Investigations of S-transnitrosylation reactions between low- and high-molecular-weight S-nitroso compounds and their thiols by high-performance liquid chromatography and gas chromatography-mass spectrometry.

S-Transnitrosylation reactions are supposed to be the basic principle by which nitric oxide-related biological activities are regulated in vivo. Mechanisms of S-transnitrosylation reactions are poorly understood and equilibria constants for physiological S-nitroso compounds and thiols are rare. In the present study we investigated S-transnitrosylation reactions of the thiols homocysteine, cysteine, glutathione, N-acetylcysteine, N-acetylpenicillamine, and human plasma albumin and their corresponding S-nitroso compounds SNhC, SNC, GSNO, SNAC, SNAP, and SNALB utilizing high-performance liquid chromatographic and gas chromatographic-mass spectrometric techniques. These methods allowed to study S-transnitrosylation reactions in mixtures of several S-nitroso compound/thiol pairs, to determine equilibria constants, and to elucidate the mechanism of S-transnitrosylation reactions. We obtained the following order for the equilibria constants in aqueous buffered solution at pH 7.4: SNhC approximately SNAC > GSNO approximately SNALB > SNAP > SNC. Our results suggest that the mechanism of S-transnitrosylation reactions of these S-nitroso compounds and their thiols involve heterolytic cleavage of the S&sbond;N bond. Incubation of SNC with human red blood cells resulted in a dose-dependent formation of GSNO in the cytosol through S-transnitrosylation of intracellular GSH by the SNC transported into the cells. This reaction was accompanied with an almost complete disappearance of the SNC fraction transported into the cells. This finding is in full agreement with the equilibrium constant Keq of 1.9 for the reaction SNC + GSH <--> Cys + GSNO in aqueous buffer.     

Characterization of arachidonic acid metabolic profiles in animal tissues by high-resolution gas chromatography-mass spectrometry

A standardized, highly specific routine method was developed for the quantitative profiling of cyclooxygenase metabolites of arachidonic acid in animal tissues. Whole homogenates were used to assess the potential capacity of tissues to metabolize endogenous arachidonic acid. Samples were analyzed by high-resolution gas chromatography-mass spectrometry in the selected ion monitoring mode. The screening of several rat tissues by this method revealed marked tissue-specificity in both the synthesis capacity and prostaglandin profile. The major products detected were: 6-ketoprostaglandin F1alpha for lung, stomach, muscle and heart; prostaglandin D2 for spleen, brain and liver; prostaglandin F2alpha for kidney and prostaglandin E2 for seminal vesicles. Marked species differences were found when guinea pig tissues were analyzed.     

Analysis of trimethyl carboxyphosphate by gas chromatography-mass spectrometry

Analysis of trimethyl carboxyphosphate samples by gas chromatography-mass spectrometry, using typical conditions resulted in significant decomposition of the analyte. Optimization of injection conditions, including conditioning of the injection port liner, produced a dramatic increase in observed peak areas and afforded an effective method for detection of trimethyl carboxyphosphate at the <1μg mL⁻¹ level.     

Characterization of glycosphingolipids by supercritical fluid chromatography-mass spectrometry

Gangliosides have been characterized by supercritical fluid chromatography-chemical ionization mass spectrometry (SFC-CIMS) as permethyl and pertrimethylsilyl derivatives, using carbon dioxide as the SFC mobile phase and CI reagent gas. Ganglioside classes and ceramide heterogeneity within each class are well resolved by SFC. Direct SFC-interfacing allows the analytical manipulations of single-ion monitoring, total-ion plots, background subtraction, library searches, and spectral reconstruction algorithms. Addition of ammonia to the CI ion chamber (NH3 as a CI reagent gas) yields abundant molecular-weight-related ions, (MH)+ and (MNH4)+ from analyte derivatives. Substitution of methanol for ammonia yields considerable parent-ion fragmentation, providing structural information on carbohydrate sequence, fatty acid, and sphingoid components. Under these latter conditions a unique alpha-cleavage fragment is observed which differentiates fatty acid from sphingosine heterogeneity. For ganglioside samples, the carboxyl group of neuraminyl residue(s) have been esterified with pentafluorobenzyl bromide and the products analyzed by negative ion chemical ionization MS. This modification improves chemical selectivity and greatly enhances detecting sensitivity. These "soft" ionization conditions provide abundant molecular-weight-related anions for collision-induced dissociation and subpicogram detection.     

Determination of urinary trans,trans-muconic acid by gas chromatography-mass spectrometry

A sensitive and specific method for the determination of trans,trans-muconic acid (t,t-MA) in urine is described. After clean-up on an anion-exchange cartridge, t,t-MA was derivatized with BF₃-methanol to the dimethyl ester and analyzed by gas chromatography-mass spectrometry (GC-MS), with 2-bromohexanoic acid as an internal standard. The limit of detection was 0.01 mg/l, the coefficient of variation for duplicate analysis in a series of urine samples (n = 50) was 2.6% and the recovery rate ranged from 93.3 to 106.3%. The between-day and within-day precision for the analysis were 7.4 and 14.6%, respectively. The method was applied to the determination of t,t-MA in urine samples from smokers and non-smokers. The mean concentration of t,t-MA in urine of 10 smokers was 0.09 ± 0.04 mg/g creatinine and was significantly (p = 0.012) higher than that found in urine of 10 non-smokers (0.05 ± 0.02 mg/g creatinine). In contrast to the results obtained with the commonly used high-performance liquid chromatographic ultraviolet detection (HPLC-UV) methods, no interference between t,t-MA and other urinary compounds was found. This GC-MS method is both specific and sensitive for biomonitoring of low environmental benzene exposure.     

Characterization of the heme of cytochrome P-450 using gas chromatography-mass spectrometry

The heme moieties of cytochromes P-450 and P₁-450 (P-448) have been characterized. CO-binding particles, devoid of cytochrome b₅, were prepared from normal or 3-methylcholanthrene-treated animals. Heme was removed by acid-acetone treatment of the CO-binding particles and crystallized. Heme isolated from hemoglobin of the corresponding animals served as a control. Reductive degradation (hydriodic acid) followed by gas chromatography/mass spectrometry analysis indicated the presence of opso-, crypto-, hemo-, and phyllopyrrole. Visible spectra of the iron-free tetrapyrroles isolated from microsomal heme and hemoglobin were identical and showed typical aetioporphyrin spectra. Finally, the mass spectra of the tetrapyrrole dimethyl esters isolated from microsomal heme and hemoglobin were identical to authentic protoporphyrin IX dimethyl ester. These data strongly suggest that the heme of cytochrome P-450 and P₁-450 are identical and are the same the same as that of hemoglobin, namely protoporphyrin IX.     

Characterization of gibberellins from dark-grown Phaseolus coccineus seedlings by gas-liquid chromatography and combined gas chromatography-mass spectrometry

Summary An extract from 6000 dark-grown Phaseolus coccineus seedlings was purified by countercurrent distribution and G-10 Sephadex followed by gradient elution from a silicic acid partition column with increasing amounts of ethyl actetate in n-hexane. 25 fractions were collected and tested with the barley-aleurone, Tan-ginbozu dwarf-rice, lettuce, cucumber, dwarf-pea, d-1, d-2, d-3 and d-5 maize, oat first-internode, and sugarcane-spindle bioassays. Major gibberellin (GA)-like activity was detected in fractions 4 (500g GA₃-equivalents) and 12–13 (270 g GA₃-equivalents) with smaller amounts in fractions 6, 8–9, 15–16, 18, 20, 23 and 25. The extracts were also applied to AMO-1618=dwarfed Ph.-coccineus seedlings. Fractions 4, 8 and 12 promoted the growth of both light- and dark-grown seedlings. GA₁, GA₃, GA₄ and GA₈ were active in the Phaseolus bioassay but GA₈-glucoside was inactive.The biological and chromatographic properties of fractions 4, 8–9 and 12–13 correspond with those of GA₄, GA₁₉ and GA₁. The identity of GA₄ in fraction 4 was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of the methyl ester and the trimethylsilyl ether of the methyl ester. Gasliquid-chromatography peaks corresponding to these derivatives of GA₁₉ and GA₁ were detected on QF-1 and SE-33 columns but their intensities were too weak to permit conclusive identification by GC-MS.Supported by an S.R.C. StudentshipSupported by a NATO Grant.Supported by NRC Grant A-5727.     

Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10(6) DNA bases can be detected using amounts of DNA as low as 2 microgram. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10(6) DNA bases, or even lower, when more DNA is used. Up to 50 microgram of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.     

Characterization of prednisone, prednisolone and their metabolites by gas chromatography—mass spectrometry

Human urinary metabolites of the synthetic corticosteroids prednisone and prednisolone were detected in the course of gas chromatographic steroid profiling as methoxime-trimethylsilyl derivatives. Metabolites were provisionaly identified by combined gas chromatography—mass spectrometry. The major metabolites were 11-keto/11-hydroxy conversion products, 20-hydroxy and 4,5-dihydro analogues of the parent drugs. Cortisone, 6-hydroxy and fully saturated A-ring compounds were minor metabolites. Retention indices and mass spectral data are presented.     

Quantitation of lipid peroxidation products by gas chromatography-mass spectrometry

A method for the quantitation of lipid peroxidation products in total hepatic lipid has been developed. Lipid extracts are reduced and subsequently transmethylated with sodium methoxide. The hydroxy fatty acid methyl esters are isolated by silicic acid chromatography and derivatized to their trimethylsilyl ethers prior to analysis by gas chromatography-mass spectrometry. Three isomers, 11-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE), are quantitated using selected ion monitoring techniques relative to the internal standard, methyl 15-hydroxyarachidate. In mice treated with carbon tetrachloride (2 ml/kg), the HETE levels in total hepatic lipid were 20-fold greater than those found in control animals. HETE levels were also elevated (5- to 10-fold) in hepatic lipid from rats treated with the same dose of carbon tetrachloride. Studies on subcellular fractions with this methodology show that these lipid peroxidation products are 5- to 6-fold higher in hepatic plasma membrane vesicles isolated from rats treated with carbon tetrachloride when compared with those isolated from control animals.     

Systematic study of high molecular weight compounds in Amazonian plants by high temperature gas chromatography-mass spectrometry

The fractions of hexane and dichloromethane extraction from marupá (Simaruba amara) and (Bertholletia excelsa) leaves were analyzed by HT-HRGC (high temperature high resolution gas chromatography) and HT-HRGC coupled to mass spectrometry (HT-HRGC-MS). Several compounds can be characterized including unusual high molecular weight compounds.     

Characterization of N-linked glycans by supercritical fluid chromatography-mass spectrometry

N-Linked glycans have been characterized by supercritical fluid chromatography (SFC) and SFC-MS using positive- and negative-ion chemical ionization. Four common oligosaccharide derivatives have been prepared and their chromatographic properties assessed on three SFC columns of varying polarity. Carbon dioxide has been used as the SFC mobile phase, with ammonia or CO2 added to the ion source for positive- and negative-ion chemical ionization, respectively. Direct SFC-MS interfacing allows the analytical manipulations of single-ion monitoring, total-ion plots, background subtraction, library searches, and spectral reconstruction algorithms. Positive ammonia chemical ionization yields abundant molecular-weight information, (MH)+, and (MNH4)+ with little or no fragmentation. To capitalize on sensitivity, samples were prepared with the pentafluorobenzyl aminobenzoate reagent, acetylated, and analyzed by SFC-NICI-MS. This modification improves column efficiency and resolution and greatly enhances detecting sensitivity. These "soft" ionization conditions provide abundant molecular-weight-related anions for collision-induced dissociation and subpicogram detection.     

Simultaneous determination of various aromatic amines and metabolites of aromatic nitro compounds in urine for low level exposure using gas chromatography-mass spectrometry

A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, alpha- and beta-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid-liquid extraction at pH 6.2-6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 microg/l (4-aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 microg/l urine (4-amino-2,6-dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 microg/l and 10 microg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70-121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 microg/l; 95th percentile: 7.9 microg/l), o- (0.12 microg/l; 2.7 microg/l), m- (0.17 microg/l; 2.2 microg/l) and p-toluidine (0.11 microg/l; 0.43 microg/l), and o-anisidine (0.22 microg/l; 0.68 microg/l). Additionally, we found that the persons investigated also excrete 3- (<0.05 microg/l; 0.55 microg/l) and 4-chloroaniline (0.11 microg/l; 0.57 microg/l) as well as 3,5-dichloroaniline (0.18 microg/l; 1.5 microg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (<0.05 microg/l; 0.12 microg/l). We did not detect alpha- or beta-naphtylamine, 4-aminobiphenyl or metabolites of explosives in the samples.     

Characterization of glycosphingolipid mixtures with up to ten sugars by gas chromatography and gas chromatography-mass spectrometry as permethylated oligosaccharides and ceramides released by ceramide glycanase

A novel, effective method for structural characterization of glycosphingolipids has been devised. It employs ceramide glycanase to release intact oligosaccharides followed by analysis using high-mass gas chromatography-mass spectrometry. The oligosaccharides and ceramides released by the glycanase were permethylated and analyzed. The capillary gas chromatography gave excellent resolution and separated, for example, two isomeric 10-sugar oligosaccharides with a molecular mass of 2150 daltons differing only by a Gal1-3GlcNAc and a Gal1-4GlcNAc linkage. The oligosaccharides released from sialic acid containing glycosphingolipids (gangliosides) were also analyzed for monosialo compounds. This analytical approach is simple, is quick, and can readily allow quantitation of individual glycosphingolipids.     

Isolation and identification of some guanidino compounds in the urine of patients with hyperargininaemia by liquid chromatography,thin-layer chromatography and gas chromatography-mass spectrometry

Liquid column chromatographic studies of monosubstituted guanidino compounds, which are excreted in the urine of patients with hyperargininaemia are reported. The guanidino-positive peaks, with the highest excretion values, were isolated from urine and the isolated compounds were identified by thin-layer chromatography and gas chromatography—mass spectrometry. Guanidinoacetic acid, N-α-acetylarginine, argininic acid, γ-guanidinobutyric acid, arginine and α-keto-δ-guanidinovaleric acid were found to be excreted at high levels in the urine of patients with hyperargininaemia compared with controls.     

光肩星天牛幼虫虫粪挥发物成分分析

天牛幼虫排出的虫粪所释放的挥发性物质成为天敌搜寻天牛所在微栖境的重要化学信息物质.本试验采用固相微萃取技术,提取光肩星天牛Anoplophora glabripennis(Motschulsky)取食垂柳和金丝柳后产生虫粪的挥发性物质,利用气相色谱-质谱联用仪(GC-MS)进行鉴定.结果表明,虫粪挥发性物质主要为萜烯类物质.两种虫粪挥发物中含共有物质19种,其中à-蒎烯的含量最高,该物质在垂柳虫粪挥发物中相对含量为51.60%,在金丝柳虫粪中的相对含量为28.84%.在金丝柳虫粪中检测到的挥发物种类稍多于垂柳虫粪中检测到的种类.     

Simultaneous determination of urinary cystathionine, lanthionine, and their cyclic compounds using liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization

A measurement system for cystathionine (Cysta) lanthionine (LT), and (AEC), and reduced products of their ketimines, perhydro-1,4-thiazapine-3,5-dicarboxylic acid (PHTZDC), 1,4-thiomorpholine-3,5-dicarboxylic acid (TMDA) and 1,4-thiomorpholine-3-carboxylic acid (TMA) in the urine samples of a patient with cystathioninuria and normal human subjects has been developed, using column liquid chromatography-mass spectrometry. The recoveries were about 90–105% for Cysta, LT and AEC, and about 77–87% for PHTZDC, TMDA and TMA after ion-exchange treatment. The concentrations of Cysta and PHTZDC in the urine of a patient with cystathioninuria were much higher compared with those in the urine of normal human subjects. The concentrations of AEC and TMDA were almost the same. LT and TMA could not be detected in the urine samples by this method. This method proved useful for the determination of sulfur-containing amino acids and their cyclic compounds in biological samples.     

Determination of cyanide and thiocyanate in blood by gas chromatography and gas chromatography-mass spectrometry

We devised a sensitive and simple method for determining cyanide And its major metabolite, thiocyanate, in blood using an extractive alkylation technique. Pentafluorobenzyl bromide was used as the alkylating agent, and tetradecyldimethylbenzylammonium chloride was used as the phase-transfer catalyst. The derivatives obtained were analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with an electron-capture detection. The detection limits of cyanide and thiocyanate were 0.01 and 0.003 μmol/ml, respectively, while the gross recovery of both compounds was 80%. The calibration curve was linear over the concentration range from 0.02 to 1.0 μmol/ml for cyanide and from 0.01 to 1.0 μmol/ml for thiocyanate. The accuracy and precision of the method were evaluated, and the coefficients of variation were found to be within 10%. Using this method, the blood levels of two victims who had died from cyanide poisoning were determined.