We report on a highly sensitive electrochemical immunoassay for the serum inflammation marker amyloid A (SAA). It is making use of a glassy carbon electrode that was modified with carboxy-endcapped polypyrrole (PPy-α-COOH), multiwalled carbon nanotubes (MWCNTs), ionic liquid and chitosan acting as the support platform. The nanocomposite increases the sensitivity and stability of the assay. Antibody against SAA was immobilized on a monolayer surface consisting of PPy-α-COOH. The electrode material was characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectra, cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry. The calibration plot for this assay, when operated at 0.16 V (vs. SCE) and applied to spiked serum samples, is linear in the 0.001 to 900 ng mL−1 SAA concentration range, and the detection limit is as low as 0.3 pg mL−1 (at an S/N ratio of 3). The electrode is stable and highly sensitive. The detection scheme is likely to be applicable to numerous other kinds of immunoassays.

In this paper, we presented a novel electrochemical immunosensor using gold nanoparticles (AuNPs) loaded on the Prussian blue (PB)-based hybrid nanocomposite film that sensitively detects beta-galactoside alpha-2, 6-sialyltransferase (ST6Gal-I). The immunosensor was fabricated based on a Prussian blue-based (PB) hybrid nanocomposite, consisting of graphene oxide (GO), multi-walled carbon nanotubes (MWCNTs), PTA (a derivative of 3, 4, 9, 10-perylenetetracarboxylicdianhydride, PTC-NH2) and chitosan (CS). With the assistance of the abundance of amino groups from CS and PTA, AuNPs were densely adsorbed onto the surface of the nanocomposite, providing a large available surface area for the immobilization of abundant anti-ST6Gal-I. As measured by cyclic voltammogram (CV) and differential pulse voltammogram (DPV), PB served as an electroactive and biocompatible redox probe in achieving electrochemical signal. Under the optimal detection conditions, the immunosensor exhibited a good linear response to ST6Gal-I in the range from 0.01 to 250 ng mL(-1) and a lower detection limit of 3 pg mL(-1) (S/N = 3). Furthermore, the method was successfully applied to analyze ST6Gal-I from human serum samples and yielded satisfactory results. In conclusion, our prepared immunosensor presented a high sensitivity and excellent selectivity to ST6Gal-I, indicating its potential in future clinical and experimental analysis. (C) 2015 Elsevier Ltd. All rights reserved.

BACKGROUND:Polycystic ovary syndrome (PCOS) is an endocrine disorder whose pathogenesis remains unclear. There are also no effective biomarkers for this disease. We evaluated the metabolic changes in PCOS patients and to investigate potential metabolic biomarkers for PCOS. METHODS:Twenty-two women with PCOS and 15 healthy controls were studied. Urine samples were assessed through ultra-performance liquid chromatography-mass spectrometry followed by principal component analysis and partial least squares discriminant analysis. RESULTS:Using the presented methods, 59 urine metabolites were found at different concentrations in PCOS patients. Moreover, two novel potential biomarkers, testosterone-glucuronide and 11α-hydroxyprogesterone, and four candidate biomarkers, benzofenap, methionyl-phenylalanine, MG(18:4(6Z,9Z,12Z,15Z)/0:0/0:0) and 2-(14,15-epoxyeicosatrienoyl) glycerol, were found to show significant differences through variance analysis (P<0.01) and were identified as target metabolites. The two potential biomarkers identified in this study highly correlate to the metabolites catalyzed by the ovarian cytochrome P450c17α. CONCLUSIONS:Two novel potential urinary biomarkers, testosterone-glucuronide and 11α-hydroxyprogesterone, and four candidate urinary biomarkers, benzofenap, methionyl-phenylalanine, MG(18:4(6Z,9Z,12Z,15Z)/0:0/0:0), and 2-(14,15-epoxyeicosatrienoyl) glycerol, were identified in PCOS patients by a metabolomic approach. Further study of the biomarkers using larger populations is needed to validate these biomarkers and thereby understand the pathogenesis of PCOS, potentially allowing for its diagnosis.

目的 探讨柴胡提取物与氯苯那敏在大鼠体内药物间的相互作用.方法 将20只SD大鼠随机分为2组,实验组10只给予20 mg/kg的柴胡提取物2周,对照组10只平行给予生理盐水,第15天分别给予氯苯那敏,于(0、0.1、0.5、1、1.5、2、3、4、6、8、10、12、24h)点采血200 μL,采用LC-MS分析方法测定氯苯那敏的血药浓度,以Graphpad prism 5.01软件计算2组的药代动力学参数.结果 实验组的AUC(0-∞)[1.40±0.27(μg·h)/mL]明显高于对照组[0.87±0.25(μg·h)/mL];而代谢清除率CLint[14.25±2.84(L·kg)/h]明显低于对照组[22.97±2.03(L·kg)/h] (P ＜0.05).结论 柴胡提取物能够降低大鼠体内氯苯那敏的代谢清除率,临床上用药时应尽量避免2种药物的联合使用.

A simple process in which a graphene oxide (GO)-Prussian Blue (PB)-3,4,9,10-perylenetetracarboxylic dianhydride derivative (PTC-NH2) nanocomposite film is spread onto a glassy carbon electrode (GCE) surface is described. Glucose oxidase (GOD) was adsorbed on the modified glass carbon electrode via the specific structure of hollow Pt nanospheres. This process was sufficient to prevent GOD from leaching away, and to improve the sensitivity of our sensor. To characterise the sensors, several techniques were employed, including transmission electron microscopy (TEM), Fourier transform infrared (Fr-IR) spectroscopy, cyclic voltammetry (CV), electric impedance spectroscopy (EIS) and chronoamperometry. The resulting amperometric glucose biosensor exhibited a fast response time (within 5 s) and a good linear calibration range from 0.01 +/- 0.06 mM to 5.23 +/- 0.04 mM with a lower limit of detection of 33 mu M (SIN = 3), Possible interferents, including ascorbic acid, 1-cysteine, dopamine and ethanol, showed almost negligible electrochemical responses, indicating the high specificity of the proposed biosensor for glucose detection. The improved performance of the proposed electrode for detecting glucose in human serum was ascribed to the high surface-to-volume ratio and the excellent conductivity of the hollow Pt nanospheres. This skilful strategy of incorporating the GO/PB/PTC-NH2 nanocomposite film with hollow Pt nanospheres enhances the performance of the electrochemical sensor, which holds promising for application in bioassay analysis. (C) 2015 Elsevier B.V. All rights reserved.

目的 体外研究没食子酸对大鼠肝微粒体CYP3A的抑制效应.方法 将不同浓度没食子酸与鼠肝微粒体及探针底物在37℃下孵育,用高效液相色谱-紫外法测定探针底物的代谢产物生成率,计算IC50值.在NADPH存在或不存在时,将没食子酸与鼠肝微粒体在37℃下分别预孵育0、15、30 min后继续孵育15 min,测定探针底物的代谢产物生成率,观察没食子酸对CYP3A抑制的时间依赖性.在无NADPH存在时,将不同浓度没食子酸与鼠肝微粒体在37℃下分别预孵育0、30 min后继续孵育15 min,测定探针底物的代谢产物生成率,计算出两实验组IC50平移值(IC50shift).观察2.5h内没食子酸在紫外-可见光光谱下的变化.在无NADPH存在时,将抗氧剂与没食子酸及鼠肝微粒体在37℃下共同预孵育,观察抗氧剂谷胱甘肽和维生素C的加入对没食子酸抑制强度的影响.结果 没食子酸对CYP3A表现出浓度依赖性抑制(P＜0.05),IC50为130.2 μg/mL.预孵育无NADPH存在时,没食子酸对CYP3A表现出时间依赖性抑制(P＜0.05),且抑制强度远大于NADPH存在的实验组.在无NADPH存在时,没食子酸与鼠肝微粒体在37℃下分别预孵育0 min和30 min,两实验组间的IC50平移值达11.9.紫外-可见光全波长扫描观察显示,没食子酸在孵育过程中生成了2种反应产物.抗氧剂的加入能显著降低没食子酸对CYP3A的抑制能力(P＜0.05).结论 没食子酸对CYP3A的活性表现出较强的不可逆性抑制,具有诱导药物-药物相互作用的风险.

α2,6-Sialylated glycans are crucial molecular targets for cancer diagnosis and clinical research. In this work, a novel ultrasensitive electrochemical biosensor was fabricated based on a graphite oxide (GO), Prussian blue (PB), and PTC-NH2 (an ammonolysis product of 3,4,9,10-perylenetetracarboxylic dianhydride) nanocomposite for the selective detection of α2,6-sialylated glycans. To increase the sensitivity of the electrochemical biosensor, gold nanoparticles (GNPs) were immobilized on a GO-PB-PTC-NH2 modified glassy carbon electrode (GCE). Sambucus nigra agglutinins (SNAs), which specifically bind with α2,6-sialylated glycans, were covalently immobilized on GNPs for the sensitive detection of α2,6-sialylated glycans in serum. This proposed method can be applied to human serum, and it worked well over a broad linear range (0.1 pg mL(-1)-500 ng mL(-1)) with detection limits of 0.03 pg mL(-1). Moreover, recovery of the spiked samples ranged from 100.2% to 105.0%, suggesting that this excellent electrochemical biosensor can be used for the practical detection of α2,6-sialylated glycans.

目的 建立体外大鼠肝微粒体中CYP2C9酶活性测定方法,并以此方法评价20种中药有效成分单体对大鼠CYP2C9酶的作用.方法 以双氯芬酸(Diclofenac)为探针,在实验组大鼠肝微粒体孵育体系中加入20种中药有效成分单体,在37℃水浴温孵15 min后冰乙腈终止反应,HPLC测定各孵育体系中探针药物的代谢产物4’-羟基双氯芬酸的转化率,并与对照组比较其差异来评价各有效成分对CYP2C9酶活性的作用.结果 4’-羟基双氯芬酸、双氯芬酸及其内标香豆素三者分离良好且无其他内源性物质干扰;动力学考察表明双氯芬酸在0.25 mg/mL的大鼠肝微粒体体系中孵育15 min,测得酶动力学参数Vmax为0.145 6 nmol/(min·mg),Km为8.270 μmol/L;抑制实验考察发现:黄芩素、蛇床子素、大黄素、獐牙菜苦苷、淫羊藿苷、白藜芦醇实验组中4’-羟基双氯芬酸转化率分别为(402.60±10.58)、(210.90±10.26)、(414.46±10.32)、(509.83±26.45)、(355.44±15.87)、(387.34±22.16) pmol/(min·mg),显著低于对照组[(735.80±17.27) pmol/(min·mg),P＜0.05],其他中药成分实验组与对照组比较,其转化率无统计学差异.结论 建立了中药有效成分对CYP2C9酶作用的体外大鼠肝微粒体研究方法,并初步评价了黄芩素、蛇床子素、大黄素、獐牙菜苦苷、淫羊藿苷、白藜芦醇对CYP2C9具有抑制作用,在临床联合用药中需注意其可能引起的药物-药物相互作用.

Paeonol and resacetophenone in rat liver microsomes were analyzed by mass technology of Q exactive and the chemical structures of metabolites of paeonol and resacetophenone were analyzed in vitro by using the metabolites software of MetWorks with build-in metabolic pathways of phase I and phase II. The results suggested that 2 ',4 '-dihydroxyacetophenone was 4 '-O-demethylation metabolites of paeonol. Other metabolites such as 2 '-hydroxy-4 '-methoxybenzaldehyde and 2 ',5 '-dihydroxy-4 '-methoxyacetophenone were determined in reaction. It was also found that 2-hydroxy-O-4 '-monoglucuronideacetophenone was the principal metabolites of resacetophenone in phase.

目的:研究柴胡提取物对氯苯那敏经肝微粒体代谢的影响.方法:采用HPLC建立氯苯那敏经肝微粒体酶代谢动力学测定方法,以Graphhpad prism 5.01软件计算Vmax与Km值及代谢清除率(CLint)值;采用体外温孵法评价柴胡提取物对氯苯那敏经大鼠肝微粒体代谢的影响.结果:氯苯那敏与其代谢产物分离良好且温孵体系内无其他内源性物质干扰;测得氯苯那敏体外酶动力学参数:Vmax为0.625 7 nmol·min-1·mg-1;Km为23.68μmol·L-1;Clint为0.083 mL·min-1·mg-1;柴胡提取物对氯苯那敏经大鼠肝微粒体的代谢产生明显抑制.结论:柴胡提取物对氯苯那敏经大鼠肝微粒体的代谢产生明显抑制,二者联合使用可能产生药物-药物相互作用.

With the growing popularity of herbal and natural medicinal products, attention has turned to possible interactions between these products and pharmaceutical drugs. In this study, we examined whether astragaloside IV (AGS‐IV) could inhibit the activity of CYP1A2 in rat liver microsomes in vitro and in vivo.

Objective To develop a HPLC method for the determination of the activity of cytochrome P450 2A5(CYP2A5) in mouse microsomes with coumarin as the probe drug.Methods An waters symmetry C 18 column was used.The mobile phase was water(0.1 % acetic acid) and acetonitrile(70∶ 30).The flow rate was 1.0 mL·min-1.The column temperature was 30 ℃ and the RF wave length was E x = 340 nm,E m = 458 nm.Coumarin was incubated with mouse liver microsomes in vitro at 37 ℃ for 20 min and stopped by addition ice acetonitrile which contained internal standard substances and centrifuged(12000 r ·min-1) for 15 min,then the production 7-hydroxycoumarin was analyzed by HPLC RF.Results The lowest detection limit of 7-hydroxycoumarin was 2 nmol·L-1(S/N≥3),and the calibration curve was linear from 8 nmol· L-1to 800 nmol·L-1.The intra-day and inter-day relative standard deviations were less than 5% respectively.The method recoveries ranged from 91.3 % ~101.4%.The kinetic parameters,K m was 2.15 μmol·L-1and V max was 9.74 pmol·(min·mg protein)-1.Conclusion This method is reliable and the results can accurately indicate CYP2A5 enzyme activity,which can be used for the kinetics study of CYP2A5 in liver microsomes.

A new electrochemical sensor based on a novel organic–inorganic material (PNFCTs) was proposed for detection of paracetamol in this paper. First, PNFCTs were prepared with multi-walled carbon nanotubes (MWNTs) and a derivative of 3,4,9,10-perylenetetracarboxylic dianhydride (PTC-NH2) via cross-linking method. Then, PNFCTs were coated onto the surface of the glassy carbon electrode (GCE) to form porous organic conducting polymer films (PNFCTs/GCE), which could not only increase the loading of paracetamol efficiently but also provide an interface with exceptional electrical conductivity for paracetamol. Finally, gold nanoparticles (GNPs) were attached to the electrode surface through electrodepositing method, which obtained GNPs/PNFCTs/GCE electrode. The electrochemical behavior of paracetamol on GNPs/PNFCTs/GCE was explored by cyclic voltammetrys (CVs) and differential pulse voltammograms (DPVs). The results showed that the GNPs/PNFCTs/GCE exhibited excellent electrocatalytic activity to paracetamol, which should be attributed to remarkable properties of the new composite nanomaterials with porous nanostructure and exceptional electrical conductivity. The wide liner range and detection limit were 0.3–575 and 0.1 μM, respectively. Finally, it was successfully used to detect paracetamol in dilution human serum and commercial tablets. The sensor shows great promise for simple, sensitive, and selective detection paracetamol and provides a promising approach in paracetamol clinical research and overdose diagnostic applications.

OBJECTIVE To optimize the condition where the reaction was catalyzed by GST and lay the fundation of the accurats determination of the GST activity with CDNB.METHODS The Welch Materials Ultimate TM XB C18 ODS column(250 mm×4.6 mm,5 μm),the mobile phase was acetonitrile-water(7:3) and the flow rate was 0.8 mL·min-1.Detection wavelength was 238 nm.Firstly,under the 0.05 mg·mL-1 protein density,to foster 10 min,and then check whether CDNB reacted with GSH by the GST enzyme or not;latter on,to set CDNB and CDNB,GSH as two kinds of reaction conditions,and compare them among the different protein density,foster time,substrate concentration;at last,to choose the appropriate reaction condition from CDNB and CDNB,GSH.RESULTS The retention time of CDNB was earlyer(about 6 min),and the peak shape was better,there was not any disturbance.CDNB can response with GSH by the GST enzyme.The response by CDNB was more than which by CDNB,GSH among the different protein density,foster time,substrate concentrations(P0.05).The response were linear with protein density and foster time by CDNB,GSH,which was the appropriate reaction condition.CONCLUSION When we determined the GST enzyme activity by CDNB,we had better to select CDNB,GSH together to start the response,by which we were able to determine accuratelly the GST enzyme activity and study on kinetics of GST in rat liver microsomes.

目的:以丹皮酚为研究对象,系统分析丹皮酚在体外经大鼠肝微粒体代谢的Ⅰ相以及Ⅱ相代谢产物.方法:借助HPLC DAD与三重四级杆复合线性离子阱高分辨质谱,采用Light Sight软件辅助建立丹皮酚及2,4-二羟基苯乙酮经肝微粒体代谢产物的pMRM-IDA分析方法,分析了丹皮酚在体外经大鼠肝微粒体代谢的Ⅰ相以及Ⅱ相代谢产物.结果:丹皮酚在大鼠肝微粒体中经Ⅰ相代谢后主要产物为4位去甲基的2,4二羟基苯乙酮和4种氧化代谢物,而2,4-二羟基苯乙酮Ⅱ相代谢主要产物为4位葡萄糖醛酸化的糖苷化代谢物.结论:建立了丹皮酚Ⅰ相以及Ⅱ相代谢产物的分析方法,为丹皮酚进一步的代谢研究以及临床合理化用药提供新的方向.

Objective: To develop a GC method to determine the menthol quickly in productions with mint. Methods: We bought two kinds peppermint and one kind mouthwash from the market, chose n-hexaneas the suitable extractant, use the AT. OV-1301 capillary column for separating the menthol. The best chromatographic operating condition was chosen. Split stream sampling was 2 μL, split ratio was 10:1, injector temperature was 220 ℃, detection temperature: 220 ℃, oven temperature: 110 ℃ for 2 min, stepped temperature: 5 ℃/min to 150 ℃, maintain for 5 min, determinated by FID. Results: Menthol have good linearity within the range of 5.0~1500 μg/mL. Within-day precisions of the method were 1.30%~1.80%. The recovery ratio of the three sample was 103.44%, 101.87% and 107.26% respectively, and the content of the three samples was (4.09±0.24) mg/g, (3.78±0.21) mg/g and (0.41±0.01) mg/mL respectively. Conclusion: The method is fast, simple and accurate in the determination of menthol.

Objective To establish a HPLC-UV method for determining the concentration of 5-HT in brain tissue of rats.Methods Adopted a SHIMADZU VP-ODS C18 reversed-phase column(250×4.6 mm,5 μm).Methanol-0.01mol/L CH3COOK buffer solution containing 10 % citric acid(1:9,V/V,pH 4.00)as mobile phase,with the flow rate was 1.00 ml/min,and the detection was wavelength of 275 nm,The column temperature was 25℃.Results A excellent linearity of 5-HT concentration curve was obtained in a rang of 0.1~20 μg/ml,and the regression equation was A = 46008.89C +1886.58(r = 0.9999).The detection limit was 40 ng/ml,the lowest detection limits of 5-HT was 100 ng/ml.And the spotting recovery was within the range of 89.62%~101.6%.Intra-day and inter-day precisions were less than 3 %.Conclusion The method is accurate,reliable,simple and feasible,which is accepted to determine the concentration of 5-HT.

Objective:To develop a stable and sensitive method with p-nitrophenol as probe drug in order to evaluate the activity of UDP-glucuronosyltransferases(UGT1A6) in rat liver microsomes by the high-performance liquid chromatograph(HPLC).Methods:An Agilent Zorbax SB-C18(250 mm×4.6 mm,5 μm) column was used.The mobile phase was methanol and 20 mmol·L-1 KH2PO4 solution(50:50,V/V).The flow rate was 0.8 mL·min-1.The UV wave length was set at 254 nm and the column temperature at 30 ℃.p-Nitrophenol was incubated with rat liver microsomes in vitro at 30 ℃ and stopped by addition ice acetonitrile and centrifuged(12000 r·min-1) for 3 min and further analyzed by HPLC.Results:The retention time of P-Nitrophenol is 8.95 min.The concentration range was 0.5～100 μmol·L-1 and the regression equation was Y=5973.12X+571.58(r=0.9999).The lowest detectable limit(LOD) was 0.1 μmol·L-1(S/N≥3) and the lower limit of quantification(LLOQ) was 0.5 μmol·L-1.The Abstraction recoveries were 104.5%-105.5%.The intra-day and inter-day relative standard deviations were all less than 15%.There were no endogenous substances existing in the incubation system which interfered with the determination of the analyses of interest.For the rat liver microsomes Km was 24.63 μmol·L-1 and Vmax was 18.87 nmol·min-1·mg·protein-1.Conclusion:The method is stable and highly sensitive for determination of P-Nitrophenol in vitro and suitable for the evaluation of UGT1A6 activity in vitro.

OBJECTIVE To provide reference for exploitation and the quality evaluation of Bupleurum smithii Wolff var.parvifolium Shan et Y.Li in Qinghai province,and to determine the content of total saponins,saikoside a and saikoside d in it.METHODS The content of total saponins was determined by spectrophotometry.The content of saikoside a and saikoside d were determined by HPLC.RESULTS The content of total saponins,saikoside a and saikoside d in Bupleurum smithii Wolff var.parvifolium Shan et Y.Li were more than 2.77%,0.54%,0.14%,respectively.The content of saikoside a and saikoside d were higher than quality criteria in China Pharmacopeia.CONCLUSION The method is simple,rapid and accurate.The mothod is suitable for quality analysis of total saponins,saikoside a and saikoside d in Bupleurum smithii Wolff var.parvifolium Shan et Y.Li in Qinghai province which possess high medicinal value.

Objective To determine the effect of astragaloside Ⅳ(AGS-Ⅳ) on the activity of CYP1A2 in rats to elucidate the possibility CYP1A2-induced interactions of drugs.Methods Analysis of theophylline,and acetamidophenol(internal standard) was achieved using waters C18 column(250 mm×4.6 mm×5 μm) and a mobile phase consisted of methanol-water(17∶83,v/v) was delivered at a flow rate of 1.0 ml/min at 30 ℃ at a wavelength of 274 nm.Seven rats were divided into 2 groups by simple random sampling.Three rats were intravenously treated with AGS-Ⅳ(3 mg/kg),and the left 4 rats were intravenously administered with an equivalent volume of 0.1% DMSO vehicle once a day for a week.On the eighth day,all of the rats were intragastrically administered theophylline.The concentration of theophylline in the plasma was determined by high performance liquid chromatography(HPLC).The distinction of pharmacokinetic parameters was compared to investigate the effect of AGS-Ⅳ on the activity of CYP1A2.Results Theophylline and acetamidophenol were separated well,the detection limit of theophylline was 80 nmol/L,and the linear rang of our method was 0.2 to 50 μmol/L.The intra-day and inter-day relative standard deviations were both less than 10%,and the recoveries were more than 91.The main pharmacokinetic parameters of area under the concentration-time cure(AUC) of theophylline were higher than control groups(131.16±3.22 vs 97.51±6.40).The apparent oral total body clearance value(CL/F) of test group and control group were 0.12±0.01 and 0.17±0.02 respectively,with the former obviously lower than the latter(P<0.05).Conclusion AGS-Ⅳ exerts obvious inhibitory effect on CYP1A2 activity in rats,so its should be cautious to co-administer with drugs metabolized by CYP1A2.