Identification of pesticides by liquid chromatography--particle beam mass spectrometry using electron ionization and chemical ionization

Liquid chromatography-mass spectrometry (LC-MS) with a particle beam (PB) interface is used to separate and identify a group of pesticides. The mass spectra obtained under the different ionization modes, electron ionization (EI) and positive and negative chemical ionization (PCI and NCI) are compared. The operating conditions under each mode, determined by studying the influence on the ion abundance of the ion source temperature of the EI mode, and the gas pressure and ion source temperature in the methane CI were optimized. EI was more sensitive than PCI and NCI and of the latter two modes, NCI gave higher responses, especially for organophosphorus compounds. When on-line solid-phase extraction-LC-PB-MS was applied to real samples, limits of detection in full scan mode were in the range of 0.5 and 10 micrograms l-1 for EI. The analysis of real samples by on-line solid-phase extraction-LC-PB-MS enabled EI detection of one of the pesticides studied and confirmation by PCI and NCI. The combined EI/CI information also enabled the detection of some non-target compounds.     

Hair analysis for nordiazepam and oxazepam by gas chromatography--negative-ion chemical ionization mass spectrometry

The sub-Tenon's technique uses blunt dissection and a blunt probe to inject local anaesthetic into the posterior sub-Tenon's space. This avoids the potentially catastrophic complications which result from passing a sharp needle blindly into the orbit and retrobulbar space. The anatomy of Tenon's capsule and the block technique is described. Results of the block quality and degree of patient comfort from 300 consecutive sub-Tenon's blocks are also described. No significant complications occurred in this series. Single-quadrant sub-Tenon's block offers an excellent quality of anaesthesia, is virtually painless to perform and avoids complications due to passage of a sharp needle into the orbit.     

Determination of four anabolic steroid metabolites by gas chromatography/mass spectrometry with negative ion chemical ionization and tandem mass spectrometry

A gas chromatography/mass spectrometry method is described which uses negative ion chemical ionization and tandem mass spectrometry for the determination of anabolic steroid metabolites. Four anabolic steroid metabolites to be derivatized by Pentafluoropropionic anhydride (PFPA) were determined using gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization (NCI) and NCI/MS/MS. The repeatability and reproducibility of this procedure were in the range of 5.3-9.7% and 6.1-10.2%, respectively. This method of derivatization with PFPA for NCI and NCI/MS/MS was useful to determine four metabolites of nandrolone, dromostanolone, methenolone and boldenone. The derivatized metabolites of boldenone could be detected to 2 ppb and the other three steroids could be detected to 25 ppb in urine at a signal-to-noise ratio of S/N = 3.     

Analysis of malachite green and metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry

Malachite green (MG), a traditional agent used in aquaculture, is structurally related to other carcinogenic triphenylmethane dyes. Although MG is not approved for use in aquaculture, its low cost and high efficacy make illicit use likely. We developed sensitive and specific methods for determination of MG and its principal metabolite, leucoMG (LMG), in edible fish tissues using isotope dilution liquid chromatography atmosphere pressure chemical ionization mass spectrometry. MG and LMG concentrations were measured in filets from catfish treated with MG under putative use conditions (ca. 250 and 1000 ppb, respectively) and from commercial trout samples (0-3 and 0-96 ppb, respectively). Concentrations of LMG in edible fish tissues always exceeded those of MG. A rapid cone voltage switching acquisition procedure was used to simultaneously produce molecular ions for quantification and diagnostic fragment ions for confirmation of MG and metabolites. The accurate and precise agreement between diagnostic ion intensity ratios produced by LMG in authentic standards and incurred fish samples was used to unambiguously confirm the presence of LMG in edible fish tissue. This suggested the validity of using LMG as a marker residue for regulatory determination of MG misuse. Additional metabolites derived from oxidative metabolism of MG or LMG (demethylation and N-oxygenation) were identified in catfish and trout filets, including a primary arylamine which is structurally related to known carcinogens. The ability to simultaneously quantify residues of MG and LMG, and to confirm the chemical structure of a marker residue by using LC/MS, suggests that this procedure may be useful in monitoring the food supply for the unauthorized use of MG in aquaculture.     

Determination of alprazolam and alpha-hydroxyalprazolam in human plasma by gas chromatography/negative-ion chemical ionization mass spectrometry

A sensitive and specific method was developed for the determination of alprazolam and its major metabolite alpha-hydroxyalprazolam in plasma. After the addition of deuterium-labeled internal standards, plasma samples were buffered to pH 9 with 1 ml of saturated sodium borate buffer, extracted with toluene-methylene chloride (7:3) and evaporated to dryness. The residues were treated with N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% of trimethylchlorosilane and analyzed on a Finnigan-MAT mass spectrometer operated in the negative-ion chemical ionization mode with methane as the reagent gas. Chromatographic separation was achieved on a Restek-200 capillary column using hydrogen as the carrier gas. The assay was linear from 0.25 to 50 ng ml-1 for both compounds. The intra-assay precision for alprazolam was 16.1% at 0.5 ng ml-1 and 4.6% at 50 ng ml-1 and that for alpha-hydroxyalprazolam was 15.8% at 0.5 ng ml-1 and 4.2% at 50 ng ml-1. The method was used to determine alprazolam and alpha-hydroxyalprazolam in human plasma samples collected after a single 2 mg oral does of alprazolam. A peak concentration of 32.9 ng ml-1 of alprazolam was detected at 1 h following the dose.     

Multi-anode detection in electrospray ionization time-of-flight mass spectrometry

An electrospray ionization ion source coupled to a time-of-flight mass analyzer incorporating a multi-anode time-to-digital converter is described. High-speed data acquisition (kHz mass spectral acquisition) rates are achieved. The four-anode detector produces a significant increase in detection/counting efficiency over that for a single-anode detector. In this work a 2.5 times increase in detection efficiency is demonstrated. The multi-anode detector is also used as a diagnostic tool to optimize transmission of the ion optics.     

Determination of tetrahydro-beta-carbolines in rat brain by gas chromatography-negative-ion chemical ionization mass spectrometry without interference from artifactual formation

This paper describes a quantitative method for neuroactive alkaloids, 1-methyl-1,2,3,4-tetrahydro-beta-carboline (MTBC) and 1,2,3,4-tetrahydro-beta-carboline (TBC), in rat brain by gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NICIMS). After addition of tetradeuterated MTBC and TBC (internal standards), the samples were subjected to deproteinization, reaction with fluorescamine, solvent extractions, trifluoroacetylation and GC-NICIMS analysis. In contrast to the other previous methods, the artifactual formation during analysis did not interfere with the determination of MTBC and TBC because their precursor tryptamine was removed as a fluorescamine derivative from the analytical system at the first step of pretreatment. MTBC and TBC were specifically and reliably determined in the range of pg-ng/sample. Application of the proposed method has revealed that the MTBC and TBC contents in rat brain significantly increase after intraperitoneal administration of MTBC and TBC, indicating their ability to easily cross the blood-brain barrier.     

Molecular species of sphingomyelin: determination by high-performance liquid chromatography/mass spectrometry with electrospray and high-performance liquid chromatography/tandem mass spectrometry with atmospheric pressure chemical ionization

In a sphingomyelin-enriched sample of polar lipids from bovine milk, molecular species of intact sphingomyelin were separated by normal-phase high-performance liquid chromatography and detected by mass spectrometry (MS) for structural information. First, by using electrospray with positive ionization (ESI), protonated molecules ([M + H]+) were detected. Second, in atmospheric pressure chemical ionisation (APCI+), in-source fragmentation of sphingomyelin ions led to the formation of ceramide ions. With the ceramide ions as precursors, ions representative of both the long-chain base (LCB) parts and the fatty acid (FA) parts were detected in APCI-MS/MS via collision-induced decomposition (CID). Using this procedure, it was possible to determine the sphingomyelin molecular masses using ESI+ and then their respective LCB-FA combinations(s) using APCI+(-)MS/MS. At least 36 protonated molecules of intact sphingomyelin were detected in the bovine milk sample. The combinations found covered a range of molecular masses from 673 to 815 Da. The 12 most common protonated molecules (constituting approximately 90% of the total ion current in ESI) were composed of at least 25 different LCB-FA combinations. Saturated and unsaturated LCBs and FAs were detected in addition to hydroxy fatty acids. The most common LCBs were 16:1, 17:1, 18:1 and 19:1, whereas the most common FAs were 16:0, 22:0, 23:0 and 24:0. LCB-FA combinations of sphingomyelin from bovine brian, bovine erythrocytes and chicken egg yolk are also presented.     

Evaluation of the uncertainty of molecular mass measurement by electrospray ionization mass spectrometry

Electrospray ionization (ES) is a novel method used in mass spectrometry (MS) for producing gas-phase ions from substances in solutions. Common practices for molecular mass estimation from ES spectra summarize the spectrum as a single peak giving no estimate of uncertainty or treat each peak as an independent molecular mass measurement. ES-MS data analysis showed that each peak in an ES spectrum does not always provide an independent measure of molecular mass. Underestimation of measurement uncertainty is a possible result. An elementary time series method, the Yule-Walker equations, was applied to molecular mass estimation from ES data.     

Electrospray ionization and collision-induced dissociation time-of-flight mass spectrometry of neutral glycosphingolipids

A series of native naturally occurring neutral glycosphingolipids has been analysed by electrospray ionization tandem mass spectrometry using a hybrid magnetic sector-TOF instrument. The collision-induced dissociation products of precursor ions were detected by an orthogonal acceleration time-of-flight mass spectrometer as the second analyser. Glycosphingolipids, with mono- to hexa-saccharide chain lengths with different ceramide constituents, were studied. The result of electrospray ionization in the positive ion mode generally showed singly charged molecular ions with Na+ as adduct, [M + Na]+. The sensitivity of the electrospray ionization was greatly enhanced by addition of NaCl, LiCl (forming [M + Li]+) or KCl (yielding [M + K]+) to the sample. A comparison between the collision-induced dissociation of precursor molecular ions of monoglycosylceramides, using Na+, Li+ and K+ as adducting species, showed that the intensity of the fragment ions and the extent of the daughter ion fragmentation of the molecular ions, are dependent on the type of adduct used. The daughter ion spectra of Li+ adduct ions showed intense sequence fragment ions, both of the saccharide chain and the ceramide moiety, and were superior to those obtained using Na+ or K+. The collision-induced dissociation spectra of the [M + Li]+ ions, of glycosphingolipids containing di- to hexasaccharides, are also presented. Proposed possible fragments, resulting from the CID of the molecular ions [M + Li]+ of monoglycosylceramides, are shown.     

Characterization of bacterial phospholipids by electrospray ionization tandem mass spectrometry

A negative ion electrospray ionization tandem mass spectrometric technique was developed for the analysis of glycerophospholipids. Examination of the product ion mass spectrum of the deprotonated molecular ion provided sufficient information to identify both the class of glycerophospholipid and the molecular weights of the two fatty acid moieties. This technique was applied to the profiling of glycerophospholipids present in the chloroform/methanol extracts of four different bacterial species. The principal bacterial phospholipids detected by this technique were phosphatidylglycerols and diphosphatidylglycerols, accompanied by small amounts of phosphatidylethanolamines for two of the bacterial species examined. The fatty acid composition of the phosphatidylglycerols for each bacteria was determined by tandem mass spectrometry and presented graphically. Differences in the fatty acid composition for each bacterial species were readily apparent from a visual examination of the data sets.     

Analysis of nonylphenol polyethoxylates and their degradation products in river water and sewage effluent by gas chromatography-ion trap (tandem) mass spectrometry with electron impact and chemical ionization

Two chromosome races of the house shrew Suncus murinus that differ from each other for five Robertsonian translocations (8.17, 9.13, 10.12, 11.16, and 14.15), heterochromatic insertions in chromosomes 7 and X, and multiple rearrangements in the Y chromosome were crossed and then intercrossed in captivity to produce a hybrid stock. Electron-microscopic analysis of synaptonemal complexes in fertile and sterile hybrid males was carried out. Meiosis in sterile males did not progress beyond pachytene and was severely disrupted. Meiotic arrest was not determined by structural heterozygosity: heterozygotes for all variant chromosomes distinguishing two parental races were found in both sterile and fertile male hybrids. Fertile hybrids demonstrated an orderly pairing of all chromosomes. In heterozygotes for Robertsonian fusions, completely paired trivalents were formed between the Robertsonian metacentrics and homologous acrocentrics. In heterozygotes for chromosome 7, bivalents with a small buckle were observed in a small fraction of pachytene cells. No differences were found in the morphology and pairing pattern of sex bivalents, composed of the X and Y chromosomes derived from the same or different parental races. Univalents, multivalents, and associations between X and Y chromosomes and autosomal trivalents, as well as associations of autosomal trivalents with each other, were observed in a small fraction of the pachytene cells of fertile males. Our results indicate that the system controlling male sterility in interracial hybrids of S. murinus is of genic rather than of chromosomal type.     

Application of gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry for analysis of DNA and protein adducts

Olive-pollen profilin has been isolated and characterized as a significant allergen. Its molecular properties, such as a molecular mass of 15 kDa; amino-acid composition; and secondary repetitive structure percentages of 15% alpha-helix, 33% beta-strand, 20% beta-turn, and 32% random coil, have been determined. Its allergenic capability, a recognition frequency estimated at 24% of olive-hypersensitive patients, and high cross-reactivity with all the pollen used have been found. The presence of conformation epitopes in the olive profilin, as well as a high structural and immunologic similarity to other pollen sources such as birch and ash, can be established from these studies.     

Capillary electrochromatography of biomolecules with on-line electrospray ionization and time-of-flight mass spectrometry

Capillary electrochromatography (CEC) is considered a hybrid of liquid chromatography and capillary electrophoresis. It is expected to combine the high peak efficiency of capillary zone electrophoresis with the versatility and loading capacity of HPLC to bring about another high-performance MS-compatible chromatographic system. This paper explores the potential of CEC coupled with the electrospray ionization and time-of-flight mass spectrometry in biochemical analysis. The packed columns used in this study were tapered at the outlet to retain the packing material, thereby obviating the need for an outlet frit. Electrosmotically driven solvent gradients were employed for the separation of phenylthiohydantoin (PTH)-amino acids by reversed-phase chromatography, and a time-of-flight (TOF) mass spectrometer was employed as the detector for the CEC column effluent. The effect of CEC operating parameters, such as gradient shape, column length, and electric field, on the analytical results from the separation and MS detection of a standard mixture of PTH-amino acids was investigated. Particular attention was paid to the effect of sheath flow-rate, sheath composition and mass spectra acquisition rate on the performance of the electrospray TOF-MS.     

The observation of chaperone-ligand noncovalent complexes with electrospray ionization mass spectrometry

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was carried out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.