液相色谱-电化学检测法测定小鼠血浆和组织线粒体中的辅酶Q和维生素E

 建立了同时测定氧化型和还原型辅酶Q以及维生素E的液相色谱 电化学检测方法。样品中氧化型和还原型辅酶Q9和Q10 以及维生素E混合物经过液相色谱分离柱分离 ,在 - 5 5 0mV的电化学调节池中将氧化型辅酶Q还原为还原型 ,再经过 15 0mV分析池将样品中原有的还原型辅酶Q和经过调节池还原的辅酶Q以及维生素E一同氧化。该方法用于小鼠组织线粒体和血浆样品中氧化型和还原型辅酶Q9和Q10 以及维生素E的同时检测 ,灵敏度高 ,选择性好 ,结果令人满意。     

Fluorescence studies of the interactions of ubiquinol-10 with liposomes

Ubiquinone-10 plays a central role in energy production and its reduced form, ubiquinol-10 is also capable of acting as a potent radical scavenging antioxidant against membrane lipid peroxidation. Efficiency of this protection depends mostly on its localization in lipid bilayer. The intrinsic fluorescence of ubiquinol-10 and of the exogenous probe, Laurdan, has been used to determine the location of ubiquinol-10 in unilamellar liposomes of egg phosphatidylcholine (EggPC) and dimyristoyl phosphatidylcholine. Laurdan fluorescence moiety is positioned at the hydrophilic-hydrophobic interface of the phospholipid bilayer and its parameters reflect the membrane polarity and microheterogeneity, which we have used to explore the coexistence of microdomains with distinct physical properties. In liquid-crystalline bilayers ubiquinol has a short fluorescence lifetime (0.4 ns) and a high steady-state anisotropy. In a concentration-dependent manner, ubiquinol-10 influences the Laurdan excitation, emission and generalized polarization measurements. In EggPC liposomes ubiquinol-10 induces a decrease in membrane water mobility near the probe, while in dimyristoyl liposomes a decrease in the membrane water content was found. Moreover the presence of ubiquinol results in the formation of coexisting phospholipid domains of gel and liquid-crystalline phases. The results indicate that ubiquinol-10 molecules are mainly located at the polar-lipid interface, inducing changes in the physico-chemical properties of the bilayer microenvironment.     

Validation and application of an HPLC-EC method for analysis of coenzyme Q10 in blood platelets

Previous studies have indicated that analysis of coenzyme Q10 (CoQ10) in platelets may be clinically useful. The study objectives are to describe, validate and provide application of an HPLC-EC method for platelet CoQ10 analysis. This method analyzes oxidized (ubiquinone-10) and reduced (ubiquinol-10) forms of CoQ10 using two separate injections with the electrochemical analytical cell set at neutral and oxidizing potentials. Results showed that chromatograms were free of interfering peaks. Calibration curves were constructed over a concentration range 116-2317 nmol/L (r(2) = 0.99). The extraction recovery was >95%. The within-run precision CV% was < or =4.2%, and the day-to-day precision was < or =9.9%. Platelets were isolated by differential centrifugation, and frozen at -70 degrees C until analysis. The application of the method was used to compare accumulation of CoQ10 in platelets vs plasma in eight adult volunteers during a 28 day supplementation period (5 mg/kg/day of ubiquinol-10). Mean platelet total CoQ10 was 164 pmol/10(9) cells, and ubiquinol-10:total CoQ10 ratio was 0.56. During supplementation platelet CoQ10 levels were more consistent and predictable than plasma CoQ10 levels. The results indicate that this validated method for platelet ubiquinol-10 and ubiquinone-10 analysis is acceptable for use in the clinical laboratory, and that platelet CoQ10 may have important advantages over plasma during CoQ10 supplementation.     

Stability of ubiquinol‐10 (reduced form of coenzyme Q10) in human blood

The ratio of ubiquinol‐10 in total coenzyme Q₁₀ (TQ₁₀) in human plasma has been proposed as a useful biomarker of oxidative stress. Since ubiquinol‐10 is easily oxidized in air, it is necessary to perform suitable processing at medical institutions prior to analysis. To establish stable storage conditions for blood to determine the ubiquinol‐10/TQ₁₀ ratios properly, the effects of temperature conditions on the stability of ubiquinol‐10 were studied. Blood samples were collected from nine male Japanese volunteers. Changes in ubiquinol‐10/TQ₁₀ ratios in blood samples were evaluated under three temperature conditions (room temperature, refrigerated and ice‐cooled). Plasma levels of ubiquinol‐10 and ubiquinone‐10 were determined by an HPLC system with electrochemical detection and the ubiquinol‐10/TQ₁₀ ratios were calculated. We found that the ubiquinol‐10/TQ₁₀ ratio was stable up to 8 or 4 h when blood samples were stored in refrigerator or ice‐cold container, respectively, and its decreases during these periods were <1.0%. We conclude that, in order to evaluate ubiquinol‐10/TQ₁₀ ratios, blood samples should be stored in a refrigerator or an ice‐cold container, and processed for plasma separation within 4 h. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of cocaine in human urine, plasma and red blood cells by gas-liquid chromatography

This paper describes a sensitive and reliable method for the determination of cocaine in human urine, plasma and red blood cells. Cocaine is extracted into cyclohexane from the biological materials at slightly alkaline pH, reduced with lithium aluminium hydride, acylated with pentafluoropropionic anhydride and detected by an electron capture detector. When compared with a gas chromatography-mass spectrometry method the results of cocaine determination correlated highly (r = 0.986). When cocaine was given intravenously to volunteer subjects only 0.2-1.4% of the administered dose was excreted as unmetabolized cocaine in the first 9 h after administration. Plasma and red blood cell levels of cocaine were also determined by this method after intravenous administration.     

High-performance liquid chromatography-fluorometry for the determination of thiols in biological samples using N-[4-(6-dimethylamino-2-benzofuranyl) phenyl]-maleimide

The selective determination of thiols in biological samples was investigated by high-performance liquid chromatography using N-[4-(6-dimethylamino-2-benzofuranyl)phenyl] maleimide, which was found to give fluorescent products when treated with certain thiols. Six kinds of thiol (reduced glutathione, cysteine, N-acetylcysteine, cysteamine, homocysteine and coenzyme A) could be separated simultaneously within ca. 12 min and determined at final level of sensitivity. The method was successfully applied to the determination of thiols in rat tissues and plasma and in human normal serum.     

Determination of 2,3-dihydro-6-[3-(2-hydroxymethyl)phenyl-2-propenyl]-5-benzofuranol in plasma using liquid chromatography with electrochemical detection

A reversed-phase column liquid chromatographic (LC) method with electrochemical detection (ED) is described for the quantification of 2,3-dihydro-6-[3-(2-hydroxymethyl)phenyl-2-propenyl]-5-benzofuranol (compound 1), a new locally active dual inhibitor of leukotriene and prostaglandin synthesis, in plasma. After a single liquid-liquid extraction of the biological specimen, the extract was analyzed using a liquid chromatograph with an amperometric detector set at an oxidation potential of +0.55 V. The resulting chromatograms are free from endogenous interference and the limit of detection is 0.2 ng/ml. Several other analogous dihydrobenzofuranols were shown to be electrochemically active, permitting their determination using LC with ED. The described analytical method has been fully validated in the concentration range 0.5-20 ng/ml of plasma and utilized in the analysis of plasma samples from human clinical studies. The analytical methodology has also been adapted for analysis of compound 1 in human skin blister fluid after topical administration of 1.     

Evaluation of a sensitive HPLC method for the determination of Malondialdehyde, and application of the method to different biological materials

Summary Reactive oxygen species (ROS), important mediators of cell and tissue injury during inflammation, are produced by several types of inflammatory cells. The formation of ROS can be monitored by detection of lipid peroxidation products. The extremely broad spectrum of biological effects of aldehydic lipid peroxidation products has necessitated the development of a technique that enables the sensitive routine quantitation of aldehydes formed in biological materials. Malondialdehyde (MDA) is a by-product of enzymatic eicosanoid formation and an end-product of nonenzymatic peroxidation of polyunsaturated fatty acids with three or more bisallylic double bonds. The determination of the thiobarbituric acid derivative of MDA (TBA-MDA) is a widely used method for estimating overall lipid peroxidation. We describe a rapid, isocratic, simple, and sensitive high-performance liquid chromatographic (HPLC) method with spectrofluorimetric detection for measurement of MDA-TBA in human biological samples such as plasma, urine, wound secretions, amniotic fluid, sputum and tissue samples. By use of this method, picomole quantities of MDA can be readily and specifically detected in different biological materials. Coefficients of variation of repeated MDA-TBA assays were 4.4% within run and 6.9% from run to run. Reference values are given for a variety of human body fluids and for rat tissues.     

Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid chromatography-mass spectrometry

A sensitive and simultaneous liquid chromatographic-mass spectrometric (LC/MS) method for the determination of current four HIV protease inhibitors (PIs), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV) and amprenavir (APV) in rat plasma and liver dialysate by a microdialysis method was described. An isocratic LC/MS method in combination with atmospheric pressure chemical ionization was developed for the determination of these four PIs in biological samples in the same run. The analytes including an internal standard were extracted from 100 microL of plasma or 150 microL of liver dialysate samples by salting-out with 100 microL of ice-cold 2 M K(3)PO(4) followed by ether extraction. The separation of analytes was carried out on a reversed-phase semi-micro column using 50% of acetonitrile containing 1% acetic acid as mobile phase at a flow rate of 0.2mL/min(-1). The separation was completed within 5 min. Precision, recovery and limits of detection indicated that the method was suitable for the quantitative determination of these PIs in rat plasma or liver dialysate. This simple, sensitive and highly specific LC/MS method is suitable for pharmacokinetic studies and therapeutic drug monitoring in AIDS patients who receive double protease therapy.     

新型纳米纤维固相萃取柱制备及其用于组织样品中柔红霉素测定研究

研制出一种新型纳米纤维固相萃取柱,并将其用于大鼠心、肝、脾、肺、肾组织及血浆中柔红霉素的净化、浓缩处理,建立了生物样品中柔红霉素的高效液相荧光检测法.以10%(V/V)HClO4为溶剂,对组织样进行匀浆和离心处理,上清液用纳米纤维小柱净化富集,以50 μL含1%(V/V)冰醋酸的甲醇洗脱,进样20μL检测.流动相为甲醇...     

Development and validation of a single HPLC method for determination of α‐tocopherol in cell culture and in human or mouse biological samples

A straightforward and common analytical method for α‐tocopherol (αT) determination in various biological samples, including plasma, red blood cells (RBC), tissues and cultured cell lines, was developed and validated, using a reverse phase‐chromatographic method (RP‐HPLC). Even though many chromatographic methods for αT determination have been reported, most of them require readjustment when applied to different types of samples. Thus, an effective and simple method for αT determination in different biological matrices is still necessary, specifically for translational research. This method was applied using a C₁₈ column (250 × 4.6 mm, 5 µm particle size) under isocratic elution with MeOH:ACN:H₂O (90:9:1 v/v/v) at a flow rate of 1 mL/min and detected using photodiode array at 293 nm. Linearity (r >0.9997) was observed for standard calibration with inter‐ and intraday variation of standard <4%. Lower limits of detection and quantification for αT in this assay were 0.091 and 0.305 µg/mL respectively. Validation proved the method to be selective, linear, accurate and precise. The method was successfully applied in great variety of biological samples, that is, human and mouse plasma, RBCs, murine tissues and human/mouse/rat cultured cell lines. More importantly, a single protocol of extraction and detection can be applied, making this method very convenient for standardization of different types of samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Supercritical fluid extraction and ultra performance liquid chromatography of respiratory quinones for microbial community analysis in environmental and biological samples

Microbial community structure plays a significant role in environmental assessment and animal health management. The development of a superior analytical strategy for the characterization of microbial community structure is an ongoing challenge. In this study, we developed an effective supercritical fluid extraction (SFE) and ultra performance liquid chromatography (UPLC) method for the analysis of bacterial respiratory quinones (RQ) in environmental and biological samples. RQ profile analysis is one of the most widely used culture-independent tools for characterizing microbial community structure. A UPLC equipped with a photo diode array (PDA) detector was successfully applied to the simultaneous determination of ubiquinones (UQ) and menaquinones (MK) without tedious pretreatment. Supercritical carbon dioxide (scCO(2)) extraction with the solid-phase cartridge trap proved to be a more effective and rapid method for extracting respiratory quinones, compared to a conventional organic solvent extraction method. This methodology leads to a successful analytical procedure that involves a significant reduction in the complexity and sample preparation time. Application of the optimized methodology to characterize microbial communities based on the RQ profile was demonstrated for a variety of environmental samples (activated sludge, digested sludge, and compost) and biological samples (swine and Japanese quail feces).     

Determination of aluminum in high fat content biological materials using emulsions and direct current plasma

A new sample dissolution method employing an emulsion has been developed and evaluated for the determination of aluminum (Al) in high fat content biological samples. An emulsion is used to disperse the residual fat left over from nitric acid digestion to form a stable homogeneous moiety before aspiration into the direct current plasma (DCP) source. The method eliminates the need to filter the fat or extract fat soluble Al species from the undigested fat before analysis. Comparisons were made between the present method and two conventional sample dissolution methods for the determination of Al present in spiked and certified biological samples. The results indicated that the emulsification procedure offers simplicity, speed and improved accuracy/precision for the DCP determination of Al, especially for biological materials containing high amounts of fat and fat soluble Al complex(es).     

High-performance liquid chromatography of coenzyme Q-related compounds and its application to biological materials

A convenient and precise method for the separation and determination of coenzyme Q (CoQ)-related compounds (CoQ homologues, plastoquinone-9, ubichromenol-9, etc.) was developed using high-performance liquid chromatography (HPLC). All compounds tested were separated using a reverse-phase column with a suitable mobile phase and detected at a wavelength of 275 nm. CoQ extracts in plasma and erythrocytes were purified by thin-layer chromatography prior to HPLC analysis, but such purification was not necessary when determining CoQ in urine and tissues. Hydroquinone forms of CoQ existing in animal tissues were oxidized to the corresponding quinone forms with potassium hexacyanoferrate(III). This HPLC method was applied satisfactorily to the determination of the contents of CoQ homologues in human and animal samples. CoQ10 was the only homologue detected in human samples, and CoQ8, CoQ9 and CoQ10 were native homologues of CoQ in rat tissues. Ubichromenol-9 and plastoquinone-9 were not detected in these samples.     

Quantitation of agmatine by liquid chromatography with laser-induced fluorescence detection

A high performance liquid chromatography (HPLC) method is described for the determination of agmatine, an endogenous neuromodulator. The method involves pre-column derivatization of the sample with a fluorescent tagging reagent, 7-fluoro-4-nitrobenzoxadiazole (NBD-F). The resulting agmatine derivative is stable and can be readily extracted into ethyl acetate at pH 8.5. The extraction enhances the quantification of low level agmatine because it eliminates chromatographic peaks caused by endogenous amino acids. The HPLC separation is carried out on a C₈ reversed phase column and completed in less than 10 min. With laser-induced fluorescence (LIF) detection, the detection limit is 5×10⁻⁹ M agmatine. Method precision (coefficient of variation) is 5% for agmatine in human plasma at the sub-μM level. This method has been validated by determination of agmatine in biological samples including human plasma and rat brain and stomach tissues.     

Total iodine in nutritional and biological reference materials using neutron activation analysis and inductively coupled plasma mass spectrometry

In a recent collaborative study intended to extend the variety of reference materials certified for iodine, three mineralization methods were developed to quantify the total iodine content of biological and nutritional materials by inductively coupled plasma mass spectrometry (ICP-MS). A mixture of water-soluble tertiary amines was used as the matrix solution for two oxygen combustion methods and for a simple extraction at room temperature. Calibrations with matrix-matched standards, internal calibration, and isotope dilution with ¹²⁹I were used. Good agreement between neutron activation analysis (NAA) and the two combustion methods was observed except for < 0.1 mg kg^–1 iodine concentrations. The amine extraction method gave the most reliable results for the mixed diet, milk powder, and infant formula samples but low recoveries for other biological materials owing to an incomplete extraction and solubilization of iodine. The NAA method, with its freedom from reagent blank, is a useful technique for the independent determination of iodine in biological, environmental and food matrices, especially for verification of iodine results obtained by ICP-MS.     

同位素稀释-气相色谱-质谱法检测大鼠血浆中的内源性胍丁胺

建立了大鼠血浆中内源性胍丁胺的同位素稀释-气相色谱-负化学电离质谱定量分析方法。大鼠血浆样品经蛋白沉淀并蒸干后,用六氟乙酰丙酮衍生化,采用Florisil固相萃取柱净化,以稳定同位素标记的d₈-胍丁胺为内标,在气相色谱-质谱仪上采用负化学电离方式电离,选择离子模式检测。胍丁胺标准溶液的检出限为0.0057 ng/mL。血浆中添加的胍丁胺在1.14~57.0 ng/mL范围内呈良好的线性关系（r=0.997）,方法回收率介于92.3%~109.8%之间,日内和日间精密度均小于15%。大鼠血浆中胍丁胺平均含量水平为（22±9）ng/mL,雌、雄大鼠血浆中的胍丁胺水平未见显著性差异（p>0.05）。该方法特异性好、灵敏度高,为生物体内胍丁胺的生理功能研究提供了高灵敏的分析方法。     

High performance liquid chromatography-mass spectrometry analysis for rat metabolism and pharmacokinetic studies of lithospermic acid B from danshen

Metabolism and pharmacokinetic studies on rat were conducted for lithospermic acid B, one of the components from Radix Salviae Miltiorrhizae (danshen) that shows many bioactivities. Liquid chromatography-electrospray ionization mass spectrometry method was applied for the determination of lithospermic acid B and its metabolites in samples from in vitro and in vivo metabolism studies. Rat plasma samples collected after intravenous administration were analyzed for obtaining pharmacokinetic data of lithospermic acid B. Four O-methylated metabolites, namely one monomethyl-, two dimethyl- and one trimethyl-lithospermic acid B, were detected when lithospermic acid B was incubated in rat hepatic cytosol. These four metabolites were also detected in rat bile, plasma and feces samples after intravenous administration of lithospermic acid B. The in vitro and in vivo results indicate that the methylation is the main metabolic pathway of lithospermic acid B. The danshen component and its methylated metabolites were excreted to rat bile and feces.     

Simultaneous measurement of tocopherols and tocopheryl quinones in tissue fractions using high-performance liquid chromatography with redox-cycling electrochemical detection

Tocopherols and tocopheryl quinones in lipid extracts of biological samples have primarily been measured using relatively insensitive ultraviolet detection methods. Oxidative electrochemical detection increases both the sensitivity and selectivity when measuring the tocopherols. We have developed an electrochemical detection system which sequentially reduces and oxidizes tocopheryl metabolites eluted from a reversed-phase high-performance liquid chromatography column, achieving sensitivities of about 0.05 pmol for both the tocopherols and their quinones. Using a rapid and mild extraction procedure, endogenous levels of alpha- and gamma-tocopherol as well as their respective quinones were measured in homogenates of chicken liver and muscle, and in dilute preparations of rat liver microsomes. The principle of the detection system could be applied to the determination of tocopheryl dihydroquinones, ubiquinols and ubiquinones with slight modifications to the mobile phase buffer and the electrode potentials of the detector.     

Determination of the enantiomeric purity of amphetamine after derivatization with Sanger’s reagent (2,4-dinitrofluorobenzene [DNFB]) by simultaneous dual circular dichroism and ultraviolet spectroscopy

Determination of Se in biological materials was attempted by microwave-induced plasma mass spectrometry (MIP-MS). (1) Serum samples were available after 10 times dilution with 0.5% nitric acid solution containing 0.1% Triton X-100. When oxygen gas was inserted into the plasma gas (nitrogen) in order to improve the combustion, the sensitivity was reduced to 45%. The detection limit of this method was 0.5 ng/mL. (2) Standard reference materials on commercial base were used to evaluate the accuracy of the Se determination by MIP-MS after microwave digestion. In samples like bovine liver and human hair with Se concentrations of more than 0.7 μg/g, the standard curve method after internal standard (IS) correction was acceptable. This procedure was unsuitable for samples with low Se concentrations such as milk powder (certified value of Se 0.11 μg/g), or plant leaf samples. (3) Instead of IS correction, the peak height of the spectrum was used for calculations from the matrix matched calibration curve. The results of all materials were close to the certified values, even at 25 ng/g. The detection limit of the MIP-MS with microwave digestion and IS correction was 0.05 ng/mL in standard solutions. The detection limit of the peak height method was 0.1 ng/mL and was estimated to be < 20 ng/g in plant materials. Received: 25 September 1998 / Revised: 15 February 1999 / Accepted: 18 February 1999