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 The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques.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

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.

Highly sensitive detection of melamine based on reversed phase liquid chromatography mass spectrometry

In this work, we developed a highly sensitive method to detect melamine based on reversed phase liquid chromatography mass spectrometry. A mass spectrometry compatible ion pair, heptafluorobu-tyric acid(HFBA), was used to separate melamine by reversed phase liquid chromatography prior to electrospray mass spectrometry. The incorporation of isotope internal standard and multiple reaction monitoring improved the accuracy and linearity of quantification. Based on this strategy, the method limit of quantification was 0.1 ng/g. The limits of quantification were 8 ng/g for liquid milk and 15 ng/g for dry milk powder. This method provided a reproducible and stable approach to sensitive detection and quantification of melamine.

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.

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。

盐酸丙卡特罗片有关物质的HPLC检查研究

目的:建立盐酸丙卡特罗片有关物质的HPLC测定法。方法:采用Inertsil ODS-3(4.6 mm×250 mm,5μm)色谱柱,以10 mmol·L^(-1)辛烷磺酸钠溶液(用10%磷酸调节pH值至2.90)为流动相A,水-乙腈(43∶57,V∶V)为流动相B,进行梯度洗脱,检测波长为254 nm,柱温为40℃,流速为1.0 mL·min^(-1),进样量为50μL。结果:盐酸丙卡特罗与各有关物质色谱分离度良好,杂质A、B、C质量浓度在10~50 ng·mL^(-1)范围内与峰面积线性关系良好(r>0.9990),校正因子分别为0.5、1.0、0.4,平均回收率为95.0%~105.0%(n=9,RSD<3.0%)。结论:所建立的HPLC方法适用于盐酸丙卡特罗片的有关物质检查,可为盐酸丙卡特罗片现有标准的进一步完善提供参考。

Characters of the Plateau of Methanol Increment in Frontal Analysis in Reversed Phase Liquid Chromatography

With insulin methanol water, and the ion pairing agent, hydrochloric acid and trifluroacetic acid (TFA), the character of the first plateau (FP) on the elution curve of frontal analysis in reversed phase liquid chromatography (RPLC) was investigated by on line UV spectrometry and identified with nuclear magnetic resonance (NMR) spectrometry and mass spectrometry. The profile of the FP is the same as that of a usual elution curve of methanol in frontal analysis (FA). When the insulin concentration was limited to a certain range, the height of the FP was found to be proportional to the insulin concentration in mobile phase and its length companying to shorten. The FP profile on the intersection of two tangents reflects the components of the microstructure in the depth direction of the bonded stationary phase layer and the desorption dynamics of the displaced components. The displaced methanol was quantitatively determined by NMR and on line UV spectrometries. TFA with high UV absorbance can not be used as an ion pairing agent for the investigation of the FP in RPLC, but it can be used as a good marker to investigate the complicated transfer process of components in the stationary phase in RPLC. A stoichiometric displacement process between solute and solvent was proved to be valid in both usual and FA in RPLC. From the point of view of dynamics of mass transfer, the solutes can only contact to the surface of stationary phase in usual RPLC, while solute can penetrate into it in FA of RPLC. The solvation of insulin in methanol and water solution as an example indicating the usage of the FP in the FA was also investigated in this paper.

Development and validation of RP-HPLC and RP-UPLC methods for quantification of parathyroid hormones (1-34) in medicinal product formulated with meta-cresol

Rapid and sensitive reversed phase high performance liquid chromatography (RP-HPLC) and ultra performance liquid chromatography (RP-UPLC) method with UV detection has been developed and validated for quantification of parathyroid hormone (PTH) in presence of meta-cresol as a stabilizer in a pharmaceutical formulation.Chromatography was performed with mobile phase containing 0.1% Trifluoroacetic acid (TFA) in MilliQ water and 0.1% TFA in acetonitrile with gradient program and flow rate at 0.3 mL/min for HPLC and 0.4 mL/min for UPLC.Quantification was accomplished with internal reference standard (qualified against innovator product and National Institute for Biological Standards and Control (NIBSC) standard).The methods were validated for linearity (correlation coefficient 0.99),range,accuracy,precision and robustness.Robustness was confirmed by considering three factors;mobile phase composition,column temperature and flow rate/age of mobile phase.Intermediate precision was confirmed on different equipments,different columns and on different days.The relative standard deviation (RSD) (<2% for RP-HPLC and <1% for UPLC,n=30) indicated a good precision.Retention time was found about 17 min and 2 min by HPLC and UPLC methods,respectively.Both methods are simple,highly sensitive,precise and accurate and have the potential of being useful for routine quality control.

Development and Validation of HPLC Method for Simultaneous Determination of Amlodipine, Valsartan, Hydrochlorothiazide in Dosage Form and Spiked Human Plasma

A simple, sensitive, and specific method was developed for simultaneous determination of Amlodipine besylate (AML), Valsartan (Vals) and Hydrochlorothiazide (HCT) by high performance liquid chromatography without previous separation. Satisfactory resolution was achieved using a RP-C18 chromatographic column, Phenomenex Kinetex (150 mm × 4.6 mm i.d) and a mobile phase consisting of acetonitrile-phosphate buffer (0.05 M) with pH 2.8 in the proportion of (40/60, v/v) at a flow rate 0.8 mL/min and the wavelength detection was 227 nm. The retention time for HCT, AML and VAls was 2.26, 3.16 and 11.19 min;respectively. The described method was linear over a range of 4-28 μg /ml, 5-40 μg /ml and 1-12 μg /ml for AML, Vals and HCT;respectively. The mean percent recoveries were 99.94%, 99.96% and 99.78% for AML, Vals and HCT;respectively. F-test and t-test at 95%con?dence level were used to check the intermediate precision data obtained under different experimental setups. The method could be used for analysis of combined dose tablet formulation containing AML, Vals, HCT as well as spiked human plasma.

Characterization of Acute Renal Allograft Rejection by Human Serum Proteomic Analysis

To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis （2-D DIGE） and reversed phase high-performance liquid chromatography （RP-HPLC） followed by electrospray ionization mass spectrometry （ESI-MS） were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups： acute rejec- tion （AR）, stable renal function （SRF） and normal volunteer （N）. Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differentially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were excised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor, apolipoprotein A-IV precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor, etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into understanding the mechanisms and potential treatment strategy of acute rejection.

Development and Validation of a Method for Simultaneous Determination of Metformin Hydrochloride and Sitagliptin Phosphate in a Formulation by RP-HPLC

Present study was aimed to develop and validate a reverse-phase high-performance liquid chromatography method for simultaneous determination of sitagliptin phosphate and metformin hy-drochloride in a marketed formulation. The drug separation was performed on Hibar-240, Li-chrosphere-100 C18 ODS (250 × 4.6 mm, 5 μm) column, at a flow rate of 1 mL/min. The mobile phase used was a mixture of methanol: potassium di-hydrogen phosphate buffer at a ratio of 70:30 v/v. The detection was carried out at a wavelength of 266 nm. The retention times of sitagliptin phosphate and metformin hydrochloride were found as 6.1 and 4.9 min respectively. Linear calibration curves with good correlation coefficients were obtained over the concentration ranges of 10 - 50 μg/mL for sitagliptin and 20 - 100 μg/mL for metformin. The limit of detection was 0.016 and 0.14 μg/mL and the limit of quantification was 0.048 and 0.42 μg/mL for sitagliptin phosphate and metformin hydrochloride respectively. Validation of the method demonstrated system selectivity, specificity, linearity, accuracy and precision. The developed method was found useful in the simultaneous analysis of sitagliptin phosphate and metformin hydrochloride in formulation.

Separation and Measurement of Three Kinds of Endogenous Hormones in Rhizome of Alhagi sparsifolia

[ Objective] The aim was to establish effective method for endogenous hormone extraction and explore conditions of chromatographic analysis for three endogenous hormones in rhizome of Alhagi sparsifolia. [ Methed ] Activol （GA3 ）, zeatin （ZR） and indole-3-acetic acid （IAA） in rhizome were separated and measured as per RP-HPLC. [Result] The average recovery rates of GA3, ZR and IAA were 98.3%, 90.3% and 101.3%, respectively, indicating that the method is suitable for quantitative analysis with little errors. The chromatographic conditions were as follows： methanol/0. 75% of acetic acid at 45：55 （mobile phase） ; flow speed at 0.7 ml/min; wavelength at 254 nm; column temperature at 25 ℃. [Conclusion] The research preliminarily established HPLC conditions for separation of endogenous hormones in rhizome of Alhagi sparsifolia.

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.