Margaret McCooeye

Mapping of Selenium Metabolic Pathway in Yeast by Liquid Chromatography−Orbitrap Mass Spectrometry

A high-resolution mass spectrometric detection method is described for the identification of key metabolites in the selenium pathway in selenium enriched yeast. Iodoacetic acid (IAA) was used as the derivatizing reagent to stabilize the selenols. Oxidized forms of selenocysteine (Se-Cys), selenohomocystine (Se-HCys), selenoglutathione (Se-GSH), seleno-γ-glutamyl-cysteine (Se-Glu-Cys), N-(2,3-dihydroxy-1-oxopropyl)-selenocysteine (Se-DOP-Cys), N-(2,3-dihydroxy-1-oxopropyl)-selenohomocysteine (Se-DOP-HCys), selenomethionine (SeMet), seleno-S-adenosyl-homocysteine (Se-AdoHcy), the conjugate of glutathione and N-(2,3-dihydroxy-1-oxopropyl)-selenocysteine (GSH-Se-DOP-Cys), and the conjugate of glutathione and N-(2,3-dihydroxy-1-oxopropyl)-selenohomocysteine (GSH-Se-DOP-HCys) were found in the selenium enriched yeast certified reference material (SELM-1). Selenols were also derivatized with a mercury tag, p-hydroxymercurybenzoate (PHMB). The selenol-PHMB complexes showed the overlapped isotopic patterns of selenium and mercury, which provided supporting information for the identification of selenols. Both methods showed good agreement (<4 ppm difference) between the theoretical masses of the target compounds and the measured masses in the yeast matrix. The method using IAA as the derivatizing reagent was used to study the response of Saccharomyces cerevisiae to three forms of selenium, Se-Met, Na(2)SeO(3) (Se(IV)), and Na(2)SeO(4)·10H(2)O (Se(VI)) (concentration of Se: 100 mg/L). The production of selenocompounds observed over a 6 h period was high in the Se-Met treated group compared to the groups treated with Se(IV) and Se(VI).

Evaluation of high-field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of the mycotoxin zearalenone

A high-field asymmetric waveform ion mobility spectrometry (FAIMS)-based method for the determination of the mycotoxin zearalenone (ZON) and its metabolites alpha-zearalenol (alpha-ZOL), beta-zearalenol (beta-ZOL), and beta-zearalanol (beta-ZAL), in a cornmeal (maize) matrix is described. Detection limits achieved using the FAIMS device coupled with electrospray ionization (ESI) and mass spectrometric (MS) detection are 0.4 ng mL(-1) for ZON and 3 ng mL(-1) for alpha-ZOL+beta-ZOL, and beta-ZAL. This represents a significant improvement when compared to detection limits determined using ESI-MS or ESI-tandem mass spectrometry (MSMS) analytical methods. The developed flow-injection (FIA)-ESI-FAIMS-MS method was applied to reference materials ERM-BC-716 and ERM-BC-717 certified for ZON and excellent agreement with the certified values was observed.

Determination of Arsenobetaine in Fish Tissue by Species Specific Isotope Dilution LC-LTQ-Orbitrap-MS and Standard Addition LC-ICPMS

An accurate and precise method for the determination of arsenobetaine (AsB, (CH(3))(3)(+)AsCH(2)COO(-)) in fish samples using exact matching species specific isotope dilution (ID) liquid chromatography LTQ-Orbitrap mass spectrometry (LC-LTQ-Orbitrap-MS) and standard addition LC inductively coupled plasma mass spectrometry (LC-ICPMS) is described. Samples were extracted by sonication for 30 min with high purity deionized water. An in-house synthesized (13)C enriched AsB spike was used for species specific ID analysis whereas natural abundance AsB, synthesized and characterized by quantitative (1)H NMR (nuclear magnetic resonance spectroscopy), was used for reverse ID and standard addition LC-ICPMS. With the LTQ-Orbitrap-MS instrument in scan mode (m/z 170-190) and resolution set at 7500, the intensities of [M + H](+) ions at m/z of 179.0053 and 180.0087 were used to calculate the 179.0053/180.0087 ion ratio for quantification of AsB in fish tissues. To circumvent potential difficulty in mass bias correction, an exact matching approach was applied. A quantitatively prepared mixture of the natural abundance AsB standard and the enriched spike to give a ratio near one was used for mass bias correction. Concentrations of 9.65 ± 0.24 and 11.39 ± 0.39 mg kg(-1) (expanded uncertainty, k = 2) for AsB in two fish samples of fish1 and fish2, respectively, were obtained by ID LC-LTQ-Orbitrap-MS. These results are in good agreement with those obtained by standard addition LC-ICPMS, 9.56 ± 0.32 and 11.26 ± 0.44 mg kg(-1) (expanded uncertainty, k = 2), respectively. Fish CRM DORM-2 was used for method validation and measured results of 37.9 ± 1.8 and 38.7 ± 0.66 mg kg(-1) (expanded uncertainty, k = 2) for AsB obtained by standard addition LC-ICPMS and ID LC-LTQ-Orbitrap-MS, respectively, are in good agreement with the certified value of 39.0 ± 2.6 mg kg(-1) (expanded uncertainty, k = 2). Detection limits of 0.011 and 0.033 mg kg(-1) for AsB with LC-ICPMS and ID LC-LTQ-Orbitrap-MS, respectively, were obtained demonstrating that the technique is well suited to the determination AsB in fish samples. To the best of our knowledge, this is first application of species specific isotope dilution for the accurate and precise determination of AsB in biological tissues.

Application of UPLC-QTOF-MS in MSE mode for the rapid and precise identification of alkaloids in goldenseal (Hydrastis canadensis)

AbstractHere, we describe a new application of ultra-performance liquid chromatography coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry operating in MSE mode (UPLC-QTOF-MSE) for the sensitive, fast, and effective characterization of alkaloids in goldenseal (Hydrastis canadensis). This approach allowed identification of alkaloids using a cyclic low and high collision energy spectral acquisition mode providing simultaneous accurate precursor and fragment ion mass information. A total of 45 compounds were separated and 40 of them characterized including one new compound and 7 identified for the first time in goldenseal. The spectral data obtained using this method is comparable to those obtained by conventional LC-MSn. However, the UPLC-QTOF-MSE method offers high chromatographic resolution with structural characterization facilitated by accurate mass measurement in both MS and MS/MS modes in a single analytical run; this makes it suitable for the rapid analysis and screening of alkaloids in plant extracts. FigureIdentification of alkaloids in goldenseal by UPLC-QTOF-MS in MSE mode

Mapping of sulfur metabolic pathway by LC Orbitrap mass spectrometry.

For the first time a liquid chromatography method with high resolution mass spectrometric detection has been developed for the simultaneous determination all key metabolites of the sulfur pathway in yeast, including all thiolic (cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), γ-glutamyl-cysteine (Glu-Cys)) and non-thiolic compounds (methionine (Met), s-adenosyl-methionine (AdoMet), s-adenosyl-homocysteine (AdoHcy), and cystathionine (Cysta)). The developed assay also permits the speciation and selective determination of reduced, oxidized and protein bound fractions of all of the five thiols. Iodoacetic acid (IAA) was chosen as the derivatizing reagent. Thiols were extracted from sub-mg quantities of yeast using hot 75% ethanol. The detection limits were in the range of 1-12 nmol L(-1) for standard solution (high femotomole, absolute), except AdoMet (116 nmol L(-1)), which was unstable. In freshly harvested yeast, most of the thiols were in the reduced forms and low levels of protein-bound GSH and Glu-Cys were found. In a selenium enriched yeast, the thiols were mainly in the oxidized forms, and a significant amount of protein-bound Cys, HCys, GSH, Cys-Gly and Glu-Cys were found. The method was also applied to the metabolic study of the adaptive response of Saccharomyces cerevisiae to hydrogen peroxide, cadmium, and arsenite, and the change in concentration of thiols in the sulfur pathway was monitored over a period of 4h.

Characterization of the alkaloids in goldenseal (Hydrastis canadensis) root by high resolution Orbitrap LC-MSn

AbstractLiquid chromatography coupled to multistage mass spectrometry (LC-MSn) is being used increasingly in pharmaceutical research and for quality control in herbal medicines because of its superior sensitivity and selectivity. In this study, a rapid, high-resolution liquid chromatography-mass spectrometry (LC-MSn) method was developed to separate and identify alkaloids in the root extract of goldenseal, which is one of the 20 most popular herbal supplements used worldwide. In total, 28 alkaloids were separated and characterized including one novel compound and 21 identified, or tentatively identified, for the first time in goldenseal. The current high-resolution LC-MSn method provides a rapid and definitive means of profiling the composition of goldenseal root and will provide a useful tool in understanding the bioactivity of this medicinal plant. FigureExtraction and Orbitrap LC-MSn analysis of Goldenseal root for alkaloid identification

Determination of underivatized amino acids in microsamples of a yeast nutritional supplement by LC-MS following microwave assisted acid hydrolysis

In this paper we describe a rapid method for microscale microwave assisted acid hydrolysis followed by quantitative amino acid analysis, using liquid chromatography mass spectrometry UPLC-ESI-MS (QTOF) without derivatization. Such an approach allows substantial decrease in the amount of yeast sample (less than 1 mg) required for effective analysis. The vapor phase hydrolysis approach was compared to condensed phase hydrolysis and all the microwave parameters were optimized for 16 amino acids. Separation was achieved on a BEH C-18 column. Limits of detection and quantitation (LODs/LOQs), using standard solutions, were in the sub-ppb range. The method was successfully applied to the determination and quantitation of methionine and selenomethionine by isotope dilution and standard addition methods in yeast on a yeast certified reference material sample.

Applications of High-Field Asymmetric Waveform Ion Mobility Spectrometry for the Certification of Reference Materials

Abstract: High-field asymmetric waveform ion mobility spectrometry (FAIMS), is a novel gas-phase separation technique, developed at the Institute for National Measurement Standards (INMS), and commercialized by Ionaltyics Corporation, ON, Canada (now ThermoFisher). The FAIMS device operates at ambient conditions and is installed between an atmospheric pressure ion source and the mass spectrometer. The first use of FAIMS data in the certification of a reference material was in support of the National Institute of Standards and Technology project to certify ephedrine alkaloids in dietary supplements. FAIMS technology has also been applied to the quantitation of amino acids in a yeast matrix with a view to producing a certified reference material.