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 The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques.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

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.

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.

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.

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。

反相高效液相色谱法(RP-HPLC)测定冷却猪肉中生物胺

应用反相高效液相色谱法（RP-HPLC）建立同时测定冷却猪肉中多种生物胺的方法。样品经0.4 mol/L高氯酸提取后用丹磺酰氯柱前衍生，流动相为乙腈和水，采用梯度洗脱，流速为1.0 mL/min，荧光检测器激发波长（Ex）340 nm，发射波长（Em）515 nm。结果表明：采用该方法色胺、苯乙胺、腐胺、尸胺、组胺、酪胺、亚精胺和精胺8种生物胺能在25 min内得到很好分离。线性范围为0.5-20μg/mL（R^2〉0.99），回收率在82.43%-97.14%之间，相对标准偏差RSD小于10%。

RP-HPLC法测定血浆中阿魏酸哌嗪浓度

目的:建立血浆中阿魏酸哌嗪浓度的反相高效液相色谱法。方法:血浆标本以西沙比利为内标,经酸化和二氯甲烷提取后,以0．02 mol·L^(-1)磷酸二氢钠(pH2．7)-甲醇(45:55)为流动相,经碳十八烷基键合硅胶柱分离,在紫外310nm处检测。结果:阿魏酸哌嗪标准曲线范围0．016～16 mg·L～(-1),(r=0．9999),最低定量限为0．016 mg·L～(-1),高、中、低4种浓度的日内精密度均＜4%,日间精密度均＜6%,方法回收率93．8%～102．3%。结论:本法简单可靠,可用于临床阿魏酸哌嗪的药动学和药效学研究。

RP-HPLC法分析测定人参、西洋参、三七中的皂苷类化合物

利用反相高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)技术对人参、西洋参和三七不同方法提取物中的8种皂苷进行定量分析。采用乙醇回流、温浸法和超声辅助提取法提取3种药材中的有效成分,并结合高效液相色谱技术进行分析。结果表明:不同提取方法比较,超声辅助法所提取的3种药材中皂苷含量较高;3种五加科人参属的药材中,三七提取物中主要皂苷含量较高,人参提取物中的皂苷种类较多。

运用RP-HPLC同时测定火龙果中7种有机酸和Vc含量的方法研究

以紫红肉火龙果为试验材料,对影响有机酸分离的主要因素进行研究,最终建立了运用反相高效液相色谱法(RP-HPLC)同时快速测定火龙果果实中草酸、酒石酸、苹果酸、乙酸、乳酸、柠檬酸、琥珀酸7种有机酸及Vc含量的方法。采用色谱条件为:柱温30℃,流速1 m L/min,色谱柱为Kromasil柱(250 mm×4.6 mm),波长215 nm,缓冲液0.05 mol/L H3PO4-Na2HPO4;测定结果显示,供试火龙果含有7种有机酸和Vc,苹果酸是其最主要的有机酸、其次是柠檬酸。此方法可以在10 min内完成1次测定,具有分析速度快、灵敏度高、重复性好等优点,为果实中有机酸及Vc的测定及相关代谢研究提供参考。

DNFB柱前衍生化RP-HPLC测定大黄种子氨基酸的含量

采用柱前衍生RP-HPLC法测定大黄种子中氨基酸的含量。6mol／L盐酸水解得到氨基酸，以2，4-二硝基氟苯（DNFB）为柱前衍生化试剂，梯度洗脱。色谱柱为Gemlmi—NX C18（4．6mm×250mm，5μm）；流动相A相为0．1M乙酸钠水溶液，B相为乙腈-水（1：1），柱温37℃，检测波长360nm。结果表明，三种正品大黄种子中均含有17种氨基酸，总量在11．30％-19．26％。该方法灵敏、准确，有良好的重复性和稳定性。

RP-HPLC法分析萱草属植物花蕾中游离氨基酸组成

目的:分析萱草属植物8个野生种和25个栽培品种花蕾中游离氨基酸的组成特性。方法:以邻苯二甲醛作为衍生试剂,采用柱前在线衍生反相高效液相色谱法对该属植物花蕾中游离氨基酸组成进行分析,利用主成分分析法对游离氨基酸进行综合评价。结果:供试样品游离氨基酸总量在6.864~21.219 mg/g之间;在萱草属植物花蕾中能检测出14~16种游离氨基酸,且含量存在差异;其中呈味氨基酸以Asp、Gly、Ser和Glu等鲜甜味氨基酸含量最为丰富。通过综合评价指数得出:萱草属植物中食用品种黄花菜、小黄花菜、北黄花菜和‘白花’萱草等的品质最优,‘金娃娃’萱草和‘春节’萱草的品质较优,‘馨口’和‘银光芙蓉’萱草的品质最差。

RP-HPLC同时测定人血清中维生素A和维生素E

目的：测定不同病理生理状态下人血清中维生素Ａ、维生素Ｅ的浓度，寻求弄清与某些疾病的关系，为临床治疗学、营养学等研究提供参考。方法：取血清２００μｌ加入含内标的无水乙醇液２００μｌ，振荡，加入正己烷５００μｌ，离心，取正己烷层１００μｌ进样；色谱条件为：色谱柱ＷａｔｅｒｓμＢｏｎｄａｐａｋＣ１８柱（３．９ｍｍ×３０ｃｍ），流动相甲醇水（９６∶４），内标维生素Ａ醋酸酯，检测波长３３０ｎｍ，２９２ｎｍ。结果：维生素Ａ在８６．８０４８～９７２．２１３０μｇ·Ｌ－１浓度范围内呈线性，ｒ＝０．９９９５，维生素Ｅ在１．２６６５～２５．３３１２ｍｇ·Ｌ－１浓度范围内呈线性关系，ｒ＝０．９９９６；维生素Ａ和维生素Ｅ的平均回收率分别为９６．４０％（ＲＳＤ＝３．９８％）和１０３．６７％（ＲＳＤ＝４．２６％），测定了２１８例人血清中维生素Ａ、维生素Ｅ的浓度。结论：方法快速、灵敏、准确，样品处理简便易行。

RP-HPLC法测定细叶杜香叶中4种活性成分的质量分数

为了建立测定细叶杜香叶中秦皮乙素、秦皮素、金丝桃苷和槲皮素质量分数的方法,采用反相高效液相色谱法(色谱条件:色谱柱为Inertsil ODS-SP(5μm4,.6mm×250mm,柱号:9KI92609)、流动相为V(甲醇)∶V(0.2%醋酸)=43∶57、流速1.0 mL.min-1、检测波长350 nm),测得秦皮乙素、秦皮素、金丝桃苷和槲皮素4种成分的平均加样回收率分别为99.71%9、8.62%1、00.13%9、8.33%,其相对标准偏差RSD分别为1.45%1、.53%、1.04%1、.69%。结果表明:反相高效液相色谱法操作简便、准确,具有良好的重复性,可用于细叶杜香中此4种成分的质量分数的测定。