反相高效液相色谱测定依替巴肽注射液中氨基酸的含量

采用异硫氰酸苯酯为柱前衍生化试剂,结合反相高效液相色谱技术,建立了定量分析依替巴肽注射液中氨基酸组分的检测方法。优化色谱条件,选用Agilent XDB-C18色谱柱(5μm,150 mm×4.6 mm),以0.1 mol/L醋酸钠(冰醋酸调至pH 6.0)-乙腈(90∶10)为流动相A,乙腈为流动相B,梯度洗脱方式,检测波长为254 nm,流速为1.0 mL/min,进样体积为5μL,柱温为30℃。结果表明,在优化条件下,各氨基酸能实现有效分离,且在1.23～3.68 mmol/L浓度范围内,各氨基酸的线性关系良好,相关系数(r)为0.999 1～0.999 6。该方法显示了良好的准确性、重复性、稳定性及选择性,能对依替巴肽注射液中的氨基酸实现准确定量分析。     

高效液相色谱法快速测定抗真菌制剂M18中的吩嗪-1-羧酸

 建立了测定抗真菌制剂M18中吩嗪 1 羧酸的反相高效液相色谱方法。流动相为甲醇 5mmol/L磷酸缓冲液(pH 5 0 ) (体积比为 6 0∶40 ) ,流速为 1mL/min ,检测波长为 2 48nm ,线性范围是 5 0mg/L～ 5 0 0mg/L ,检测限是 30mg/L ,回收率为 97 5 3% ,RSD为 1 5 %。该方法具有快速、简便、灵敏、重现性好的特点。     

高效液相色谱手性流动相添加剂分离西孟坦对映体

以 β 环糊精作为手性流动相添加剂 ,研究了DL 西孟坦在反相HPLC系统中的拆分。考察了缓冲盐的浓度、pH、β 环糊精的浓度、流动相中甲醇的比例、流动相流速和温度对手性分离的影响 ,建立了 β 环糊精动态手性固定相法分离西孟坦对映体的方法。色谱条件为 :ZirchromKromasilODS 1(5 μm ,15 0mm× 4 .6mm)色谱柱 ,流动相为 2 0mmol/L磷酸盐缓冲液 (pH 6 .0 )含 12mmol/Lβ 环糊精∶甲醇 (70∶30 ,V/V) ,流速为 0 .8mL/min ,温度为 17℃。DL 西孟坦对映体的保留时间分别为 2 2 .5和 2 4 .5min ,分离度为 1.5 7。     

固相萃取/反相高效液相色谱法对血清中胰岛素的测定

采用固相萃取纯化血清样品,荧光胺柱前衍生,建立了反相高效液相色谱/荧光检测血清中胰岛素的方法.色谱条件:LunaC5柱(100 mm×4.6 mm,5 μm)分离,流动相为乙腈-Tris缓冲溶液(10 mmol/L,pH 8.5)梯度洗脱,流速:1 mL/min.激发波长(λex)273 nm,发射波长(λem)476 nm,进样量20 μL.结果表明:胰岛素在10 ～800 μg/L范围内峰面积和质量浓度呈良好的线性关系,相关系数r为0.997 1;检出限为5 μg/L.日内、日间精密度分别为1.5%、1.0%,回收率为92%.     

反相高效液相色谱法测定抗生素AGPM发酵液中的主要成分

主要探讨了高效液相色谱法(HPLC)测定新型抗生素AGPM发酵液中主要成分的可行性,优化了反相高效液相色谱法分析发酵液组分的色谱条件。在Waters TM Nova- Pak C18 色谱柱上,以甲醇-0.01 mol/L 乙酸铵溶液(pH 5.20)为流动相,其中甲醇的体积分数按线性梯度方式在60 min内从30%升至50%,流动相流速为1.0 mL/min ,在330 nm处进行紫外检测,发酵液中各主要组分均能很好地分离。采用该方法对AGPM发酵液中5种主要组分的线性范围、检出限、相对标准偏差     

固相萃取-反相高效液相色谱法测定隐形眼镜护理液中聚胺丙基双胍

建立了固相萃取-反相高效液相色谱法测定隐形眼镜护理液中聚胺丙基双胍的方法.用Spe-ed WCX固相萃取柱(500 rag/3 mL)将样品浓缩富集后,以Shiseido C18色谱柱(150 min×4.6 mm,5 pm)为分离柱,以pH 4.0的乙腈-20 mm01.L-1乙酸铵(体积比16比84)为流动相,检测波长为235 nm,7 min内测定样品中聚胺丙基双胍.方法检出限(S/N=3)为0.046 mg·L-1,相对标准偏差为3.3%(n=7),线性范围为100～800 mg·L-1,加标回收率93.5%～110.9%.     

超高效液相色谱法同时测定碳酸饮料中的22种食品添加剂

建立了超高效液相色谱(UPLC)同时测定碳酸饮料中22种食品添加剂的方法.采用Waters BEH C18(1.7 μm,2.1 mmx50mm)色谱柱,以0.01 mol/L乙酸铵(pH 5.8 ～6.0)和乙腈为流动相进行梯度洗脱,在35℃柱温,0.2 ml/min流速下,采用二极管阵列检测器在217、232、25...     

高效液相色谱-质谱法测定发酵液中的喷司他丁

建立了测定发酵液中喷司他丁含量的反相高效液相色谱-质谱分析方法。采用的色谱条件: 色谱柱为Hypersil ODS2柱(250 mm×4.6 mm, 5 μm);流动相为甲醇/乙腈/10 mmol/L乙酸铵(pH 7.6)(2.5/2.5/95, v/v/v),流速为1.0 mL/min;检测波长为280 nm;柱温为40 ℃;进样量为10 μL。喷司他丁在1.0～100 mg/L范围内有良好的线性关系,相关系数为0.9999。该方法精密度好,稳定性高,能简便、快速、准确地测定发酵液中喷司他丁的含量。     

反相离子对色谱-蒸发光闪射法测定α-复方酮酸片中的5种有效成分

建立了反相离子对色谱结合蒸发光散射检测器同时测定α-复方酮酸片中消旋羟蛋氨酸钙(HMACa),酮缬氨酸钙(KVCa),消旋酮异亮氨酸钙(KILCa),酮亮氨酸钙(KLCa)和酮苯丙氨酸钙(KPACa)含量的方法。液相色谱条件:以乙腈-20 mmol/L乙酸铵缓冲溶液(含30 mmol/L正戊胺,pH 7.0)为流动相进行梯度洗脱,流速为0.6 mL/min,柱温为35℃。蒸发光散射检测器(ELSD)漂移管温度为50℃,载气流速为1.0 mL/min。方法的线性范围为20~150 mg/L,5种物质的相关系数(r)均大于0.999,检出限分别为7.05,16.66,8.00,8.00和14.28 mg/L,样品加标回收率在87.5%~109.8%之间,相对标准偏差(RSD)均小于3.2%(n=5)。方法适用于α-复方酮酸片中5种有效成分的同时测定。     

以L-酒石酸正己酯-硼酸配合物作为反相液相色谱手性流动相添加剂拆分5种β-受体阻滞剂

本文以L-酒石酸正己酯-硼酸配合物为手性流动相添加剂,建立了普萘洛尔、艾司洛尔、美托洛尔、比索洛尔、索他洛尔和阿替洛尔六种β-受体阻滞剂的反相高效液相色谱手性分离方法。对影响对映体分离的主要因素：L-酒石酸正己酯、硼酸浓度,缓冲溶液种类、浓度、pH值和有机改性剂-甲醇含量等进行了详细考察。最佳色谱条件为：Venusil MP-C18色谱柱（4.6 mm × 250 mm,5 μm）,流动相为15 mmol/L乙酸铵-甲醇（体积比为20: 80或30: 70,含60 mmol/L硼酸,70 mmol/L L-酒石酸正己酯,醋酸调节pH值6.00）,检测波长214 nm。在最佳分离条件下,五对对映体（普萘洛尔、艾司洛尔、美托洛尔、比索洛尔、索他洛尔）可以分别获得基线分离。     

Investigation of zinc binding metallothioneins' polymerization in tris(hydroxymethyl)-aminomethane buffer by coupling of size exclusion chromatography with electrospray ionization mass spectrometry

Polymerization of metallothioneins (MTs) is one of the commonly encountered puzzles in researching the structure and function of metallothioneins. In this work, a method involving SEC coupled with negative ion electrospray ionization mass spectrometry (ESI-MS) detection has been developed for the study of zinc binding MTs’ polymerization in tris(hydroxymethyl)-aminomethane (TRIS) acetate buffer at physiological pH. This hyphenated technique allows separating the different polymeric states of MTs by SEC, followed by on-line identification of the individual MT subisoforms in each polymeric peak by ESI-MS detection. Purified MT subisoforms (MT-2d and MT-2a), MT-2d and MT-2a mixture and rabbit liver MT complexes were investigated in the experiments to confirm the results obtained. From the results, both oxidative polymerization and non-oxidative oligomerization were found. The cystein-dependent oxidation results in the tetrameric peak as shown in the chromatograms of oxidized MT-2d, and stable dimeric and monomeric of MT were detected in this peak by MS. For the dimeric and trimeric peaks, different MT subisoforms were detected. In the five major subisoforms detected in rabbit liver MT complexes, MT-2a and MT-2c exist primarily as trimer, while MT-2e, MT-2d and MT-1a exist mainly as dimer. Our results suggest that in the three kinds of polymers, dimer, trimer and tetramer that were found in samples, the tetramer comes from the oxidation of MT molecular; for the dimer and trimer resulting from cystein independent oligomerization, they are closely associated with the charge of subisoform.     

Separation and characterization of rabbit liver apothioneins by capillary electrophoresis coupled to electrospray ionization time-of-flight mass spectrometry

The present study establishes a method for the separation and characterization of rabbit liver metallothionein (MT) subisoforms by capillary electrophoresis coupled to electrospray ionization time-of-flight mass spectrometry (CE-ESI-TOF-MS) via a sheath-flow interface. Directly coupled-CE-MS enables the extraction of specific molecular weight information and thereby facilitates the identification of peaks when no reference materials are available, as in the case of MT subisoforms. The analysis described here revealed the presence of the apothioneins MT-1a, MT-2d, and MT-2e, belonging to MT-I sample, and MT-2a, MT-2b, and MT-2c, belonging to MT-II. Several non-N-acetylated forms were also detected as traces appearing with their respective acetylated forms in both samples. Similar results were found when MALDI-TOF experiments were performed, identifying all the sequenced rabbit liver MTs as apo-MT-forms, as in the CE-ESI-MS coupling.     

金属硫蛋白异构体及亚型异构体的液相色谱分离与质谱鉴别

建立了金属硫蛋白(MT)异构体及亚型异构体的色谱分离与质谱鉴别方法。将金属硫蛋白混合物通过弱阴离子DEAE Sephadex A-25离子交换柱,结合离线电感耦合等离子体质谱(ICP-MS)对锌诱导金属硫蛋白的两个异构体MT-1和MT-2进行分离和检测;利用Sephadex G-25凝胶排阻色谱柱对得到的两个金属硫蛋白异构体进行脱盐;探索脱盐后的金属硫蛋白异构体在不同色谱条件下的C18反相色谱柱上的保留行为,进而实现各个亚型异构体的分离;通过在线电喷雾质谱检测实现了对金属硫蛋白各个亚型异构体的鉴别。结果表明,通过优化色谱条件,由离子交换色谱及凝胶排阻色谱得到的金属硫蛋白各亚型异构体在酸性条件下均得到了良好的分离,质谱检测结果与前人的文献报道结果一致。该方法可使金属硫蛋白各异构体均达到最佳的分离效果。     

大鼠肾脏汞结合金属硫蛋白的联用技术表征研究

通过凝胶色谱(SEC)及反相液相色谱(RPC)分别与电感耦合等离子体质谱(ICP-MS)和电喷雾电离质谱(ESI-MS)联用的技术，对灌喂HgCl₂大鼠的肾组织中诱导的多金属结合金属硫蛋白(MT)的结构进行表征分析。通过SEC-ICP-MS曲线上的金属信号及紫外检测的MT光谱吸收特征，可确认组织提取液中MT在色谱曲线上的位置。与对照组相比，灌喂HgCl₂大鼠肾中Hg-MT的诱导量显著增加，与此同时，Cu-MT在染毒组的诱导量也相应增加，说明MT在染汞大鼠肾脏中起着对汞的解毒和对铜的调节作用。MT样品提取物经葡聚糖凝胶(G-75)柱分离纯化并用透析法脱盐后，用细内径反相色谱柱分离，进行梯度洗脱，并与ESI-MS联用，可获得总离子流色谱图及与色谱峰对应的质谱图，通过对质谱信号的解析及参考脱金属MT构型的有关报道，对汞诱导大鼠肾组织MT的不同亚型和次亚型分子结构进行推断。结果证明，在灌喂HgCl₂大鼠的肾组织中存在着一系列汞结合的MT形态。     

Pressure-assisted capillary electrochromatography with electrospray ionization-mass spectrometry based on silica-based monolithic column for rapid analysis of narcotics

A pressure-assisted CEC (pCEC) with ESI-MS based on silica-based monolithic column was developed for rapid analysis of narcotics. Combining the extremely high permeability and separation efficiency of silica-based monolithic column with the high selectivity and sensitivity of pCEC-ESI-MS, the developed system exhibited its prominent advantages in separation and detection. A systematic investigation of the pCEC separation and ESI-MS detection parameters was performed. Experiment results showed that the optimized separation efficiency could be obtained at 8 bar assisted pressure with 25 kV separation voltage, using the solution containing 65% ACN v/v and 20 mmol/L ammonium acetate with pH 6.0 as running buffer. 3 microL/min of sheath liquid was considered as the optimized flow rate since it could provide the maximum signal intensity. Under the optimum conditions, the tested five narcotics could be completely separated within 10 min with the detection limit in the range of 2.0-80 nmol/L. The proposed method has been successfully used for detection of narcotics in real urine samples.     

Production of Metallothionein Polyclonal Antibodies Using Chickens as Model

The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.     

Characterization of the metal composition of metallothionein isoforms using reversed-phase high-performance liquid chromatography with atomic absorption spectrophotometric detection

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to separate metallothionein (MT) isoforms and on-line atomic absorption spectrophotometric (AAS) detection was used to quantitatively determine their metal content. With this coupled system (HPLC-AAS), it was possible to determine the zinc, cadmium and copper content of individual horse kidney MT isoforms. When rabbit liver MT and the purified isoforms (MT-1 and MT-2) were subjected to RP-HPLC and the zinc-containing peaks of the MT sample to MT-1 or MT-2. HPLC-AAS was used to identify zinc-induced MT in heat-treated cytosol from turkey hen liver, thereby demonstrating its application to the analysis of crude tissue extracts. A standard curve was established using turkey liver MT for the quantitative determination of the zinc content of MT isoforms. There was excellent linear correlation between the micrograms of zinc bound to MT injected onto the column (ranging from 0.34 to 3.43 micrograms of MT-bond zinc) and the integrated peak area of the atomic absorbance for zinc. Using this standard curve, it was possible to quantitate the amount of MT-bound zinc in cytosol extracts of cultured turkey embryo hepatocytes exposed to varying levels of supplemental zinc in the culture medium.     

Separation of metallothionein isoforms of mouse liver cytosol by capillary zone electrophoresis

Metallothionein (MT) isoforms of mouse liver cytosol were separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH, samples prepared from non-treated, heat-treated, and ethanol-precipitated specimens were compared. The liver was homogenized in three kinds of media, 0.25 M sucrose containing 100 mM Tris-HCl buffer at pH 7.4 (BS), BS containing 1% ascorbic acid (BS-C), and BS containing 5 mM beta-mercaptoethanol (BS-M). Mouse liver was used 24 h after subcutaneous injection of 50 mg Zn kg(-1). In the non-treated specimen of the cytosol fraction, the MT-2 isoform was separated in all three media, while the MT-1 isoform was difficult to identify. In the ethanol-precipitated specimen, MT isoforms were separated well using either BS or BS-C. However, when BS-M was used, a small MT-2 peak was obtained the MT-1 peak could not be identified. MT-1 isoform in the heat-treated specimen was difficult to identify. In contrast, MT-2 isoform was separated well in all three kinds of media. In the non-treated specimen of the control liver cytosol, the MT-2 isoform was detected using all three media, the MT-1 peak was undetected. Based on these results, MT isoforms can be detected in the crude cytosol fraction of liver using CZE combined with a polyacrylamide-coated tube at neutral pH.     

兔肝金属硫蛋白亚型异构体的高效液相色谱分离与ESI-MS,MALDI-TOF-MS鉴定

由于金属硫蛋白(MT)基因的多态性,决定不同亚型的MT异构体的存在,MT亚型异构体的结构是MT功能研究的基础.通过离子交换柱可将MT分成MT-1和MT-2两个异构体,用不同条件的反相高效液相色谱(RP-HPLC)可将MT-1和MT-2分成不同的亚型异构体,并利用MALDI-TOFMS和LC-ESI-MS对比确定了它们的分子量.结果表明,兔肝MT在不同的pH条件下分离得到不同分子量的亚型异构体.在酸性条件下,MT-1可分为2个主要亚型异构体,分子量分别为6149.0和6244.5,而MT-2主要分为3个亚型异构体,分子量分别为6149.0,6244.0和6127.0.MT-1和MT-2有2个亚型异构体分子量相同的异构体存在.在酸性条件下,MT-1的2个异构体及MT-2分子量为6127的亚型异构体可稳定存在.     

Species analysis of metallothionein isoforms in human brain cytosols by use of capillary electrophoresis hyphenated to inductively coupled plasma-sector field mass spectrometry

A new approach for the speciation of metallothioneins (MT) in human brain cytosols is described. The analysis is performed by application of a newly developed coupling of capillary electrophoresis (CE) with inductively coupled plasma-sector field mass spectrometry (ICP-SFMS). Isoforms of metallothioneins are separated from 30-100 microliter sample volumes by CE and the elements Cu, Zn, Cd, and S are detected by use of ICP-SFMS. The extraction of cytosols is the first step in the analytical procedure. Tissue samples from human brain are homogenized in a buffer solution and submitted to ultra-centrifugation. The supernatant is defatted and the cytosol pre-treatment is optimized for CE separation by matrix reduction. The buffer concentration and pH used for capillary electrophoretic separation of metallothionein from rabbit liver were optimized. CE with ICP-MS detection is compared to UV detection. In the electropherograms obtained from the cytosols three peaks can be assigned to MT-1, MT-2, and MT-3. As an additional method, size-exclusion chromatography (SEC) is applied. Fractions from an SEC separation of the cytosol are collected, concentrated, and then injected into the CE. The detection of sulfur by ICP-SFMS (medium resolution mode) and quantification by isotope dilution have also been investigated as a new method for the quantification of MT isoforms. The analytical procedure developed has been used for the first time in comparative studies of the distributions of MT-1, MT-2, and MT-3 in brain samples taken from patients with Alzheimer's disease and from a control group.