化学发光分析与高效液相色谱联用检测酪氨酸

在强碱性条件下,KMnO4氧化酪氨酸产生自由基,自由基氧化Luminol产生强化学发光,酪氨酸浓度与相对发光强度呈线性关系。选用C18反相色谱柱、CH3OH、KH2PO4混合液为流动相,酪氨酸能很好分离。基于此,建立CL-HPLC检测酪氨酸的新方法,线性范围为1.2×10-3～5.0×10-2g/L、线性相关系数为0.9994、RSD为2.37%、检测限为3.3×10-4g/L。方法已于检测实际样品氨基酸口服液,并与UV光度法,结果一致。本文探讨了酪氨酸增强KMnO4-Luminol化学发光机理。     

高效液相色谱-荧光法同时测定人体尿液中的酪氨酸和色氨酸

建立了高效液相色谱-荧光检测法同时测定人体尿液中的酪氨酸(Tyr)和色氨酸(Typ)。采用Diamonsil C18柱(250×4.6mm,5μm),以水-乙腈(80∶20,V/V)为流动相,流速1.0mL/min,荧光检测波长为λex=260nm、λ_(em)=340nm。Tyr和Typ的含量分别在0.10~6.0μg/mL及0.050~6.0μg/mL范围内与色谱峰面积呈良好的线性关系(r=0.9993),检测限分别为0.05μg/mL和0.02μg/mL。在三个添加水平的回收中,两种氨基酸的平均回收率在86.3%~122.3%之间,相对标准偏差(RSD)小于8.51%。将所建立的方法应用于健康人、普通病人和癌症患者晨尿检测,结果发现:癌症患者尿样中色氨酸的水平与健康人和普通病人相比偏低。     

饲料中生物素的高效液相色谱测定

介绍了用液相色谱测定饲料中生物素的方法,探讨了流动相中缓冲液、pH值、有机溶剂等对分离的影响,确定的较佳色谱条件:HypersilODS柱,流动相为甲醇-0.1mol·L-1KH2PO4(H3PO4调pH＝3.5)(体积比25∶75),流速1.0mL／min,紫外210nm检测;方法的相对标准偏差在3.5%以内,回收率在92%～102%,检出限为1mg／kg;实验表明该法简便、快速,适应性好;方法应用于饲料样品中生物素的测定,取得了很好的结果。     

高效液相色谱发展史

以１９７１年为界，评述了高效液相色谱分析逐步发展和完善的历史过程。     

亚氨基二乙酸的高效液相色谱测定

利用高效液相色谱，采用外标法测定样品中亚氨基二乙酸的含量，紫外检测器检测波长为210nm，色谱柱为SAX强阴离子交换柱，用0．005mol.L^-1磷酸二氢钾缓冲液（PH2．5）为流动相，对样品进行分析，其线性相关系数为0．9996，标准偏差为0．035，回收率为98．5％－101．5％。     

新型杀菌剂施保克有效成分的高效液相色谱测定

建立了测定施保克乳油中有铲成分ｐｒｏｃｈｌｏｒａｚ含量的反相高效液相色谱法,用Ｎｏｖａ－Ｐａｋ Ｃ８柱分离,乙腈－磷酸盐（ｐＨ６．４）为流动相,紫外波长２３０ｎｍ检测,方法的相对标准偏差为０．５７％,加标回收率为９８．５％～１００．４％,质量浓度在２００～１２００ｍｇ／Ｌ范围内与峰面积呈现良好的线性,ｒ＝０．９９９８。该法给出了令人满意的结果,可作为检验进口或国内同类产品的借鉴。     

间接液相色谱电化学法测定酯类化合物

本文用间接液相色谱电化学的方法对非电活性的酯类混合物甲酸甲酯、甲酸乙酯、乙酸甲酯和乙酸乙酯进行了分离和测定,其线性范围分别是3.2×10~(-9)～4.4×10~(-8)mol、2.4×10~(-9)～2.8×10~(-8)mol、2.0×10~(-9)～4.0×10~(-8)mol和2.2×10~(-9)～2.4×10~(-8)mol。对甲酸甲酯8次平行测定的相对标准偏差为0.89％。     

高效液相色谱柱前衍生化法测定牛磺酸含量

高效液相色谱柱前衍生化法测定牛磺酸含量谢继红,陈玉仁,唐少珍（大连市医药科学研究所大连ｌ１６０１３）１前言牛磺酸又称牛胆硷（Ｔａｕｒｉｎｅ）,其化学名为２－氨基乙磺酸,普遍存在于动物体内,特别是海洋生物体内,据文献报道￣［１－３］,牛磺酸以游离形式存...     

高效液相色谱电化学发光法测定丙酮酸

基于在恒电流条件下电解产生不稳定化学发光试剂Mn(Ⅲ)能直接氧化丙酮酸产生化学发光,将高效液相色谱法与电化学发光法相结合,建立了高效液相色谱电化学发光法测定丙酮酸的检测方法。在优化的实验条件下,提高了测定的选择性,对一些复杂的样品如:啤酒、人体血清、尿液中丙酮酸进行了分析,本方法的检出限:3×10-8g/mL,相对标准偏差2.5%(n=11,ρ=5.0×10-5g/mL),线性范围1×10-7～1×10-4g/mL。     

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.     

Assay for tyrosine hydroxylase by high-performance liquid chromatography with fluorescence detection

A sensitive assay method for tyrosine hydroxylase in rat brain and adrenal medulla by high-performance liquid chromatography with fluorescence detection is described. L-DOPA formed enzymatically from the substrate L-tyrosine and alpha-methyldopa (internal standard), after clean-up with small cartridges of an activated alumina and a cation exchanger, Toyopak IC-SP M, are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-120T. The detection limit for L-DOPA formed enzymatically is 2 pmol per assay tube.     

Determination of acrylamide and methacrylamide by normal phase high performance liquid chromatography and UV detection

A method using normal phase high performance liquid chromatography (NP-HPLC) with UV detection was developed for the analysis of acrylamide and methacrylamide. The method relies on the chromatographic separation of these analytes on a polar HPLC column designed for the separation of organic acids. Identification of acrylamide and methacrylamide is approached dually, that is directly in their protonated forms and as their hydrolysis products acrylic and methacrylic acid respectively, for confirmation. Detection and quantification is performed at 200 nm. The method is simple allowing for clear resolution of the target peaks from any interfering substances. Detection limits of 10 microg L(-1) were obtained for both analytes with the inter- and intra-day RSD for standard analysis lying below 1.0%. Use of acetonitrile in the elution solvent lowers detection limits and retention times, without impairing resolution of peaks. The method was applied for the determination of acrylamide and methacrylamide in spiked food samples without native acrylamide yielding recoveries between 95 and 103%. Finally, commercial samples of french and roasted fries, cookies, cocoa and coffee were analyzed to assess applicability of the method towards acrylamide, giving results similar with those reported in the literature.     

Determination of acyclovir in plasma by high performance liquid chromatography with UV detection. Method development and method validation

A HJPLC method for the determination of acyclovir in plasma is described. The method is simple and sensitive enough for bioequiva-lence studies, where a large number of plasma samples with low acyclovir concertration are involved. The procedure is based on the deproteinization of plasma with perchloric acid and separation of acyclovir on a Hypersil ODS Column at pH 5.6 with UV detection. The calibration standards are linear up to at least 4000 ng/mL and the limit of quantification is 10 ng/mL.     

反相高效液相色谱法测定玉米中玉米赤霉烯酮

建立了反相高效液相色谱法检测玉米中玉米赤霉烯酮的分析方法.该方法的标准曲线回归方程为y=2.97×103 5.23×104ρ(r=0.9998);玉米样品3个水平的加标回收率分别为84.8%,86.7%和89.2%;相对标准偏差分别为4.0%,5.2%和4.4%;加标浓度为50 ng/g的玉米样品日内相对标准偏差为0.69%;玉米样品日间相对标准偏差为2.6%;方法检出限为3.0 ng/g.     

高效液相色谱化学发光法测定血清中左羟丙哌嗪

在H2SO4介质中, KMnO4氧化左羟丙哌嗪产生化学发光, 甲醛可增敏上述化学发光, 在此基础上建立了反相高效液相色谱(RP-HPLC)分离, 柱后化学发光检测左羟丙哌嗪的新方法, 并成功应用于血清中左羟丙哌嗪的测定. 在优化的实验条件下, 该法测定左羟丙哌嗪的线性范围为1.0×10-7～1.0×10-5 g/mL, 检出限(3σ)为3×10-8 g/mL. 对2.0×10-6 g/mL的左羟丙哌嗪进行11次平行测定, 其相对标准偏差为2.3%.     

Simple assay procedure for tyrosine hydroxylase activity by high-performance liquid chromatography employing coulometric detection with minimal sample preparation

A simple assay procedure for tyrosine hydroxylase activity in crude tissue samples was devised that requires minimal sample preparation and use of high-performance liquid chromatography with coulometric electrochemical detection. After incubation of enzyme samples, such as human brain homogenates or rat pheochromocytoma PC12h cells, with L-tyrosine and a tetrahydropterin cofactor, in the presence or absence of p-bromobenzyloxyamine, an inhibitor of aromatic L-amino acid decarboxylase, the reaction was terminated by addition of an equal volume of 0.1 M perchloric acid. For quantitation of L-DOPA produced, the sample was centrifuged, filtered and directly applied to the chromatographic apparatus connected to a coulometric electrochemical detector. This method makes redundant a time-consuming step in the previous methods, purification and concentration of L-DOPA or dopamine using alumina. The reaction conditions for the assay of tyrosine hydroxylase activity in brain homogenates and PC12h cells were re-examined by this method. Both tyrosine hydroxylase samples required a naturally occurring cofactor, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6R)BH4], catalase and NSD-1055 for the full activity, and tyrosine hydroxylase in human brain homogenates required Fe2+ ions for its full activity. (6R)BH4 proved to be a more effective cofactor than a synthetic cofactor, (6RS)-methyl-5,6,7,8-tetrahydropterin, which is commonly used for this assay.     

Determination of phylloquinone (vitamin K1) by high performance liquid chromatography with UV detection and with particle beam-mass spectrometry

A reversed-phase PB LC-MS method was used for the determination and unequivocal identification of vitamin K(1) in some vegetable samples. The method was also applied to the identification of other constituents in the food analyzed. LC-MS experiments were carried out in both EI and negative-ion CI modes. Preliminary sensitivity studies on vitamin K(1) showed a detection limit of 2 ng (signal-to-noise ratio=3) in negative-ion operation and in single-ion monitoring at m/z 450. Quantitative results obtained with LC-MS for the extracts of four vegetables were compared with those of LC-UV analyses performed at 247 nm.     

Determination of phenolic compounds and hydroxymethylfurfural in meads using high performance liquid chromatography with coulometric-array and UV detection

The objective of this study was the determination of 25 phenolic compounds in different mead samples (honeywines) using high performance liquid chromatography (HPLC) with coulometric-array detection and in case of hydroxymethylfurfural with UV detection. Our method was optimized in respect to both the separation selectivity of individual phenolic compounds and the maximum sensitivity with the electrochemical detection. The method development included the optimization of mobile phase composition, the pH value, conditions of the gradient elution and the flow rate using a window-diagram approach. The developed method was used for the determination of limits of detection and limits of quantitation for individual compounds. The linearity of calibration curves, accuracy and precision (intra- and inter-day) at three concentration levels (low, middle and high concentration range) were verified. Mead samples were diluted with the mobile phase at 1:1 to 1:50 ratio depending on the concentration and filtered through a PTFE filter without any other sample pre-treatment. Phenolic compounds concentration was determined in 50 real samples of meads and correlated with meads composition and hydroxymethylfurfural concentration. The most frequently occurred compounds were protocatechuic acid and vanillic acid (both of them were present in 98% samples), the least occurred compounds were (+)-catechin (10% samples) and sinapic acid (12% samples). Vanillin and ethylvanillin, which are used as artificial additives for the taste improvement, were found in 60% and 42% samples, respectively. Hydroxymethylfurfural concentration, as an indicator of honey quality, was in the range from 2.47 to 158 mg/L. Our method is applicable for the determination of 25 phenolic compounds in mead, honey and related natural samples.