高效液相色谱法测定精浆中过氧化脂质水平及其在男性不育症研究中的应用

采用高效液相色谱法测定精浆中过氧化脂质(lipid peroxidation,LPO)含量,研究了有正常生育能力的男子和不育症患者精浆中LPO含量水平差异及其对男子不育症的影响。精浆样品经酸化后,分解生成的丙二醛(malondialdehyde,MDA)与硫代巴比妥酸(thiobarbituric acid,TBA)缩合反应形成紫红色产物,以Lichrospher C18化学键合硅胶为固定相,0.025 mol/L KH2PO4 (pH 6.2)-甲醇(体积比为58∶42)为流动相进行色谱等度分离     

Quantification of malondialdehyde by HPLC‐FL – application to various biological samples

Malondialdehyde (MDA) is stabile product of lipid peroxidation (LPO), and therefore MDA is frequently used as a biomarker of LPO. To determine MDA level in various biological samples (human plasma, fish liver tissue and cells in culture), we used an HPLC method with fluorescent detection based on 2‐thiobarbituric acid (TBA) assay. The method was validated by the use of spiked pooled plasma samples. In tested concentration range (0.15–3.0 µmol/L) the method was linear (R² = 0.9963), the between‐day variability (coefficient of variations, CVs) was between 4.7 and 7.6%, the within‐day variability CVs was between 2.6 and 6.4% and recovery was between 91.2 and 107.6%. The level of MDA in human plasma (healthy male, non‐smokers, 46.3 ± 4.7 years; N = 38) was 2.2 ± 1.4 µmol/L; that in liver tissue of common carp (Cyprinus carpio; N = 12) was 0.02 ± 0.004 µmol/g tissue, and in cultured cells (human laryngeal carcinoma cells; N = 10) it was 0.18 ± 0.02 nmol/mg proteins. The HPLC‐FL method is rapid, accurate and reliable to follow the extent of LPO in various biological samples, particularly in samples in which a low level of MDA is expected, such as cells in culture. Owing to the rapid analytical process and run time, it can be used for routine analysis of MDA in clinical laboratory. Copyright © 2014 John Wiley & Sons, Ltd.     

High-performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct in biological materials (plasma and human cells) using a commercially available reagent.

The assay of malondialdehyde (MDA) is widely used in clinical chemistry laboratories to investigate lipid peroxidation in oxidative pathologies. In the present work, the thiobarbituric acid (TBA) reaction was carried out on plasma, human erythrocytes and fibroblasts. The reagents used were those of the fluorimetry MDA kit manufactured by Sobioda. We have defined the application of this kit to high-performance liquid chromatography. This adaptation satisfied the criteria of good analytical practice. The detection limit was 2.5 pmol per injection. The retention time of the MDA-TBA2 peak (4.96 +/- 0.07 min) led to excellent resolution of the complex. The within-assay (6-12%) and between-assay (11-12%) precisions were satisfactory. The analytical recovery of MDA after spiking samples of human plasma with tetraethoxypropane standards varied from 70 to 100%. The mean lipoperoxide concentration determined in 32 healthy adults (20-40 years) was 1.04 +/- 0.23 mumol l-1 in plasma. Applied to the erythrocytes of fifteen laboratory workers, the method furnished physiological values of 0.59 +/- 0.21 mumol l-1. Concentrations were significantly higher in chronic renal dialysis patients (4.15 +/- 2.35 mumol l-1. The MDA content of fibroblasts cultured in standard medium was 0.38 +/- 0.04 mumol per g of protein and increased (5.78 +/- 1.38 mumol per g of protein) if the cells were grown in an iron-enriched medium. This accurate high-performance liquid chromatographic method for detection of MDA is the first one which can be applied to plasma, red blood cells and cultured cells. This technique will prevent false positives and should make inter-laboratory comparisons possible.     

Detection of malondialdehyde in vivo using microdialysis sampling with CE-fluorescence

Oxidative damage is a naturally occurring process where reactive oxygen species (ROS) attack and disrupt normal cellular function; however, these effects become elevated during a stress event, such as ischemia/reperfusion or seizure. One result of oxidative stress is lipid peroxidation, where ROS attack free unsaturated fatty acids forming lipid hydorperoxides, which then break down to form secondary products acrolein, 4‐hydroxynonenal, and malondialdehyde (MDA) resulting in irreversible membrane damage and ultimately cell death. Described here is a CE‐fluorescence method for the determination of MDA in conjunction with in vivo microdialysis sampling. MDA was derivatized with thiobarbituric acid under acidic conditions for 20 minutes and injected directly into the capillary without any pretreatment. This method provided a limit of detection of 25 nM (S/N=3) and a linear range of 25–2400 nM (1.8–174 ng/mL). This method was used to quantify MDA in rat heart, muscle, liver, and brain dialysate.     

Comparison of spectrophotometric and HPLC methods for determination of lipid peroxidation products in rat brain tissues

Two methods for determination of lipid peroxidation (LPX) products in rat brain homogenates were compared. The thiobarbituric acid (TBA) test and HPLC assay for analysis of malondialdehyde (MDA) were applied. Rat brain homogenate dissolved in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) was mixed with TBA and H₃PO₄ and heated at 100°C to form colored complex that was extracted into butanol. No significant differences were found between the contents of TBA-reacting substances and their amount deduced from the MDA-TBA analysis. The presented results show that LPX products in brain homogenates can be determined without interferences also by the TBA test. Moreover, a survey of various methods used for the sample preparation before analysis of LPX products originating from different brain areas was made and compared with the obtained results.     

Determination of acrolein in serum by high‐performance liquid chromatography with fluorescence detection after pre‐column fluorogenic derivatization using 1,2‐diamino‐4,5‐dimethoxybenzene

Acrolein is a major unsaturated aldehyde that is generated during the lipid peroxidation process. The measurement of acrolein in biological samples should be useful to estimate the degree of lipid peroxidation and to evaluate the effect of hazardous properties of acrolein on human health. In this study, a highly sensitive and selective high‐performance liquid chromatography with fluorescence detection method was developed for the determination of acrolein in human serum. The proposed method involves the pre‐column fluorogenic derivatization of acrolein with 1,2‐diamino‐4,5‐dimethoxybenzene (DDB) as a reagent. The fluorescent derivative of acrolein could be detected clearly without any interfering reagent blank peaks because DDB does not have intrinsic fluorescence itself, and the detection limit was 10 nM (signal‐to‐noise ratio = 3). The proposed method could selectively detect acrolein in human serum with a simple protein precipitation treatment. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of malondialdehyde by capillary electrophoresis, application to human plasma and relation of its levels with prematurity.

Malondialdehyde (MDA) is considered as the most important marker for monitoring lipid peroxidation, which is strongly associated with the development of serious diseases in adults and premature neonates. In this paper we report a method for determination of free MDA in human plasma using capillary zone electrophoresis. MDA was separated and determined as conjugate with tetrabutylammonium hydrogen sulphate (TBAS). Analysis was performed using 20 mM borare, pH = 9.3, as operating buffer and detection of the MDA-TBAS adduct at 267 nm. The method has a linear range up to 80 microM with a detection limit of 0.2 microM. The method was applied to the analysis of MDA in plasma of healthy adults, normal-gestation infants and of preterm neonates. Plasma proteins were successfully removed following centrifugation through a centricon-3 membrane. Results showed that the method can be easily and accurate applied for the determination of MDA in human plasma and that the level of MDA in pretern neonates is significantly higher (p 相似文献   

New high-performance liquid chromatographic method for sensitive determination of pheomelanin in biological materials by precolumn fluorescence derivatization with naphthalene-2,3-dicarboxaldehyde

Pheomelanin is an important type of melanin distributed in the skin, eye and hair in the mammal, which is of great social, clinical and cosmetic significance. In this study, a new HPLC method with fluorescence detection is described originally for the sensitive determination of pheomelanin in biological materials. The pheomelanin polymer is decomposed into two specific degradation products, 3-amino-4-hydroxyphenylalanine (3-AHP) and 4-amino-3- hydroxyphenylalanine (4-AHP) with hydriodic acid. Then the two AHP isomers are derivatized using a fluorescent probe naphthalene-2,3-dicarboxaldehyde in the presence of cyanide. The resulting highly stable 2-substituted 1-cyanobenz[f] isoindole derivatives were separated on a 5NH(2)-MS aminopropyl packed HPLC column with binary isocratic elution profile and detected fluorimetrically. The assay shows high sensitivity of 0.11nM (2.2fmol per injection, the lowest reported) at signal-to-noise ratio of 3 for each AHPs, good accuracy and precision (RSDs<3.1%), and linearity (range of 0.02-10microM, r>0.995). The results obtained by using fluorescence detection have been compared with other detection systems (electrochemical and UV). The sensitivity can increase from 100 to respect electrochemical detection and 30000 times respect UV detection. The method has been used for the quantitative determination of pheomelanin in various biological samples, including cell cultures from five types of melanoma cell lines of human and rat origin, hair samples of various colors, melanoma tissue and the urines from human melanoma patients and healthy subjects. This original application of HPLC-fluorescence detection represents a powerful tool for investigating pheomelanin synthesis in vitro and in vivo under physiological and pathophysiological conditions.     

New fluorimetric method of liquid chromatography for the determination of the neurotoxin domoic acid in seafood and marine phytoplankton

Domoic acid (DA) is a neurotoxic amino acid that is responsible for the human toxic syndrome, amnesic shellfish poisoning (ASP). A new rapid, sensitive liquid chromatographic (LC) method has been developed for the determination of DA in various marine samples. DA in marine biological materials was derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and analysed using isocratic reversed-phase LC with fluorimetric detection. The calibration, based on standard DA solutions, was linear in the range 0.04-2 microg/ml (r2=0.998) and the detection limit (3:1, signal/noise) was better than 1 ng/ml. Using the certified reference material (MUS-1B), recoveries of DA from shellfish tissue were >95% (n=5). When a strong anion exchange SPE cartridge was used for sample clean-up the detection limit was 6 ng DA/g mussel tissue. Good reproducibility was achieved with RSD values ranging from 3% for 8 microg DA/g (n=5), to 5% for 0.04 microg DA/g (n=5). This new method was successfully applied to the determination of DA in naturally contaminated shellfish and in marine phytoplankton cultures of Pseudonitzschia sp.     

Field-amplified online sample stacking capillary electrophoresis UV detection for plasma malondialdehyde measurement

Malondialdehyde (MDA) determination is the most widely used method for monitoring lipid peroxidation. Here, we describe an easy field-amplified sample injection (FASI) CE method with UV detection for the detection of free plasma MDA. MDA was detected within 8 min by using 200 mmol/L Tris phosphate pH 5.0 as running buffer. Plasma samples treated with ACN for protein elimination were directly injected on capillary without complex cleanup and/or sample derivatization procedures. Using electrokinetic injection, the detection limit in real sample was 3 nmol/L, thus improving of about 100-fold the LOD of the previous described methods based on CE. Precision tests indicate a good repeatability of our method both for migration times (CV = 1.11%) and for areas (CV = 2.05%). Moreover, a good reproducibility of intra- and inter-assay tests was obtained (CV = 2.55% and CV = 5.14%, respectively). Suitability of the method was tested by measuring MDA levels in 44 healthy volunteers.     

Detection of oxidative stress. Interest of GC-MS for malondialdehyde and formaldehyde monitoring

Ischemia-reperfusion syndrome is a condition where the role of oxygen free radicals is important. Malondialdehyde (MDA) and formaldehyde (FA), products of lipid peroxidation, are the presumptive markers for the development of oxidative stress in tissues and plasmas. A GC-MS method for the determination of MDA and FA in rat brain extract is described. Rat brains were homogenized with deionized water. The homogenates were derivatized with 2,4-dinitrophenylhydrazone (DNPH) to obtain hydrazines derivatives of MDA and FA. The hydrazine derivatives were analyzed by GC-MS and quantitation was by single ion monitoring (SIM). The retention times of FA and MDA were, respectively, 13.75 and 14.20 min, and for SIM quantitation, ion at m/z 210 for FA, and m/z 158 for MDA were used. The results showed that it is possible to estimate the products of lipid peroxidation in brain and to monitor the oxidative stress developed during the ischemia-reperfusion syndrome compared to the normal values.     

A specific sensitive HPLC method for determination of plasma dopamine

Summary We describe here a sensitive, selective and rapid method to quantitate plasma catecholamines, especially dopamine, using high-performance liquid chromatography with electrochemical detection. This method requires a 10-minute run time and has a threshold for detection of 2 picograms, (10pg/ml).A number of commonly employed mobile phases for catecholamine analysis have been tested and have failed to detect dopamine in biological samples. Neither acetonitrile (3–7%) or methanol, (5–8%) in the mobile phase has produced consistently interpretable data either due to inability to detect or interference from co-eluting substances. Optimal detection was achieved with a mobile phase containing sodium acetate (6.8g), citric acid (5.9g), EDTA (48mg), di-n-butylamine (270l), Na-1-octane sulfate (850mg), methanol (100 ml) (amounts refer to 1 liter aqueous solution) (pH 4.3). The mobile phase was passed through a Waters 5 resolve C₁₈ column using a Waters 590 pump and m460 electrochemical detector and 740 data module, Flow rate was 0.9ml/min. Using this method, normal values in human and swine left ventricular myocardium and human and swine plasma have been established for norepinephrine, epinephrine, and dopamine.     

High-performance liquid chromatographic determination of desmosine and isodesmosine after phenylisothiocyanate derivatization.

A new sensitive and selective high-performance liquid chromatographic method for the analysis of desmosine and isodesmosine in human and rat tissues is described. This method requires a purification step with column chromatography, followed by precolumn derivatization phenylisothiocyanate. The reaction products are then separated by isocratic chromatography on a C18 column and quantitated by ultraviolet detection at 254 nm. The recovery of standards of both compounds added to tissue samples and analysed by this method is usually greater than 90%, and the absolute detection limit is 0.5 ng for both compounds. The method is sensitive enough to measure both substances in tissue fragments of 30 mg of wet mass, which means that it can be used to study elastin in small human biopsies.     

Antioxidant Effect of Human Selenium-containing Single-chain Fv in Rat Cardiac Myocytes

Reactive oxygen species(ROS) plays a key role in human heart diseases.Glutathione peroxidase(GPX) functions as an antioxidant as it catalyzes the reduction of hydroperoxide.In order to investigate the antioxidant effect of human selenium-containing single-chain Fv(Se-scFv-B3),a new mimic of GPX,a model system of hydrogen peroxide(H2O2)-induced rat cardiac myocyte damage was established.The cardiac myocyte damage was characterized in terms of cell viability,lipid peroxidation,cell membrane integrity,and intracellular H2O2 level.The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability,the decline of malondialdehyde(MDA) production,lactate dehydrogenase(LDH) release,and intracellular H2O2 level.So Se-scFvB3 may have a great potential in the treatment of human heart diseases induced by ROS.     

Development and application of an analytical method for the determination of storage lipids, fatty acids and fatty alcohols in Calanus finmarchicus

A method was developed for the determination of the major storage lipids, wax ester and triglycerides, in the copepod Calanus finmarchicus. A variation of the Folch method was used to extract the lipid. The method was scaled down to enable the extraction of either pooled (-1 mg) or individual (approximately 200 microg) copepods. The major lipid classes were identified using TLC and quantified using HPLC coupled with evaporative light scattering detection. Analysis of laboratory reference materials indicated that this method underestimated the minor triglyceride component, but gave a good estimate of the major wax ester component. The fatty acid and fatty alcohol composition of the C. finmarchicus were determined following trans-esterification of the lipid extract in methanol. Fatty acids and fatty alcohols were initially identified by comparison with authentic standard and by mass spectroscopy. Using GC with flame ionisation detection the normalised area percentage of the fatty alcohols and fatty acid methyl esters was determined simultaneously in one run for either pooled or individual copepod samples. These methods were applied to C. finmarchicus collected from the Irminger Sea, North Atlantic in 2001 and 2002.     

Spectrophotometric determination of a thiobarbituric acid-reactive substance in human hair.

A simple and sensitive spectrophotometric method for the determination of a thiobarbituric acid-reactive substance (TBARS) in human hair has been developed. The proposed method is based on the formation of a red-colored product by the reaction of products of lipid peroxidation with thiobarbituric acid in an acidic medium. The absorbance of the resulting red product was measured at 534 nm. The linear dynamic range was between 1.0 and 20 micromol/L. The recoveries were 98.3-105.0%, and the relative standard deviations (RSD) were 0.32-1.24, respectively. TBARS in digested hair sample was stable for 3 days at room temperature. It was found that, using this method, the hair TBARS concentration in smokers (0.116 +/- 0.030 micromol/g, n = 30) was significantly higher than that in non-smokers (0.096 +/- 0.015 micromol/g, n = 30) (p < 0.05).     

Development and validation of a sensitive method for simultaneous determination of rosuvastatin and N‐desmethyl rosuvastatin in human plasma using liquid chromatography/negative electrospray ionization/tandem mass spectrometry

A sensitive and specific liquid chromatography tandem mass spectrometric method was developed and validated for the simultaneous determination of rosuvastatin (ROS) and N‐desmethyl rosuvastatin (NOR‐ROS) in human plasma using deuterium‐labeled internal standards. The plasma samples were prepared using liquid–liquid extraction with diethyl ether. Chromatographic separation was accomplished on an Xterra MS C₁₈ column. The mobile phase consisted of a gradient mixture of 15 µmol/L ammonium acetate in water and in methanol, maintained at a flow rate of 0.4 mL/min. Mass spectrometric detection was carried out in negative electrospray ionization mode and monitored by quantification and qualification transitions for each analyte. Using 300 μL plasma samples, the lower limits of quantification of ROS and NOR‐ROS were 0.05 and 0.02 µg/L respectively. The linearity of ROS and NOR‐ROS ranged from 0.05 to 42 and 0.02 to 14 µg/L respectively. The relative standard deviations of ROS and NOR‐ROS were <13 and 9%, respectively, while the deviations from expected values were within ?4.7–9.8 and ?5.2–4.6%, respectively. The present method offered high sensitivity and was successfully applied to a 24 h pharmacokinetic study of ROS and NOR‐ROS in healthy subjects receiving a single dose of 10 mg ROS. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid,sensitive, and widely applicable method for quantitative analysis of underivatized amino acids in different biological matrices by UHPLC‐MS/MS

A rapid, sensitive, and widely applicable method for the simultaneous quantitative analysis of 20 underivatized amino acids in different biological matrices, including serum, plasma, and tissue homogenates, using ultra high performance liquid chromatography with tandem mass spectrometry was developed and validated. Only 4 µL of serum, plasma, or tissue homogenate was extracted with 996 µL of solution (1.7 mM ammonium formate in 85% acetonitrile containing 0.1% formic acid) containing 100 ng/mL phenylalanine‐d5 as an internal standard without any further derivatization step. In addition, the matrix effects were small because a large volume of extraction solution was used. The total run time including reequilibration was 13 min. The results of linearity, accuracy, repeatability, precision, limits of detection, limits of quantification, and sample stability were sufficient to allow the measurement of the amino acids in different biological matrices. We conclude that our method is rapid, sensitive, and widely applicable and represents an improvement over other currently available technologies.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the estimation of nicorandil in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of nicorandil in human plasma. Nicorandil was extracted from human plasma using solid-phase extraction technique. Imipramine was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at nanogram levels. The proposed method has been validated for a linear range of 1.0-500.0 ng/mL with a correlation coefficient of > or =0.9993. The intra-run and inter-run precision and accuracy was within 10.0%. The overall recovery for nicorandil was 63.81%. The total run time was just 3.0 min.     

Development of analytical procedures to study changes in the composition of meat phospholipids caused by induced oxidation

Lipid peroxidation affects quality of meat products. The aim of this study was to develop a model system and analytical procedures for evaluating the oxidation level of meat samples, by studying the changes in meat phospholipids (PL) composition and the compounds generated by induced oxidation. Different techniques (liquid-, dry column-, accelerated solvent extraction) were investigated to identify a suitable lipid extraction system for extracting PL from bovine meat and to induce lipid oxidation by using tert-butyl hydroperoxide, 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP) or Fe(2+) and Cu(2+) salts. Accelerated solvent extraction (ASE) gave results not significantly different from the other extraction methods, but offered the advantage of being a rapid and solvent-saving procedure. The method using a silica column proved to be valid in eluting and separating the components of the phospholipidic fraction and the PL standard mixture. The analytical techniques used to analyse oxidation products of PL included GC-FID, HPLC with corona charged aerosol detector (CAD), MDA determination and the spectrophotometric measurement of peroxide levels (PxL). By means of CAD, PL were quantified and their concentration in the lipid extract was 0.98%+/-0.17 (w/w+/-SD, n=10). The oxidation method induced by ABAP proved to be fast and did not produce any artifacts. Three oxidation times were monitored (0, 90 and 180 min). The oxidation levels after 180 min correlated with a significant increase in the peroxide levels PxL (+71%), MDA (+29%) and aldehydes (+75%), whereas a decrease or even total disappearance of some unsaturated fatty acids was observed. The results obtained demonstrate that the model used in this work is useful for studying oxidation of meat phospholipids. Also, the use of the innovative detector CAD proved to be a good complementary technique in the investigation of lipids.