Identification and quantification of 8,5'-cyclo-2'-deoxy-adenosine in DNA by liquid chromatography/ mass spectrometry

Recent studies suggested that 8,5'-cyclo-2'-deoxyadenosine may play a role in diseases with defective nucleotide-excision repair. This compound is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the sugar moiety of 2'-deoxyadenosine. It is likely to be repaired by nucleotide-excision repair rather than by base-excision repair because of a covalent bond between the sugar and base moieties. We studied the measurement of 8,5'-cyclo-2'-deoxyadenosine in DNA by liquid chromatography/isotope-dilution mass spectrometry. A methodology was developed for the analysis of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography in DNA hydrolyzed to nucleosides by a combination of four enzymes, i.e., DNase I, phosphodiesterases I and II, and alkaline phosphatase. Detection by mass spectrometry was performed using atmospheric pressure ionization-electrospray process in the positive ionization mode. Results showed that liquid chromatography/isotope-dilution mass spectrometry is well suited for identification and quantification of 8,5'-cyclo-2'-deoxyadenosine in DNA. Both (5'R)- and (5'S)-diastereomers of 8,5'-cyclo-2'-deoxyadenosine were detected. The level of sensitivity of liquid chromatography/mass spectrometry with selected-ion monitoring amounted to 2 fmol of this compound on the column. The yield of 8,5'-cyclo-2'-deoxyadenosine was measured in DNA in aqueous solution exposed to ionizing radiation at doses from 2.5 to 80 Gray. Gas chromatography/mass spectrometry was also used to measure this compound in DNA. Both techniques yielded similar results. The yield of 8,5'-cyclo-2'-deoxyadenosine was comparable to the yields of some of the other major modified bases in DNA, which were measured using gas chromatography/mass spectrometry. The measurement of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography/mass spectrometry may contribute to the understanding of its biological properties and its role in diseases with defective nucleotide-excision repair.     

Application of FRIT fast atom bombardment liquid chromatography/mass spectrometry for the determination of acetylcholine levels in rat brain regions

This report describes the use of FRIT fast atom bombardment (FAB) liquid chromatography/mass spectrometry for the analysis of acetylcholine in rat brain regions. Direct assessment of acetylcholine levels is possible without the need for either derivatization or extensive sample preparation. Quantification is accomplished by monitoring intact molecular cations of acetylcholine and a deuterated internal standard. The results are compared with those obtained by conventional pyrolysis gas chromatography/mass spectrometry and by liquid chromatography with electrochemical detection.     

Purification and properties of pyridoxal kinase from bovine brain

A 27,000-fold purification of pyridoxal kinase from bovine brain tissue has been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-150 gel filtration, Blue Sepharose CL-6B chromatography, and Phenyl-Superose chromatography. The final chromatography step yields a homogeneous preparation of high specific activity (2105 nmol/min/mg protein). The molecular mass of the native enzyme was estimated to be approximately 80,000 on gel filtration. The subunit molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 39,500. This indicates that pyridoxal kinase is a dimeric enzyme.     

Enzymatic isomerization and epimerization of D-erythrose 4-phosphate and its quantitative analysis by gas chromatography/mass spectrometry

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate. The presence of D-erythrulose 4-phosphate and D-threose 4-phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of D-erythrulose and D-threose was confirmed by thin-layer chromatography, gas-liquid chromatography and an enzymatic method depending upon D-erythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m/z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that D-threose 4-phosphate was also converted into D-erythrulose 4-phosphate and D-erythrose 4-phosphate. At the equilibrium, about 90% of the tetrose 4-phosphate existed in the form of D-erythrulose 4-phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin-layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4-phosphate.     

Sensitive determination of pethidine in body fluids by surface ionization organic mass spectrometry

We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography (GC)/surface ionization organic mass spectrometry (SIOMS). Good linearity was obtained in the range of 0.625–25 ng/ml of whole blood and urine by mass chromatography, and in the range of 0.05–2 ng/ml of whole blood by selected ion monitoring (SIM). Pethidine and diphenylpyraline (internal standard) were extracted from body fluids with Bond Elut Certify cartridges; their recoveries were above 95%. The detection limits (signal-to-noise ratio=3) were estimated to be 0.2 ng/ml of whole blood or urine by mass chromatography, 0.02 ng/ml of whole blood by SIM.     

Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method

Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-(15)N(2)] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M-H)(-) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08-0.18% and between-set CVs of 0.02-0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

Rapid analysis of naphthalene and its metabolites in biological systems: Determination by high-performance liquid chromatography/fluorescence detection and by plasma desorption/chemical ionizations mass spectometry

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.     

Biotransformation and toxicity of aniline and aniline derivatives in cyanobacteria

Agmenellum quadruplicatum strain PR-6 and Oscillatoria sp. strain JCM grown photoautotrophically in the presence of aniline metabolized the aromatic amine to formanilide, acetanilide and p-aminophenol. The metabolites were isolated by either thin-layer, gas-liquid or high pressure liquid chromatography and identified by comparison of their chromatographic, ultraviolet absorbance and mass spectral properties with those of authentic compounds. The toxicity of aniline derivatives towards Agmenellum quadruplicatum strain PR-6 indicated that the cyanobacterium was extremely sensitive to o-, m- and p-aminophenols, and phenylhydroxylamine.Abbreviations TLC thin layer chromatography - HPLC high pressure liquid chromatography - GC/MS gas chromatography/mass spectrometry - m/e mass to charge ratio     

Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS

An overview is presented of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), the two major hyphenated techniques employed in metabolic profiling that complement direct 'fingerprinting' methods such as atmospheric pressure ionization (API) quadrupole time-of-flight MS, API Fourier transform MS, and NMR. In GC/MS, the analytes are normally derivatized prior to analysis in order to reduce their polarity and facilitate chromatographic separation. The electron ionization mass spectra obtained are reproducible and suitable for library matching, mass spectral collections being readily available. In LC/MS, derivatization and library matching are at an early stage of development and mini-reviews are provided. Chemical derivatization can dramatically increase the sensitivity and specificity of LC/MS methods for less polar compounds and provides additional structural information. The potential of derivatization for metabolic profiling in LC/MS is demonstrated by the enhanced analysis of plant extracts, including the potential to measure volatile acids such as formic acid, difficult to achieve by GC/MS. The important role of mass spectral library creation and usage in these techniques is discussed and illustrated by examples.     

Cytochrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin

The cytochrome P450 enzyme is one of the most versatile redox proteins and it is responsible for the oxidative metabolism of a wide variety of endogenous and exogenous compounds. The cytochrome P450 gene, CYP105F2, from Streptomyces peucetius was subcloned into the pET-32a(+) vector to overexpress the protein in E. coli BL21 (DE3) pLysS. The expressed enzyme was purified by fast protein liquid chromatography with a DEAE and UNO Q column. A 3D model was constructed based on the known crystallographic structures of cytochrome P450, and comparison with PikC and MoxA signified broad substrate specificity toward structurally diverse compounds. In addition, the in vitro hydroxylation of oleandomycin by purified CYP105F2 observed in liquid chromatography/mass spectrometry and mass/mass spectrometry indicated its flexibility towards alternative polyketides for the structural diversification of the macrolide by post-polyketide synthase hydroxylation.     

Gallidermin: a new lanthionine-containing polypeptide antibiotic

Gallidermin is a new member of the class of lanthionine-containing peptide antibiotics, which are summarized under the common name lantibiotics. The lantibiotic gallidermin is produced by Staphylococcus gallinarum (F16/P57) Tü3928, and it exhibits activities against the Propionibacteria, involved in acne disease. Gallidermin differs from the recently discovered tetracyclic 21-residue peptide antibiotic epidermin only in a Leu/Ile exchange in position 6. The isolation procedures for gallidermin included adsorption directly from the culture broth, ion-exchange chromatography of the amphiphilic and basic polypeptide followed by desalting, and final purification by reversed-phase HPLC. The structural elucidation of the polypeptide containing four thioether bridges involved mainly a combination of automated gas-phase sequencing, thermospray liquid chromatography/mass spectrometry and fast-atom-bombardment mass spectrometry.     

Incorporation of tandem mass spectrometric detection to the analysis of peptide mixtures by continuous flow fast atom bombardment mass spectrometry

The ability to acquire structurally informative daughter ion spectra for individual peptides undergoing separation and analysis by continuous flow fast atom bombardment (CF FAB) is demonstrated. To illustrate the potential of this methodology, tryptic and chymotryptic digests of the 29-residue peptide glucagon were analyzed by CF FAB using mass spectrometric and tandem mass spectrometric detection in consecutive analyses. Daughter ion spectra were recorded using B/E linked scans for the major hydrolysis products observed by liquid chromatography/mass spectrometry. The peptide mixtures were separated by gradient capillary high-performance liquid chromatography with the FAB matrix being added post-column using a coaxial flow interface between the column and flow probe. The entire effluent (3 microl min(-1)) was sampled by the mass spectrometer. Results obtained using less than 300 pmol of digested glucagon indicated several advantages to tandem mass spectrometric detection including the ability to confirm identities for products of enzymatic digestion and the potential use of this method for tandem sequence analysis of peptide mixtures.     

The use of negative ion thermospray liquid chromatography/tandem mass spectrometry for the determination of bile acids and their glycine conjugates.

A study was carried out on the negative ion thermospray liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry of a group of bile acids and their glycine conjugates. The liquid chromatographic/tandem mass spectrometric experiments were performed using low-energy collisions on a triple-quadrupole mass spectrometer. It was found that relatively high collision energies and collision gas pressures were required to produce fragmentation and that some unusual fragments were produced.     

Identification of 2-guanidinoethanol in human urine

Guanidino compounds in normal human urine were analyzed by high-performance liquid chromatography; an unknown peak was observed in the chromatogram that was identical to the peak of synthetic 2-guanidinoethanol. In another experiment, the substance was purified from human urine by successive use of strongly acidic ion-exchanger, thin-layer chromatography and then weakly acidic ion-exchanger. After this it was reacted with acetylacetone to form dimethylpyrimidyl derivative. After further reaction of this derivative with trifluoroacetic anhydrate, it was analyzed by gas chromatography/mass spectrometry. The mass chromatogram and mass spectrum were identical to those of the trifluoroacetylated dimethylpyrimidyl derivative of synthetic 2-guanidinoethanol. This is the first report on the identification of 2-guanidinoethanol in human urine. The concentration of 2-guanidinoethanol in the urine of healthy humans was 5.7 +/- 1.8 (mean +/- SD) mumol/g creatinine.     

Characterization of 2,7-anhydro-N-acetylneuraminic acid in human wet cerumen

A molecular species of sialic acid was isolated in a free form from cerumen of the wet type, but not of the dry type, by an ion-exchange column chromatography and preparative high-performance liquid chromatography. Structural analysis of this sialic acid was performed by gas-liquid chromatography/mass spectrometry with chemical ionization (CI) and electron ionization (EI). In the CI mass spectra, the protonated molecular ion of the trimethylsilyl derivative was observed at m/z 580. and that of the methyl ester-trimethylsilyl derivative was at m/z 522. In the EI mass spectrum, the methyl ester-trimethylsilyl derivative gave characteristic ions at m/z 506, 462, 418, 416, 328, 316, 238, 228, 205, 186, and 173. This mass spectrum was identical with that of 2,7-anhydro-N-acetylneuraminic acid, which was reported by Lifely and Cottee (Carbohydr. Res. 107, 187-197, 1982) as the mass spectrum of a by-product prepared from N-acetylneuraminic acid by methanolysis. These results indicate that the compound in the wet cerumen is 2,7-anhydro-N-acetylneuraminic acid. Since this sialic acid species could not be detected in cerumens of the dry type, its formation in the wet type may be controlled by an autosomal dominant gene.     

Novel O-linked carbohydrate chains in the cellulase complex (cellulosome) of Clostridium thermocellum. 3-O-Methyl-N-acetylglucosamine as a constituent of a glycoprotein

Alkaline borohydride treatment of the cellulosome of Clostridium thermocellum yielded two major oligosaccharide-alditols, namely D-Galp-beta(1----4)-D-GalOH and (formula; see text) The compounds, isolated via gel permeation chromatography and high performance liquid chromatography, were analyzed by monosaccharide analysis, methylation analysis, gas-liquid chromatography/mass spectrometry, fast atom bombardment/mass spectrometry, and one- and two-dimensional 500-MHz (COSY, HOHAHA, ROESY) 1H NMR spectroscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with blotting technology indicated that the tetrasaccharide is mainly associated with one of the cellulosome subunits.     

Preparative purification of dipeptidyl peptidase IV.

Dipeptidyl-Peptidase IV was purified from pig kidney by ammonium sulfate fractionation, gel filtration, QAE-cellulose chromatography and affinity columns with Gly-Pro- and Concanavalin A-Sepharose. The specific activity of the purified enzyme is 41.8 units/mg. Polyacrylamide gel electrophoresis and silver staining show a single band. The enzyme preparation is free of aminopeptidase and dipeptidase activity, proved fluorimetrically and by gas chromatography/mass spectrometry. The most important procedure for removal of contaminating enzyme activities is a stepwise NaCl-gradient on a QAE-ZetaPrep ion exchange disk.     

Trennung und charakterisierung saurer harnbestandteile

Separation and characterization of acidic urine constituentsThe acidic compounds of urine were separated by thin-layer chromatography in eight fractions. Each fraction was investigated separately by the combination glass capillary gas chromatography—mass spectrometry. About 500 compounds were detected, 2/5 of these could be characterized by their mass spectra. Retention data and key fragments of the mass spectra were tabulated. Many of the detected compounds are still unknown.     

Assay of bromhexine in human plasma by capillary gas—liquid chromatography with nitrogen-selective detection and selected ion monitoring

A specific, sensitive method for the determination of bromhexine in human plasma is described. It comprises a selective extraction procedure and a specific determination with capillary gas—liquid chromatography and nitrogen-selective flame ionization detection. The detection limit of the assay is about 0.5 ng/ml. The specificity of the assay was checked by gas chromatography—mass spectrometry. The method is applied to the pharmacokinetics of bromhexine in humans.     

Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography–electron impact mass spectrometry

A common feature of most peroxisomal disorders is the accumulation of very-long-chain fatty acids (VLCFAs) and/or pristanic and phytanic acid in plasma. Previously described methods utilizing either gas chromatography alone or gas chromatography–mass spectrometry are, in general, time-consuming and unable to analyze VLCFAs, pristanic and phytanic acid within a single analysis. We describe a simple, reproducible and rapid method using gas chromatography/mass spectrometry with deuterated internal standards. The method was evaluated by analysing 30 control samples and samples from 35 patients with defined peroxisomal disorders and showed good discrimination between controls and patients. This method is suitable for routine screening for peroxisomal disorders.