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.

INTERSECTION POINT RULE OF THE RETENTION VALUE AND NORMAL BOILING POINT OF THE HOMOLOGUES IN REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

In this paper, a linear relationship between the logarithm of capacity factor k and normal boiling point to of the homologues has been derived, based on the basic retention equation of liquid chromatography according to statistical thermodyoamics proposed by professor Ln Peizhang and others, This equation has been verified by a large number of experimental data, all the strsight lines of lnk- of bumologues for different mobile phass coaiposltion cross each other at the same point, So the intereection point equation van proposed, wbich was used to prodict the retention valu, the result was satisfactory.

INTERSECTION POINT RULE OF THE RETENTION VALUE AND MOBILE PHASE COMPOSITION OF THE HOMOLOGUES IN REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Based on the intersection point rule of the retention value and normal boiling point of homologues in reversed-phase high-performance liquid chromatography(RPLC), the intersection point rule of the retention value of homologues and mobile phase composition has been derived, and was testified by a lot of experimental data from the literature. With this newly proposed equation, we can use the retention value of the compound in one mobile phase composition to predict its retention value in any other mobile phase composition. For fourteen groups of homologues in five mobile phase compositions on five Kinds of columns, the overall average absolute error of 721 data sets is 2.8%.

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.

A highly sensitive SPE-liquid/liquid extraction-RPLC analytical method for the determination of 6β-hydroxycortisol and cortisol in cancer patients' urine

A highly sensitive SPE-liquid/liquid extraction RPLC method has been developed for the analysis of 6β-hydroxycortisol and cortisol in the urine of cancer patients. Methods： After SPE column purification and liquid-liquid extraction, the sample test solutions were analyzed with RPLC using a C18 analytical column. This improved analytical method has been validated for linearity, accuracy （recovery from urine）, repeatability （within-day and between-day precision）, specificity, sensitivity, and stability. This SPE-liquid/liquid extraction-RPLC is rapid, simple, accurate and reproducible. The technique is particularly useful for monitoring the CYP3A activity of cancer patients in clinical settings. The results are expressed as the ratio of 6β-hydroxycortisol to cortisol. Results： The CYP3A activity from a total of 153 samples was measured using this improved method. Considerable variation in the CYP3A activity of different cancer patients has been documented. Thus, personalized medical treatment based on the individual metabolic enzyme activity level is necessary. Conclusion： This new analytical method facilitates such individualized medical treatments.

A liquid chromatography with tandem mass spectrometry method for quantitating total and unbound ceritinib in patient plasma and brain tumor

A rapid, sensitive, and robust reversed-phase liquid chromatography with tandem mass spectrometry method was developed and validated for the determination of total and unbound ceritinib, a secondgeneration ALK inhibitor, in patient plasma and brain tumor tissue samples. Sample preparation involved simple protein precipitation with acetonitrile. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C_(18) column using a 4-min gradient elution consisting of mobile phase A(0.1% formic acid in water) and mobile phase B(0.1% formic acid in acetonitrile), at a flow rate of 0.4 m L/min. Ceritinib and the internal standard([^(13)C_6]ceritinib) were monitored using multiple reaction monitoring mode under positive electrospray ionization. The lower limit of quantitation(LLOQ) was 1 n M of ceritinib in plasma. The calibration curve was linear over ceritinib concentration range of 1–2000 n M in plasma. The intra-and interday precision and accuracy were within the generally accepted criteria for bioanalytical method( o15%).The method was successfully applied to assess ceritinib brain tumor penetration, as assessed by the unbound drug brain concentration to unbound drug plasma concentration ratio, in patients with brain tumors.

Ion-pair Reversed-phase×Low-pH Reversed-phase Two-dimensional Liquid Chromatography for In-depth Proteomic Profiling

High-resolution liquid chromatography separation is essential to in-depth proteomic profiling of complex biological samples.Herein,we established an ion-pair reversed-phase×reversed-phase two-dimensional liquid chromatography(IPRP×RP 2DLC)strategy for comprehensive proteomic analysis.Both RPLC separation dimensions were performed at low pH,with trifluoroacetic acid(TFA)and formic acid(FA)as mobile phase addictive,respectively.As the good separation resolution offered by ion-pairing effect of TFA,the fractionation efficiency was greatly improved with 74.0%peptides identified in just one fraction.Comparing with conventional high pH RP fractionation,the overall separation rate of IPRP was about 1.6 times that of high-pH RP,which increased the number of identified peptides by 21%.Further,2169 proteins and 8540 peptides were confidently identified from crude serum sample by our IPRP×RP 2DLC strategy,exhibiting great potential in clinical proteomics in the future.

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。

高温正相色谱/反相色谱(HTNPLC/RPLC)二维联用系统的构建与应用

为解决含水正相色谱与反相色谱联用过程中的谱带展宽问题,以高温正相色谱(HTNPLC)为第一维,室温反相色谱(RPLC)为第二维,分别建立了切割模式的二维液相色谱(HTNPLC-μRPLC)和全二维液相色谱(HTNPLC×RPLC)系统。样品首先在第一维(CN色谱柱)进行正相分离,第一维洗脱产物选择性地或者交替存储在十通阀上的定量环中,然后切换十通阀将定量环内存储的样品组分转移到第二维(C18色谱柱)进行反相分离。该系统降低了第一维切割组分在第二维柱头的扩散,提高了分离度以及分离效率。采用多环芳烃混合物和天然植物甘草提取液对该二维液相色谱系统进行了评价。

混合离子对试剂体系下寡核苷酸的IP-RPLC保留行为

利用离子对反相液相色谱(IP-RPLC)对寡核苷酸在混合离子对试剂三乙胺/丙胺-乙酸盐(TEA/PAAA)体系下的保留行为进行了研究,并与经典离子对试剂三乙胺乙酸盐(TEAA)体系下的保留行为进行了对比.实验结果表明,相同离子对试剂浓度下,寡核苷酸在TEA/PA-AA体系下的保留均弱于TEAA体系下的保留,且寡核苷酸的保留均随着离子对试剂浓度(20~120 mmol/L)的增加而增强.同型寡核苷酸(dC)_n作为特例,当n>10时,保留基本趋于稳定,这是由于(dC)_n随着离子对试剂浓度的增加保留增长较快,在较低的离子对试剂浓度下即可达到最大保留.同时发现,短链同型寡核苷酸(dT)_n和异型寡核苷酸在TEA/PA-AA体系下的分离均优于TEAA体系,而同型寡核苷酸(dA)_n和(dC)_n的分离则在TEAA体系下更优.通过研究流动相中离子对试剂总浓度c_p与寡核苷酸保留因子k之间的关系,推断出2种体系下寡核苷酸的保留机理均以离子对模型占主导地位.总体而言,分离相同长度的异型寡核苷酸时,TEA/PA-AA混合离子对体系尤其在中等离子对浓度下比TEAA体系具有明显优势,低的离子对试剂浓度可增加与后续电喷雾质谱(ESIMS)的兼容性,有利于寡核苷酸的定性分析.

Preparation and Evaluation of Silicon Quantum Dots-Bonded Silica Stationary Phase for Reversed-Phase Chromatography

In this paper,silicon quantum dots(SiQDs)with green fluorescence are synthesized by solvothermal reaction of 3-(2,3-epoxypropoxy)propyltrimethoxysilane(GPTMS)and ethylenediaminetetraacetic acid(EDTA),and then SiQDs are bonded to the surface of silica to obtain a new nano-on-micro stationary phase(SiO_(2)-SiQDs)for reversed-phase chromatography.The successful preparation of SiO_(2)-SiQDs stationary phase is demonstrated by a variety of characterizations,such as transmission electron microscopy,laser confocal microscopy,elemental analysis and Fourier infrared spectroscopy.In addition,the chromatographic performance of the prepared stationary phase is evaluated and it shows good separation performance for non-polar substances such as alkylbenzene,aniline and polycyclic aromatic hydrocarbons in reversed-phase liquid chromatography.It is also verified that the stationary phase has good methyl selectivity and shape selectivity.More interestingly,the separation of prednisolone and hydrocortisone isomers can also be achieved at a low ratio of organic solvents,indicating that this new stationary phase has a good application prospect in isomer separation.

Predicting a, c indices in reversed-phase high-performance liquid chromatography (RP-HPLC) from Kovats index in gas chromatography (GC)

KOVATS index is the most precise index system reflecting the interaction between the molecules of solutes and stationary phase in gas chromatography at present. Large quantity of Kovats in dex data have been published. It is a good way to use Kováts index in gas chromatography to predict the retention value in liquid chromatography, which is significant in theory and apphcation.

Purification of RhIFN- in Bacteria Bodies with Acetic Acid-Water as Mobile Phase in RPLC

Thr extract of E. containing recombinant human interferon- (rhIFN-) with 7.0 mol/L guanidine hydrochloride (Gu . HCl) was directly injected into a column of reverse phase liquid chromatography (RPLC) to separate and purify rhIFN- with acetic acid-water as mobile phase. Gu I-ICI and most impure proteins can be separated by this way. Compared with the usual dilution method, the bioactivity recovery of the purified rhIFN- was found to be over 500%. In addition, compared to common organic solvents employed ill RPLC, acetic acid has higher freezing point, and therefore, it is easy to concentrate the aim-protein by freeze-drying when acetic acid-water is used as mobile phase ill RPLC.

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.

扇贝多肽对淋巴细胞的保护作用和反相液相色谱分析

为进一步研究扇贝多肽 (海洋肽 ,PCF)对淋巴细胞的作用及其成分。采用地塞米松 ( DEX)与脾脏和胸腺淋巴细胞共同培养 ,建立了淋巴细胞免疫抑制实验模型。并利用反相液相色谱法 ( RPLC)对 PCF进行了分析。结果表明 ,DEX能显著降低脾脏和胸腺淋巴细胞的活性 ,DEX组吸收度值明显降低 ( P<0 .0 1) ;而 PCF不仅能显著地减轻其对免疫细胞的抑制作用 ,同时还可以促进免疫细胞的活性 ,使 DEX+PCF组吸收度值明显增高( P<0 .0 1)。相对分子质量为 80 0～ 10 0 0 D的 PCF经 RPL C分析含有保留时间为 3.92 3,4 .865,5.0 33,5.4 4 1等4个组分。实验证明 ,PCF对 DEX引起的淋巴细胞抑制具有保护作用。

亲水/反相二维色谱法制备桔梗中的三萜皂苷

建立了亲水/反相二维色谱用于制备桔梗中三萜皂苷单体的方法。桔梗经水煮醇沉、反相和亲水两种模式的固相萃取后得到三萜皂苷类组分。选定XAmide色谱柱（150 mm×20 mm,5μm）,以乙腈和水为流动相,在亲水色谱模式下进行组分制备。选择时间触发模式,以1 min为单位进行馏分收集,得到6-25 min之间的20个三萜皂苷精细组分。以第18个馏分（JG23）为例,在反相色谱模式下采用Atlantis Prep T3色谱柱（100 mm×30 mm,5μm）制备,得到两个单体化合物。通过质谱和核磁共振对其进行定性,确定分别为deapi-platycoside E和platycoside E。实验结果表明,该制备方法具有好的正交选择性,对于复杂样品中三萜皂苷类化合物的分离纯化有一定的借鉴意义。

氟环唑外消旋体在纤维素-三(3,5-二甲基苯基氨基甲酸酯)手性固定相上的高效液相色谱法拆分

在纤维素-三(3,5-二甲基苯基氨基甲酸酯)(CDMPC)手性固定相上,分别采用正相、反相及极性有机相色谱模式对氟环唑外消旋体进行了拆分,并考察了流动相组成在手性识别中对手性分离的影响。氟环唑在Chiralcel OD-H手性色谱柱(填充CDMPC手性固定相)上采用反相色谱模式,以甲醇-水(体积比为80∶20)为流动相,获得了最佳的拆分,其两对对映异构体的分离度Rs分别为1.64和6.50。

混合模式色谱分离材料的研究及其应用进展

近年来混合模式色谱以其独特的分离特性受到人们越来越多的关注。混合模式色谱的种类主要集中在反相/离子交换混合模式色谱(reversed-phase/ion-exchange mixed-mode chromatography,RPLC/IEX),亲水作用/离子交换混合模式色谱(hydrophilic interaction/ion-exchange mixed-mode chromatography,HILIC/IEX),反相/亲水作用混合模式色谱(reversed-phase/hydrophilic interaction mixed-mode chromatography,RPLC/HILIC)等混合模式。两种或多种机理混合使用,往往在分离选择性和色谱峰形等方面能得到不同于单一模式操作所得到的效果,分离选择性以及色谱峰形等都能得到极大的改善与提高,这使得混合模式色谱渐渐进入研究者们的视野。混合模式色谱的研究多数集中在色谱填料的设计。混合模式色谱填料的应用主要针对生物样品的分离分析。该文综述了近年来混合模式色谱的研究及其应用进展,并展望了混合模式色谱的发展。

正相模式/反相模式的二维液相色谱系统的构建与应用

以50mm×4.6mm的Hypersil SiO2正相色谱柱为第一维色谱系统,250mm×4.6mm的Kromasil C18反相色谱柱为第二维色谱系统,通过升高第二维色谱温度增加两维色谱流动相间互溶性的方法构建了定量环-阀切换接口的二维液相色谱系统(NPLC×RPLC)。根据有机溶剂的特征,在第一维正相色谱流动相中加入二氧六环,第二维反相色谱流动相中加入异丙醇,由此改善流动相兼容性的同时,有效地调整了分离选择性。采用此系统对中成药正天丸样品进行分离分析,峰容量达到1220的色谱系统。