Quantitative gas chromatography mass spectrometric analysis of 2'-deoxyinosine in tissue DNA

Several studies examining DNA deamination have published levels of 2'-deoxyinosine that illustrated a large variation between studies. Most of them are the result of artifactual DNA deamination that occurs during the process of sample preparation, particularly acid hydrolysis. This protocol for measurement of 2'-deoxyinosine describes the use of nuclease P1 and alkaline phosphatase to achieve release of nucleosides from DNA, followed by HPLC prepurification with subsequent gas chromatography-mass spectrometry analysis of the nucleosides. It has been used in the measurement of the levels of 2'-deoxyinosine in DNA of commercial sources and DNA from cells and animal tissues, and gives values ranging from 3 to 7 2'-deoxyinosine per 10(6) 2-deoxyadenosine. This protocol should take approximately 7 days to complete.     

A capillary gas chromatography/mass spectrometric method for the quantification of hydroxysteroids in human plasma

A specific and sensitive methodology for the quantitative determination of hydroxysteroids dehydroepiandrosterone and pregnenolone and their main metabolites in human plasma is described. Hydroxysteroids were extracted using methanol and steroids were further separated by reverse-phase high-performance liquid chromatography, allowing for minimization of the possible chromatographic interferences. Eluted fractions were collected, pooled, and analyzed by gas chromatography-mass spectrometry as trimethylsilyl ether derivatives. The quantification was performed with single-ion monitoring of the highly abundant m/z 129 or m/z 358 fragments. The combination of the chromatographic characteristics to the specific fragments ensured the selectivity and specificity of the method. Under these conditions the method was linear (typical R2 is superior to 0.98 for all hydroxysteroids studied) over the concentration range of 2 x 10(-9) to 10(-6)M with good precision and accuracy.     

The analysis of trenbolone and the human urinary metabolites of trenbolone acetate by gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry.

The electron impact mass spectrometric properties of trimethylsilyl ether and fluoroacyl ester derivatives of trenbolone, combined or not combined with a methoxime group, are presented. Some derivatization problems were observed and were due to the formation of enol derivatives at the 3C-position in several tautomeric forms, which in their turn were not stable and lost two or four hydrogens under the conditions studied. The enolization could be minimized by carefully selecting the reaction conditions or could be prevented by the introduction of a methoxime group at the 3C-position. The limits of detection and identification of the methoxime heptafluorobutyryl ester and the methoxime trimethylsilyl ether derivative of trenbolone were determined using a mass selective detector in the electron impact mode and a triple-stage quadrupole in the methane positive chemical ionization mode. Selected reaction monitoring in tandem mass spectrometry did not improve the limit of detection, but because of the gain in selectivity did improve the limit of identification. The glucuronides of trenbolone and epitrenbolone could be identified in three urine specimens out of 200 samples in routine doping control.     

Capillary gas chromatographic/mass spectrometric analysis of abnormal metabolites in hypoglycin-treated rat urine

Numerous abnormal metabolites were identified in large amounts in the urine of hypoglycin-treated rats using capillary gas chromatography/mass spectrometry-computer analysis. These metabolites are not detectable in significant amounts in normal rats' urine. Ten of them have not been previously associated with hypoglycin administration: these are several hydroxy compounds, including those from the valine and isoleucine pathways, 2-oxo-adipic acid, n-butyrylglycine and isovaleryl glucuronide. These results indicate that the pathways of isoleucine and valine metabolism are inhibited at their respective acyl-CoA dehydrogenation steps, as is the case for fatty acid, leucine and lysine metabolism, as previously shown. The mass spectra of the trimethylsilyl derivatives of cis, cis-4,7-decadiene-1,10-dioic, cis-4-decene-1,10-dioic, cis-4-octene-1,8-dioic acids, and (methylenecyclopropyl)acetylglycine, which were previously identified using nuclear magnetic resonance and oxidative cleavage or acid hydrolysis, are presented for the first time.     

Gas chromatography chemical ionization mass spectrometric analysis of diminazene in plasma

A quantitative and sensitive method was developed for the determination of diminazene in plasma. The assay involves the reduction of diminazene to 4-aminobenzamidine and 4-hydrazinobenzamidine. The latter is further reduced to give an additional mole of 4-aminobenzamidine which is extracted, acetylated and condensed with hexafluoroacetylacetone to form a volatile derivative that is subsequently analyzed by gas chromatography chemical ionization mass spectrometry. 4-Aminobenzylamidine was synthesized and used as an internal standard. The method is reproducible and its sensitivity limit using 1 ml of plasma is 0.1 microgram diminazene ml-1. This sensitivity limit is sufficient to detect plasma levels in cattle following therapeutic doses of the drug.     

Enantiomeric analysis of anatabine, nornicotine and anabasine in commercial tobacco by multi-dimensional gas chromatography and mass spectrometry

A fully automated multi-dimensional gas chromatography (MDGC) system with a megabore precolumn and cyclodextrin-based analytical column was developed to analyze the enantiomeric compositions of anatabine, nornicotine and anabasine in commercial tobacco. The enantiomer abundances of anatabine and nornicotine varied among different tobacco. S-(-)-anatabine, as a proportion of total anatabine, was 86.6% for flue-cured, 86.0% for burley and 77.5% for oriental tobacco. S-(-)-nornicotine, as a proportion of total nornicotine, was 90.8% in oriental tobacco and higher than in burley (69.4%) and flue-cured (58.7%) tobacco. S-(-)-anabasine, as a proportion of total anabasine, was relatively constant for flue-cured (60.1%), burley (65.1%) and oriental (61.7%) tobacco. A simple solvent extraction with dichloromethane followed by derivatisation with trifluoroacetic anhydride gave relative standard deviations of less than 1.5% for the determination of the S-(-)-isomers of all three alkaloids. The study also indicated that, a higher proportion of S-(-)-nornicotine is related to the more active nicotine demethylation in the leaf.     

Prenatal diagnosis for organic acid disorders using two mass spectrometric methods, gas chromatography mass spectrometry and tandem mass spectrometry

We performed prenatal diagnosis of organic acid disorders using two mass spectrometric methods; gas chromatography mass spectrometry (GC/MS) and tandem mass spectrometry (ESI/MS/MS). Of 28 cases whose amniotic fluid was tested, 11 cases were diagnosed as "affected". All cases whose samples were diagnosed as "unaffected" were confirmed to have no symptoms or abnormalities in urinary organic acid analysis after birth. Of the 11 "affected" cases, two cases were missed by ESI/MS/MS but not by GC/MS. When the stability of metabolites in amniotic fluid was checked, it was found that acylcarnitines degraded in one week at room temperature, whereas organic acids such as methylmalonate or methylcitrate were stable for at least 14 days. Prenatal diagnosis by analysis using simultaneous two or more methods may be more reliable, though attention should be paid to sample transportation conditions.     

Stable isotope dilution analysis of salicylic acid and hydroquinone in human skin samples by gas chromatography with mass spectrometric detection

A sensitive and accurate gas chromatographic-mass spectrometric (GC-MS) method has been developed for the quantitative determination of salicylic acid (SA) and hydroquinone (HQ) from human skin samples and cosmetic emulsions. Deuterium labeled SA-d(6) and HQ-d(6) were used as internal standards (IS). The samples were extracted with methanol, dried under nitrogen and derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+1% trimethylchlorosilane (TMCS). Quantification was performed in SIM mode with a limit of quantification (LOQ) of 50 ng ml(-1) for SA and 10 ng ml(-1) for HQ. The inter-day variation (R.S.D.) was less than 5% and the accuracy was better than 13.3% for both compounds. The recoveries from the different matrices ranged between 93.1 and 103.3% for SA, and 97.3 and 100.8% for HQ.     

Rapid identification of microbial VOCs from tobacco molds using closed-loop stripping and gas chromatography/time-of-flight mass spectrometry

Several microbial volatile organic compounds (MVOCs) that can serve as potential chemical markers for microbial contamination in tobacco have been identified. Four different fungal species, Aspergillus niger (AN), A. ornatus (AO), Pencillium chrysogenum (PC) and Rhizopus stolonifer (RS), commonly reported in moldy tobacco were cultured and screened for MVOCs. Because the MVOCs emitted by a microbial species are substrate specific, the fungal strains were separately grown on potato dextrose agar (PDA) and tobacco products. MVOCs from the mold cultures grown on PDA and tobacco products were extracted using closed-loop stripping analysis (CLSA) and identified by gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). Some of the prominent tobacco mold markers identified by this method include: 1-octen-3-ol; 2-octen-1-ol; 2-methyl-1-butanol; 3-methyl-1-butanol; 1-octene and 2-pentanone. In particular, 1-octen-3-ol was detected in all the mold cultures and moldy tobacco samples analyzed. Olfactory evaluation of 1-octen-3-ol indicated a characteristic musty odor and the odor threshold was determined to be approximately 200 ng/ml. The limits of detection for 1-octen-3-ol using GC/TOF-MS and GC/mass selective detector (MSD) in the full-scan mode and selected ion monitoring (SIM) mode were investigated. The CLSA-GC/TOF-MS demonstrates a fast, sensitive and semi-quantitative analytical technique for screening tobacco materials for the presence of mold via chemical markers of microbial contamination.     

The decomposition of benzodiazepines during analysis by capillary gas chromatography/mass spectrometry

A capillary gas chromatography column directly interfaced to a mass spectrometer was used for the analysis of sixteen benzodiazepines. The thermal stability of the drugs was found to be related to their chemical structure. Nine of the benzodiazepines were thermally unstable indicating that care should be taken in the interpretation of gas chromatographic data from this class of drugs. The unstable benzodiazepines were: ketazolam which decomposes to diazepam; N-4 oxides (chlordiazepoxide and demoxepam) which lose an oxygen radical; aromatic 7-nitro compounds (nitrazepam and clonazepam) which are partially reduced to the corresponding amine; alpha-hydroxy ketones (lorazepam and oxazepam) which decompose with the loss of water and N-methyl-alpha-hydroxy ketones (lormetazepam and temazepam) which partially decompose with the loss of a hydrogen molecule to produce the corresponding alpha, beta-diketones. Few problems were encountered in distinguishing the drugs by their mass spectra, the exceptions being ketazolam which decomposes to diazepam and demoxepam which decomposes to desmethyldiazepam. In general, good spectra were obtained from 20-50 ng of drug injected. However, for those compounds where the decompositions were not quantitative (nitrazepam, clonazepam, lormetazepam, temazepam) detection limits were poor.     

Comprehensive analysis of metabolites in Corynebacterium glutamicum by gas chromatography/mass spectrometry

An analytical method based on gas chromatography/mass spectrometry was developed for metabolome investigation of Corynebacterium glutamicum. For the first time a fast method for metabolic screening that can be automated is described for this organism. More than 1000 compounds could be detected per experiment, ca. 330 of those showed a peak area significantly above background. Out of these 164 compounds were identified so far, representing derivatives of 121 different metabolites, which were quantified in one sample. In spite of the different chemical nature of metabolites and high matrix content, a measurement reproducibility in the range of 6% error was achieved. The application of this method for the analysis of the adaptation of C. glutamicum to different growth conditions is demonstrated.     

A high-throughput method for microbial metabolome analysis using gas chromatography/mass spectrometry

An analytical high-throughput method based on gas chromatography/mass spectrometry (GC/MS) was developed for fast metabolome investigation. By parallelization and partial automation the time needed for the preanalytical steps could be reduced. In addition a strong decrease of the relative standard deviation of metabolite concentrations from independent samples on the same microtiter plate from 25 to 13% was achieved. Between different plates the relative standard deviation is comparable to the one observed in standard experiments with shaking flasks. Using a fast GC the time need for the full GC/MS-based metabolome analysis could be decreased from 60 to 18 min per run, allowing the measurement of 72 single samples per day and GC/MS machine. In samples of the model organism Corynebacterium glutamicum more than 1000 peaks in the total ion current could be detected in a single fast GC/MS run of which 650 were strong enough to be quantified. Approximately 150 compounds of these were identified using our metabolite MS-library. Correlation analysis of the concentration vectors of independent wild-type samples raised under the same conditions show very high correlations of 0.99+/-0.01 (logs). In conclusion this method allows screenings of large mutant libraries for genetically induced metabolic perturbations.     

Streamlined F2-isoprostane analysis in plasma and urine with high-performance liquid chromatography and gas chromatography/mass spectroscopy

F2-Isoprostanes in plasma and urine are generally determined by labor-intensive methods requiring sample purification by solid-phase extraction and thin-layer chromatography (TLC). A streamlined and more sensitive method for the measurement of esterified plasma F2-isoprostanes was developed by replacing these steps with high-performance liquid chromatography (HPLC) using an amino column with a hexane/2-propanol gradient. Pentafluorobenzyl esters of F2-isoprostanes were prepared and purified by HPLC, silylated, and then analyzed by gas chromatography (GC) with negative chemical ionization mass spectroscopy (NCI-MS). This method permits analysis with lower plasma volumes (100 microL) and greater sensitivity (to 10 pg; allowing detection to 50 pg/mL) than provided by other methods. Urinary F2-isoprostanes can also be efficiently quantified by this method, with 8-iso-PGF2alpha being identified as a major isomer. With this procedure, esterified plasma F2-isoprostanes were found to be 8.3-fold higher in an end-stage renal failure patient on hemodialysis and urinary 8-iso-PGF2alpha was 7.1-fold higher in a cigarette smoker than respective control subjects. This method, particularly the substitution of the TLC step common to other methods with HPLC, results in a more sensitive and reproducible assay.     

Enantioselective analysis of levetiracetam and its enantiomer R-α-ethyl-2-oxo-pyrrolidine acetamide using gas chromatography and ion trap mass spectrometric detection

A gas chromatographic–mass spectrometric method was developed for the enantioselective analysis of levetiracetam and its enantiomer (R)-α-ethyl-2-oxo-pyrrolidine acetamide in dog plasma and urine. A solid-phase extraction procedure was followed by gas chromatographic separation of the enantiomers on a chiral cyclodextrin capillary column and detection using ion trap mass spectrometry. The fragmentation pattern of the enantiomers was further investigated using tandem mass spectrometry. For quantitative analysis three single ions were selected from the enantiomers, enabling selected ion monitoring in detection. The calibration curves were linear from 1 μM to 2 mM for plasma samples and from 0.5 mM to 38 mM for urine samples. In plasma and urine samples the inter-day precision, expressed as relative standard deviation was around 10% in all concentrations. Selected ion monitoring mass spectrometry is suitable for quantitative analysis of a wide concentration range of levetiracetam and its enantiomer in biological samples. The method was successfully applied to a pharmacokinetic study of levetiracetam and (R)-α-ethyl-2-oxo-pyrrolidine acetamide in a dog.     

Design of a stable isotope dilution gas chromatography/mass spectrometric assay for cAMP: Comparison with standard protein-binding and radioimmunoassay methods

A stable isotope dilution assay is presented in which picomole quantities of cAMP can be determined with high precision and selectivity using gas chromatography and electron impact mass spectrometry with multiple ion detection techniques. Using synthetic [2,8-²H₂,6-¹⁵N]-cAMP as the internal standard, suitable specificity was obtained by monitoring the (MCH₃)⁺ fragment ions of the trimethylsilyl derivatives of cAMP and the internal standard at $\text{m}\text{z}$ 530 and $\text{m}\text{z}$ 533, respectively. The sensitivity of the assay as judged from the lower limit of detection of the mass spectrometer was 3.0 pmol. Rat liver and human urine cAMP levels were assayed using gas chromatography/mass spectrometry and were compared with levels determined by protein-binding assays and radioimmunoassays for the same samples. The intraassay coefficients of variation of the gas chromatography/mass spectrometry assay were 5.3% for the rat liver sample (cAMP level 832 pmol/g) and 6.0% for the urine sample (cAMP level 2.50 μmol/liter). Comparison of the levels of cAMP determined by the three assay methods showed correlation to within 10% variation.     

A rapid microwave-assisted derivatization of bacterial metabolome samples for gas chromatography/mass spectrometry analysis

Analysis of metabolome samples by gas chromatography/mass spectrometry requires a comprehensive derivatization method to afford quantitative and qualitative information of a complex biological sample. Here we describe an extremely time-effective microwave-assisted protocol for the commonly used methoxyamine and N-methyl-N-trimethylsilylfluoracetamide silylation method of primary metabolites. Our studies show that microwave irradiation can decrease the sample preparation time from approximately 120 min to 6 min without loss of either qualitative or quantitative information for the tested synthetic metabolite mixtures and microbial-derived metabolome samples collected from Bacillus subtilis and Staphylococcus aureus. Comparisons of metabolic fingerprints and selected metabolites show no noticeable differences compared with the commonly used heating block methods.     

Method for analysis of polybrominated biphenyls by gas chromatography mass spectrometry

Gas chromatography using a short packed column (45 cm, 0.2 cm i.d., 2% OV-101 on Gas-Chrom Q) with mass spectrometric detection in the selected ion monitoring mode has been found satisfactory for the analysis of lower as well as higher polybrominated biphenyls. Acceptable sensitivity (< 1 ng) may be achieved for this method by focusing selectively at either the low (m/z 20-600) or the high m/z 600-1000) range of the quadrupole filter (low range for mono- through hexabromobiphenyl, high range for hexa- through decabromobiphenyl). A tuning technique has been developed for low range and high range polybrominated biphenyls using the ion abundances of perfluorotributylamine as a standard. Standard ions for the quantitation of mono- through decabromo-biphenyls were selected and validated. The technique was applied to the analysis of a variety of environmental samples.