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 The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques.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

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 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.

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 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.

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.

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。

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.

A reversed phase/hydrophilic interaction/ion exchange mixed-mode stationary phase for liquid chromatography

A reversed phase (RP)/hydrophilic interaction (HILIC)/ion exchange (IEX) mixed tri-mode stationary phase (TMSP) has been prepared via a divergent synthesis scheme starting from propylamine on silica then by amine-epoxy reactions with 1,4-butanedioldiglycidyl ether and tertiary amines (N,Ndimethyldecylamine, DMDA). Its retention mechanism was found to follow RP/HILIC/IEX mixed-mode.The stop-flow test revealed that TMSP had good compatibility with 100% aqueous mobile phase. It demonstrated effective separation towards several kinds of compounds or drug molecules and their counterions within a single run.

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.

Determination of Methanol Increment in Mobile Phase Consisting of Methanol and Water by On-line UV Spectrometry in Reversed Phase Liquid Chromatography

An on-line UV spectrometric method for the quantitative determination ofmethanol increment of methanol-water in the mobile phase (i.e., of greater concentration than thatof the mobile phase) by frontal analysis (FA) of insulin in reversed phase liquid chromatography(RPLC) was presented. When the methanol increment concentration ranged from 0.05% to 0.50%,V(CH_3OH)/V(H_2O), a set of elution curves could be obtained at 198 nm by a diode-array detector inthe presence of 47% methanol, V(CH_3OH)/V(H_2O) containing 0.03% hydrochloric acid,V(CH_3OH-H_2O)/V(HCl) in the mobile phase. The plateau height of the elution curves of the methanolincrement was found to be proportional to the methanol increment in the mobile phase. The methanolincrement could be determined on a quantitative basis. When the method was used to investigate theelution curve of insulin by FA in RPLC, a small plateau, being the methanol increment, was detectedbefore the usual insulin plateau of each elution curve. In this case the methanol increment wasfound to vary with insulin concentration in the mobile phase. When that concentration was between0.025 mg/mL and 0.30 mg/mL, the methanol increment could be determined in the range from 0.03% to0.19% with a deviation of ±10%. A nuclear magnetic resonance spectrometer (NMR) was also employedto confirm the obtained result. A methodology with a very rigorous experimental procedure forobtaining results of such accuracy is also included. The presented result may be used to prove thata displacement process definitely occurs as insulin is adsorbed by the RPLC stationary phase inFA.

RETENTION PREDICTION OF CONJUGATED BILE ACIDS AND BARBITUR ATES BY USING INTER ACTION INDEX IN REVERSED PHASE LIQUID CHROMATOGRAPHY

Ⅰ. INTRODUCTION Recently, reversed phase liquid chromatography (RPLC) has become a very efficient standard technique for the modern analyses and is widely used for the analysis of many types of compounds. Retention prediction plays an important part in the thermodynamic studies in RPLC. Many attempts have been made to establish Kováts-like retention

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.

A Direct Evidence of Stoichiometric Displacement between Insulin and Methanol in Reversed Phase Liquid Chromatography

With four kinds of mobile phases, methanol water, ethanol water, 2 propanol and acetonitrile water (all containing 0 1% triflu roacetic acid), the displacement between solute and solvent in RPLC was proved to be universal in frontal analysis (FA). Based on the measured Z value in usual RPLC to be a constant and the quantitative determination of methanol increment in mobile phase in FA, the stoichiometric displacement (SD) between insulin and methanol was directly proved by the experiment. The SD was also proved to occur only on about the one fourth of the maximum amount of adsorbed methanol in the bonded phase layer (BPL) without any dynamic problem of mass transfer, while in FA, the SD firstly occurs on the surface of the BPL and then gradually sinks into the deeper sites companied with a dynamic problem. Although the displaced solvent by the same solute is less in the former case, the SD is independent of how deep of the solute enters the BPL. In addition, the adsorbed amount of solute on an adsorbent not only depends on the numbers of the adsorbed layer on the adsorbent surface, but also on the extent of the complete removal of the displaceable solvent in the BPL. The physical fundamental of the SD and the methodology for investigation were also discussed.

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。

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.

混合型离子交换反相吸附固相萃取-超高效液相色谱-三重四极杆质谱法测定水中23种非甾体抗炎药的残留

非甾体抗炎药(NSAIDs)作为一种新型有机污染物,在环境水体中普遍检出,对生态系统及人体健康造成潜在的威胁,开发准确、可靠的测定水中痕量非甾体抗炎药的检测方法至为重要.本文采用Oasis MCX固相萃取柱对水样进行富集,建立测定水中23种非甾体抗炎药的超高效液相色谱-三重四极杆质谱分析方法.采用酸性条件(pH=4)萃取水样,上样的流速为2 mL·min^(−1),用4 mL甲醇-4 mL5%氨水甲醇进行洗脱,浓缩定容后用ACQUITY UPLCTM BEH C18柱(2.1 mm×100 mm,1.7μm),以2 mmol·L^(−1)乙酸铵+0.05%(V/V)甲酸水溶液-乙腈作为流动相进行梯度洗脱,多反应选择离子监测(MRM)模式进行检测,内标法定量.23种NSAIDs在相关线性范围内线性良好(r=0.9951—0.9992),回收率为80.2%—120%,相对标准偏差为0.4%—12.5%,方法检出限为0.20—4.84 ng·L^(−1).将该方法应用于10份地表水的检测,结果显示有10种NSAIDs检出,质量浓度在ND—83.5 ng·L^(−1)之间.该方法简单、灵敏、高效,可应用于环境水体中NSAIDs的检测.