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 The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques.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

初榨橄榄油中多酚类化合物含量随油橄榄果生长发育的累积变化

旨在为甘肃陇南橄榄油的质量评价和油橄榄鲜果采收时间提供指导,探究了不同品种初榨橄榄油(VOO)中多酚类化合物(PPs)随油橄榄果生长发育的累积变化。以甘肃陇南不同成熟度的8个品种油橄榄鲜果为原料,采用压榨法获得VOO,利用反相高效液相色谱法同时测定VOO中的9种PPs含量。结果表明:VOO中总多酚的含量主要由酪醇和橄榄苦苷决定,橄榄苦苷是含量最高的PPs,且其含量随油橄榄果成熟度指数(MI)变化最明显;VOO中木犀草苷含量均较低,阿魏酸和芹菜素仅在个别品种中检测到,芦丁在所测油品中均未检测到;‘阿斯’‘鄂植8号’‘中山24号’和‘佛奥’4个品种VOO的PPs评估数据表现良好,分别在MI为0~1.0、2.0~4.0、0~2.0和5.0~7.0表现最佳,建议这4个品种油橄榄鲜果的采收时间分别为9月下旬、11月上旬、9月下旬至10月上旬以及11月下旬,而‘切姆拉尔’VOO在PPs种类和总含量等评估数据中表现不佳。综上,可以依据不同MI的油橄榄鲜果制备的VOO中多酚含量,尤其是酪醇和橄榄苦苷含量,确定不同品种油橄榄鲜果的采收时间,并进行VOO的品质评价。

高效液相色谱法测定异烟酸辛酰肼含量

目的:异烟肼及其衍生物作为治疗肺结核的一线药物,其含量测定在药物的研发及应用中至关重要。然而,异烟肼的含量测定方法虽常有报道,但是针对异烟肼衍生物的含量测定方法却鲜有报道。因此本研究构建一种适用于测定异烟酸辛酰肼(INH-CHO)的反相高效液相色谱法(reverse-phase high performance liquid chromatography,RP-HPLC),用于INH-CHO的含量检测。方法:采用Capcell Pak C18 MG II S5色谱柱,柱温为30℃,以pH=6.0磷酸盐缓冲液:甲醇=30:70为流动相进行洗脱,流速为1 mL/min,进样0.02 mL,在262 nm处检测,考察该方法学的线性关系、精密度、准确度及定量限。结果:INH-CHO的保留时间为9.2 min,在1~240μg/mL、0.1~1μg/mL浓度范围内都具有较好的线性关系(R~2>0.9990),在该色谱条件下测定INH-CHO的日内精密度、日间精密度、准确度较好,定量限为0.1μg/mL。结论:在实验浓度范围内,该方法线性良好、操作简便、结果精确、可重现,适用于INH-CHO的含量检测。

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

Objective: 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.

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。

反相超高效液相色谱分析小麦麦谷蛋白组分研究

为利用反相超高效液相色谱(RP-UPLC)分离、定量分析小麦麦谷蛋白组分,研究了流动相比例、洗脱时间、柱温、流速和进样量对色谱分离结果的影响,并利用新建立的分离麦谷蛋白亚基的方法分析了基因型及环境对麦谷蛋白组分的影响。结果表明,最优的洗脱程序为79%的流动相A(含0.08%三氟乙酸的超纯水)和21%的流动相B(含0.08%三氟乙酸的乙腈溶液)洗脱5 min,在25 min内,流动相A逐渐降低至48%,流动相B逐渐增加至52%,最后维持5 min,柱温、流速和进样量分别为55℃、0.5 mL·min^(-1)和16μL,该方法可以精确分离至少11种高分子量麦谷蛋白亚基(HMW-GS)。单个HMW-GS的相对含量和HMW-GS与低分子量麦谷蛋白亚基(LMW-GS)的比值均受基因型和环境的影响,且基因型的影响大于环境。

反相高效液相色谱法测定大豆磷脂中磷脂酰胆碱含量

建立一种通过反相高效液相色谱-紫外测定大豆磷脂中磷脂酰胆碱含量的方法。结果表明:方法的流动相为异丙醇-甲醇-水,且大豆磷脂酰胆碱在0.4615~2.3075 mg/mL范围内线性关系良好(R^(2)=0.9977);方法的检出限为1.4 mg/g、平均回收率为99.8%、相对标准偏差(RSD)为2.41%。方法的精确度高、重复性好,适用于大豆磷脂中磷脂酰胆碱含量的测定。

大豆低聚肽的分离纯化及呈鲜序列鉴定

为了分析大豆低聚肽中鲜味肽成分,本文通过纳滤分离结合感官评价对大豆低聚肽进行了初步分离得到鲜味最佳组分Pb-3,通过凝胶过滤层析从Pb-3中分离出鲜味和增鲜效果最强的Fb-2,进一步采用反相高效液相色谱分离出鲜味效果最佳的Kb-2,采用超高效液相色谱-串联质谱法从Kb-2中鉴定出鲜味肽.结果显示:与对照组相比,相对分子质量为300~500的大豆低聚肽Kb-2具有较强的鲜味和增鲜效果;鉴定出3种鲜味肽,分别为2个四肽Val-Thr-Val-Glu(VTVE)、Leu-Glu-Lys-Asp(LEKD)和1个三肽Trp-Glu-Arg(WER).本研究为大豆低聚肽生产复合调味品提供了理论支持.

反向高效液相色谱法同时测定复方氨酚烷胺片中三种主要组分的含量

目的建立复方氨酚烷胺片中三种主要组分含量测定的反向高效液相色谱(RP-HPLC)法。方法色谱柱选用ZORBAX Eclipse XDB-C18色谱柱(4.6 mm×150 mm,5μm),流动相选用85∶15的戊烷磺酸钠-甲醇溶液(0.05 mol/L),流速及柱温分别设置为1.0 ml/min、30℃,检测波长选取216 nm,进样量设定20μl,进样测定复方氨酚烷胺片中马来酸氯苯那敏、对乙酰氨基酚、咖啡因的含量,并进行方法学考察。结果马来酸氯苯那敏在2.25~42.90 mg/L浓度范围内呈良好的线性关系(r=0.9971),对乙酰氨基酚在30.10~526.71 mg/L浓度范围内呈良好的线性关系(r=0.9983),咖啡因在4.50~96.82 mg/L浓度范围内呈良好的线性关系(r=0.9989)。马来酸氯苯那敏、对乙酰氨基酚、咖啡因的加样回收率为分别为98.49%(RSD=0.844%)、99.39%(RSD=0.607%)、99.70%(RSD=0.159%)。结论应用RP-HPLC测定复方氨酚烷胺片中三种主要组分的含量,具有较好的准确性和重现性,且方法简单迅速,可用于其主要组分的含量测定及质量控制。

反相HPLC快速测定全反式维生素A含量的方法探究

目的:建立了反相高效液相色谱法(High Performance Liquid Chromatography,HPLC)测定全反式维生素A含量,采用碘试液反应生成的全反式维生素A棕榈酸酯及其异构体在本方法条件下分离度为1.62,检测方法专属性好。方法:本方法前处理简单,利用涡旋振荡器提取目标成分,再利用反相HPLC进行反式维生素A的定量测定。结果:适用于全反式维生素A及其异构体的快速分离检测。结论:对主要含维生素A棕榈酸酯的食品原料及其制剂中该成分的含量检测具有指导性作用。

反相高效液相色谱法测定加味四妙勇安汤中绿原酸和梓醇的含量

目的:建立反相高效液相色谱法测定加味四妙勇安汤浸膏中绿原酸和梓醇含量的方法。方法:色谱柱为Merck Purospher STARLP RP-18 endcapped柱(250 mm×4.6 mm,5μm),流速为1.0 mL/min,柱温为35℃。(1)绿原酸的色谱条件,流动相为乙腈-0.4%磷酸(V∶V=11∶89),检测波长为327 nm。(2)梓醇的色谱条件,流动相A为乙腈,流动相B为0.1%磷酸,梯度洗脱(0~15 min,0.8%A;15~60 min,2%A),检测波长为210 nm。结果:绿原酸在4.420~442.0μg/mL(r=0.9998)范围内质量浓度与峰面积成良好线性关系;平均回收率为99.38%(RSD=1.26%)。梓醇在9.760~488.0μg/mL(r=0.9999)范围内质量浓度与峰面积成良好线性关系,平均回收率为99.34%(RSD=1.43%)。绿原酸和梓醇的最低检测限分别为0.40和0.82 ng,定量限分别为1.20和2.44 ng,精密度RSD分别为0.71%(n=6)和0.45%(n=6)。结论:本方法操作简便,分离效果和重复性好,准确度高,可用于加味四妙勇安汤提取物的质量评价。

PPTA回收溶剂NMP中PPD残留量的测定

采用反相高效液相色谱法测定聚对苯二甲酰对苯二胺(PPTA)回收溶剂N-甲基吡咯烷酮(NMP)中对苯二甲酰氯与对苯二胺(PPD)的残留量,探讨了高效液相色谱仪的色谱条件,并研究了分析方法的精密度、准确度、检出限。结果表明:采用反相高效液相色谱法测定回收溶剂NMP中PPD的含量,适宜的色谱条件为色谱柱采用十八烷基硅烷键合硅胶柱,流动相中水/改性剂(体积比为991)与乙腈体积比为955,紫外波长为280 nm,进样量为10μL;该分析方法精密度优、重复性好、线性方程的相关性好,相对标准偏差为0.85%,加标回收率为98.1%~101.1%,检出限为0.35μg/g,能满足PPTA回收溶剂NMP中微量PPD含量的测定需求。

反相高效液相色谱法测定铁叶酸片中叶酸含量

依据《2020版中国药典》二部叶酸片项下含量测定检测复方铁叶酸片中叶酸含量,叶酸结果由于受到亚铁离子的影响,检测结果会比理论值显著偏低。本文对药典方法进行优化,通过采用加入乙二胺四乙酸二钠(EDTA)络合剂将亚铁离子掩蔽排除亚铁离子的影响,采用超声提取和高效液相色谱-二极管阵列检测器(High Performance Liquid Chromatography with Diode-Array Detection, HPLC-DAD)检测叶酸含量,可以显著改善叶酸的检测结果准确性。方法简单、重复性好,可以满足含铁叶酸片中叶酸含量的检测。

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。