Search

Here, we describe a new application of ultra-performance liquid chromatography coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry operating in MS(E) mode (UPLC-QTOF-MS(E)) 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-MS(n). However, the UPLC-QTOF-MS(E) 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.

Liquid chromatography coupled to multistage mass spectrometry (LC-MS(n)) 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-MS(n)) 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-MS(n) 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.

Absolute quantitation of peptides/proteins in dilute calibration solutions used in various diagnostic settings is a major challenge. Here we report the absolute quantitation of peptides by non-species-specific isotope dilution liquid chromatography-inductively coupled plasma mass spectrometry (ID LC-ICPMS) based on stoichiometric Eu tagging. The method was validated by species-specific isotope dilution gas chromatography/mass spectrometry (GC/MS) analysis of constituent amino acids of the target peptide. Quantitative labeling of bradykinin peptide was accomplished with a commercially available 2',2″-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-NHS-ester) and subsequently tagged with Eu. A (151)Eu-enriched spike was used for the non-species-specific ID LC-ICPMS determination of bradykinin. The non-species-specific ID LC-ICPMS method was cross-validated by a species-specific ID GC/MS approach, which is based on the determination of phenylalanine in bradykinin to derive the concentration of the peptide in the sample. The hydrolysis of the peptide into amino acids was achieved by microwave digestion with 4 M methanesulfonic acid, and derivatization of phenylalanine with methyl chloroformate (MCF) was performed prior to its detection by GC/MS based on a (13)C-enriched phenylalanine spike. The accuracy of the method was confirmed at various concentration levels with a typical precision of better than 5% relative standard deviation (RSD) at 20 pmol for non-species-specific ID LC-ICPMS and 500 pmol for species-specific ID GC/MS. A detection limit (3 SD) of 7.2 fmol estimated for ID LC-ICPMS with a 10 μL injection volume from three procedure blanks was obtained for bradykinin, confirming the suitability of the method for the direct determination of peptides at trace levels. To the best of our knowledge, the proposed method is the first ICPMS peptide quantification strategy which employs an independent validation strategy using species-specific ID GC/MS for amino acid quantitation.

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., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

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.

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).

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.

The National Institute of Standards and Technology, the U.S. Food and Drug Administration, Center for Drug Evaluation and Research and Center for Food Safety and Applied Nutrition, and the National Institutes of Health, Office of Dietary Supplements, are collaborating to produce a series of Standard Reference Materials (SRMs) for dietary supplements. A suite of ephedra materials is the first in the series, and this paper describes the acquisition, preparation, and value assignment of these materials: SRMs 3240 Ephedra sinica Stapf Aerial Parts, 3241 E. sinica Stapf Native Extract, 3242 E. sinica Stapf Commercial Extract, 3243 Ephedra-Containing Solid Oral Dosage Form, and 3244 Ephedra-Containing Protein Powder. Values are assigned for ephedrine alkaloids and toxic elements in all 5 materials. Values are assigned for other analytes (e.g., caffeine, nutrient elements, proximates, etc.) in some of the materials, as appropriate. Materials in this suite of SRMs are intended for use as primary control materials when values are assigned to in-house (secondary) control materials and for validation of analytical methods for the measurement of alkaloids, toxic elements, and, in the case of SRM 3244, nutrients in similar materials.

Twenty proteinogenic amino acids (AAs) were determined without derivatization using flow injection analysis followed by electrospray ionization mass spectrometry and tandem mass spectrometry (ESI-MS and ESI-MS/MS) and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry (ESI-FAIMS-MS and ESI-FAIMS-MS/MS), in positive and negative ionization modes. Three separate sets of ESI-FAIMS conditions were used for the separation and detection of the 20 AAs. Typically ESI-FAIMS-MS showed somewhat improved sensitivity and significantly better signal-to-noise ratios than ESI-MS mainly due to the elimination of background noise. However, the difference between ESI-FAIMS-MS and ESI-MS/MS was significantly less. ESI-FAIMS was able to partially or completely resolve all the isobaric amino acid overlaps such as leucine, isoleucine and hydroxyproline or lysine and glutamine. Detection limits for the amino acids in ESI-FAIMS-MS mode ranged from 2 ng/mL for proline to 200 ng/mL for aspartic acid. Overall, ESI-FAIMS-MS is the preferred method for the quantitative analysis of AAs in a hydrolyzed yeast matrix., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

A method for accurate determination of ephedrine (E) alkaloids in natural health products (NHP) is described. The NIST dietary supplement standard reference materials (SRMs) were selected for these studies. These SRMs comprise ground Ma Huang herb (Ephedra sinica Stapf.), a spray dried extract of the former, and commercial formulations derived from gel caps and a protein drink. The efficiency of sonication-assisted extraction and Soxhlet extraction was studied using both ammonium formate and potassium phosphate in 3% methanol as extraction media. The efficiency of SPE clean-up of the extract deteriorated rapidly when increasing amounts of sample matrix or analyte were processed, because of limited cartridge capacity. Quantitation by the method of additions was required to ensure the highest accuracy using both LC-UV and ES-LC-MS-MS techniques. Whereas the LC-UV method is more convenient and precise, the results are more questionable than ES-LC-MS-MS, because species-specific detection is not possible.

A suite of five ephedra-containing dietary supplement Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST) with certified values for ephedrine alkaloids, synephrine, caffeine, and selected toxic trace elements. The materials represent a variety of natural, extracted, and processed sample matrixes that provide different analytical challenges. The constituents have been determined by multiple independent methods with measurements performed by NIST and by three collaborating laboratories. The methods utilized different sample extraction and cleanup steps in addition to different instrumental analytical techniques and approaches to quantification. In addition, food-matrix proximates were determined by National Food Processor Association laboratories for one of the ephedra-containing SRMs. The SRMs are primarily intended for method validation and for use as control materials to support the analysis of dietary supplements and related botanical materials.

A method is described for the determination of ephedrine (E) and pseudoephedrine (PE) and their metabolites norephedrine (NE), norpseudoephedrine (NPE), methylephedrine (ME), and methylpseudoephedrine (MPE) alkaloids in natural health products by flow injection-electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry (FI-ESI-FAIMS-MS). The determination of the six alkaloids requires the separation of diastereomic pairs of E-PE, NE-NPE, and ME-MPE. FAIMS was able to resolve/separate these isomeric pairs based on their gas-phase ion mobility differences. The FAIMS-based separation and detection approach has been tested on over-the-counter diet pills. Following the extraction of the tablets, either by pressurized fluid extraction developed in-house or with sonication, the ephedra alkaloids were quantified using a modified isotope dilution approach. Detection limits for the alkaloids ranged from 0.1 to 3 ng/mL, and a linear range of at least 2 orders of magnitude was observed for the six analytes. The throughput of the current configuration of the FI-ESI-FAIMS-MS system is 2 min/sample, which is significantly higher than conventional chromatographic approaches. The developed FI-ESI-FAIMS-MS method has been compared with a conventional LC-UV analysis, and good agreement has been found for the major alkaloids.