A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

Simultaneous Determination of Amlodipine with H₁-Receptor Antagonists by Reversed Phase High Performance Liquid Chromatography and Application to Interaction Studies

A rapid, fast and precise method has been developed and validated for the simultaneous determination of amlodipine with H1-receptor antagonists (cetirizine, fexofenadine, and buclizine) from dosage forms. The chromatography was performed on a Purospher? Star, C18 (5 mm, 250 × 4.6 mm) column using acetonitrile: buffer (0.01 mM) (40:60, v/v, pH adjusted to 3.0), as a mobile phase. The mobile phase was pumped at a flow rate of 1.0 mL·min-1 and UV detection was performed at 240 nm. The method was validated for linearity, accuracy, precision and specificity. The method was applied to study the interaction between amlodipine and H1-receptor antagonists. These interactions were carried out in simulated gastric juice (pH 1), simulated full stomach (pH 4), blood pH (pH 7.4) and simulating GI (pH 9). The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques. In most cases the in vitro availability of amlodipine was decreased. It was observed that the change in in vitro availability was pH dependent.

Quantitative analysis by reversed-phase high-performance liquid chromatography and retinal neuroprotection after topical administration of moxonidine

AIM:To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography(RP-HPLC),and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure(IOP)model.METHODS:The eyes of albino rabbits were administered topically and ipsilaterally with 0.2%moxonidine.A RPHPLC method was employed for the identification and quantification of moxonidine between 2 and 480 min,which presented in the aqueous humor and iris-ciliary body.Flash electroretinography(F-ERG)amplitude and superoxide dismutase(SOD)level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP.Histological and ultrastructural observation underwent to analyze the changes of retinal morphology,the inner retinal layers(IRL)thickness,and retinal ganglion cell(RGC)counting.RESULTS:Moxonidine was detectable between 2 and 480 min after administration,and the peak concentration developed both in the two tissues at 30 min,0.51μg/m Lin aqueous humor and 1.03μg/g in iris-ciliary body.In comparison to control,F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15(P<0.01)in the high IOP model;SOD levels were significantly higher at all time-points(P<0.01)with a maximum level of 20.29 U/mgprot at day 15;and RGCs were significantly higher(P<0.05).CONCLUSION:Moxonidine is a viable neuroprotective agent with application to high IOP model.All layers of retina,including RGC layer,retinal nerve fiber layer and INL,are more preserved after moxonidine administration.SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.

Isolation and Purification of Unstable Iridoid Glucosides from Traditional Chinese Medicine by Preparative High Performance Liquid Chromatography Coupled with Solid-phase Extraction

An efficient preparative method was successfully developed for isolation and purification of unstable components from medicinal plant extracts, using a combined method of preparative high performance liquid chro-matography(HPLC) and solid-phase extraction(SPE). The aim of this study was to obtain an effective method with high preparative efficiency and importantly to avoid the transformation of unstable compounds. The preparative HPLC system was based on an LC/MS controlled four-channel autopurification system. The SPE method was performed with a C18 packing material to trap the target compounds and to remove the acidic additive derived from the mobile phase. Using this method, the unstable iridoid glucosides(IGs) as model compounds were successfully isolated and purified from the extract of Hedyotis diffusa Willd. Six IGs(including one new minor IG) and one nucleotide compound were simultaneously obtained, each with a purity of 91% as determined by HPLC. The structures of the isolated compounds were identified by UPLC/Q-TOF MS, UV, 1D and/or 2D NMR. It was demonstrated that the combination of preparative HPLC with SPE is a versatile tool for preparative purification of unstable compounds from complex natural products.

THYMINE BONDED-STATIONARY PHASE FOR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A new type of HPLC stationary phase containing thymine derivative was successfully prepared.It was found to give selective separation of nucleic acid bases and several purine derivatives,such as caffeine and theophylline.The retention behaviour and elution order of the solutes were interpreted in terms of molecular structure.

Determination of Quinolone Antibiotics in Water Using Solid Phase Extraction-High Performance Liquid Chromatography-Fluorescence Method

[Objective] To develop a solid phase extraction-high performance liquid chromatography-fluorescence method for determination of quin- olone antibiotics in water. [ Metbod] The standard curves of four quinolones （norfloxacin, ciprofloxacin, Iomefloxacin and enrofloxacin） were pre- pared. The detection limit in water and recovery were determined. The water samples collected from different areas, river and tap water were trea- ted using solid-phese extraction method and analyzed by high performance liquid chromatography. Then the concentration of quinolones antibiotics was determined by fluorescence method. [ Result] The detection limit of quinolone antibiotics in water was 0.083 -0.248 μg/L, and their recovery was 63.7% -134.1%. The four quinolone antibiotics at different levels were detected in various water samples, and the total concentration of quin- olone antibiotics was 0.045 -3.969 μg/L. The total concentration of quinolone antibiotics was higher in the water samples collected from rivers in Shenzhen area than in the sewage samples. The four quinolone antibiotics could be detected in all tap water samples. [ CoaduLsion ] The solid phase extraction-high performance liquid chromatography-fluorescence method is feasible and effective to detect quinolones in water. In addition, this method needs low cost and can meet requirements of daily monitorina and analysis.

Trace Determination of Tamoxifen in Biological Fluids Using Hollow Fiber Liquid-Phase Microextraction Followed by High-Performance Liquid Chromatography-Ultraviolet Detection

The applicability of hollow fiber liquid-phase microextraction (HF-LPME) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was evaluated for the extraction and determination of tamoxifen (TAM) in biological fluids including human urine and plasma. The drug was extracted from a 15 mL aqueous sample (source phase;SP) into an organic phase impregnated in the pores of the hollow fiber (membrane phase;MP) followed by the back-extraction into a second aqueous solution (receiving phase;RP) located in the lumen of the hollow fiber. The effects of several factors such as the nature of organic solvent, compositions of SP and RP solutions, extraction time, ionic strength and stirring rate on the extraction efficiency were examined and optimized. An enrichment factor of 360 along with substantial sample clean up was obtained under the optimized conditions. The calibration curve showed linearity in the range of 1 - 500 ng?mL–1 and the limit of detection was found to be 0.5 ng?mL–1 in aqueous medium. A reasonable relative recovery (≥89%) and satisfactory intra-assay (3.7% - 4.2%, n = 3) and inter-assay (7.5% - 7.8%, n = 3) precision illustrated good performance of the analytical procedure in spiked human urine and plasma samples.

Porous Monoliths: Stationary Phases of Choice for High Performance Liquid Chromatography in Various Formats

Modern porous monoliths have been conceived as a new class of stationary phases for high performance liquid chromatography(HPLC) in classical columns in the early 1990s and later extended to the capillary format.These monolithic materials are prepared using simple processes carried out in an external mold(inorganic monoliths) or within the confines of the column(organic monoliths and all capillary columns).These methods afford macroporous materials with large through-pores that enable applications in a rapid flow-through mode.Since all the mobile phase must flow through the monolith,the convection considerably accelerates mass transport within the monolithic separation medium and improves the separations.As a result,the monolithic columns perform well even at very high flow rates.The applications of monolithic capillary columns are demonstrated on numerous separations in the HPLC mode.

Determination of seven active components in Salvia miltiorrhiza herb by matrix solid phase dispersion combined with ion liquid extraction followed by high performance liquid chromatography

A low cost,rapid and sensitive preparation method of silica gel supported ionic liquid(SGSIL)combined with matrix solid phase dispersion(MSPD)followed by high performance liquid chromatography(HPLC)with ultraviolet detection(UV)is proposed,and it was applied to determine the seven active compounds in Salvia Miltiorrhiza herb.SGSIL and ionic liquid[BMIM]BF4 were used as the adsorbent and the green elution reagent in the MSPD procedure.Several extraction conditions including type of filler and elution solvent,the volume of elution solvent,material liquid ratio were optimized.Under the optimum conditions,the SGSIL-MSPD-HPLC method showed a low limit of detection(LOD,S/N=3)of 0.0122-0.8788μg/mL for standard solution,limit of quantification(LOQ,S/N=10)of 0.0406-2.9292μg/mL for standard solution,wide linear range from 1.56 to 2000μg/mL for all compounds for standard solution,correlation coefficients(r)of more than 0.9990,acceptable reproducibility(relative standard deviations,RSDs<3.54%),and precision of RSDs<3.36%for intra-day,RSDs<3.50%for inter-day.The satisfactory recoveries ranged from 96.4 to 102.5,with RSDs less than 3.45%.The developed SGSIL-MSPD method is easier and more suitable for the determination of the seven active compounds in Salvia Miltiorrhiza herb than the traditional ultrasonic extraction.It was an effective and efficient method for the extraction and quantification of the seven active compounds in traditional Chinese herbal samples.

Molecularly Imprinted Micro-Solid-Phase Extraction for the Selective Determination of Phenolic Compounds in Environmental Water Samples with High Performance Liquid Chromatraphy

2,4,6-trichlorophenol molecularly imprinted suspension polymer has been prepared and applied to the molecularly imprinted micro-solid-phase extraction procedure for selective preconcentration of phenolic compounds from environmental water samples. The influence of functional monomer, cross-linker, polymerization condition, porogen, and the ratio of template molecule and functional monomer to cross-linker on the size of the obtained particles were investigated. It was found that methyacrylic acid as functional monomer, divinylbenzene as cross-linker, the molar ratio of template molecule and functional monomer to cross-linker was 1:4:20, the amount of AIBN was 100 mg, ultraviolet radiation at 365 nm were the optimal conditions, and at these conditions, the polymers had the best adsorption efficiency and had the monodispersity of 2 - 3 μm microsphere particles. The characteristics of the MIMSPE method were valid by high performance liquid chromatography. This MIMSPE-HPLC method has been successfully applied to the direct preconcentration and determination of phenolic compounds (phenol, 4-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol) in environmental water samples.

固相萃取结合高效液相色谱-串联质谱测定胎便中14种全氟和多氟烷基化合物

全氟和多氟烷基化合物(PFASs)是一类人工合成的物质,由于其热稳定、疏水、疏油等优良性质而被广泛使用于生活和生产中.PFASs具有环境持久性、生物累积性、多种毒性等特性,且可以通过胎盘屏障进入到胎儿体内,进而对胎儿健康产生潜在危害.胎便中积累了妊娠期间暴露于胎儿的外源性化合物,可用于监测PFASs对胎儿的宫内暴露特征.本研究基于固相萃取结合高效液相色谱-串联质谱技术,建立了胎便中14种PFASs的分析方法.采用乙腈/水(9∶1,V/V)对0.2 g冻干胎便样品进行超声提取,提取液经Envi-carb和Oasis WAX小柱固相萃取,0.1%氨甲醇洗脱.以10 mmol·L^(−1)乙酸铵水溶液和乙腈作为流动相对目标化合物进行梯度洗脱,采用Acquity UPLC BEH C18色谱柱进行分离,基于多反应监测负离子模式采集,内标法定量.结果表明,在2、5、20 ng·g^(−1)的加标浓度下,14种PFASs的回收率为65%—149%,相对标准偏差为3%—22%,方法检出限(MDLs)为0.001—0.149 ng·g^(−1),方法定量限(MQLs)为0.003—0.495 ng·g^(−1).使用该方法测定了10个胎便样品,ΣPFASs浓度范围为<MDLs—2.49 ng·g^(−1).该方法操作简单、便捷、灵敏度高且定量准确,为系统性研究胎便中PFASs的赋存特征及暴露风险提供了技术基础.

分子印迹固相萃取-高效液相色谱法测定果蔬中4种有机磷类农药的残留量

以4种目标物丙溴磷、毒死蜱、二嗪磷、辛硫磷的混合物作为模板分子,乙二醇二甲基丙烯酸酯为交联剂、甲基丙烯酸为功能单体,通过水热聚合法制备分子印迹聚合物(MIPs),将MIPs填充在聚丙烯空柱中制备MIPs固相萃取(MIPs-SPE)柱。将果蔬样品洗净、晾干、粉碎后,分取5.00 g,加入2 g无水硫酸钠,研磨均匀。加入30 mL乙腈,超声1.5 h,离心10 min。分取1 mL上清液过活化好的MIPs-SPE柱,用4 mL正己烷淋洗,6 mL体积比9∶1的甲醇-乙酸混合溶液洗脱。收集洗脱液,于35℃氮气吹干,用1 mL乙腈定容,用高效液相色谱法分析。结果显示:MIPs可特异性识别4种目标物,对目标物的吸附量约非分子印迹聚合物(NIPs)的2.5倍,对硫磷、马拉硫磷、甲拌磷、甲基毒死蜱的吸附量显著低于目标物的;经MIPs-SPE柱净化后,样品大部分基质成分被去除,目标物测定无干扰。4种目标物的浓度在0.005~2.0μmol·L^(−1)内和对应的峰面积呈线性关系,检出限(3S/N)为0.0015~0.0040μmol·L^(−1);在0.01,0.5,1.0,2.0 mg·kg^(−1)加标浓度水平下,4种目标物的回收率为85.9%~102%,测定值的相对标准偏差(n=5)为3.0%~7.1%。方法用于4种果蔬样品的分析,在草莓样品中检出了丙溴磷(检出量为0.07 mg·kg^(−1)),甘蓝样品中检出了辛硫磷(检出量为0.05 mg·kg^(−1)),其他样品中均未检出这4种目标物。

分散固相萃取净化-超高效液相色谱-串联质谱法测定蜂蜜中双甲脒、杀虫脒及其代谢物

该研究建立了分散固相萃取-超高效液相色谱-串联质谱(dispersive solid phase extraction-ultra-performance liquid chromatography,UPLC-MS-MS)检测蜂蜜中双甲脒、杀虫脒及其代谢物残留的分析方法。蜂蜜样品2 g加入10 mL水溶液充分混匀,经1%(体积分数)氨化乙腈超声辅助提取后离心,利用N-丙基乙二胺、C18、氨基键合硅胶混合材料进行分散固相萃取净化,采用ACQUITY UPLC BEH C18(2.1 mm×50 mm,1.7μm)色谱柱分离,以甲醇和0.1%(体积分数)甲酸水溶液为流动相进行梯度洗脱,多反应监测模式正离子扫描分析。6种农药及其代谢物平均回收率为84.1%~113.8%,相对标准偏差为0.9%~6.0%,检出限为0.2~0.8μg/kg,定量限为0.7~2.5μg/kg。实验结果表明该方法快速简便、灵敏度高,适用于蜂蜜中双甲脒、杀虫脒及其代谢物残留同时分析。

固相萃取-一标多测高效液相色谱法测定化妆品中5种苯并三唑类防晒剂

建立了固相萃取技术结合高效液相色谱测定化妆品中5种苯并三唑类防晒剂的一标多测定量分析方法。该方法待测样品使用HLB固相萃取小柱净化处理,以亚甲基双-苯并三唑基四甲基丁基酚为内参物,通过建立该物质与另外4种物质的相对校正因子,计算出各物质的含量。研究了不同进样量、不同流动相流速及不同柱温对相对校正因子的影响,比较了一标多测法和外标法的计算结果。结果表明,5种物质的线性关系良好(R^(2)≥0.9997),检测限和定量限分别为4.00~26.43μg/L和10.00~60.42μg/L;各物质的相对校正因子重复性较好,其相对标准偏差(RSD)为0.25%~1.16%;两种方法计算结果无明显差异,其相对平均偏差(RAD)为0.12%~2.19%。该方法操作简单,使用标准物质少,检测效率高,可用于化妆品中5种防晒剂的含量测定和质量控制。

三相中空纤维-液相微萃取-高效液相色谱法快速测定烟用香精中9种防腐剂

烟用香精不仅可应用于传统卷烟,其在电子烟、加热卷烟及口含烟等新型烟草制品中均有着广泛应用。烟用香精的含水量较高,为避免香精产品在生产、储存和运输环节发生霉变,往往加入一定量的防腐剂(如山梨酸、苯甲酸及对羟基苯甲酸酯类化合物),以抑制细菌的生长。然而,若超标添加上述防腐剂,亦会对消费者健康带来不良影响。本研究基于三相中空纤维-液相微萃取(3P-HF-LPME)-高效液相色谱法,建立了一种同时测定烟用香精中山梨酸、苯甲酸及7种对羟基苯甲酸酯类防腐剂的分析方法。对仪器参数和HF-LPME条件进行优化,采用Agilent Poroshell 120 EC-C18色谱柱(100 mm×3 mm,2.7μm)对9种防腐剂进行分离,以甲醇、0.02 mol/L乙酸铵水溶液(含0.5%乙酸)和乙腈作为流动相进行梯度洗脱,检测波长为226 nm(苯甲酸)和258 nm(山梨酸和7种对羟基苯甲酸酯类化合物)。以二己醚为萃取溶剂,15 mL样品溶液(pH 4)为样品相,氢氧化钠水溶液(pH 12)为接受相,在800 r/min下搅拌萃取30 min。实验结果表明,9种目标分析物在各自的线性范围内线性关系良好,相关系数(r)≥0.9967,检出限为0.02~0.07 mg/kg,定量限为0.08~0.24 mg/kg,富集倍数为30.6~91.1,在3个加标水平下的回收率为82.2%~115.7%,相对标准偏差(n=5)≤15.0%。该方法简单高效,准确可靠,灵敏度高,适用于烟用香精中防腐剂的快速筛查。

基于分散固相萃取-超高效液相色谱-串联质谱法的大豆农药残留测定研究

本研究建立了一种分散固相萃取-超高效液相色谱-串联质谱测定吡虫啉、烯啶虫胺等6种常见农药残留的方法。方法学验证结果显示,所有农药的标准曲线线性良好(R^(2)>0.999),方法灵敏度良好;加标回收试验中不同加标浓度的相对标准偏差均小于10%,验证了方法的稳定性和可靠性。应用此方法对20份市售大豆样品进行农药残留检测,结果显示所有样品中农药残留均未超出或远低于国家规定的最大残留限量,合格率为100%。

PEAK IDENTIFICATION FROM INTERACTION INDEX IN REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

The interaction index c which was derived from the fundamental retention equationlogk’ =a + cC_B in reversed--phase high--performancc liquid chromatography (RP-HPLC) quan-titatively describes the difference in the interaction between solute--strong. solvent andsolute--weak solvent; it has shown to be a constant for a specific solute even when columnsystems with different C18 packings are used. The theoretical basis for peak identificationby using interaction index has been proposed, which was based on the a,c values on stan-dard C18 column by utilizing linear a-a plots on column pairs and the linear relationship be-tween parameters a and c for the structural related compounds. Through the establishmentof parameters a,c data based on the standard C18 column for a certain type of compounds,the retention of thesc compounds on various C18 columns can be predicted. Typical exam-ples have been given to verify the correctness of this method.

高效液相色谱-质谱技术在蛋白质组学中的应用

蛋白质组学研究在生物医学领域发挥了重要作用,然而研究面临的主要难点在于其研究对象的复杂性和多样性。随着质谱技术的快速发展,高效液相色谱-质谱(HPLC-MS)分离分析复杂生物样品已经成为蛋白质组学研究的基础工具。蛋白质组学的研究从肽段分离,延伸到蛋白质和蛋白质复合物的分离,随着分析物的分子质量不断增大,其结构和组成复杂性也持续增加,分子特性也发生改变。面对不同的蛋白质组学研究对象,选择不同的分离模式、分离条件以及固定相参数是进行深度蛋白质组学研究的关键。本文综述了实验室常用的液相色谱分离模式,包括反相色谱(RPLC)、亲水相互作用色谱(HILIC)、疏水相互作用色谱(HIC)、离子交换色谱(IEC)和体积排阻色谱(SEC),以及其不同的组合模式与质谱联用在自下而上(bottom-up)分析、自上而下(top-down)分析、蛋白-蛋白相互作用分析中应用的研究。具体分析了色谱流动相与被分析对象之间的兼容性问题、色谱流动相与质谱兼容性问题,以及多维色谱中不同色谱模式之间流动相的兼容性问题。重点关注存在不兼容问题时研究者所提出的解决方案。此外,本文还评述了HPLC-MS结合样本前处理的方法在外泌体和单细胞蛋白质组学中的应用研究。总之,文章聚焦于近年来HPLC-MS技术在蛋白质组学中的研究进展,旨在为未来蛋白质组学领域的研究提供参考。

固相萃取-高效液相色谱法同时测定水产品中15种磺胺类药物残留

建立固相萃取-高效液相色谱法(solid phase extraction-high performance liquid chromatography,SPE-HPLC)同时检测水产品中15种磺胺类药物(sulfonamides,SAs)残留。样品用乙酸乙酯提取,正己烷脱脂,PCX(polymer cation exchange)固相萃取柱净化,采用高效液相色谱法检测,色谱条件为:Waters C18(4.6 mm×250 mm,5.0μm)色谱柱分离,以甲醇和含体积分数1.1%乙酸的PBS缓冲液(0.01 mol/L)为流动相进行梯度洗脱,检测波长为270 nm。15种SAs在65 min内达到完全分离,在0.5~10.0μg/m L时线性关系良好,相关系数均大于0.9900;方法检出限为10~20μg/kg,定量限为20~60μg/kg;对空白鱼肉样品进行了3个质量分数水平(500、1000、2000μg/kg)的加标实验,测定其空白加标回收率为73.1%~109.6%,相对标准偏差(relative standard deviations,RSDs)在1.0%~6.7%。实验建立的检测方法重现性和稳定性好、检出限低。对江西10个不同品种的水产品中磺胺类药物残留进行了采样检测,结果均未检出,表明江西水产品中磺胺类药物残留情况并不严重。

地表水中34种新兴污染物的超高效液相色谱-四极杆-飞行时间质谱快速筛查

目的 建立基于超高效液相色谱-四极杆-飞行时间质谱(ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry,UHPLC-Q-TOF-MS)技术快速筛查地表水中34种新兴污染物的方法。方法 通过正交试验设计优化固相萃取(solid phase extraction,SPE)前处理条件,样品经Oasis~? HLB和Oasis~? MCX固相萃取柱串联富集萃取。萃取物采用Kinetex~? EVO C18色谱柱分离,0.1%甲酸水溶液和0.1%甲酸甲醇溶液梯度洗脱,Q-TOF-MS “fullscan”和“targeted MS/MS”模式检测34种新兴污染物,建立34种新兴污染物母离子、子离子及保留时间的数据库。结果 地表水中34种新兴污染物在各自线性范围内呈良好的线性关系,相关系数(r)大于0.97。检出限为0.2~10 ng/L,回收率为81.2%~119.2%,日间精密度为0.78%~18.70%。将方法应用于多份地表水样品的检测,共检出6种新兴污染物,质量浓度为1.93~157.71 ng/L。结论 该方法能简单、快速地对地表水中的多种痕量级新兴污染物进行筛查分析。