Simultaneous quantification of total eicosapentaenoic acid,docosahexaenoic acid and arachidonic acid in plasma by high‐performance liquid chromatography–tandem mass spectrometry

A method for the simultaneous quantification of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) in human plasma by HPLC–tandem mass spectrometry (HPLC‐MS/MS) was developed and validated. Free and esterified forms of fatty acids were hydrolysed from plasma samples in the presence of an internal standard and subjected to liquid–liquid extraction. The chromatographic run time was 3.5 min per sample. The assay was linear from 0.5 to 300 mg/L (r² > 0.997, n = 18). Based on matrix addition, accuracy deviation was <15%, except for AA at 10 mg/L (30–90%), whereas precision was <8% for all fatty acids studied. The method was applied to the measurement of these omega‐3 fatty acids in a fish oil supplement study with healthy volunteers. Healthy males (n = 4) were administered a supplement containing 465 mg EPA and 375 mg DHA per capsule (Omacor®). A dose of two capsules was given daily over a 4 week period. Pre‐treatment concentrations varied between subjects for EPA (17–68 mg/L), DHA (36–63 mg/L) and AA (121–248 mg/L). During the dosing period EPA increased 460–480% from the baseline concentration, while DHA increased 150–160%. The EPA–AA ratio increased from 0.07–0.56 to 0.3–3.1 after 4 weeks of dosing. In conclusion, the method described could be suitable for monitoring EPA, DHA and AA in clinical studies that may aid in achieving optimal concentrations of these fatty acids in patients who could be at risk of sudden cardiac death. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of docosahexaenoic acid and eicosapentaenoic acid in common seafood using ultrasonic cell crusher extraction combined with gas chromatography

An effective method for the simultaneous determination of docosahexaenoic acid and eicosapentaenoic acid in common seafood by gas chromatography was developed and validated. Total docosahexaenoic acid and eicosapentaenoic acid were extracted from seafood by ultrasonic cell crusher assisted extraction and methyl esterified for gas chromatography analysis in the presence of the internal standard. The linearity was good (r > 0.999) in 9.59 ∼ 479.5 μg/mL for docosahexaenoic acid and 9.56 ∼ 477.8 μg/mL for eicosapentaenoic acid. The intrarun and interrun precisions were both within 4.8 and 6.1% for the two analytes, while the accuracy was less than 5.8%. The developed method was applied for determination of docosahexaenoic acid and eicosapentaenoic acid in six kinds of seafood. The result showed the content of docosahexaenoic acid and eicosapentaenoic acid was all higher than 1 mg/g in yellow croaker, hairtail, venerupis philippinarum, mussel, and oyster. Our work may be helpful for dietary optimization and production of docosahexaenoic acid and eicosapentaenoic acid.     

Simultaneous determination of two anti-inflammatory drugs in serum using isopotential fluorimetry

The simultaneous determination of 6-methoxy-2-naphthylacetic acid (6MNA) and diflunisal in serum samples using the combination of matrix isopotential synchronous fluorescence (MISF) and first derivative technique is proposed. 6MNA and diflunisal exhibit overlapped spectra and serum produces background fluorescence that precludes the direct determination of these anti-inflammatory drugs by conventional fluorimetry. This method provides good analytical results for determination of compounds in samples with unknown background fluorescence. The method was applied for the simultaneous determination of 6MNA and diflunisal in serum samples at concentrations between 20-200 and 100-1000 ng mL⁻¹, respectively, by means of absolute values of first derivative of synchronous scan at 247.9/364.0 and 262.6/392.4 nm for 6MNA and diflunisal, respectively. In order to obtain maximum sensitivity and adequate selectivity, factors affecting fluorescence intensity were studied. As a result, the analyses were performed in water at a pH of 7.2, adjusted by using sodium dihydrogen phosphate/hydrogen phosphate (0.1 M) as a buffer solution. Serum samples were diluted 200 times. Analytical parameters of the proposed method were calculated according to the error propagation theory. The limit of detection calculated according to Clayton was 15.8 and 63.0 ng mL⁻¹ for 6MNA and diflunisal, respectively. The sensitivity, repeatability and reproducibility achieved with the proposed method were adequate for the determination of these anti-inflammatory agents in serum samples.     

在重组痘苗病毒中共表达HIV-1p24gag与hIL-6基因

以ＨＩＶ－１ ｐ２４ｇａｇ 与ｈＩＬ－６ 基因在重组痘苗病毒中的共表达研究为目的,将痘苗病毒复制非必需区血凝素（ＨＡ） 基因作为侧翼,把编码人白细胞介素６（ｈＩＬ－６）的基因片段克隆到真核表达质粒ｐ１６ＱＦ１ －２４ 的下游,构建成含有ＨＩＶ－ １ｐ２４ｇａｇ 和ｈＩＬ－６ 两种外源基因的重组表达质粒ｐ１６ＱＦ２４ －ＩＬ６ ,经同源重组和血凝素阴性空斑筛选,获得了重组痘苗病毒ｖ１６ＱＦ２４ －ＩＬ６．经间接免疫荧光试验、Ｄｏｔ－ ＥＬＩＳＡ 和Ｗｅｓｔｅｒｎ ｂｏｌｔ 等检测证明,重组病毒能同时表达ｐ２４ｇａｇ 蛋白和ＩＬ－６ 蛋白,表达产物的相对分子质量分别为２－４×１０４ ,２－６×１０４ ．此种方式表达的外源蛋白持续释放,可产生更强的免疫效果,可望用于ＨＩＶ疫苗的研究．     

LC-APCI-MS/MS assay for quantitation of ethyl esters of eicosapentaenoic acid and docosahexaenoic acid in human plasma and its application in a pharmacokinetic study

A simple and specific LC–MS/MS method was developed and validated for the determination of ethyl ester of eicosapentaenoic acid (EPAEE) and ethyl ester of docosahexaenoic acid (DHAEE). After deproteinized with acetonitrile, the plasma samples were separated on a C18 column using a gradient elution system consisted of methanol and 1.0 mM ammonium acetate in water. The detection used an atmospheric-pressure chemical ionization ion source in positive mode with multiple reaction monitoring for the quantitation of EPAEE and DHAEE. The acceptable linearity was achieved over the concentration ranges of 1.00~1000 ng/mL for EPAEE and 2.50~2500 ng/mL for DHAEE. The method was successfully applied to a pharmacokinetic study of EPAEE and DHAEE in healthy Chinese volunteers after the oral administration of 4 g omega-3-acid ethyl esters 90 soft capsule. The pharmacokinetic profiles of EPAEE and DHAEE were observed for the first time in Chinese volunteers, which reached a maximum concentration of 499 ± 243 ng/mL and 1596 ± 476 ng/mL for EPAEE and DHAEE, respectively. The areas under the plasma concentration–time curve were 1290 ± 765 ng/mL·h for EPAEE and 4369 ± 1680 ng/mL·h for DHAEE, respectively.     

高效液相色谱法分离纯化微生物油脂中的花生四烯酸

为满足花生四烯酸样品制备的需要,将微生物油脂中制得的脂肪酸先经尿素包合(脲包法)和甲酯化后,再利用反相高效液相色谱法分离纯化其中的花生四烯酸。在C18反相柱上,以甲醇-水(体积比为95∶5)为流动相,采用示差折光检测器检测,流速为5 mL/min时,目标产物的分离度较佳。用气相色谱/质谱法(GC/MS)进行分析的结果表明,花生四烯酸甲酯在纯化后的样品中的质量分数由38%提高到99%以上,基本达到标准物质的质量要求。     

Deciphering the Spectral Tuning Mechanism in Proteorhodopsin: The Dominant Role of Electrostatics Instead of Chromophore Geometry

Proteorhodopsin (PR) is a photoactive proton pump found in marine bacteria. There are two phenotypes of PR exhibiting an environmental adaptation to the ocean's depth which tunes their maximum absorption: blue-absorbing proteorhodopsin (BPR) and green-absorbing proteorhodopsin (GPR). This blue/green color-shift is controlled by a glutamine to leucine substitution at position 105 which accounts for a 20 nm shift. Typically, spectral tuning in rhodopsins is rationalized by the external point charge model but the Q105L mutation is charge neutral. To study this tuning mechanism, we employed the hybrid QM/MM method with sampling from molecular dynamics. Our results reveal that the positive partial charge of glutamine near the C₁₄−C₁₅ bond of retinal shortens the effective conjugation length of the chromophore compared to the leucine residue. The derived mechanism can be applied to explain the color regulation in other retinal proteins and can serve as a guideline for rational design of spectral shifts.     

衍生化-气相色谱-三重四极杆质谱法同时测定粮食作物中三氯甲基吡啶及其代谢物残留

建立了粮食作物中三氯甲基吡啶及其代谢物6-氯吡啶甲酸的衍生化-气相色谱-三重四极杆质谱(GC-MS/MS)检测方法。选取高粱、小麦、玉米及爆谷等典型的粮食作物,采用酸性乙腈提取,以浓硫酸作为三氯甲基吡啶代谢物的衍生化试剂,优化了衍生化反应的最佳反应条件,并使用GC-MS/MS分析,以内标法对三氯甲基吡啶及其代谢物进行定量检测。结果表明,在0.025～0.4 mg/L范围内,线性关系良好,相关系数(r²)均大于0.995,在3个不同添加水平下的平均回收率在80.4%～98.4%之间,相对标准偏差(RSD)在1.0%～10.1%之间。该方法操作简单、高效,灵敏度高,抗干扰能力强,回收率和重复性良好,能够满足粮食作物中三氯甲基吡啶及其代谢产物残留量的检测要求。     

Simultaneous analysis of naltrexone and its major metabolite, 6-β-naltrexol, in human plasma using liquid chromatography-tandem mass spectrometry: Application to a parent-metabolite kinetic model in humans

We developed a method for simultaneously determining naltrexone, an opioid antagonist, and its major metabolite (6-β-naltrexol) in plasma using LC/MS/MS. Three compounds, and naloxone as an internal standard, were extracted from plasma using a mixture of methyl-tertiary-butyl ether. After drying the organic layer, the residue was reconstituted in a mobile phase (0.1% formic acid-acetonitrile:0.1% formic acid buffer, 95:5, v/v) and injected onto a reversed-phase C₁₈ column. The isocratic mobile phase was eluted at 0.2 ml/min. The ion transitions monitored in multiple reaction-monitoring modes were m/z 342 → 324, 344 → 326, and 328 → 310 for naltrexone, 6-β-naltrexol, and naloxone, respectively. The coefficient of variation of the assay precision was less than 11.520%, and the accuracy exceeded 93.465%. The limit of quantification was 2 ng/ml for naltrexone and 7.2 ng/ml for 6-β-naltrexol. And the limit of detection was 0.1 ng/ml for naltrexone and 0.36 ng/ml for 6-β-naltrexol. This method was used to measure the plasma concentration of naltrexone and 6-β-naltrexol in healthy subjects after a single oral 50 mg dose of naltrexone. This analytical method is a simple, sensitive, and accurate way of determining the pharmacokinetic profiles of naltrexone and its metabolites. The pharmacokinetic parameters were analyzed using both non-compartmental analysis performed for each subject according to standard methods and compartmental analysis with a parent-metabolite pharmacokinetic model that was fitted to the data, simultaneously, using the program ADAPT II. The tested parent-metabolite pharmacokinetic model successfully described the relationship between the plasma concentration of naltrexone and one of its major metabolites, 6-β-naltrexol.     

12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid suppresses UV-induced IL-6 synthesis in keratinocytes, exerting an anti-inflammatory activity

12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid (12- HHT) is an enzymatic product of prostaglandin H(2) (PGH(2)) derived from cyclooxygenase (COX)-mediated arachidonic acid metabolism. Despite the high level of 12-HHT present in tissues and bodily fluids, its precise function remains largely unknown. In this study, we found that 12-HHT treatment in HaCaT cells remarkably down-regulated the ultraviolet B (UVB) irradiation-induced synthesis of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cutaneous inflammation. In an approach to identify the down-stream signaling mechanism by which 12-HHT down-regulates UVB-induced IL-6 synthesis in keratinocytes, we observed that 12-HHT inhibits the UVB-stimulated activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB). In addition, we found that 12-HHT markedly up-regulates MAPK phosphatase-1 (MKP-1), a critical negative regulator of p38 MAPK. When MKP-1 was suppressed by siRNA knock-down, the 12-HHT-mediated inhibitory effects on the UVB-stimulated activation of p38 MAPK and NF-κB, as well as the production of IL-6, were attenuated in HaCaT cells. Taken together, our results suggest that 12-HHT exerts anti-inflammatory effect via up-regulation of MKP-1, which negatively regulates p38 MAPK and NF-κB, thus attenuating IL-6 production in UVB-irradiated HaCaT cells. Considering the critical role of IL-6 in cutaneous inflammation, our findings provide the basis for the application of 12-HHT as a potential anti-inflammatory therapeutic agent in UV-induced skin diseases.     

Role of edge-to-face interaction between aromatic rings in clathrate formation of 1-benzoyl-2-hydroxyindoline derivatives with benzene. X-ray crystal and PM6 analyses of the interaction

(4-Nitrophenyl- and 4-chlorophenyl)(2-hydroxy-3,3-dimethylindolin-1-yl)methanone (4a,b) serve as clathrate hosts for benzene guests. X-ray crystal analyses of the inclusion compounds of 4a and 4b with benzene indicate that the ‘edge-to-face interaction’ plays an important role in the formation of the inclusion complexes with benzene as well as in the host-host interactions. PM6 molecular orbital calculations were found to reproduce the characteristic structural features of both intra- and intermolecular edge-to-face interactions.     

Potential Application of Gambogic Acid for Retarding Renal Cyst Progression in Polycystic Kidney Disease

Abnormal cell proliferation and accumulation of fluid-filled cysts along the nephrons in polycystic kidney disease (PKD) could lead to a decline in renal function and eventual end-stage renal disease (ESRD). Gambogic acid (GA), a xanthone compound extracted from the brownish resin of the Garcinia hanburyi tree, exhibits various pharmacological properties, including anti-inflammation, antioxidant, anti-proliferation, and anti-cancer activity. However, its effect on inhibiting cell proliferation in PKD is still unknown. This study aimed to determine the pharmacological effects and detailed mechanisms of GA in slowing an in vitro cyst growth model of PKD. The results showed that GA (0.25–2.5 μM) significantly retarded MDCK cyst growth and cyst formation in a dose-dependent manner, without cytotoxicity. Using the BrdU cell proliferation assay, it was found that GA (0.5–2.5 μM) suppressed MDCK and Pkd1 mutant cell proliferation. In addition, GA (0.5–2.5 μM) strongly inhibited phosphorylation of ERK1/2 and S6K expression and upregulated the activation of phosphorylation of AMPK, both in MDCK cells and Pkd1 mutant cells. Taken together, these findings suggested that GA could retard MDCK cyst enlargement, at least in part by inhibiting the cell proliferation pathway. GA could be a natural plant-based drug candidate for ADPKD intervention.     

毛细管气相色谱法测定油田水中短链有机酸

The evaporation of water phase has been used to remove a vast amount of inorganic salts and interferingorganic components. The distillate concentrated by rotary evaporator was directly analyzed by means of wide-bore capillary GC column, The coefficients of variation for C_2～C_5 organic acids were 1.9～6.4%. The recov-eries of the acids in the oilfield water were 81.0～106%.     

环丁酮光化学反应中S1/T1/S0交叉点的重要作用

势能面间的交叉在光化学反应中起着重要的作用 ,是由激发态反应物到基态产物发生无辐射跃迁的机制 .在本文中 ,我们用 CASSCF和态平均 CASSCF方法分别对环丁酮光化学反应的势能剖面及 S1,T1和 S0三个势能面间交叉进行了研究 .结果发现 ,基态和三态产物的形成是通过 S1,T1和 S0三个势能面交叉于同一区域 (称为 S1/T1/S0交叉点 )这一有效途径完成的 .　　60年代末 ,实验 [1－ 9]发现环丁酮和其它烷基酮 ,如丙酮、环戊酮的光化学反应机理很不一致 .主要体现在 ,i)环丁酮 (n,π态 )的α解离发生在 S1态势能面上 ,而其它烷基酮 (n,π态 …     

特种液压油添加剂的GC-MS定量分析

摘要：采用选择离子监测（SIM）模式作为气相色谱/质谱法（GC/MS）检测方式测定了某超高压液压油中两种添加剂2,6-二叔丁基对甲酚的质量比和磷酸三苯酯的质量比。测得的校正因子F值和添加剂质量比均有较高的精确度。还对SIM模式中有关离子的选择作了简单的探讨。     

榴莲中脂肪酸成分的色谱-质谱分析

报道了用色谱－质谱分析榴莲中的脂肪酸及其含量。结果表明榴莲含有丰富的对人体有益的脂肪酸,是一种具有较高食用价值和药用价值的水果。     

毛细管气相色谱法分析虾蛄肉中的脂肪酸

以快速脂肪提取器分别提取雄、雌虾蛄肉中脂肪,利用５０ｍ×０．３５ｍｍｉ．ｄ．ＰＥＧ－２０Ｍ玻璃毛细管柱分离测定了雄、雌虾蛄肉中的脂肪酸,结合气相色谱／质谱法,鉴定出４５个峰,分别占脂肪酸总量的９９．２％和９８．０％,其中不饱和脂肪酸分别为７４．７％和７１．５％。     

Dried and Fermented Powders of Edible Algae (Neopyropia yezoensis) Attenuate Hepatic Steatosis in Obese Mice

Edible algae Neopyropia yezoensis is used as “Nori”, its dried sheet product, in Japanese cuisine. Its lipid components reportedly improve hepatic steatosis in obese db/db mice. In this study, we prepared “Nori powder (NP)” and “fermented Nori powder (FNP)” to utilize the functional lipids contained in “Nori” and examined their nutraceutical effects in vivo. Male db/db mice were fed a basal AIN-76 diet, a 10% NP-supplemented diet, or a 10% FNP-supplemented diet for 4 weeks. We detected eicosapentaenoic acid (EPA) present in both NP and FNP in the serum and liver of db/db mice in a dose-dependent manner. The NP diet reduced hepatic triglyceride accumulation (by 58%) in db/db mice by modulating gene expression, which resulted in the inhibition of lipogenic enzyme activity. Additionally, NP intake significantly suppressed the expression of inflammatory genes in the liver and hepatic injury marker levels in the sera (by 26%) of db/db mice. The FNP diet also led to a marked reduction in hepatic triglyceride accumulation (by 50%) and hepatic injury (by 28%) in db/db mice, and the mechanism of these alleviative actions was similar to that of the NP diet. Although the EPA content of FNP was one-third that of NP, metabolomic analysis revealed that bioactive betaine analogs, such as stachydrine, betaine, and carnitine, were detected only in FNP. In conclusion, we suggest that (1) mechanical processing of “Nori” makes its lipid components readily absorbable by the body to exert their lipid-lowering effects, and (2) fermentation of “Nori” produces anti-inflammatory molecules and lipid-lowering molecules, which together with the lipid components, can exert hepatic steatosis-alleviating effects.     

1－己烯羰化制庚醛过程中的气相色谱法分析

用交联ＳＥ－５４ＳＣＯＴ玻璃毛细管柱分离了１－己烯羰化制庚醛过程的原料、产品及杂质，并采用气相色谱－质谱（ＧＣ－ＭＳ）技术和纯样对照法鉴定组分。与填充柱定量结果对比表明，毛细管气相色谱法简便、准确，具有实用价值。