Workshop on DNA polymorphisms.
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Cloning, Molecular↗
Biomedical subjects
S Naylor
Publications and source records attributed to S Naylor.
Detection of corticosteroid in an over-the-counter product.
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Clobetasol↗
Membrane preconcentration-capillary electrophoresis-mass spectrometry in the analysis of biologically derived metabolites and biopolymers.
On-line capillary electrophoresis-mass spectrometry (CE-MS) is finding increased use in the analysis of a wide variety of chemically diverse complex mixtures. It is characterized by minimal sample loss and enhanced separation efficiencies as compared to conventional techniques such as high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, the major limitation of both CE and CE-MS is the limited sample loading capacity of conventional CE capillaries. Typical loading volumes are approximately 1-100 nL, which afford optimal CE performance, and this is in stark contrast to the 1-100 microL commonly injected onto capillary HPLC columns. The limited loading of CE leads to relatively poor concentration limits of detection. In this work a unique method for analyte preconcentration with CE is described. A cartridge containing an impregnated membrane is installed at the inlet of the CE capillary, and we term this approach membrane preconcentration-CE (mPC-CE), and in conjunction with mass spectrometry, mPC-CE-MS. It allows both on-line sample concentration and cleanup and concentration limits of detection of fg/mL are possible using this approach. The analysis of in vivo derived metabolites, peptides, and proteins is described. This demonstrates the wide applicability of the technology in the analysis of any class of compounds, ranging in molecular weight from 100 to 70,000 Da with loading capacities of approximately 1- > 100 microL sample volumes.
Adult↗
Rapid HPLC screening method for contaminants found in implicated L-tryptophan associated with eosinophilia myalgia syndrome and adulterated rapeseed oil associated with toxic oil syndrome.
In 1981 a massive food-borne epidemic, termed the toxic oil syndrome (TOS), occurred in Spain. Eight years later a closely related disease, the eosinophilia myalgia syndrome (EMS), was reported in the USA with many additional cases being reported worldwide. Although EMS was linked to the ingestion of contaminated L-tryptophan and TOS to aniline denatured rapeseed oil, the etiological agent(s) responsible for both diseases remains unknown. However, contaminants in both the oil and the dietary supplement are believed to have triggered these diseases, and there has been much speculation that a common contaminant may have caused both epidemics. In this report, methods for the facile preparation and HPLC analysis of EMS-implicated L-tryptophan and adulterated rapeseed oil samples associated with TOS are described which allow a direct comparison between the contaminants of both foodstuffs. A combination of solvent and solid phase extraction methods are demonstrated along with the application of C18 reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with on-line UV and MS detection. These methods have allowed us to determine for the first time, based upon this work, that there are no detectable common contaminants that possess a UV response, between EMS implicated L-tryptophan and TOS implicated rapeseed oil samples.
Chromatography, High Pressure Liquid↗
Characterization of N-benzylcarbazole and its metabolites from microsomal mixtures by tandem mass spectrometry.
The metabolism of N-benzylcarbazole (NBC) was studied in vitro using hamster hepatic microsomes to establish whether the corresponding amide is formed. This work was carried out in order to see if the extremely low pka characteristic of such a benzylic amine would allow the formation of the carbonyl derivative. No amide formation was observed. However, a number of metabolic products were detected using HPLC, including the oxidative debenzylation products, namely carbazole and benzaldehyde, together with 2 phenolic isomers of NBC. These products were tentatively characterized by their UV spectra using a rapiscan UV detector connected to HPLC equipment. The structural characterization of these 4 metabolites, together with unchanged substrate, was carried out using desorption electron impact tandem mass spectrometry (DEI-MS/MS) on a hybrid instrument with EBQ1Q2 configuration.
Animals↗
Capillary electrophoresis and capillary electrophoresis-mass spectrometry in drug and metabolite analysis.
The structural diversity of modern therapeutic agents can lead to labour intensive method development for each drug and the structural characterization of their metabolites. In this work, we show the benefits of the high resolution capabilities of capillary electrophoresis (CE) and demonstrate that nonaqueous CE and on-line CE-mass spectrometry (CE-MS) leads to enhanced resolution and recovery of mixtures containing the prototype H2-antagonist, mifentidine, and putative metabolites. Furthermore, the usefulness of CE-tandem MS (CE-MS/MS) is also demonstrated by the structural characterization of the novel N2-hydroxylamine metabolite of mifentidine.
Electrophoresis↗
Every patient deserves an nurse.
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Humans↗
Origin of tetrahydrotetrols derived from human hemoglobin adducts of benzo[a]pyrene.
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Benzo(a)pyrene↗
Benzo[a]pyrene diol epoxide adduct formation in mouse and human hemoglobin: physicochemical basis for dosimetry.
The interactions of human hemoglobin (hHb) with the anti-diol epoxide of benzo[a]pyrene (aBaPDE) and with the corresponding tetrols formed by hydrolysis of the epoxide were investigated with the aim of characterizing the covalent adducts formed by reaction of the epoxide with the protein. The major product (80% of the total) was determined to be an ester resulting from oxirane ring opening by one or several (unidentified) carboxylate group(s). Minor products were characterized as adducts formed by reaction with amino or heterocyclic nitrogen by comparison of their UV spectra with those of model compounds. There was no evidence for reaction with cysteine. Formation of ester adducts by aBaPDE with mouse hemoglobin (mHb) following administration of BaP to mice was also investigated. It was found that esters constituted the majority of the adducts formed by aBaPDE and a substantial fraction of the total adducts formed. The esters formed by mHb were significantly less stable than those formed by hHb, both in vivo and in vitro. The instability of mHb ester adducts is believed by the responsible for differences among previous descriptions of the in vivo binding of BaP to mHb.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Conversion of a hemoglobin alpha chain aspartate(47) ester to N-(2,3-dihydroxypropyl)asparagine as a method for identification of the principal binding site for benzo[a]pyrene anti-diol epoxide.
Human hemoglobin was alkylated with (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and then treated with aqueous (+/-)-3-amino-1,2-propanediol to convert alkylated carboxyl side chains to N-(2,3-dihydroxypropyl) amides. Tryptic peptides produced from the modified protein were subjected to affinity chromatography on phenylboronic acid. The bound fraction was further purified by HPLC on C-4 reverse-phase medium to yield one modified peptide, which was identified as the Thr(41)-Lys(56) peptide of the alpha chain by amino acid analysis, Edman sequencing analysis, and FAB-MS. Limited direct evidence from this study and further indirect evidence from previous work identify Asp(47) alpha as the amino acid reacting with BPDE. The only other likely sites would be the C-terminal carboxyl groups of either the alpha or beta chain. Possible reasons for the site selectivity of the alkylation of human hemoglobin by BPDE are discussed.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Identification of cyclophosphamide-DNA adducts in rat embryos exposed in vitro to 4-hydroperoxycyclophosphamide.
Cyclophosphamide and other bifunctional alkylating agents are potent animal teratogens inducing a variety of malformations. Although cyclophosphamide-induced DNA damage is implicated as a primary mechanism underlying the teratogenesis initiated by cyclophosphamide, additional insights into the complex nature of the teratogenic process have been hampered by the inability to analyze the primary teratogenic lesions, i.e., cyclophosphamide-DNA adducts. Using tandem mass spectrometry, we show that the monofunctional adduct N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine (NOR-G) and bifunctional adduct N,N-bis[2-(7-guaninyl)ethyl]amine (G-NOR-G) can be detected in the DNA of organogenesis-stage rat embryos after an in vitro exposure to an embryotoxic concentration of activated cyclophosphamide, i.e., 4-hydroperoxycyclophosphamide.
Animals↗
Fluoranthene metabolism: human and rat liver microsomes display different stereoselective formation of the trans-2,3-dihydrodiol.
The metabolism of the environmental carcinogen fluoroanthene by human liver microsomes was compared to that by liver microsomes from rats treated with Aroclor 1254. Although the human-derived system gave primarily one product, similar metabolites were noted from each system. Enantiomers of the major metabolic product, in both cases the trans-2,3-dihydrodiol, were separated by chiral stationary-phase chromatography. Absolute configurations were assigned by application of the benzoate exciton chirality rules to the CD spectra of the 4-(dimethylamino)benzoyl esters. Liver microsomes from Aroclor 1254-treated rats produced the R,R enantiomer of the diol in 75-78% enantiomeric excess, while human liver microsomes produced this enantiomer in only 6-12% excess. The activities of these enantiomers were compared in Salmonella typhimurium strain TM677 mutagenicity assays employing the 9000g supernatant of Aroclor 1254-induced rat liver homogenates. Both the syn- and anti-2,3-dihydrodiol 1,10b-epoxides, which had only been inferred to be metabolites in previous studies, were isolated from the microsomal incubations by preparative reverse-phase HPLC. The evident exceptional aqueous stabilities of these diol epoxides were further examined by half-life determination experiments. Their tetrahydrotetrol hydrolysis products were also noted in the metabolite HPLC profiles. The structures of the tetrahydrotetrols were confirmed by total synthesis.
Animals↗
Biotransformation of 3-(phenylamino)-1,2-propanediol to 3-(phenylamino)alanine: a chemical link between toxic oil syndrome and eosinophilia-myalgia syndrome.
During late 1989, the eosinophilia-myalgia syndrome (EMS) developed as an epidemic in the United States, with numerous additional cases reported in several other countries worldwide. Eight years earlier, a closely-related disease, the toxic oil syndrome (TOS), occurred in Spain as a massive food-borne epidemic. Although EMS was linked to the ingestion of tainted L-tryptophan, and TOS to aniline-denatured rapeseed oil, the etiologic agent(s) responsible for both diseases remains undetermined. Contaminants in these foodstuffs are believed to have triggered the diseases. Aniline contaminants, including 3-(phenylamino)-1,2-propanediol (PAP), have been reported in oil used by patients who developed TOS. A related aniline derivative, 3-(phenylamino)-L-alanine (PAA), was recently isolated from L-tryptophan associated with the onset of EMS. Here, we demonstrate the biotransformation of PAP into PAA by both rat hepatocytes and human liver tissue. The structural characterization of PAA was unequivocally determined using on-line HPLC coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS). This finding is the first reported chemical link between TOS and EMS and suggests that these two related diseases share a common etiology, namely, PAA.
Alanine↗
Manpower planning. Eight. Muddle in the Midlands.
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Health Planning↗
Systematic development of on-line membrane preconcentration-capillary electrophoresis-mass spectrometry for the analysis of peptide mixtures.
A unique method for analyte preconcentration on-line with CE and CE-mass spectrometry (CE-MS) that can concentrate large sample volumes (> 100 microL) is described. A cartridge containing a suitably impregnated membrane is installed at the inlet of the CE capillary, and this approach is termed membrane preconcentration-CE-MS (mPC-CE-MS). The authors describe the mPC-CE-MS analysis of dilute peptide mixtures (approximately 1 fmol/nL) that are concentrated onto the membrane and subsequently subjected to on-line sample cleanup prior to elution and ultimately CE-MS. The systematic development of mPC-CE-MS is demonstrated, and the necessity of minimizing the adsorptive phase in order to ensure efficient peptide analyte recovery and minimization of organic elution solvent is described. The authors compare and contrast mPC-CE and mPC-CE-MS with PC-CE and solid-phase preconcentration-CE-MS (PC-CE-MS), and describe several limitations of the latter approach, which includes analyte zone broadening and compromised analyte resolution. Finally, the authors show that the use of mPC-CE-MS in conjunction with transient isotachophoresis after the peptides have been eluted from the adsorptive membrane affords optimal performance, and theoretical plate values of up to approximately 2.6 x 10(6) are observed for the analysis of a nine-component peptide mixture using this approach. It is conservatively estimated that, in conjunction with an MS array detector, a practical concentration limit of detection (CLOD) for peptides is approximately 500 fg-1 pg/mL using mPC-CE-MS.
Electrophoresis, Capillary↗
Enhancement of concentration limits of detection in CE and CE-MS: a review of on-line sample extraction, cleanup, analyte preconcentration, and microreactor technology.
The techniques of CE and on-line capillary electrophoresis-mass spectrometry (CE-MS) have been widely used for the analysis of many chemically diverse molecules. These methods of analysis allow analyte separations in aqueous solutions that are complementary to classical techniques such as HPLC and HPLC-MS. However, the one major limitation of CE is the fact that the best performance is normally obtained in analyzing small sample volumes (typically < 50 nL for a 50-micron-i.d. capillary). Ultimately, this leads to a relatively poor concentration limit of detection (CLOD) for CE and CE-MS when compared to that of HPLC or HPLC-MS. Recently, the analyte concentrator and membrane preconcentration cartridge have been described for use on-line with the CE capillary. Common to many of the approaches that led to the development of these devices is the incorporation of a suitable stationary phase at the inlet of the CE capillary. This relatively simple modification permits the introduction of much larger sample volumes (> 100 microL) into the CE capillary, and lowers both the CE and CE-MS CLOD. Detection of analytes present in complex mixtures at concentrations of < 200 fg/mL is reported to be possible when using such techniques in conjunction with CE and CE-MS. Additionally, the analyte concentrator has been developed to analyze microreactions on-line with the CE capillary. Recently, analyte derivatization and enzymatic protein digestion on-line with CE separations were reported. The purpose of this manuscript is to discuss and critically review these techniques and all described attempts at improving the CLOD of CE and CE-MS techniques using an adsorptive mechanism at the inlet of the CE capillary.
Animals↗