John M. Halket

Deconvolution gas chromatography/mass spectrometry of urinary organic acids – potential for pattern recognition and automated identification of metabolic disorders

The National Institute of Standards and Technology (NIST) Automated Mass Spectral Deconvolution and Identification System (AMDIS) is applied to a selection of data files obtained from the gas chromatography/mass spectrometry (GC/MS) analysis of urinary organic acids. Mass spectra obtained after deconvolution are compared with a special user library containing both the mass spectra and retention indices of ethoxime-trimethylsilyl (EO-TMS) derivatives of a set of organic acids. Efficient identification of components is achieved and the potential of the procedure for automated diagnosis of inborn errors of metabolism and for related research is demonstrated.

Manipulation of Phytoene Levels in Tomato Fruit: Effects on Isoprenoids, Plastids, and Intermediary Metabolism

In tomato (Solanum lycopersicum), phytoene synthase-1 (PSY-1) is the key biosynthetic enzyme responsible for the synthesis of fruit carotenoids. To further our understanding of carotenoid formation in tomato fruit, we characterized the effect of constitutive expression of an additional tomato Psy-1 gene product. A quantitative data set defining levels of carotenoid/isoprenoid gene expression, enzyme activities, and metabolites was generated from fruit that showed the greatest perturbation in carotenoid content. Transcriptional upregulation, resulting in increased enzyme activities and metabolites, occurred only in the case of Psy-1, Psy-2, and lycopene cyclase B. For reactions involving 1-deoxy-d-xylulose5-phosphate synthase, geranylgeranyl diphosphate synthase, phytoene desaturase, ζ-carotene desaturase, carotene isomerase, and lycopene β-cyclase, there were no correlations between gene expression, enzyme activities, and metabolites. Perturbations in carotenoid composition were associated with changes in plastid type and with chromoplast-like structures arising prematurely during fruit development. The levels of >120 known metabolites were determined. Comparison with the wild type illustrated that key metabolites (sucrose, glucose/fructose, and Glu) and sectors of intermediary metabolism (e.g., trichloroacetic acid cycle intermediates and fatty acids) in the Psy-1 transgenic mature green fruit resembled changes in metabolism associated with fruit ripening. General fruit developmental and ripening properties, such as ethylene production and fruit firmness, were unaffected. Therefore, it appears that the changes to pigmentation, plastid type, and metabolism associated with Psy-1 overexpression are not connected with the ripening process.

Isatin: identity with the purified endogenous monoamine oxidase inhibitor tribulin

Abstract: Purified tribulin, an endogenous monoamine oxidase (MAO) inhibitor, has been identified by direct probe insertion mass spectrometry as the indole‐2,3‐dione, isatin. A gas chromatographic‐mass spectrometric assay for isatin has been developed and used to measure its relatively high concentrations in unpurified human urine, and in rat heart and brain. Isatin is a known compound with a broad range of biological activity; this is the first report of its presence in the animal body. Isatin is a potent inhibitor of MAO, particularly of MAO B (IC50, 3 μM), and also binds to central benzodiazepine receptors (IC50 against clonazepam, 123 μM).

Derivatization in Mass Spectrometry—1. Silylation:

This is the first of a series of reviews on the application of derivatization in mass spectrometry. A description is given of advances in silylation as a powerful tool used for increasing the volatility, thermal and thermo-catalytic stability, and chromatographic mobility of polar and unstable organic compounds. In addition to chemical aspects of silylation, mass spectral properties of silyl derivatives useful for structure determination and quantitation of various organic and biologically-active compounds, mainly by GC/MS, are described. Practically all tested and widely used silylating agents are described. The role of comprehensive libraries containing reference mass spectra for various silyl derivatives and search systems in structure determination is emphasized. Applications of silylation for particular analyses are summarised.

Brain quinolinic acid in Huntington's disease.

Abstract: Concentrations of the endogenous neurotoxic tryptophan metabolite, quinolinic acid (QA), were measured in postmortem brain tissue obtained from patients with Huntingtons disease (HD) and matched controls, using a gas chromatography/mass spectrometry method. There was no significant difference in either the putamen or the frontal cortex between the HD and control groups. These results do not support the hypothesis that increased QA is responsible for neuronal degeneration in HD.

A Proteomic-Based Approach for Detection of Chicken in Meat Mixes

A proteomic-based method has been developed for the detection of chicken meat within mixed meat preparations. The procedure is robust and simple, comprising the extraction of myofibrillar proteins, enrichment of target proteins using OFFGEL isoelectric focusing, in-solution trypsin digestion of myosin light chain 3, and analysis of the generated peptides by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Using this approach, it was possible for example to detect 0.5% contaminating chicken in pork meat with high confidence. Quantitative detection of chicken meat was done by using AQUA stable isotope peptides made from the sequence of previously selected species-specific peptide biomarkers. Linearity was observed between the amount of the peptide biomarker and the amount of chicken present in the mixture; further independent replication is required now to validate the method. Apart from its simplicity, this approach has the advantage that it can be used effectively for the detection of both raw and cooked meat. The method is robust, reliable, and sensitive, representing a serious alternative to methods currently in use for these purposes. It is amenable to highly processed foods which can be particularly problematic, as the tertiary protein structure is often affected in processed food precluding immunoassays. In addition, this proteomic analysis will permit the determination of definitive discriminatory sequence, unlike the DNA PCR based methods used presently. The present article also demonstrates the translation of the technology to routine mass spectrometry equipment, making the methodology suitable for public analysts.

Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography-tandem mass spectrometry

Current analytical methods used for screening drugs and their metabolites in biological samples from victims of drug-facilitated sexual assault (DFSA) or other vulnerable groups can lack sufficient sensitivity. The application of liquid chromatography, employing small particle sizes, with tandem mass spectrometry (MS/MS) is likely to offer the sensitivity required for detecting candidate drugs and/or their metabolites in urine, as demonstrated here for ketamine. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was performed following extraction of urine (4 mL) using mixed-mode (cation and C8) solid-phase cartridges. Only 20 microL of the 250 microL extract was injected, leaving sufficient volume for other assays important in DFSA cases. Three ion transitions were chosen for confirmatory purposes. As ketamine and norketamine (including their stable isotopes) are available as reference standards, the assay was additionally validated for quantification purposes to study elimination of the drug and primary metabolite following a small oral dose of ketamine (50 mg) in 6 volunteers. Dehydronorketamine, a secondary metabolite, was also analyzed qualitatively to determine whether monitoring could improve retrospective detection of administration. The detection limit for ketamine and norketamine was 0.03 ng/mL and 0.05 ng/mL, respectively, and these compounds could be confirmed in urine for up to 5 and 6 days, respectively. Dehydronorketamine was confirmed up to 10 days, providing a very broad window of detection.

A library of atmospheric pressure ionization daughter ion mass spectra based on wideband excitation in an ion trap mass spectrometer

A searchable library of MS/MS spectra obtained using a quadrupole ion trap mass spectrometer and electrospray or atmospheric pressure chemical ionization is presented. The application of wideband excitation (activation) and normalized collision energy leads to highly reproducible mass spectra which are searched using the NIST algorithm. Flow injection and LC/MS/MS applications of this powerful technique in the biomedical (diastereoisomeric steroids, morphine glucuronides, isovalerylcarnitine) and environmental (pirimicarb and desmethyl-pirimicarb) areas are described.

Derivatization in mass spectrometry - 8. Soft ionization mass spectrometry of small molecules

This is the first of two reviews devoted to derivatization approaches for “soft” ionization mass spectrometry (fast atom bombardment, matrix assisted laser desorption/ionization, electrospray ionization and atmospheric pressure chemical ionization) and deals, in particular, with small molecules. The principles of the main “soft” ionization mass spectrometric methods as well as the reasons for derivatizing small molecules are briefly described. Derivatization methods for modification of amines, carboxylic acids, amino acids, alcohols, carbonyl compounds, monosaccharides, thiols, unsaturated and aromatic compounds etc. to improve their ionizability and to enhance structure information content are discussed. The use of “fixed”-charge bearing derivatization reagents is especially emphasized. Chemical aspects of derivatization and “soft” ionization mass spectrometric properties of derivatives are considered.

Isatin, regional distribution in rat brain and tissues

Isatin has recently been identified in rat tissues and normal human urine, where it forms the major proportion of the endogenous monoamine oxidase inhibitor, tribulin. In this paper, we show that isatin, measured by gas chromatography/mass spectrometry, has a distinct regional distribution in rat tissues, with highest concentrations in seminal vesicles (1.6 ?g/g) and vas deferens (3.4 ?g/g). There was also a discontinuous distribution within rat brain, concentrations being highest in the hippocampus (0.13 ?g/g).