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1.
Arnis Kuksis Jukka-Pekka Suomela Marko Tarvainen Heikki Kallio 《Molecular biotechnology》2009,42(2):224-268
Thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography (LC) in combination with mass spectrometry
(MS) have been adopted for the isolation and identification of oxolipids and for determining their functionality. TLC provides
a rapid separation and access to most oxolipids as intact molecules and has recently been effectively interfaced with time-of-flight
(TOF) MS (TOF-MS). GC with flame ionization (FI) (GC/FI) and electron impact (EI) MS (GC/EI-MS) has been extensively utilized
in the analysis of isoprostanes and other low-molecular-weight oxolipids, although these methods require derivatization of
the analytes. In contrast, LC with ultraviolet (UV) absorption (LC/UV) or evaporate light scattering detection (ELSD) (LC/ELSD)
as well as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) MS (LC/ESI-MS) or LC/APCI-MS has
proven to be well suited for the analysis of intact oxolipids and their conjugates without or with minimal derivatization.
Nevertheless, kit-based colorimetric and fluorescent procedures continue to serve as sensitive indicators of the presence
of hydroperoxides and aldehydes.
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Arnis KuksisEmail: |
2.
Mariusz A. Bromke Anton Hochmuth Takayuki Tohge Alisdair R. Fernie Patrick Giavalisco Asdrubal Burgos Lothar Willmitzer Yariv Brotman 《The Plant journal : for cell and molecular biology》2015,81(3):529-536
Quantification of fatty acids has been crucial to elucidate lipid biosynthesis pathways in plants. To date, fatty acid identification and quantification has relied mainly on gas chromatography (GC) coupled to flame ionization detection (FID) or mass spectrometry (MS), which requires the derivatization of samples and the use of chemical standards for annotation. Here we present an alternative method based on a simple procedure for the hydrolysis of lipids, so that fatty acids can be quantified by liquid chromatography mass spectrometry (LC‐MS) analysis. Proper peak annotation of the fatty acids in the LC‐MS‐based methods has been achieved by LC‐MS measurements of authentic standard compounds and elemental formula annotation supported by 13C isotope‐labeled Arabidopsis. As a proof of concept, we have compared the analysis by LC‐MS and GC‐FID of two previously characterized Arabidopsis thaliana knock‐out mutants for FAD6 and FAD7 desaturase genes. These results are discussed in light of lipidomic profiles obtained from the same samples. In addition, we performed untargeted LC‐MS analysis to determine the fatty acid content of two diatom species. Our results indicate that both LC‐MS and GC‐FID analyses are comparable, but that because of higher sensitivity and selectivity the LC‐MS‐based method allows for a broader coverage and determination of novel fatty acids. 相似文献
3.
Berdyshev EV 《Biochimica et biophysica acta》2011,1811(11):680-693
Fatty aldehydes are important components of the cellular lipidome. Significant interest has been developed towards the analysis of the short chain α,β-unsaturated and hydroxylated aldehydes formed as a result of oxidation of polyunsaturated fatty acids. Multiple gas chromatography-mass spectrometry (GC/MS) and subsequently liquid chromatography-mass spectrometry (LC/MS) approaches have been developed to identify and quantify short-chain as well as long-chain fatty aldehydes. Due to the ability to non-enzymaticaly form Schiff bases with amino groups of proteins, lipids, and with DNA guanidine, free aldehydes are viewed as a marker or metric of fatty acid oxidation and not the part of intracellular signaling pathways which has significantly limited the overall attention this group of molecules have received. This review provides an overview of current GC/MS and LC/MS approaches of fatty aldehyde analysis as well as discusses technical challenges standing in the way of free fatty aldehyde quantitation. 相似文献
4.
Maralikova B Weinmann W 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,811(1):21-30
Recently, high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) has become a powerful tool for quantitative confirmatory analysis of drugs of abuse and has begun to spread in the field of forensic toxicology. Guidelines for confirmatory analysis by GC/MS and LC/MS/MS have been published recently by several organizations (WADA, IOC, SOFT, GTFCh, EU). However, these guidelines have not yet been included in procedures for drug analysis with LC/MS/MS. The prerequisites for forensic confirmatory analysis by LC/MS/MS with respect to EU guidelines are chromatographic separation, a minimum number of two MS/MS transitions to obtain the required identification points and predefined thresholds for the variability of the relative intensities of the MS/MS transitions (MRM transitions) in samples and reference standards. LC/MS/MS methods for determination of several classes of drugs of abuse including some basic drugs (opiates, stimulants), cannabinoids and some of their phase-I- and phase-II-metabolites (especially glucuronides) in urine and serum of drug abusers and/or crime offenders or victims have been developed and validated following current recommendations and are presented in this paper. At least two MRM transitions for each substance were monitored to provide sufficient identification of drugs, deuterated analogues of analytes were used as internal standards for quantitation where possible and chromatographic separation has been performed on reversed-phase columns with gradient elution. Validation data obtained and the application to real samples show that the requested criteria for confirmatory analysis of drugs of abuse by EU guidelines can be fulfilled with a total number of four identification points by LC/MS/MS methods using a triple-quadrupole mass spectrometer. Furthermore, the methods are sufficiently sensitive to meet current requirements for confirmatory analysis of drugs of abuse in driving under the influence of drugs (DUID) cases established by the Society of Toxicological and Forensic Chemistry (GTFCh). 相似文献
5.
Schumacher M Liere P Akwa Y Rajkowski K Griffiths W Bodin K Sjövall J Baulieu EE 《Neurochemistry international》2008,52(4-5):522-540
Pregnenolone sulfate (PREGS) has been shown, either at high nanomolar or at micromolar concentrations, to increase neuronal activity by inhibiting GABAergic and by stimulating glutamatergic neurotransmission. PREGS is also a potent modulator of sigma type 1 (sigma1) receptors. It has been proposed that these actions of PREGS underlie its neuropharmacological effects, and in particular its influence on memory processes. On the other hand, the PREGS-mediated increase in neuronal excitability may become dangerous under particular conditions, for example in the case of excitotoxic stress or convulsions. However, the physiopathological significance of these observations has recently been put into question by the failure to detect significant levels of PREGS within the brain and plasma of rats and mice, either by direct analytical methods based on liquid chromatography/mass spectrometry (LC/MS) or enzyme linked immunosorbent assay (ELISA) with specific antibodies against PREGS, or by indirect gas chromatography/mass spectrometry (GC/MS) analysis with improved sample workup. These recent results have not come to the attention of a large number of neurobiologists interested in steroid sulfates. However, although available direct analytical methods have failed to detect levels of PREGS above 0.1-0.3 ng/g in brain tissue, it may be premature to completely exclude the local formation of biologically active PREGS within specific and limited compartments of the nervous system. In contrast to the situation in rodents, significant levels of sulfated 3beta-hydroxysteroids have been measured in human plasma and brain. Previous indirect measures of steroid sulfates by radioimmunoassays (RIA) or GC/MS had detected elevated levels of PREGS in rodent brain. The discrepancies between the results of different assay procedures have revealed the danger of indirect analysis of steroid sulfates. Indeed, PREGS must be solvolyzed/hydrolyzed prior to RIA or GC/MS analysis, and it is the released, unconjugated PREG which is then quantified. Extreme caution needs to be exercised during the preparation of samples for RIA or GC/MS analysis, because the fraction presumed to contain only steroid sulfates can be contaminated by nonpolar components from which PREG is generated by the solvolysis/hydrolysis/derivatization reactions. 相似文献
6.
Lotte Joosten Patrick P. J. Mulder Klaas Vrieling Johannes A. van Veen Peter G. L. Klinkhamer 《Phytochemical analysis : PCA》2010,21(2):197-204
Introduction – Pyrrolizidine alkaloids (PAs) serve an important function in plant defence. Objective – To compare different extraction methods and detection techniques, namely gas chromatography with nitrogen phosphorus detection (GC‐NPD) and liquid chromatography tandem mass spectrometry (LC‐MS/MS) with quadrupole analysers for analysing PAs in Jacobaea vulgaris. Methodology – Both formic acid and sulfuric acid were tested for PA extraction from dry plant material. For GC‐NPD, reduction is required to transform PA N‐oxides into tertiary amines. Zinc and sodium metabisulfite were compared as reducing agents. Results – The lowest PA concentration measured with GC‐NPD was approximately 0.03 mg/g and with LC‐MS/MS 0.002 mg/g. The detection of major PAs by both techniques was comparable but a number of minor PAs were not detected by GC‐NPD. With the LC‐MS/MS procedure higher concentrations were found in plant extracts, indicating that losses may have occurred during the sample preparation for the GC‐NPD method. Zinc proved a more effective reducing agent than sodium metabisulfite. The sample preparation for LC‐MS/MS analysis using formic acid extraction without any reduction and purification steps is far less complex and less time consuming compared to GC‐NPD analysis with sulfuric acid extraction and PA N‐oxide reduction with zinc and purification. Conclusions – In terms of sensitivity and discrimination, formic acid extraction in combination with LC‐MS/MS detection is the method of choice for analysing PAs (both free and N‐oxides forms) in plant material. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
7.
Phospholipids containing polyunsaturated fatty acyl chains are prevalent among brain lipids, and regional differences in acyl chain distribution appear to have both functional and pathological significance. A method is described in which the combined application of GC and multiple reaction monitoring (MRM) MS yielded precise relative quantitation and approximate absolute quantitation of lipid species containing a particular fatty acyl chain in milligram-sized tissue samples. The method uses targeted MRM to identify specific molecular species of glycerophosphocholine lipids, glycerophospho-ethanolamine lipids, glycerophosphoinositol lipids, glycerophosphoserine lipids, glycero-phosphoglycerol lipids, and phosphatidic acids that contain esterified arachidonate (AA) and docosahexaenoate (DHA) separated during normal phase LC/MS/MS analysis. Quantitative analysis of the AA and DHA in the LC fractions is carried out using negative ion chemical ionization GC/MS and stable isotope dilution strategies. The method has been applied to assess the glycerophospholipid molecular species containing AA and DHA in microdissected samples of murine cerebral cortex and hippocampus. Results demonstrate the potential of this approach to identify regional differences in phospholipid concentration and reveal differences in specific phospholipid species between cortex and hippocampus. These differences may be related to the differential susceptibility of different brain regions to neurodegenerative disorders. 相似文献
8.
8-Hydroxyadenine (8-OH-Ade) is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the C-8 position of adenine followed by oxidation. We describe the measurement of the nucleoside form of this compound, 8-hydroxy-2'-deoxyadenosine (8-OH-dAdo) in DNA by liquid chromatography/mass spectrometry (LC/MS). The developed methodology enabled the separation by LC of 8-OH-dAdo from intact and modified nucleosides in enzymic hydrolysates of DNA. Measurements by MS were performed using atmospheric pressure ionization-electrospray process. Isotope-dilution MS was applied for quantification using a stable isotope-labeled analog of 8-OH-dAdo. The level of sensitivity of LC/MS with selected-ion monitoring (SIM) for 8-OH-dAdo amounted to approximately 10 femtomol of this compound on the LC column. This level of sensitivity is similar to that previously reported using LC-tandem MS (LC/MS/MS) with multiple-reaction monitoring mode (MRM) (7.5 femtomol). This compound was quantified in DNA at a level of approximately one molecule/10(6) DNA bases using amounts of DNA as low as 5 microg. The results suggested that this lesion may be quantified in DNA at even lower levels, when more DNA is used for analysis. In addition, gas chromatography/isotope-dilution mass spectrometry with SIM (GC/IDMS-SIM) was applied to measure 8-OH-Ade in DNA following its removal from DNA by acidic hydrolysis. The background levels of 8-OH-dAdo and 8-OH-Ade measured by LC/IDMS-SIM and GC/IDMS-SIM, respectively, were nearly identical. In addition, DNA samples, which were exposed to ionizing radiation at different radiation doses, were analyzed by these techniques. Nearly identical results were obtained, indicating that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results. The level of sensitivity of GC/MS-SIM for 8-OH-Ade was also measured and found to be significantly greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS-MRM for 8-OH-dAdo. The results show that the LC/MS technique is well suited for the measurement of 8-OH-dAdo in DNA. 相似文献
9.
九种植物精油对腌肉及咸鱼害虫大头金蝇的杀卵作用 总被引:1,自引:0,他引:1
为开发腌肉和咸鱼害蝇的安全性杀卵剂,采用改进的浸卵法,进行了9种植物精油对大头金蝇的杀卵活性测定.结果表明:肉桂、丁香、八角茴香3种植物精油较为有效,它们的杀卵毒力(LC50)依次为0.428、1.605和2.489mg.ml-1,以肉桂油的毒力最强.通过GC/MC成分分析,从肉桂油检出22个成分,主成分桂皮醛占92.33%.合成桂皮醛(含量97.33%)的毒力为0.281mg.ml-1,略高于肉桂油,表明桂皮醛是肉桂油的主要杀卵活性成分,可替代肉桂油作为害蝇杀卵剂利用. 相似文献
10.
Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS 总被引:7,自引:0,他引:7
Halket JM Waterman D Przyborowska AM Patel RK Fraser PD Bramley PM 《Journal of experimental botany》2005,56(410):219-243
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. 相似文献
11.
D'Arcy-Moskwa E Weston L Noble GN Raidal SL 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(30):3668-3671
Mucosal integrity may be objectively assessed by determination of the absorption of exogenous substances such as sucrose. Gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) have been reported for the accurate quantification of low concentrations of sucrose in serum. LC/MS offered the advantage of high sensitivity and mass selectivity without the need for extensive sample derivatization required for GC/MS methods. However, the high polarity and non-volatile nature of the sucrose molecule renders LC/MS techniques challenging. Previously published reports lacked sufficient detail to permit replication of methodology. Problems encountered with existing protocols included poor peak resolution and weak fragmentation of the parent molecule. This communication describes a LC/MS protocol developed to provide improved resolution and product detection. 相似文献
12.
Neurosteroids and neurosterols are found in brain at low levels (ng/g-microg/g) against a high background of cholesterol (mg/g). As such their analysis can be challenging. Traditionally, these molecules have been analysed by gas chromatography (GC)-mass spectrometry (MS), however, the absence of molecular ions in GC-MS spectra, even from derivatised molecules, can make the discovery and identification of novel neurosteroids/sterols difficult. To avoid this scenario, liquid chromatography (LC) combined with desorption ionisation methods are employed. In this review we discuss the application of LC-MS and LC-tandem mass spectrometry (MS/MS) for the identification of neurosteroids/sterols, paying particular attention to the use of low-flow-rate LC to maximise chromatographic and mass spectrometric performance. 相似文献
13.
Masukawa Y Tsujimura H Imokawa G 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,823(2):131-142
A systematic method for the sensitive, precise and accurate determination of hair lipids, including trace amounts of intrinsic endogenous cholesterol (CH), ceramide/N-palmitoyl-DL-dihydrosphingosine (CER/PDS), cholesterol sulfate (CS) and chemically bound 18-methyl eicosanoic acid (18-MEA), has been developed in combination with TLC/FID (flame ionization detection), LC/MS and GC/MS. TLC/FID was used for the simultaneous determination of squalene (SQ), wax esters (WEs), triglycerides (TGs) and free fatty acids (FFAs). Optimal conditions for LC/MS to determine CS and 18-MEA were developed using selected ion monitoring (SIM) under the negative ion mode of electrospray ionization. An alternative procedure for the determination of 18-MEA was also established using commercially available heneicosanoic acid (HEA). In GC/MS, the optimal selection of ions for SIM of trimethylsilylated CH and CER/PDS, and the use of on-column injection has enabled their simultaneous detection. This newly developed method has been used to characterize the hair lipid composition from the proximal root end to the distal tip of chemically untreated hair fibers from two different females, and specific changes of hair lipids probably due to its origin and individuals have been demonstrated for the first time. This method may be useful for clarifying the important roles of intrinsic endogenous 18-MEA, CS, CH and CERs in the function of the cell membrane complex of hair fibers. 相似文献
14.
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. 相似文献
15.
Developmental regulation of galactoglycerolipid and galactosphingolipid sulphation during mammalian spermatogenesis. Evidence for a substrate-selective inhibitor of testicular sulphotransferase activity in the rat. 总被引:1,自引:1,他引:0
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C A Lingwood 《The Biochemical journal》1985,231(2):393-400
The synthesis of sulphatoxygalactosylacylalkylglycerol (SGG) is a differentiation marker of mammalian spermatogenesis. Maximal sulphation is observed in rat testis at about 20 days after birth and rapidly declines to low levels as the testis matures. The present data show that this decline in SGG synthesis is due to the appearance of an inhibitor of galactolipid sulphation. The inhibitor is a soluble testicular factor which is first detected at about 25 days after birth. Testicular homogenate can sulphate exogenous galactosylacylalkylglycerol (GG), galactosylceramide (GC) and lactosylceramide (LC) in vitro. The testicular inhibitor is most effective in preventing GG sulphation and inhibits GC and LC sulphation to a lesser extent; this correlates with the finding that glycolipid sulphation shifts from SGG production in 20-day-old testis to GC and LC sulphation at later stages of testicular development. The effect of the inhibitor on sulphotransferase activity from brain and kidney was also determined. The inhibitor decreased the sulphation of GG in vitro by both testis and kidney, inhibited testicular sulphation of GC less effectively and had no effect on GC sulphation by kidney and brain homogenates. A 9500-fold purification of the inhibitory activity has been obtained in a fraction isolated by h.p.l.c. 相似文献
16.
Castro-Perez J Previs SF McLaren DG Shah V Herath K Bhat G Johns DG Wang SP Mitnaul L Jensen K Vreeken R Hankemeier T Roddy TP Hubbard BK 《Journal of lipid research》2011,52(1):159-169
High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r2 = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with 2H2O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of 2H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols. 相似文献
17.
The role of urinary metabolic profiling in systems biology research is expanding. This is because of the use of this technology for clinical diagnostic and mechanistic studies and for the development of new personalized health care and molecular epidemiology (population) studies. The methodologies commonly used for metabolic profiling are NMR spectroscopy, liquid chromatography mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS). In this protocol, we describe urine collection and storage, GC/MS and data preprocessing methods, chemometric data analysis and urinary marker metabolite identification. Results obtained using GC/MS are complementary to NMR and LC/MS. Sample preparation for GC/MS analysis involves the depletion of urea via treatment with urease, protein precipitation with methanol, and trimethylsilyl derivatization. The protocol described here facilitates the metabolic profiling of ~400-600 metabolites in 120 urine samples per week. 相似文献
18.
Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance
liquid chromatography-mass spectrometry: comparison with measurement
by gas chromatography-mass spectrometry
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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 106 DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 106 DNA bases, or even lower, when more DNA is used. Up to 50 µg 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. 相似文献
19.
Jaysing Mahavirsing Dinore Harshal Shivaji Patil Samreen Farooqui Vidya Pradhan Mazahar Farooqui 《化学与生物多样性》2023,20(2):e202200048
Vigna unguiculata (L. Walp) or Cowpea pod methanolic extracts phytochemical analysis, total phenolic content (TPC), and secondary metabolite profiling were determined using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) analysis. GC/MS analysis revealed twenty compounds in the extract, while LC/MS analysis identified twenty-four compounds. GC/MS chromatogram analysis suggested the presence of opioid α-N-Normethadol a major constituent found in methanolic extract and fatty acid esters carotenoid is found second major constituent. LC/MS chromatogram and the mass spectral analysis demonstrated the presence of flavonoids, carotenoids, and alkaloids as major phytochemicals. We investigated the antibacterial, anti-fungal, and anti-oxidant activity of pod methanolic extract. The extract was found equally effective against E. coli, S. pyogenes, and P. aeruginosa with MIC 100 μg/mL similar to the standard Ampicillin (MIC 100 μg/mL). C. albicans were found to be most susceptible to Vign unguiculata pods methanolic extract with a MIC of 250 μg/mL. The pod extract showed significant DPPH scavenging activity (IC50=78.38±0.15) which suggests its antioxidant potential. 相似文献
20.
Ivandini TA Sarada BV Terashima C Rao TN Tryk DA Ishiguro H Kubota Y Fujishima A 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,791(1-2):63-72
Boron-doped diamond thin film (BDD) electrodes have been used to study the oxidation reactions and to detect leucine-enkephalinamide (LEA) and its metabolites, tyrosine (T), tyrosyl-alanine (TA), tyrosyl-alanine-glycine (TAG) and leucine-enkephalin (LE) using cyclic voltammetry (CV), flow-injection analysis (FIA), and gradient liquid chromatography (LC) with amperometric detection. At diamond electrodes, well-defined and highly reproducible cyclic voltammograms were obtained with signal-to-background (S/B) ratios 5-10 times higher than those observed for glassy carbon (GC) electrodes. The analytical peaks of LC for LEA and its metabolites were well resolved. No deactivation of BDD electrodes was found after several experiments with standard as well as plasma samples, indicating high stability of the electrode. Calibration curves were linear over a wide range from 0.06 to 30 microM with regression coefficients of 0.999 for all compounds. The limits of detection obtained based on a signal-to-noise ratio of 3:1 were 3, 2.2, 2.7, 20 and 11 nM for T, TA, TAG, LE and LEA, respectively. These values were at least one order lower than those obtained at GC electrodes, which has given limits of detection of 22.88, 20.64, 89.57, 116.04 and 75.67 for T, TA, TAG, LE and LEA, respectively. Application of this method to real samples was demonstrated and validated using rabbit serum samples. This work shows the promising use of conducting diamond as an amperometric detector in gradient LC, especially for the analysis of enkephalinamide and its metabolites. 相似文献