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 The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques.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.

Determination of isotretinoin in pharmaceutical formulations by reversed-phase HPLC

The development of facile and rapid quantification of biologically active biomolecules such as isotretitoin in therapeutic drugs contained in many generic formu- lations is necessary for determining their efficiency and their quality to improve the human health care. Isotretritoin finds its applications in the maintenance of epithelial tissues. Different processes to date such as normal phase HPLC, or gas chromatrography am- ong others are able to separate and quantify isote- troin. However, the extraction is quite complex and in the case of HPLC, the analysis requires long retention times. In such context, an isocratic reversed- phase high-performance liquid chromatography (HP- LC) technique coupled with an UV-vis detector is described here for easy separation and quantification of 13-cis-retinoic (isotretinoin) from soft gelatin capsule formulations. The isotretinoin was extracted from three different commercial drug samples with tetrahydrofuran (THF) solvent by a procedure that can be completed in less than 10 minutes. Subsequent separation and quantification were accomplished in less than 5 minutes under isocratic reversed-phase conditions on a Lichrospher RP18 column and a mobile phase consisting of 0.01% TFA/acetonitrile (15/85, v/v) at a flow rate of 1.0 mL/min. Isotretoin was detected for the three samples via its UV-vis absorbance at 342 nm. The method was validated and the results showed good linearity, precision and accuracy for sensitive and selective quantitative determination of isotretinoin in the different pharmaceutical formulations. We found that the average isotretinoin content in two of the three commercial pro- ducts fell outside the 90-110% United States Pha- rmacopeia specifications. Consequently, the facile extraction and the precise method for the biomole- cule quantification open up tremendous possibilities in improving the quality control of drugs which can exist as different generic brands.

RP-HPLC-ELSD法测定逍遥颗粒中柴胡皂苷c、a、d的含量

目的:采用反相高效液相色谱-蒸发光散射检测(RP-HPLC-ELSD)法同时测定逍遥颗粒中柴胡皂苷c、a、d三种成分的含量。方法:采用安捷伦(Agilent)SB-C18色谱柱(4.6mm×250mm,5μm),流动相为乙腈(A)-水(B)。梯度洗脱程序为0~18min,30%A;18~26min,35%~45%A;26~35min,45%~55%A。流速为1.0ml/min,柱温为30℃,气体流速为2.5L/min,蒸发温度为60℃,进样量为10μl。结果:柴胡皂苷c、a、d分别在0.950~19.000μg、1.088~21.760μg、1.520~30.400μg的范围内线性关系良好。柴胡皂苷c平均回收率为99.49%,RSD为0.35%(n=9);柴胡皂苷a平均回收率为99.99%,RSD为0.66%(n=9);柴胡皂苷d平均回收率为99.12%,RSD为0.47%(n=9)。结论:该方法准确、灵敏、重复性好,可作为逍遥颗粒中柴胡皂苷c、a、d的含量测定方法。

RP-HPLC法同时测定紫苏叶提取物没食子酸、迷迭香酸、木犀草素含量研究

目的建立安徽地区紫苏叶中没食子酸、迷迭香酸和木犀草素成分的含量同时检测方法。方法采用反相高效液相色谱法,Symmetry C18(250 mm×4.6 mm,5μm)色谱柱,以甲醇(A)-0.1%磷酸溶液(B)为流动相梯度洗脱,流速1.0 mL/min,柱温30℃,检测波长272 nm同时检测没食子酸、迷迭香酸和木犀草素含量。采用SPSS 17.0对测定结果进行分析,比较本地主产区紫苏叶中各成分的含量差异。结果没食子酸、迷迭香酸和木犀草素分别在33.80~101.2μg/mL,33.60~100.8μg/mL,34.00~102.0μg/mL呈线性关系,平均加样回收率分别为98.04%(RSD=1.50%,n=6),96.40%(RSD=0.53%,n=6)和96.49%(RSD=1.89%,n=6)。结论此方法简单方便,稳定可重复,可作为安徽地区主产地紫苏叶没食子酸、迷迭香酸和木犀草素成分含量的检测依据。

RP-HPLC法同时检测石榴皮3种多酚类提取物含量研究

采用RP-HPLC法,Symmetry C18(250 mm×4.6 mm,5μm)色谱柱,以甲醇(A)-0.2%磷酸溶液(B)为流动相梯度洗脱,流速1.0 mL/min,柱温30℃,检测波长272 nm同时检测安石榴苷、鞣花酸和没食子酸含量。采用SPSS 17.0对测定结果进行分析,比较不同品种石榴皮中各成分的含量差异。结果表明,安石榴苷、鞣花酸、没食子酸分别在13.120~209.92μg/mL,13.325~213.20μg/mL和13.275~212.40μg/mL与峰面积呈线性关系,平均加样回收率为97.58%(RSD:1.02%,n=6),99.31%(RSD:0.94%,n=6)和98.63%(RSD:0.70%,n=6)。白玉石榴皮中安石榴苷、鞣花酸的含量分别为7.13%和1.00%,比红酸石榴皮中的含量高。安徽地区石榴皮中安石榴苷、鞣花酸、没食子酸3种成分含量同时检测方法的灵敏度高、准确度好、重现性一致,可用于安徽本地石榴皮部位中安石榴苷、鞣花酸、没食子酸成分含量同时检测。

Development of a Rapid and Efficient Liquid Chromatography Method for Determination of Gibberellin A4 in Plant Tissue, with Solid Phase Extraction for Purification and Quantification

A new, rapid and efficient reverse phase Liquid Chromatography (RP-LC) method was developed for determination of Gibberellin A4 (GA4) in samples of flower stalk of Dasylirion cedrosanum and vegetative tissue of Epithelantha micromeris. Purification of GA4 was carried out by solid phase extraction (SPE), in Epithelantha micromeris. In the chromatography method was obtaining a retention time of 2.1 min, using Hypersil GOLD C-18 column (100 × 4.6 mm dim and size particle 5 μ), mobile phase 50/50 acetonitrile/water and a flow 1.0 ml/min. Detection was carried out by a UV detector set at 205 nm, and a quantization limit of 0.4 mg/L. The obtained correlation coefficient was 0.995.

A Stability Indicating Reverse Phase-HPLC Method Development and Validation for the Estimation of Rucaparib in Bulk and Pharmaceutical Dosage Form

The research was carried out for establishing a new reverse phase-HPLC stability indicating method for the quantification of Rucaparib. The experiment was determined on Waters HPLC instrument using 996 photo-diode array detector. The separation was done by using symmetry C-18 ODS (25 cm × 0.46 cm internal diameter) 5 μm analytical column containing mobile phase of Phosphate buffer (0.02 M) and methanol [65:35% v/v] adjusted pH to 4.8 by adding dilute ortho phosphoric acid. The method was run at 1 ml·min-1 at 286 nm detection. The drug was eluted at 5.484 min. After developing the method, it was assured for the intended use by validation which was done according to ICH Q2B guidelines. The analytical parameters checked were linearity, accuracy, repeatability, intermediate precision, limit of detection, limit of quantitation, ruggedness and robustness. It was observed that the response of the detector was linear in the range of 6 - 14 μg/ml with correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability indicating assay method was established by using the samples generated by forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative and photolytic degradation and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the analysis of quality of the rucaparib drug.

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.

基于RP-HPLC双波长法的蓝芩口服液中主要成分的检测分析

目的基于反相高效液相色谱(RP-HPLC)双波长法测定蓝岑口服液中主要成分[包括栀子苷、(R,S)-告依春、盐酸小檗碱和黄岑苷]的检测价值。方法采用色谱柱Shimadzu C18柱,检测波长0~12 min为245 nm,12~20 min为275 nm;流速为1.0 ml/min;柱温为30℃;进样量为20μl;栀子苷、(R,S)-告依春、盐酸小檗碱检测波长240 nm,黄芩苷检测波长为275 nm。结果4种成分浓度与峰面积的线性回归方程分别是Y=1011X-31.41、Y=6408X-1.798、Y=4401X+28.61、Y=3755X-82.02,且在各自浓度线性范围内均呈现良好线性关系;平均加样回收率分别为98.31%、97.77%、98.33%和97.84%,相对标准差分别为1.12%、1.33%、0.74%和1.21%。结论基于RP-HPLC双波长法可以同时测定蓝岑口服液中栀子苷、(R,S)-告依春、盐酸小檗碱和黄岑苷的含量,且具有良好的稳定性、重复性和精密性,可以作为一种可靠检测方式应用于蓝岑口服液的质量控制中。

A Stability Indicating Reverse Phase-HPLC Method Development and Validation for the Estimation of Bimatoprost 0.3% &Timolol 0.5% Pharmaceutical Ophthalmic Dosage Form

The research was carried out to establish a new reverse phase-HPLC stability indicating method for quantifying Bimatoprost & Timolol in ophthalmic solution. The experiment of Bimatoprost & Timolol in ophthalmic solution method development was determined on Waters HPLC instrument using a UV Detector. The separation was done by using L11, Zorbex SB phenyl (4.6 mm × 250 mm internal diameter) 5 μm analytical column, containing mobile phase of Phosphate buffer (0.02 M), methanol, and acetonitrile [50:30:20 % v/v]. The method was run at 1 ml·min-1 at 210 nm for Bimatoprost and 295 nm for Timolol for detection. The drug was eluted at 10.81 min for Bimatoprost and 3.77 min for Timolol. After developing the method, it was assured for the intended use by validation, which was done according to ICH Q2B guidelines. The analytical parameters checked were Specificity/Selectivity, linearity, Range, accuracy, ruggedness, and robustness. It was observed that the response of the detector was linear in the range of 6 - 18 μg/ml with a correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability-indicating assay method was established by using the samples generated by the forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative, and photolytic degradation, and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the quantitative analysis of Bimatoprost 0.3% + Timolol 0.5% Ophthalmic Solution drugs for pharmaceutical use. Currently, there is no official method for Bimatoprost & Timolol combination products in USP or BP. Available research work related to single Bimatoprost or Timolol products was not suitable for testing Bimatoprost and Timolol combination drugs. Additionally, there is no stability-indicating method to test Bimatoprost & Timolol combination products which insist us to do research and develop a new reverse phase-HPLC indicating method which will be faster and more accurate.

烟草中甲霜灵的手性分离方法差异研究

对比反相液相色谱-串联质谱(RPLC-MS/MS)和超临界流体色谱-串联质谱(SFC-MS/MS)手性分离烟草中甲霜灵的差异。烟草样品经乙腈提取、盐析分层、快速滤过型净化(multi-Plug Filtration Cleanup,m-PFC)柱净化后,分别采用RPLC-MS/MS和SFC-MS/MS进行手性分离。从多个性能参数(分离效率、线性、选择性、回收率、重复性、灵敏度、基质效应等)对两种方法进行了全面比较。采用不同的分离方法,在10~500 ng/mL范围内,甲霜灵的不同异构体均可呈现良好的线性关系(R^(2)≥0.9993)。在各异构体加标浓度为0.1、0.5、2.0 mg/kg水平下,采用不同的分离方法均可获得满意的回收率(88.7%~96.2%)和良好的重复性(RSD<7.0%)。结果表明:RPLC-MS/MS和SFC-MS/MS具有互补性,均适用于手性分离和测定烟草基质中的甲霜灵。

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。

Dependence of Elution Curve and Adsorption Isotherms of Insulin on Composition of Mobile Phase of Frontal Analysis in Reversed Phase Liquid Chromatography

With frontal analysis (FA), the dependence of adsorption isotherms of insulin on the composition of mobile phase in reversed phase liquid chromatography (RPLC) has been investigated. This is also a good example to employ the stoichiometric displacement theory (SDT) for investigating solute adsorption in physical chemistry. Six kinds of mobile phase in RPLC were employed to study the effects on the elution curves and adsorption isotherms of insulin. The key points of this paper are: (1) The stability of insulin due to delay time after preparing, the organic solvent concentration, the kind and the concentration of ion pairing agent in mobile phase were found to affect both elution curve and adsorption isotherm very seriously. (2) To obtain a valid and comparable result, the composition of the mobile phase employed in FA must be as same as possible to that in usual RPLC of either analytical scale or preparative purpose. (3) Langmuir Equation and the SDT were employed to imitate these obtained adsorption isotherms. The expression for solute adsorption from solution of the SDT was found to have a better elucidation to the insulin adsorption from mobile phase in RPLC.

RP-HPLC法对乳制品中主要牛奶蛋白的分离及定量测定

建立一种基于反相高效液相色谱(RP-HPLC)的分析方法,对牛奶及其乳制品进行定量分析。此方法可一次分离牛奶及乳制品中的7种主要蛋白成分(κ-CN、αs2-CN、αs1-CN、β-CN、α-La、β-LgB、β-LgA),解决了加工后乳制品中变性蛋白定量不准确的问题和κ-CN、αs2-CN以及乳清蛋白中α-La、β-LgB和β-LgA难以分离的困难。各组分分离度均大于1,达到基线分离,回收率达88.9%～97.1%,相对标准偏差为0.8%～5.1%。该方法不需对样品进行离心,在测定清蛋白组分时,也不需提前去除优势蛋白酪蛋白,与传统的检测方法相比,具有准确、灵敏和快速等特点,可用于牛奶及其乳制品的质量控制和定量检测。

RP-HPLC法同时测定延胡索药材中延胡索乙素和原阿片碱

建立了反相高效液相色谱(RP HPLC)法同时测定延胡索药材中延胡索乙素与原阿片碱含量的方法,并对不同产地延胡索药材中延胡索乙素与原阿片碱含量进行了考察.结果表明,使用PhenomenexLunaC18色谱柱,甲醇 磷酸盐缓冲溶液(pH=7 68,V∶V=65∶35)流动相,在检测波长281nm下,测定延胡索乙素与原阿片碱的线性范围分别为0 13～4 68μg和0 09～3 24μg,平均回收率分别为101 86%和98 13%,RSD分别为1 42%和1 36%.结果表明,此法简便、准确、快速,可用于药材中延胡索乙素与原阿片碱含量的测定,也可用于含这两种生物碱的药材的质量评价;两种生物碱含量在不同产地及来源延胡索药材中有较大差别.

RP-HPLC法测定东北地区6种红树莓果实中有机酸组成

建立一种反相高效液相色谱法分离和测定6种红树莓果实中草酸、酒石酸、柠檬酸、DL-苹果酸和乳酸5种有机酸的方法。色谱条件为：采用Sino Chrom DS-BP C18色谱柱（150 mm×4.6 mm,5μm）,流动相为甲醇-0.01 mol/L KH2PO4溶液（p H 2.60,97∶3,V/V）,流速0.6 m L/min,柱温30℃,检测波长210 nm。结果表明：在此条件下5种有机酸都被有效地分离,各种有机酸的质量浓度与峰面积在测定范围内呈良好的线性关系,标准曲线相关系数在0.999 3-0.999 9之间;精密度实验相对标准偏差在0.20%-1.53%（n=5）范围内;回收率为98.83%-105.42%,相对标准偏差为0.06%-1.00%。测得6种红树莓果实中有机酸以柠檬酸为主,含量为1 058.41-1 825.45 mg/100 g,草酸、乳酸、DL-苹果酸含量较低,未检测到酒石酸。该方法简单、高效、准确、重复性好,可用于红树莓果实中有机酸的分离测定。

RP-HPLC法测定蔬菜、水果及食用菌中9种农药残留的研究

建立了反相高效液相色谱同时测定蔬菜、水果和食用菌中9种农药（吡虫啉、啶虫脒、多菌灵、甲基硫菌灵、嘧霉胺、除虫脲、灭幼脲、辛硫磷、阿维菌素）残留的可变波长检测器检测的分析方法。样品经乙腈提取,PSA粉固相分散萃取净化,取净化液氮吹至近干后,以1 mL甲醇定容,采用C18（250 mm×4.6mm,5μm）色谱柱分离,以甲醇-乙腈（体积比3∶1）和水二元流动相梯度洗脱,采用紫外可变波长检测器（270,258,275,254,280,245 nm）检测,流速1.0 mL/min,柱温40℃。结果表明9种农药在0.05～10.0mg/L浓度范围内线性关系良好;相关系数均大于0.999;检出限为0.006～0.07 mg/kg;平均加标回收率为81.0%～115.2%,相对标准偏差（RSD）为0.5%～10.2%。该方法具有快速、灵敏、准确、重现性好以及操作简单等特点,适用于蔬菜、水果以及食用菌中上述9种农药残留的分析。

RP-HPLC法测定苹果树枝 叶中根皮苷的含量

建立了一种快速测定苹果树枝、叶中根皮苷含量的RP-HPLC检测方法。色谱条件为Waters Symmetry C18柱(150mm×4·6mm,Φ5μm),流动相为甲醇和pH为2·6的磷酸水溶液(50∶50),等度洗脱,柱温为30℃,287nm紫外检测,流速为0·6mL·min-1,在10min内可实现苹果树枝、叶中根皮苷的快速分离。分析结果表明,根皮苷在0·30～4·50μg范围内呈良好的线性关系,相关系数R2=0·9993,加标回收率98·95%,RSD为1·22%,说明该方法准确可靠。