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.

INTERSECTION POINT RULE OF THE RETENTION VALUE AND NORMAL BOILING POINT OF THE HOMOLOGUES IN REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

In this paper, a linear relationship between the logarithm of capacity factor k and normal boiling point to of the homologues has been derived, based on the basic retention equation of liquid chromatography according to statistical thermodyoamics proposed by professor Ln Peizhang and others, This equation has been verified by a large number of experimental data, all the strsight lines of lnk- of bumologues for different mobile phass coaiposltion cross each other at the same point, So the intereection point equation van proposed, wbich was used to prodict the retention valu, the result was satisfactory.

INTERSECTION POINT RULE OF THE RETENTION VALUE AND MOBILE PHASE COMPOSITION OF THE HOMOLOGUES IN REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Based on the intersection point rule of the retention value and normal boiling point of homologues in reversed-phase high-performance liquid chromatography(RPLC), the intersection point rule of the retention value of homologues and mobile phase composition has been derived, and was testified by a lot of experimental data from the literature. With this newly proposed equation, we can use the retention value of the compound in one mobile phase composition to predict its retention value in any other mobile phase composition. For fourteen groups of homologues in five mobile phase compositions on five Kinds of columns, the overall average absolute error of 721 data sets is 2.8%.

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.

STUDY ON THE SEPARATION AND EXTRACTION MECHANISM OF RARE EARTH ELEMENTS BY MEANS OF REVERSED-PHASE PAPER CHROMATOGRAPHY

Reversed-phase paper chromatography technique is used for study on the extraction mechanism and sep- aration of rare earth elements.As the stationary phase,chromatographic paper strips are impregnated with a solution of monomyristyl phosphoric acid (MPA) in chloroform.Mineral acids are used as developers. The effect of concentration of acids and/or salts upon R_f has been investigated.According to the re- sults of R_f values for a given rare earth element in various acids,the order of extraction ability is HCl>HNO_3>H_2SO_4.A tetrad effect is clearly observed.for the R_f value of rare earth elements.The effects of other parameters on the R_f value,such as the quantities of extractant retained by the paper and the temperature are also examined.Based on the determination of the molar ratio of MPA to rare earth elements and the number of H^+ ions released in extraction reaction,a reasonable mechanism is proposed.The mutual separation of heavy rare earth elements will be better than that of the light rare earth group because of the larger separation coefficient of the former.A mixture of Ho-Er-Tm-Lu is successfully separated by the present method.

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.

Preparation and Evaluation of Silicon Quantum Dots-Bonded Silica Stationary Phase for Reversed-Phase Chromatography

In this paper,silicon quantum dots(SiQDs)with green fluorescence are synthesized by solvothermal reaction of 3-(2,3-epoxypropoxy)propyltrimethoxysilane(GPTMS)and ethylenediaminetetraacetic acid(EDTA),and then SiQDs are bonded to the surface of silica to obtain a new nano-on-micro stationary phase(SiO_(2)-SiQDs)for reversed-phase chromatography.The successful preparation of SiO_(2)-SiQDs stationary phase is demonstrated by a variety of characterizations,such as transmission electron microscopy,laser confocal microscopy,elemental analysis and Fourier infrared spectroscopy.In addition,the chromatographic performance of the prepared stationary phase is evaluated and it shows good separation performance for non-polar substances such as alkylbenzene,aniline and polycyclic aromatic hydrocarbons in reversed-phase liquid chromatography.It is also verified that the stationary phase has good methyl selectivity and shape selectivity.More interestingly,the separation of prednisolone and hydrocortisone isomers can also be achieved at a low ratio of organic solvents,indicating that this new stationary phase has a good application prospect in isomer separation.

Ion-pair Reversed-phase×Low-pH Reversed-phase Two-dimensional Liquid Chromatography for In-depth Proteomic Profiling

High-resolution liquid chromatography separation is essential to in-depth proteomic profiling of complex biological samples.Herein,we established an ion-pair reversed-phase×reversed-phase two-dimensional liquid chromatography(IPRP×RP 2DLC)strategy for comprehensive proteomic analysis.Both RPLC separation dimensions were performed at low pH,with trifluoroacetic acid(TFA)and formic acid(FA)as mobile phase addictive,respectively.As the good separation resolution offered by ion-pairing effect of TFA,the fractionation efficiency was greatly improved with 74.0%peptides identified in just one fraction.Comparing with conventional high pH RP fractionation,the overall separation rate of IPRP was about 1.6 times that of high-pH RP,which increased the number of identified peptides by 21%.Further,2169 proteins and 8540 peptides were confidently identified from crude serum sample by our IPRP×RP 2DLC strategy,exhibiting great potential in clinical proteomics in the future.

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.

INVESTIGATION ON SEPARATION OF INORGANIC ANIONS BY REVERSED-PHASE ION-PAIR LIQUID CHROMATOGRAPHY

Ⅰ. INTRODUCTIONReversed-phase ion-pair liquid chromatography is widely used in the separation of ionized organic compounds. In recent years, the separation of inorganic ions by the reversedphase ion-pair liquid chromatography with indirect UV detection or conductivity

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.

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.

反相液相色谱中冲洗剂组成和性质对有机酸保留行为的影响

通过对五种有机酸在不同 pH 值下,用不同浓度的甲醇-水和乙腈-水作冲洗剂时,容量因子变化规律的系统考察,验证了原来所推导的反相液相色谱保留值基本方程的正确性,对方程中各常数的意义作了阐明,找到了分离它的最佳冲洗剂组成和 pH 值。

Assay and Evaluation of Flavonoid Content in Chinese Celery

Flavonoids were the most important secondary metabolites in celery （Apium graveolens L.）.In this study,the flavonoid （apigenin and luteolin） contents of leaves and petioles in 44 Chinese celery varieties,an American celery variety ‘Ventura＇ and an European celery variety ‘Princer＇ were assayed by reversed-phase high-perfor mance liquid chromatography （RP-HPLC）.The results showed that significant differences in the apigenin and luteolin contents of the leaves and petioles were found among 46 celery varieties,and the variation coefficients of leaf apigenin content,leaf luteolin content,leaf flavonoid content,petiole apigenin content,petiole luteolin content and petiole flavonoid content were 30％,10％,30％,20％,30％ and 20％,respectively.There was no correlation between the leaf flavonoid content and petiole flavonoid content.The apigenin content was about 18 to 50 times of luteolin in celery leaf and about 19 to 40 times of luteolin in celery petiole.The flavonoids in most of the Chinese celery varieties were higher than in ‘Ventura＇ and ‘Princer＇.The flavonoid contents of Chinese celery varieties from the Yangtze Valley and southern China were higher than those from northern China.Our results suggested that Chinese celery varieties with high petiole apigenin contents could be used for vegetable breeding because the petiole was the main edible part,and Chinese celery varieties with high leaf apigenin contents could be used for apigenin production.

变异胰岛素症

变异胰岛素症是在糖尿病患者中发现的分子病。对变异胰岛素的研究是近年在胰岛素结构与功能,临床医学和分子遗传学方面的新课题。本文就变异胰岛素的发现及其特点、变异胰岛素的鉴定技术以及变异胰岛素与糖尿病的关系作一综述。

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

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.

LC-UV and LC-MS evaluation of stress degradation behavior of desvenlafaxine

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of desvenlafaxine in bulk sample and pharmaceutical dosage form in the presence of degradation products. Forced degradation studies were performed on bulk sample of desvenlafaxine as per ICH prescribed stress conditions using acid, base, oxidative and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed under acidic stress condition and the degradation product formed was identified by LC-MS and a degradation pathway for drug has been proposed. Successful separation of drug from degradation products formed under stress conditions was achieved on a SymmetryShield column C 18 (5 mm, 250 mm 4.6 mm, i.d.) using the mobile phase consisting of a mixture of 0.2% (v/v) triethylamine in ammonium acetate (0.05 M; pH 6.5) and methanol using isocratic gradient.

Establishment of inherent stability of pramipexole and development of validated stability indicating LC-UV and LC-MS method

Pramipexole belongs to a class of nonergot dopamine agonist recently approved for the treatment of early and advanced Parkinson＇s disease.A validated specific stability indicating reversed-phase liquid chromatographic method has been developed for the quantitative determination of pramipexole in bulk as well as in pharmaceutical dosage forms in the presence of degradation products.Forced degradation studies were performed by exposition of drug to hydrolytic（acidic and basic）,oxidative and photolytic stress conditions,as defined under ICH guideline Q1A（R2）.Significant degradation was observed under hydrolytic,oxidative and photolytic conditions and the degradation products formed were identified by LC-MS.

新型吡咯烷键合反相高效液相色谱固定相的制备与研究（英文）

A new ionic liquid-based high-performance liquid chromatography stationary phase is reported.A derivative of N-methyl pyrrolidinium tetrafluoroborate was covalently immobilized on the surface of silica particles to prepare silica-based N-methyl pyrrolidinium tetrafluoroborate(SilprMP BF4)stationary phase.The obtained ionic liquid-modified silica was evaluated and confirmed by elemental analysis,infrared spectroscopy,and thermogravimetric analysis.A column was packed with the modified particles.The retention behavior of aromatic compounds,alkyl benzenes,and acidic and basic compounds on the SilprMP BF4 stationary phase was studied under reversed-phase liquid chromatography conditions.The effect of the eluent pH on the separation of the acidic and basic compounds was also studied.The new stationary phase involves multiple retention mechanisms,such as electrostatic,hydrophobic,ion-dipole,and anion-exchange interactions,which might lead to multipurpose separation media.