A method was developed to profile the major constituents of St John's wort extracts using high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS). The objective was to simultaneously separate, identify and quantify hyperforin, hypericin, pseudohypericin, rutin, hyperoside, isoquercetrin, quercitrin and chlorogenic acid using HPLC-MS. Quantification was performed using an external standardisation method with reference standards. The method consisted of two protocols: one for the analysis of flavonoids and glycosides and the other for the analysis of the more lipophilic hypericins and hyperforin. Both protocols used a reverse phase Luna phenyl hexyl column. The separation of the flavonoids and glycosides was achieved within 35 min and that of the hypericins and hyperforin within 9 min. The linear response range in ESI-MS was established for each compound and all had linear regression coefficient values greater than 0.97. Both protocols proved to be very specific for the constituents analysed. MS analysis showed no other signals within the analyte peaks. The method was robust and applicable to alcoholic tinctures, tablet/capsule extracts in various solvents and herb extracts. The method was applied to evaluate the phytopharmaceutical quality of St John's wort preparations available in the UK in order to test the method and investigate if they contain at least the main constituents and at what concentrations.

Nanoscale reversed-phase liquid chromatography (LC) combined with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used as a method for the direct identification of multiple cytochrome P450 (P450) isoforms found in male and female rat liver. In this targeted proteomic approach, rat liver microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel tryptic digestion of the proteins present in the 48- to 62-kDa bands. The resultant peptides were extracted and analyzed by LC-ESI-MS/MS. P450 identifications were made by searching the MS/MS data against a rat protein database containing 21,576 entries including 47 P450s using Sequest software (Thermo Electron, Hemel Hempstead, UK). Twenty-four P450 isoforms from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, CYP17, and CYP19 were positively identified in rat liver.

We describe the direct identification of multiple cytochrome P450 (CYP) enzymes in healthy and cancerous tissue. CYPs in human liver colorectal metastases were compared with those in the surrounding liver using a mass spectrometry-based proteomic approach. Nano-scale reversed phase liquid chromatography combined with electrospray ionisation tandem mass spectrometry has been used to identify CYPs with no pre-selection of the proteins anticipated. Fourteen distinct CYP enzymes from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, 8B and 27A were positively identified; 13 in the liver samples and 12 in the tumour tissue. It was found that three of the colorectal metastases expressed essentially the same drug-metabolising pattern of CYPs as the surrounding liver, whilst three tumour samples from different individuals showed no CYP expression. This was likely in at least one case to be due to low sample mass. The CYP expression profile in an individual tumour is likely to be an important determinant in predicting the outcome of cancer chemotherapy.

Cytochrome P450 (CYP) isoforms are known to be expressed in human cancers, including colon, breast, lung, liver, kidney and prostate. This raises the possibility of the use of CYP isoforms in tumour-specific prodrug activation (Patterson et al 1999). As a prerequisite for this work, protocols must be established for the analysis of CYPs. Nanoelectrospray ionisation tandem mass spectrometry (NSIMS/MS) has been adopted as the method of choice because of the large amount of protein sequence information that can be obtained from very small volumes of sample. Recombinant human CYP isoforms 1A2, 2E1 and 3A4 were analysed intact, digested with trypsin in solution or subjected to 1D SDS-PAGE followed by in-gel tryptic digestion and extraction. Approximately 50 pmol of each protein was loaded onto the gels; solution digests contained protein concentrations of 1.7 pmolmL. MS/MS experiments were performed on a ThermoFinnigan LCQ with a nanospray ion source. Flow rates were less than 50 nLmin. Samples were analysed as mixtures with no prior chromatographic separation. Protein digest identifications were made using ThermoFinnigan TurboSequest software. Peptides from each digest have been identified with high confidence using TurboSequest Xcorr and DelCn values (Ducret et al 1998). Table 1 shows the percentage sequence coverages obtained.

AQ4N (1,4-bis-[[2-(dimethylamino-N-oxide)ethyl]amino]5,8-dihydroxyanthracene-9,10-dione) is in a class of bioreductive agents incorporating the aliphatic N-oxide functionality and is well documented as a very effective enhancer of radiotherapy and chemotherapy. The compound is shortly to enter Phase I clinical trials in the United Kingdom, and this study describes the preclinical pharmacokinetics and metabolism of AQ4N in mice. AQ4N was administered by i.v. injection at doses of 200, 100, and 20 mg/kg and was quantified by high-performance liquid chromatography and liquid chromatography/mass spectroscopy. There was a linear increase in the maximum plasma concentration (Cmax) proportional to dose with a Cmax of 1171 microg/ml at the maximum tolerated dose of 200 mg/kg. The area under plasma concentration versus time curve (AUC) increased disproportionately with dose from 14.1 microg/h/ml at 20 mg/kg to 247 microg/h/ml at 200 mg/kg with a subsequent decrease in clearance. Terminal elimination half-lives ranged from 0.64 to 0.83 h. The spectra of the two major metabolites matched those from authentic standards with the molecular ions [M + H]+ being detected at m/z 445.4 (AQ4N), m/z 429.5 (AQ4 mono-N-oxide) and m/z 413.5 (AQ4). Only low concentrations of the toxic metabolite (AQ4) were detected in plasma at all three doses, with the AUC and Cmax at 200 mg/kg being 3.54 microg/h/ml and 3.7 microg/ml, respectively, representing <2% of AQ4N. Concentrations of the intermediate AQ4 M represented 8, 10, and 18% of those for AQ4N at the doses of 20,100, and 200 mg/kg. The concentrations necessary for a therapeutic response in vivo have been described in this pharmacokinetic study.

Although much research has been completed on the application of matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS) to the analysis of bacteria, no definitive studies have yet been performed on the analysis of fungi. Preliminary studies on the application of the MALDI-MS methodology, previously developed for the analysis of bacteria, to the analysis of intact fungal spores are described here. MALDI-MS and electrospray mass Spectrometry enable the analysis of high molecular-weight proteins, glycoproteins, oligosaccharides and oligonucleotides. Using MALDI-MS with bacteria has enabled the production of ‘fingerprints» of the intact cells; the ions observed are associated with the proteinaceous components of the cell wall. This study reports the adaptation of this technique to the direct analysis of fungal cells. Because of the large amount of carbohydrate in the fungal cell wall, the ions observed in the mass spectrometric experiments might be of carbohydrate origin. Penicillium spp., Scytalidium dimidiatum and Trichophyton rubrum have been studied in this preliminary investigation and all furnish individually distinctive spectra which seem to provide a profile of the cellular material with discrete peaks being observed over the mass range 2 to 13 kDa. The spectra obtained are reproducible within the method used but, as shown in our previous studies on bacteria, washing might selectively release components from the fungal cell wall.

A considerable volume of research has now been completed on the application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the analysis of bacteria; however, to date no definitive studies have been made using this technique on fungi. Preliminary studies on the application of the MALDI-MS methodology, previously developed for the analysis of bacteria, to the analysis of intact fungal spores are described here. MALDI-MS and electrospray mass spectrometry enable the high molecular weight analysis of proteins, glycoproteins, oligosaccharides and oligonucleotides. Using MALDI-MS with bacteria has demonstrated the ability to produce 'fingerprints' of the intact cells with the ions observed being associated with the proteinaceous components of the cell wall. This paper reports the adaptation of this technique to the direct analysis of fungal cells. The high percentage of carbohydrate in the fungal cell wall indicates that the ions observed in the mass spectrometric experiments may be of carbohydrate origin. Penicillium spp., Scytalidium dimidiatum and Trichophyton rubrum have been studied in this preliminary investigation and all show individually distinctive spectra which would appear to provide a profile of the cellular material with discrete peaks being observed over the mass range 2 to 13 kDa. The spectra obtained are reproducible within the method used but, as shown in our previous studies on bacteria, washing may selectively release components from the fungal cell wall.

We have studied a number of commonly used matrices and a range of solvent systems, of widely varying polarity, used in matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectra in an attempt to devise an optimum analytical strategy for the rapid characterization of pathogenic microorganisms. Several groups have recognized the usefulness of MALDI-TOF mass spectrometry in the characterization of these microorganisms, however, no consistent methodology has yet been developed which is in general use. Escherichia coli ATCC 9637 organisms were initially washed to remove growthmedium contaminants and then extracted with one of a range of solvents before admixing with either a single matrix ([2-(4-hydroxyphenylazo)]-benzoic acid, 2-mercaptobenzothiol, 2-amino-4-methyl-5-nitropyridine, or 2,6-dihydroxyacetophenone) or binary and ternary combinations of the four matrices. The results indicated that a binary combination of [2-(4-hydroxyphenylazo)]-benzoic acid and 2-mercaptobenzothiol (1:1) as matrix provides the most useful data with the organisms being extracted with 17% formic acid, 33% isopropyl alcohol and 50% water.

A variety of gram-positive and gram-negative intact bacterial cells have been analysed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and shown to provide fingerprint mass spectra with discrete peaks being observed over the mass range from 3 to 40 kDa. The spectra show both more peaks and peaks at a higher mass/charge ratio than have hitherto been reported for these micro-organisms and would appear to provide a profile of cellular proteinaceous material. The spectra are shown to be reproducible over variable time periods of up to three months and factors affecting reproducibility are discussed. The procedure, which requires minimal sample preparation, yields results in 30-40 minutes and allows visual identification of species- and strain-specific biomarkers for the characterization of the organisms. The importance of accurately defining sample preparation methodologies is central to the ability of the technique to generate reliable and reproducible data.

Conference Article| February 01 1995 Identification of an endogenous inhibitor of AMP-activated protein kinase CHRISTINE M. TOOMEY; CHRISTINE M. TOOMEY 1Dept of Pharmaceutical Chemistry, School of Pharmacy, University of London, Brunswick Square, London WC1N 1AX, UK Search for other works by this author on: This Site PubMed Google Scholar KEVIN J. WELHAM; KEVIN J. WELHAM 1Dept of Pharmaceutical Chemistry, School of Pharmacy, University of London, Brunswick Square, London WC1N 1AX, UK Search for other works by this author on: This Site PubMed Google Scholar MICHAEL R. MUNDAY MICHAEL R. MUNDAY 1Dept of Pharmaceutical Chemistry, School of Pharmacy, University of London, Brunswick Square, London WC1N 1AX, UK Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (1): 142S. https://doi.org/10.1042/bst023142s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation CHRISTINE M. TOOMEY, KEVIN J. WELHAM, MICHAEL R. MUNDAY; Identification of an endogenous inhibitor of AMP-activated protein kinase. Biochem Soc Trans 1 February 1995; 23 (1): 142S. doi: https://doi.org/10.1042/bst023142s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

S-Adenosyl-l-methionine (SAM) is a methyl-donor compound which is actively involved in a variety of biochemical reactions. An assay has been developed permitting the quantitative measurement of SAM and its related metabolites (S-adenosylhomocysteine, decar☐ylated SAM, methylthioadenosine, adenosine and adenine) in liver and cell cultures. As gradient reversed-phase chromatographic or cation-exchange chromatographic methods often resulted in overlapping peaks, a two-dimensional high-performance liquid chromatographic (HPLC) procedure was developed involving gradient reversed-phase chromatographic separation followed by ion-exchange chromatography. After precipitating large molecules in the sample by perchloric acid, gel permeation was carried out on a Sephadex G 25 column to separate small water-soluble metabolites from proteins and membrane fragments. The freeze-dried sample was injected onto an ODS column and a 0–10% acetonitrile gradient in 10 m M ammonium formate buffer (pH 2.9) (20 min, linear) was applied. The relevant fractions were collected and injected onto a cation-exchange column (Partisil SCX, 10 μm, 250 mm × 4.6 mm I.D.). Elution and quantification were carried out using ammonium formate buffers of various concentration (15–400 m M), pH 2.9. The detector response (254 nm) as a function of concentration was linear over the concentration range 30–500 pmol. The detection limits of the compounds after the two-dimensional chromatographic procedure ranged from 10 to 60 pmol and the recovery was higher than 70%. The reproducibility of the results obtained from given samples was within 9–22% for rat liver and 6–24% for mast cells.

A series lipidic amide conjugates (2b, c, 3b, c, 4b-d, 5b and c) of β-lactam antibiotics were synthesised using mixed anhydride methods to couple the Boc-protected lipidic amino acids (1a and b) and oligomer (1c) to a variety of penicillins and cephalosporins. Conjugates (2b, c, 3b, 4b-d and 5b) showed weak to moderate activity in vitro and were only weakly active in vivo against the non-β-lactamase producing stain S. aureus 663E.

Each amino acid in a peptide has a characteristic immonium ion (H2N+ = CHR), the presence of which in a mass spectrum can indicate the presence of that amino acid. High-energy collision-induced decomposition studies on small peptide ions formed by fast atom bombardment showed the relative intensities of these immonium ions to be dependent on the relative positions of the amino acids in the peptide chain: C-terminal, N-terminal or in-chain. Evidence in favour of competition in the formation of immonium ions is presented.

AbstractAb initio molecular orbital calculations have been used to determine the degree of charge localization associated with the formation of radical cations on electron impact of formamide, thioformamide and their N‐methyl and N, N‐dimethyl analogues. The charge favours the nitrogen of formamides and the sulphur of thioformamides, but the degree of localization is calculated to be lower than in the radical cations of urea and thiourea.

A chiral effect has been observed for formation of protonated dimers of dialkyl tartrates under fast atom bombardment, analogous to the chemical ionization behaviour reported previously. Homochiral dimers show greater stability than the heterochiral dimers, as is evidenced by their enhanced formation and lesser tendency to undergo metastable breakdown. The difference in stability is related to the size of the alkyl group.

Organic Mass SpectrometryVolume 18, Issue 4 p. 176-177 New Techniques Fast atom bombardmetn using a commercial field ionization/field desorption source Michael A. Baldwin, Michael A. Baldwin School fo Pharmacy, University of London, 29/39, Brunswick Square, London WC1N 1AX, UKSearch for more papers by this authorDavid M. Carter, David M. Carter School fo Pharmacy, University of London, 29/39, Brunswick Square, London WC1N 1AX, UKSearch for more papers by this authorKevin J. Welham, Kevin J. Welham School fo Pharmacy, University of London, 29/39, Brunswick Square, London WC1N 1AX, UKSearch for more papers by this author Michael A. Baldwin, Michael A. Baldwin School fo Pharmacy, University of London, 29/39, Brunswick Square, London WC1N 1AX, UKSearch for more papers by this authorDavid M. Carter, David M. Carter School fo Pharmacy, University of London, 29/39, Brunswick Square, London WC1N 1AX, UKSearch for more papers by this authorKevin J. Welham, Kevin J. Welham School fo Pharmacy, University of London, 29/39, Brunswick Square, London WC1N 1AX, UKSearch for more papers by this author First published: April 1983 https://doi.org/10.1002/oms.1210180410Citations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume18, Issue4April 1983Pages 176-177 RelatedInformation