急性淋巴细胞白血病患儿红细胞中硫嘌呤甲基转移酶活性的HPLC分析

建立了急性淋巴细胞白血病患儿血液中红细胞中的硫嘌呤甲基转移酶活性(TPMT)的HPLC测定法。采用反相高效液相色谱法直接测定酶促反应的产物浓度,从而计算红细胞中TPMT的活性。以S-腺苷-L-甲硫氨酸(SAM)作为甲基供给体,6-硫鸟嘌呤(6-TG)作为酶反应底物,TPMT催化6-TG生成2-氨基-6-甲基巯基嘌呤(6-MTG),采用HClO4溶液终止反应及沉淀蛋白,分离上清液进行色谱分析。色谱柱为AichromBond-1 C18柱(5μm,4.6mm i.d.×150mm);等梯度洗脱,流动相为V(乙腈)∶V(0.01mol/L磷酸钾缓冲溶液)=6∶94(用HCl调pH2.74),荧光检测器检测,激发波长为310nm,发射波长为390nm。结果表明,6-MTG在0~250μg/mL范围内呈良好的线性关系,相关系数r=0.9999,检出限为0.093μg/L(S/N=3),回收率为81.4%~106.3%。方法能用地巯基嘌呤类药物药代研究和临床用药监测常规分析。     

6,7-二氧代-4-芳胺香豆素的设计、合成及分子对接研究

茶酚-氧位-甲基转移(COMT)酶抑制剂在治疗帕金森病中起到重要作用.通过对现有COMT酶抑制剂托卡朋和恩托卡朋结构与活性关系分析,推断含有儿茶酚结构的香豆素类化合物可能具有潜在的COMT酶抑制活性,因此设计合成了一类新型的6,7-二氧代-4-芳胺香豆素,通过理论计算,研究了此类化合物对COMT酶抑制活性.结果表明,设计的10种6,7-二氧代-4-芳胺香豆素与COMT酶的对接效果均较好,其中具有甲氧基乙基保护的儿茶酚结构化合物6,7-二[2-(甲氧基)乙氧]-4-(苯胺)香豆素(6b4)和6,7-二[2-(甲氧基)乙氧]-4-[(3-乙炔基)苯胺]香豆素(6b5)与COMT酶的对接效果尤为显著.     

海兔毒素10关键合成子Dap的立体专一性合成

以N-丙酰基-(3R,4S)-3-甲基-4-苯基-2-羰基噁唑啉与N-(叔丁氧羰基)-S-脯氨醛为原料,经羟醛缩合反应合成了高立体专一性、具有三个手性中心的(4S,5S,2'R,3'R,2"S)-3-[3'-(N-叔丁氧羰基-2"-吡咯烷基)-3'-羟基-2'-甲基丙基)]-4-甲基-5-苯基-2-羰基噁唑烷酮(6);6脱去手性辅基,甲基化3'-羟基合成了抗肿瘤活性肽海兔毒索10的关键合成子--(2S,3S,2's)-3-(N-叔丁氧羰基-2'-吡咯烷基)-3-甲氧基-2-甲酸丙酸,其结构经1H NMR,13C NMR,m和MS表征.     

一种新型Hsp90抑制剂的合成及其抑制活性

对氨基苯甲酸经重氮化后再与2-氯乙酰乙酸乙酯反应制得(Z)-4-[2-(1-氯-2-乙氧基-2-羰亚甲基)肼基]苯甲酸(1);1与二甲基环己二酮成环后再与对羟基环己胺完成酰胺化反应合成了一种新型Hsp90抑制剂——N-(4-羟基环己基)-4-[1-(6,6-二甲基-4-氧-3-乙酰氧基-4,5,6,7-四氢吲唑)]苯甲酰胺(3),其结构经1HNMR表征。荧光偏振法研究结果表明3对Hsp90α具有明显的抑制活性。     

电聚合修饰碳纤维电极及乳酸脱氢酶活性的测定

用电聚合方法将亚甲基绿 (MG)修饰在碳纤维电极 (直径 7μm)上 ,并用该修饰电极测定了乳酸脱氢酶 (LDH)活性。在烟酰胺腺嘌呤二核苷酸 (NAD+ )浓度为 6 .0× 10 - 4mol L ,乳酸浓度为 5 .0× 10 - 3mol/L的pH7.0NaOH KH2 PO4 缓冲介质中 ,电位恒定在 +0 .10V下 ,用电流法测定乳酸脱氢酶活性 ,线性范围为 15～ 2 4 0U/mL ,检测限为 10U/mL ,响应时间为 15s。该修饰微电极稳定性好、灵敏度高、测定干扰小     

固相萃取-高效液相色谱法测定水果和果酱中的6种对羟基苯甲酸酯

建立了固相萃取-高效液相色谱(SPE-HPLC)同时测定水果和果酱中6种对羟基苯甲酸酯(对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸异丙酯、对羟基苯甲酸丙酯、对羟基苯甲酸异丁酯和对羟基苯甲酸丁酯)含量的方法。经Oasis HLB固相萃取柱净化的样品采用HPLC分离,优化的色谱条件为采用Symmetry-C18色谱柱分离,流动相为柠檬酸缓冲液-甲醇(体积比为48:52),流速1.0 mL/min,检测波长258 nm,测定温度40 ℃。6种对羟基苯甲酸酯的线性范围为0.1～20.0 mg/L(r=0.9999),回收率为82.8%～115.5%,相对标准偏差为0.2%～6.8%(n=6)。对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸异丙酯、对羟基苯甲酸丙酯的检出限(S/N=3)为0.1 mg/kg,定量限(S/N=10)为0.3 mg/kg;对羟基苯甲酸异丁酯和对羟基苯甲酸丁酯的检出限为0.2 mg/kg,定量限为0.6 mg/kg。该方法简便快速、结果准确、重现性好,可作为测定水果及果酱中多种对羟基苯甲酸酯的有效方法。     

(S)-(4-溴-2-甲苯)(3-甲基吗啉)甲酮的合成

以(S)-2-氨基丙醇和氯乙酰氯为起始原料,经酰化和环合反应制得(S)-5-甲基吗啉-3-酮（4）; 4经还原制得(S)-3-甲基吗啉(5); 5与4-溴-2-甲基苯甲酸酰化缩合合成了(S)-(4-溴2-甲基苯基)(3-甲基吗啉)-甲酮,总收率57%,其结构经¹H NMR 和 ¹³C NMR确证。     

合成SGLT2抑制剂埃格列净的新方法

以D-(+)-葡萄糖酸内酯为原料,经三甲硅基保护羟基后与5-溴-2-氯-4′-乙氧基二苯甲烷偶联制得(2S,3R,4S,5S,6R)-2-［4-氯-3-(4-乙氧苄基)苯基］-6-(羟甲基)-2-甲氧基四氢-2H-吡喃-3,4,5-三醇（2）; 2经羟基保护、氧化和羟醛缩合等5步反应制得(3S,4S,5R,6S)-3,4,5-三(苄氧基)-6-［4-氯-3-(4-乙氧苄基)苯基］-2-(羟甲基)-6-甲氧基四氢-2H-吡喃-2-甲醛（7）; 7经还原、脱苄同时关环制得埃格列净(1S,2S,3S,4R,5S)-5-［4-氯-3-(4-乙氧苄基)苯基］-1-(羟甲基)-6,8-二氧杂二环［3.2.1］辛烷-2,3,4-三醇,其结构经¹H NMR和LC-MS表征。     

高效液相色谱-串联质谱法同时测定盐酸拉贝洛尔中三种杂质

建立高效液相色谱-串联质谱法同时测定盐酸拉贝洛尔中2-羟基-5-[1-羟基-2-[(1-甲基-3-苯丙基)氨基]乙基]苯甲酸、2-羟基-5-[1-羟基-2-[(1-甲基-3-苯丙基)氨基]乙基]苯甲酸甲酯和2-羟基-5-(2-(4-苯基丁-2-氨基)乙酰)苯甲酰胺的含量。采用Agilent Eclipse XDB-C18柱（250 mm×4.6 mm,5μm）为分离柱,以0.1%（质量分数）的乙酸溶液为流动相A,乙腈-0.1%的乙酸溶液（体积比为1∶1）为流动相B,流量为1.5 mL/min,梯度洗脱,柱温为40℃,进样体积为10μL,离子源为大气压化学电离源,采用多反应监测模式。三种杂质的质量浓度分别在1.0160～203.20、1.0320～206.40、1.0370～207.40 ng/mL范围内与色谱峰面积线性相关,相关系数均大于0.999,3种化合物的检出限均为0.03ng/mL,定量限均为0.10 ng/mL。样品平均回收率分别为95.3%、92.4%、91.9%,测定结果的相对标准偏差分别为1.44%、1.46%、1.11%(n=9)。该方法可用于同时测定盐酸拉贝洛尔中3...     

棘孢曲霉中分离的2个新化合物

从土壤真菌棘孢曲霉(Aspergillus aculeatus)中分离得到2个新化合物和2个已知化合物,采用质谱(MS)、一维核磁共振波谱(1D NMR)、异核多键相关谱(HMBC)、异核单量子相关谱(HSQC)和旋转坐标NOE谱(ROESY)等方法对化合物的结构进行了鉴定,化合物2的顺反构型通过ROESY数据进一步确定.鉴定结果表明,2个新化合物分别为2-(2'-4'-6'-三羟基)-7-羟基-5-甲基色原酮{2-(2',4',6'-trihydroxyphenyl)-(7-hydroxy-5-methyl)chromone}(1)和(E)-4-羟基-3-[(3-甲基-4-羟基-2-丁烯)氧代]苯甲酸{(E)-4-hydroxy-3-[(4-hydroxy-3-methylbut-2-en-1-yl)oxy]benzoic acid}(2);2个已知化合物分别为(S)-2-[(2'-羟基)丙基]-5-甲基-7-羟基色原酮{2-(2'-hydroxypropyl)-5-methyl-7-hydroxychromone}(3)和(3R,4S)-8-羟基-3,4,5-三甲基-6酮-4,6-二氢-3H-异色烯-7-羧酸{(3R,4S)-8-hydroxy-3,4,5-trimethyl-6-oxo-4,6-dihydro-3H-isochromene-7-carboxylic acid}(4).活性测试结果表明,这4个化合物均表现出一定的抗氧化活性.     

L-dopa as substrate for human duodenal catechol-O-methyltransferase and aromatic L-amino acid decarboxylase

Catechol-O-methyltransferase (COMT) and aromatic L-amino acid decarboxylase (AADC) activities were determined in human gastrointestinal samples. L-dopa was used as the substrate and the reaction products 3-O-methyldopa (3OMD) and dopamine were separated by reversed phase HPLC and detected by electrochemical or UV detection. COMT activities varied between 40-350 pmol/mg/min and AADC activities between 100-3300 pmol/mg/min in different parts of the gastrointestinal tract. COMT inhibitors nitecapone (OR-462) and OR-611 effectively inhibited human gastrointestinal COMT activity in vitro, the IC50 values ranging from 10-20 nM and 5-75 nM, respectively. In vitro carbidopa inhibited AADC slightly more effectively than benserazide.     

Simultaneous analysis of catechol‐O‐methyl transferase activity,S‐adenosylhomocysteine and adenosine

Novel HPLC method utilizing UV‐detection was developed to analyse catechol‐O‐methyltransferase (COMT) products, vanillic acid and isovanillic acid, S‐adenosylhomocysteine (SAH) and adenosine formed from dihydroxybenzoic acid and S‐adenosyl‐L‐methionine (SAM) by incubation of the rat tissues. Entacapone, a COMT inhibitor, prevented the formation of SAH only partially in the striatal homogenate whereas in the kidney homogenate the increase of SAH was prevented by entacapone. In conclusion, this method was reliable, rapid and simple. COMT seemed to be partially responsible on the SAM utilizing methylations in the striatal homogenates while in the high COMT activity tissue, COMT was the main SAH producing methyltransferase. Copyright © 2009 John Wiley & Sons, Ltd.     

On‐line high‐performance liquid chromatography coupled with biochemical detection method for screening of α‐glucosidase inhibitors in green tea

An on‐line high‐performance liquid chromatography–biochemical detection (HPLC‐BCD) method, in which compounds separated by HPLC were on‐line reacted with enzyme and substrate solutions delivered by flow injection and the enzyme inhibition signal was collected by UV detection, was developed to rapidly screen α‐glucosidase inhibitors from green tea extracts in this study. The chromatographic fingerprints and enzyme inhibition profiles of the different brands of green tea could be simultaneously detected by the on‐line HPLC‐BCD method. Enzyme inhibition profiles were detected by the UV detector at 415 nm based on the reaction of α‐glucosidase and p‐nitrophenyl α‐d ‐glucopyranoside (PNPG). PNPG (1.25 mm ), α‐glucosidase (0.4 U/mL) and the flow rate 0.07 mL/min were applied as optimized parameters to detect α‐glucosidase inhibitors in green tea. Four components in green tea showed α‐glucosidase inhibition action and three of them were identified as HHDP‐galloyl glucose, (−)‐epigallocatechin‐3‐gallate and (−)‐epicatechin‐3‐gallate by HPLC–fourier‐transform mass spectrometry (HPLC‐FTMS). Two brands of green tea derived from Mengding and Enshi mountainous areas might be superior to the other samples in the prevention and treatment of diabetes owing to their stronger activities of enzyme inhibitors. The proposed on‐line HPLC‐BCD method could be used to rapidly identify the potential enzyme inhibitors in complex matrixes.     

An automated analyzer for vancomycin in plasma samples by column-switching high-performance liquid chromatography with UV detection

An automated analyzer for vancomycin in rat plasma by column-switching high-performance liquid chromatography (HPLC) with UV detection was developed. The method includes in-line extraction of vancomycin by ion-exchange cartridge column and a separation on a reversed-phase column with UV detection at 215 nm. Plasma samples were diluted by mobile phase solution and directly injected to HPLC. Vancomycin was quantitatively recovered from rat plasma samples. The separation was completed within 15 min. The calibration curve was linear over the range from 0.5 to 100 microg/mL with the detection and quantification limits of 0.5 microg/mL (2.5 ng on column; signal-to-noise ratio = 3). The values of precision in intra- and inter-day assays (n = 3) were less than 1.92 and 3.69%, respectively. This method does not require time-consuming pre-treatment and is suitable for the routine assay of plasma samples.     

Determination of catechol-O-methyltransferase activity in erythrocytes by high performance liquid chromatography with electrochemical detection

A rapid assay employing HPLC with electrochemical detection for catechol-O-methyltransferase (COMT) activity in red blood cells is described. Enzyme activity is determined from erythrocyte lysates using S-adenosyl-L-methionine as methyl donor and 3,4-dihydroxybenzoic acid as substrate. The 3-O- and 4-O-methylated reaction products are measured by high-performance liquid chromatography with electrochemical detection. Human erythrocyte soluble form of COMT had Km values of 6.1 microM and 26.0 microM for S-adenosyl-L-methionine and dihydroxybenzoic acid, respectively. The mean O-methylation ratio for the soluble form of COMT was 5.3. An O-methylation ratio of 15.5 was estimated in the membrane fraction of an erythrocyte pool from three samples. The activities of soluble COMT in erythrocytes of some animal species are also reported. The procedure is easily automated, and a large number of samples can be processed during one working day.     

Quantitative HPLC method and pharmacokinetic studies of ergosta‐4,6,8(14),22‐tetraen‐3‐one,a natural product with diuretic activity from Polyporus umbellatus

A simple and specific HPLC method with dual wavelength UV detection for the determination of ergosta‐4,6,8(14),22‐tetraen‐3‐one (ergone) in rat plasma was developed and proved to be efficient. The method used ergosterol as internal standard (IS). Following a single‐step protein precipitation, the analyte and IS were separated on an Inertsil ODS‐3 column with a mobile phase containing methanol–water (99:1, v/v) at a flow rate of 1 mL/min. The analytes were detected by using UV detection at wavelength of 350 (ergone) and 283 (IS) nm, respectively. The calibration curve was linear over the range of 0.1–2.0 µg/mL and the lower limit of quantification was 0.1 µg/mL. The intra‐day and inter‐day precision studies showed good reproducibility with RSD less than 8.5%. The intra‐day and inter‐day accuracy ranged from 95.6 to 104%. Mean extraction recovery was above 95% at the low, medium and high concentrations. The present HPLC‐UV method was simple and reliable. The method described herein had been successfully applied for the pharmacokinetic studies in male SD rats after administration of 20 mg/kg dose of solution of ergone. Copyright © 2010 John Wiley & Sons, Ltd.     

Neuraminidase Based Electro-Nano Diagnostic Platforms: Development of Model Systems for Cancer Diagnosis

An electrochemical biosensor that monitored neuraminidase (NEU3), activity was developed. The analysis platform included a graphene-platinum hybrid modified gold screen printed electrode as a transducer. The detection protocol was based on observation of NEU3 activity which was used to remove sialic acid from the GD3 ganglioside. Examination of analytical characteristics resulted with two linear ranges of 10⁻⁸ U/mL–10⁻¹ and 10⁻¹ U/mL–2.53 U/mL with limit of detection values of 10⁻⁸ U/mL and 10⁻¹ U/mL, respectively. The selectivity of the developed NEU3 activity based electrochemical biosensor was tested with HeLa, VERO and A549 cell lines.     

Suitability of ionic liquids as mobile‐phase additives in HPLC with fluorescence and UV detection for the determination of heterocyclic aromatic amines

The effects of several ionic liquids (ILs) as mobile‐phase additives in HPLC with fluorescence and UV–Vis detection for the determination of six heterocyclic aromatic amines were evaluated using two different C₁₈ stationary phases with moderate silanol activity. The studied ILs were 1‐butyl‐3‐methylimidazolium tetrafluoroborate, 1‐hexyl‐3‐methylimidazolium tetrafluoroborate and 1‐methyl‐3‐octylimidazolium tetrafluoroborate. The optical behaviour of heterocyclic aromatic amines in presence of ILs was studied and the silanol‐suppressing potency of ILs was evaluated for the two stationary phases studied. Several chromatographic parameters were evaluated in the presence or absence of ILs, or using triethylamine, the most common mobile‐phase additive. The best results were achieved using 1 mM 1‐butyl‐3‐methylimidazolium tetrafluoroborate as mobile‐phase additive and NovaPak^® column. In these conditions and with 18% of ACN in the mobile phase, analytical performance of the chromatographic methods using fluorescence and UV–Vis were evaluated, obtaining good precision in all cases (RSD lower than 6.6%) and low LOD (0.001–0.147 μg/mL with UV–Vis and 0.001–0.006 ng/mL with fluorescence detection).     

2,3-吡啶二甲酰亚胺及其工程菌转化产物的高效液相色谱检测

建立了利用含D-海因酶的基因工程菌转化吡啶二甲酰亚胺（PDI）生成3-氨基甲酰基-α-吡啶甲酸（α-3CP）的高效液相色谱（HPLC）检测方法。将工程菌pET3a-hyd/BL21（DE3）诱导表达后收集菌体,以PDI为底物,37℃摇床反应30 min后,以13000 r/min离心,取上清液进行HPLC检测。色谱条件：Hypersil^TM GOLD C18色谱柱（250 mm×4.6 mm,5 μm）;流动相为H₂O-乙腈（体积比为90：10,含0.1%（体积分数）三氟乙酸）;检测波长为254 nm。当底物PDI达到饱和浓度时,测得工程菌pET3a-hyd/BL21（DE3）的比活力为0.61 U/（mL·10OD_{600 nm}）。该研究为今后利用生物法制备复杂半酰胺有机物提供了坚实的理论基础。     

小白菜中残留虫酰肼的超临界流体萃取条件的研究

将超临界流体萃取(supercritical fluid extraction,SFE)技术与高效液相色谱分析相结合,建立了特异性杀虫剂虫酰肼的萃取分离方法。SFE对虫酰肼的萃取条件:压力48.3 MPa(7000 psi),温度60 ℃,静态萃取时间20 min,CO2体积10 mL,改性剂甲醇添加量0.04 mL/g,丙酮为收集溶剂。在此条件下,SFE对虫酰肼的萃取率为100.75%,所得样品可直接用于高效液相色谱分析。色谱条件：紫外-二极管阵列检测器(检测波长为245 nm),C18键合色谱柱,乙腈