confirmation, aflatoxin B1 in the extract from 3 g sample (20 ng/g) was purified by TLC and applied by direct on-column injection at 40 degrees C into a 6 m fused silica capillary gas chromatographic column. The column was connected directly to the ion source. After injection, the temperature was rapidly raised to 250 degrees C, and the purified extract was analyzed by negative ion chemical ionization MS.