液相色谱/质谱联用法分析不同年龄鼠皮肤中I型、III型胶原蛋白相对含量

哺乳动物皮肤中真皮内的胶原蛋白含量约占70%,主要是I型、III型和少量V型胶原蛋白。利用稀酸溶解和酶法提取了大鼠皮肤中的总胶原蛋白,将胶原蛋白粗提品在60℃变性后用胰蛋白酶进行降解,液相色谱/质谱联用法分析两种胶原蛋白的特征多肽,并研究不同生长期大鼠皮肤中I型和III型胶原蛋白相对含量。结果表明,大鼠皮肤中的III型胶原蛋白的相对含量随生长期延长逐渐降低,而I型胶原蛋白的相对含量逐渐升高,8周后两种胶原蛋白的比例趋于稳定。表明使用高效液相色谱/质谱联用法分析组织中的胶原蛋白类型及其动态变化具有可行性,为更好的临床应用提供实验基础。     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.     

Methods for determination of lipid peroxidation in biological samples

Interest in the pathological consequences of lipid peroxidation has led to the development of a number of analytical approaches to the quantitation of products. However, the various analytical methodologies employed often do not measure the same chemical classes of products, and apparent discrepencies have been observed, particularly in studies of lipid peroxidation in biological systems. This review provides a brief discussion of some of the strengths and weakness of methods currently used for the determination of lipid peroxidation in biological tissues.     

Detection of cholesteryl ester hydroperoxide isomers using gas chromatography-mass spectrometry combined with thin-layer chromatography blotting

Oxidative modification of low-density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. During the oxidation of LDL, cholesteryl esters, the major lipid components in LDL, are oxidized to cholesteryl ester hydroperoxides (CEOOH). The isomers of CEOOH may reflect the reactive species that initiate the peroxidation reaction. In the current study, a novel analytical method for the determination of CEOOH isomers, especially cholesteryl linoleate hydroperoxide isomers, was developed using the combination of two chromatographic techniques: (i) thin-layer chromatography blotting with diphenyl-1-pyrenylphosphine (DPPP) fluorescent detection (DPPP-TLC blotting) and (ii) gas chromatography-electron ionization-mass spectrometry (GC-EI-MS). CEOOH was applied to DPPP-TLC blotting, the obtained DPPP-derived fluorescent spots containing cholesteryl ester hydroxides were extracted and derivatized (hydrogenation, transmethylation, and trimethylsilylation), and the formed methyl ester/trimethylsilylether derivatives of hydroxyoctadecenoic acid were then analyzed by GC-EI-MS. The CEOOH isomers were determined by selected ion monitoring of isomer-specific fragment ions originated from the alpha-cleavage of the trimethylsilyloxyl group. Using these two chromatographic techniques, we were able to detect isomeric CEOOH in the oxidized human LDL. Our results indicated that GC-EI-MS analysis combined with DPPP-TLC blot is a specific method for analyzing cholesteryl ester hydroperoxide isomers in biological samples such as oxidized LDL.     

The isoprostanes: Unique bioactive products of lipid peroxidation

The development of a specific, reliable and noninvasive method for measuring oxidative stress in humans is essential for establishing the role of free radicals in human diseases. Currently, accurate techniques to assess oxidant injury in vivo are extremely limited although a number of approaches are being investigated. Of these, the measurement of specific products of nonenzymatic lipid peroxidation, the F₂-isoprostanes (F₂-IsoPs), appears to be a more accurate marker of oxidative stress in vivo in humans than other available methods. The purpose of this brief review is to acquaint the reader with the IsoPs from a biochemical perspective and to provide information regarding the utility of quantifying these compounds as indicators of oxidant stress.     

Minimizing artifactual elevation of lipid peroxidation products (F2-isoprostanes) in plasma during collection and storage

F₂-isoprostanes (F₂-IsoP’s) are reliable measures of in vivo lipid oxidation, but care is required to prevent artifactual elevation. We examined the effects of blood collection and storage on plasma F₂-IsoP’s. Blood was collected into EDTA/butylated hydroxytoluene/reduced glutathione (EDTA/BHT/GSH) or EDTA, at 4 °C or room temperature. Plasma was stored at −20 or −80 °C for 1 or 6 months before F₂-IsoP’s were assayed by GC–MS. The temperature of blood collection did not affect F₂-IsoP’s. However, storage at −20 °C or collection into EDTA resulted in significant increases in F₂-IsoP’s. Blood collection into EDTA/BHT/GSH and storage at −80 °C minimizes artifactual elevation of plasma F₂-IsoP’s.     

高效液相色谱-串联质谱检测基因组DNA甲基化方法的建立

目的:建立高效液相色谱-串联质谱(HPLC-ESI-MS/MS)检测基因组DNA甲基化水平的方法。方法:以5-mdC和dG为标准品,采用全自动高效液相色谱系统进行分离,串联电喷雾质谱检测,选择多反应监测模式(MRM)测定标准品,绘制标准工作曲线。结果:在MRM模式下选取5-mdC(m/z 241.9→126.3)和dG(m/z 268.1→152.3)分别作为定量检测的母子离子对,各化合物能实现良好的基线分离;5-mdC和dG碰撞能均为15 eV,去簇电压分别为40和45 V,最低定量限分别为1.65和2.47 fmol;标准品的响应值比为90%~110%;5-mdC含量的天内相对标准偏差和天间相对标准偏差均小于8%。结论:HPLC-ESI-MS/MS是能应用于检测基因组DNA甲基化的一种高通量、高准确率、高分辨率、高灵敏度且重复性好的方法。     

Quantitative determination of Aconitum alkaloids in blood and urine samples by high-performance liquid chromatography

An HPLC method has been developed for the simultaneous determination of the toxic Aconitum alkaloids, aconitine, mesaconitine and hypaconitine in blood and urine samples. The samples were initially subjected to solid phase extraction using Oasis MCX cartridges, and the alkaloids were separated on an XTerra RP18 column, gradient-eluted with acetonitrile: ammonium hydrogen carbonate buffer. Calibration curves were linear in the range 2.75-550 ng for aconitine and hypaconitine, and 3-600 ng for mesaconitine: the limit of detection was 0.1 ng (signal-to-noise ratio of 3) for each alkaloid. The described analysis proved to be sensitive, rapid and economical, and will be applied in the identification and determination of these alkaloids in forensic and therapeutic drug monitoring.     

Measurement of muramic acid in marine sediments by high performance liquid chromatography

Bacterial biomass in marine sediments may be estimated from the amount of muramic acid present. A method for determining muramic acid by high performance liquid chromatography is described, which is simpler and faster than other methods. Muramic acid is released from sediment by acid hydrolysis, and assayed as an o-phthaldialdehyde derivative.     

Plasma phospholipid metabolic profiling and biomarkers of mouse IgA nephropathy

IgA nephropathy is the most common form of glomerulonephritis (GN) and it could progress to end-stage renal failure within 10 years. Participating in biological processes in various pathways, phospholipids as a class of important constituents in the biomembranes have been paid increasing attention in many fields. However, phospholipids metabolism in glomerular disease was not clear, especially in IgA nephropathy. In this paper, the plasma phospholipid metabolic profile in mouse IgA nephropathy was investigated to discover the potential biomarkers on the progression of this disease by using high performance liquid chromatography/mass spectrometry (HPLC/MS) and the principal components analysis (PCA) as well as partial least squares-discriminant analysis (PLS-DA). The experimental mouse models of IgA nephropathy were established by oral immune and BSA injection. It was found that expression of intercellular adhesion molecule-1 (ICAM-1) in the glomeruli had a significant correlation with proteinuria in mouse IgA nephropathy. The association between plasma phospholipids and expression of ICAM-1 in the glomeruli of IgA nephropathy suggested C18:0/C18:0 PS (phosphatidylserine), C18:0/C22:5 PS (phosphatidylserine) and C18:0/C20:4 PI (phosphatidylinositol) were possible biomarkers of IgA nephropathy. The results show that the plasma phospholipid metabolic profiles from HPLC/MS combining with PCA and PLS-DA can be used not only to differentiate the IgA nephropathy from the controls, but also to discover and identify the potential biomarkers.     

Determination of trace lead in biological and water samples with dispersive liquid-liquid microextraction preconcentration

A new method for the determination of trace lead was developed by dispersive liquid–liquid microextraction preconcentration and graphite furnace atomic absorption spectrometry. In the proposed approach, 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) was used as a chelating agent, and carbon tetrachloride and ethanol were selected as extraction and dispersive solvents. Some factors influencing the extraction efficiency of lead and its subsequent determination, including extraction and dispersive solvent type and volume, pH of sample solution, concentration of the chelating agent, and extraction time, were studied and optimized. Under the optimum conditions, the enrichment factor of this method for lead was reached at 78. The detection limit for lead was 39 ng L⁻¹ (3σ), and the relative standard deviation (RSD) was 3.2% (n = 7, c = 10 ng mL⁻¹). The method was successfully applied to the determination of trace amounts of lead in human urine and water samples.     

Detection of nitrous oxide in the neuronal nitric oxide synthase reaction by gas chromatography-mass spectrometry

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.     

高效液相色谱法同时检测棉花根中的多种植物激素

目的：探寻高效液相色谱同时检测棉花根中多种植物激素含量的方法。方法：采用WatersC18反相色谱柱（4.6×250mm,5μm）,在柱温为35℃、流速为1mL.min-1的条件下,以乙腈和三乙胺溶液为流动相梯度洗脱,在每种物质的保留时间附近切换至最大吸收峰（GA3除外）波长作为检测波长,并与254nm同一波长检测多种植物激素含量的方法进行比较,分离和检测棉花根中的玉米素（Z）、玉米素核苷（ZR）、赤霉酸（GA3）、生长素（IAA）和脱落酸（ABA）的含量。结果：切换波长法检测5种植物激素的灵敏度和回收率均较高,检出限均较低。回收率为：Z 96.82%、ZR 94.14%、GA3 92.75%、IAA 93.38%、ABA 95.57%;检出限为：Z 0.1μg.mL-1、ZR 0.1μg.mL-1、GA3 0.5μg.mL-1、IAA 0.3μg.mL-1;ABA 0.05μg.mL-1,能准确检测出棉花根中Z、ZR、GA3、IAA和ABA的含量。结论：采用Waters C18反相色谱柱（4.6×250mm,5μm）,在柱温为35℃、流速为1mL.min-1的条件下,以乙腈和三乙胺溶液为流动相梯度洗脱,结合切换波长法能同时检测出植物组织中多种植物激素含量。     

Strategy for the investigation of O-linked oligosaccharides from mucins based on the separation into neutral, sialic acid- and sulfate-containing species

A method for the separation of O-linked oligosaccharides into neutral, sialic acid-containing and sulfated species was applied to oligosaccharides released by alkaline borohydride from mucin glycopeptides from porcine small intestine. The released mixture of reduced oligosaccharides was applied to an anion exchange column, and the neutral oligosaccharides were collected as the unretarded fraction. A mixture of dimethyl sulfoxide and iodomethane was passed through the column to convert the sialic acid-containing oligosaccharides into methyl esters that were eluted and converted to methyl amides by methyl amine. Finally the sulfated oligosaccharide fraction was eluted with salt. The neutral and the derivatized sialic acid-containing oligosaccharides were analysed by gas chromatography-mass spectrometry after permethylation and the sulfated oligosaccharide fraction was analysed by high performance anion exchange chromatography.Abbreviations GC gas chromatography - GC/MS gas chromatography-mass spectrometry - HPAEC-PAD high performance anion exchange chromatography-pulsed amperometric detection - Hex hexose - HexNAc N-acetyl hexosamine - HexNAcol N-acetyl hexosaminitol - Fuc Fucose - NeuAc N-acetyl neuraminic acid - NeuGc N-glycolyl neuraminic acid     

Non-protein-bound iron detection in small samples of biological fluids and tissues

Interest in the pro-oxidative nature of non-protein-bound-iron (NPBI) led to the development of an assay for its detection. The aim was to set up a reliable method of detecting NPBI in small samples of biological fluids and tissue. The method was based on preferential chelation of NPBI by a large excess of the low-affinity ligand nitrilotriacetic acid. To separate NPBI, a two-step filtration procedure was used. All glassware and plasticware were treated to minimize iron contamination. Measurements were performed in plasma, amniotic fluid, bronchoalveolar lavage, and brain tissues. The analytic system detected iron as ferric nitrate standard down to a concentration of 0.01 μM. The 1,2-dimethyl-3-hydroxy-4(1H)-pyridone-Fe(DHP-Fe) complex eluted with a retention time of about 2.6 min. The standard curve for the DHP-Fe complex was linear between 0.01 and 400 μM in water as well as in plasma, bronchoalveolar lavage, brain tissue, and amniotic fluid. The detection limit was 0.01 μM for all biological fluids and brain tissue. The data show that reliable measurements of NPBI are possible in studies on oxidative stress under experimental and clinical conditions. The possibility of investigating NPBI involvement in free-radical injury might be useful in all human diseases in which oxidative stress occur.     

刺五加叶中金丝桃苷含量的测定

用高效液相色谱法测定了刺五加(Acanthopanax senticosus(Rupr.&Maxin)Harms)叶中金丝桃苷(hyperin)的含量。色谱柱为C18柱,流动相为甲醇和0.025mol L-1磷酸,比例为V甲醇:V0.025mol.L-1磷酸.=55:45(混合后用三乙胺调pH3.0￣3.2),检测波长360nm。样品用甲醇超声提取。结果表明,金丝桃苷峰形良好,与其他组分的色谱峰达到基线分离。加样回收率为104%,相对标准偏差RSD为5.38%。本研究为刺五加叶中金丝桃苷含量的测定建立了一种可靠的方法。     

Detection of differential gene copy number using denaturing high performance liquid chromatography

Genomic rearrangements leading to deletion or duplication of gene(s) resulting in alterations in gene copy number underlie the molecular lesion in several genetic disorders. Methods currently used to determine gene copy number including real time PCR, southern hybridization, fluorescence in situ hybridization, densitometric scanning of PCR product etc. have certain disadvantages and are also expensive and time consuming. Herein, we describe a simple and rapid method to assess gene copy number using denaturing high performance liquid chromatography (dHPLC). We used X chromosome genes as model to compare the gene copy numbers present on this chromosome in males and females. DNA from these samples were amplified by biplex PCR using primer pairs specific for X chromosome genes only (target gene) and for genes present on both X and Y chromosomes (internal control). Amplified products were analyzed using HPLC under non-denaturing conditions. The ratio of peak areas (target gene/internal control) of the amplified products was approximately twice in female samples than male samples (p < 0.001) demonstrating that the differential gene copy number can be easily detected using this method. This method can potentially be used for diagnostic purpose where the need is to distinguish samples based on the differential gene copy numbers.     

高效液相色谱法测定红景天根中红景天甙的含量

建立了一种高效液相色谱法测定红景天根中红景天甙的含量。色谱柱为Hypersil—C18（5μm,150mm×4．6mm）,甲醇-水为流动相,梯度洗脱,柱温25℃,在250nm波长处检测。研究结果表明：红景天甙的检测限为50μg/L,线性范围为4—40mg／L,回归方程为Y=5．6348X-4．0931,r=0．9996,加样回收率为98．05％。方法操作简便、快速、准确。     

Detection and quantification of glucuro- and sulfoconjugated metabolites in human urine following oral administration of xenobiotic 19-norsteroids

Recently, the endogenous origin of nandrolone (19-nortestosterone) and other 19-norsteroids has been a focus of research in the field of drug testing in sport. In the present study, we investigated metabolites conjugated to a glucuronic acid and to a sulfuric acid in urine following administration of four xenobiotic 19-norsteroids. Adult male volunteers administered a single oral dose (10 mg) of each of four 19-norsteroids. Urinary samples collected from 0 to 120 h were subjected to methanolysis and beta-glucuronidase hydrolysis and were derivatized by N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) before gas chromatography-mass spectrometry analysis. We confirmed that 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) were present in both glucuronide (g) and sulfate (s) conjugates and 19-norepiandrosterone (19-NEA) was excreted exclusively as a sulfate fraction in urine of all 19-norsteroids tested. The overall levels of the three metabolites can be ranked as follows: 19-NA(g+s)>19-NE(g+s)>19-NEA(s). The concentration profiles of these three metabolites in urine peaked between 2 to 12h post-administration and declined thereafter until approximately 72-96 h. 19-NA was most prominent throughout the first 24 h post-administration, except for a case in which an inverse relationship was found after 6h post-administration of nandrolone. Furthermore, we found that sulfate conjugates were present in both 19-NA and 19-NE metabolites in urine of all 19-norsteroids tested. The averaged total amounts of metabolites (i.e. 19-NA(s+g)+19-NE(s+g)+19-NEA(s)) excreted in urine were 38.6, 42.9, 48.3 and 21.6% for nandrolone, 19-nor-4-androsten-3,17-dione, 19-nor-4-androsten-3beta,17beta-diol and 19-nor-5-androstene-3beta,17beta-diol, respectively. Results from the excretion studies demonstrate significance of sulfate-conjugated metabolites on interpretation of misuse of the 19-norsteroids.     

Detection and Pharmacokinetics of Alginate Oligosaccharides in Mouse Plasma and Urine after Oral Administration by a Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) Method

A sensitive and simple liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the detection of alginate oligosaccharides (AOs) in mouse plasma and urine after oral administration. In an AO mixture, dimer, trimer, and tetramer were detected by LC-MS/MS equipped with an anion-exchange column with extremely high sensitivity. By this method, we detected certain levels of AOs in samples prepared from mouse plasma and urine after a single oral administration of the AO mixture. Based on a calibration curve made with an AO trimer peak area as a standard, the maximum plasma and urine concentrations of AOs were estimated to be 24.5 μg/ml at 5 min and 425.5 μg/ml at 30 min, respectively. These results suggest that the LC-MS/MS method is well suited to pharmacokinetic analysis of AOs in an in vivo system, and that some of orally administered AOs, at least from dimer to tetramer, are absorbed by digestive organs promptly, and that unaltered, these oligomers were excreted into an urine after a single oral administration to a mouse.