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%.

Determination of gouty arthritis' biomarkers in human urine using reversed-phase high-performance liquid chromatography

Creatinine, uric acid, hypoxanthine and xanthine are important diagnostic biomarkers in human urine for gouty arthritis or renal disease diacrisis. A simple method for simultaneous determination of these biomarkers in urine based on reversed-phase high-performance liquid chromatography （RP-HPLC） with ultraviolet （UV） detector was proposed. After pretreatment by dilution, centrifugation and filtration, the biomarkers in urine samples were separated by ODS-BP column by elution with methanol/50 mM NaH2PO4 buffer solution at pH 5.26 （5：95）. Good linearity between peak areas and concentrations of standards was obtained for the biomarkers with correlation coefficients in the range of 0.9957-0.9993. The proposed analytical method has satisfactory repeatability （the recovery of data in a range of creatinine, uric acid, hypoxanthine and xanthine was 93.49-97.90%, 95.38-96.45%, 112.46-115.78%and 90.82-97.13%with standard deviation of o5%, respectively） and the limits of detection （LODs, S/N Z 3） for creatinine, uric acid, hypoxanthine, and xanthine were 0.010, 0.025, 0.050 and 0.025 mg/L, respectively. The established method was proved to be simple, accurate, sensitive and reliable for the quantitation of gouty arthritis＇ biomarkers in human urine samples. The ratio of creatinine to uric acid was found to be a possible factor for assessment of gouty arthritis.

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.

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.

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.

Advancements in the preparation of high-performance liquid chromatographic organic polymer monoliths for the separation of small-molecule drugs

The various advantages of organic polymer monoliths, including relatively simple preparation processes,abundant monomer availability, and a wide application range of pH, have attracted the attention of chromatographers. Organic polymer monoliths prepared by traditional methods only have macropores and mesopores, and micropores of less than 50 nm are not commonly available. These typical monoliths are suitable for the separation of biological macromolecules such as proteins and nucleic acids, but their ability to separate small molecular compounds is poor. In recent years, researchers have successfully modified polymer monoliths to achieve uniform compact pore structures. In particular, microporous materials with pores of 50 nm or less that can provide a large enough surface area are the key to the separation of small molecules. In this review, preparation methods of polymer monoliths for high-performance liquid chromatography, including ultra-high cross-linking technology, post-surface modification, and the addition of nanomaterials, are discussed. Modified monolithic columns have been used successfully to separate small molecules with obvious improvements in column efficiency.

Dispersive Liquid-liquid Microextraction Combined with High-performance Liquid Chromatography for the Determination of Clozapine and Chlorpromazine in Urine

A simple method has been proposed for the determination of clozapine （CLZ） and chlorpromazine （CPZ） in human urine by dispersive liquid-liquid microextraction （DLLME） in combination with high-performance liquid chromatography-ultraviolet detector （HPLC-UV）. All important variables influencing the extraction efficiency, such as pH, types of the extraction solvent and the disperser solvent and their volume, ionic strength and centrifugation time were investigated and optimized. Under the optimal conditions, the limit of detection （LODs） and quantification （LOQs） of the method were 13 and 39 ng/mL for CLZ, and 2 and 6 ng/mL for CPZ, respectively. The relative standard deviations （RSDs） of the targets were less than 5.1% （C=0.100 μg/mL, n=9）. Good linear behaviors over the tested concentration ranges were obtained with the values of R20.999 for the targets. The absolute extraction efficiencies of CLZ and CPZ from the spiked blank urine samples were 98.3% and 97.8%, respectively. The applicability of the technique was validated by analyzing urine samples and the mean recoveries for spiked urine samples ranged from 93.3% to 105.0%. The method was successfully applied for the determination of CLZ and CPZ in real human urine.

Quantification of six bioactive compounds in Zhenqi Fuzheng preparation by high-performance liquid chromatography coupled with diode array detector and evaporative light scattering detector

A simple and accurate high-performance liquid chromatography（HPLC）coupled with diode array detector（DAD）and evaporative light scattering detector（ELSD）was established for the determination of six bioactive compounds in Zhenqi Fuzheng preparation（ZFP）.The monitoring wavelengths were 254,275 and 328 nm.Under the optimum conditions,good separation was achieved,and the assay was fully validated in respect of precision,repeatability and accuracy.The proposed method was successfully applied to quantify the six ingredients in 31 batches of ZFP samples and evaluate the variation by hierarchical cluster analysis（HCA）,which demonstrated significant variations on the content of these compounds in the samples from different manufacturers with different preparation procedures.The developed HPLC method can be used as a valid analytical method to evaluate the intrinsic quality of this preparation.

Establishing a protein expression profile database for the normal human pituitary gland using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry

In this study, we selected adult normal pituitary gland tissues from six patients during operations for pituitary microadenomas via the transsphenoidal approach for extended normal pituitary tissue resection around the tumor, and analyzed the protein expression of human normal pituitary using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry proteomics technology. The ten most highly expressed proteins in normal human pituitary were： alpha 3 type VI collagen isoform 5 precursor （abundance among tall pituitary proteins 1.30%）, fibrinogen beta chain preproprotein （0.99%）, vimentin （0.73%）, prolactin （0.69%）, ATP synthase, H~ transporting and mitochondrial F1 complex beta subunit precursor （0.52%）, keratin I （0.49%）, growth hormone （0.45%）, carbonic anhydrase I （0.40%）, heat shock protein 90 kDa I （0.31%）, and annexin V （0.30%）. Based on the biological function classifications of these proteins, the top three categories by content were neuroendocrine proteins （abundance among all pituitary proteins, 40.1%）, catalytic and metabolic proteins （28.3%）, and cell signal transduction proteins （9.8%）. Based on cell positioning classification, the top three categories were cell organelle （24.5%） membrane （20.8%）, and cytoplasm （13.0%）. Based on biological process classification, the top three categories of proteins are involved in physiological processes （42.9%）, cellular processes （40.4%）, and regulation of biological processes （9.1%）. Our experimental findings indicate that a protein expression profile database of normal human pituitary can be precisely and efficiently established by proteomics technology.

Studies on Chromatography Fingerprint of Hongqi by High-performance Liquid Chromatography

Chromatography fingerprint (CFP) of 10 samples of hongqi were studied. 23 common peaks were analyzed, their average similarity was 97.29%. CFP were positioned with main index composition such as formononetin, calycosin and then the contents of index composition were determined. The character and exclusive of CFP of 10 samples of hongqi were clear. CFP and content determination of index composition of hongqi could be used to evaluate the quality of hongqi comprehensively.

Biological Fingerprinting Analysis of Interaction Between Taxoids in Taxus and Microtubule Protein by Microdialysis Coupled with High-performance Liquid Chromatography/Mass Spectrometry for Screening Antimicrotubule Agents

Some natural products, such as traditional Chinese medicines（TCMs）, contain compounds with anticancer activity and have attracted a great interest in recent years as alternative anticancer therapies. A quick and convenient assay for screening antimicrotubule compounds in which in vitro microdialysis/high-performance liquid chromatography （HPLC） is used to monitor the binding of the compounds extracted from TCM Taxus cuspidata Siebold ＆ Zucc（Taxus） to microtubules is reported. It was observed that the extract of Taxus contains at least five compounds which have affinity interaction with microtubules by biological fingerprinting analysis, and they were identified as the taxoids of taxol, baccatin III, 10-deacetylbaccatin Ⅲ（10-DAB）, cephalomannine and 7-epi-10-deacetyltaxol （7-epi-10-DAT） based on the comparison of their high-performance liquid chromatographic/mass spectrometric and UV spectra with those of the standard samples, both assembly-promoting and disassembly-inhibiting characteristics of those compounds were evaluated. It was observed that baccatin Ⅲ and 10-DAB bound to microtubules and the binding degrees were influenced by GTP. Competitive binding behavior of taxol with other four taxoids to microtubules was also investigated.

High-performance Liquid Chromatographic Determination of Urinary Trans,Trans-Muconic Acid Excreted by Workers Occupationally Exposed to Benzene

Objective To investigate the relationship between trans, trans-muconic acid （ttMA） as benzene metabolite of occupational workers and benzene concentration in air. Methods A rapid and sensitive high-performance liquid chromatography was developed to determine the level of urinary ttMA. ttMA was extrated from urinary samples in liquid-liquid phase a ODS （2） （5u） column （Ф4.6 min× 150 mm） and detected at wavelength 264 nm in a UV detector using vanillic acid as an internal standard. The mobile phase was acetaticacid/tetrahydrofuran/methanol/water （v/v, 1：2：10：87）. The method was validated with 56 urine samples collected from occupationally benzene-exposed individuals. Results A correlation coefficient （r = 0.9963 ） was found for ttMA ranging 0.10-10.00 μg/mL. The limit of detection was 0.10μ g/mL. The recovery and reproducibility were generally over 90%. There was a positive correlation between ttMA and benzene level in air. The equation was Y=0.859＋0.108C （before work, r=0.6200） or Y=1.980＋0.179C （after work, r=0.7930）. Conclusion This method can be used to determine and control the level of urinary ttMA in those who are occupationally exposed to benzene.

Determination of Gastrodin Content in Gastrodia elata in Bomi of China by High-Performance Liquid Chromatography

[Objective] To select excellent varieties of Gastrodia elata and to determine the most suitable picking season of the plant. [Method] The gastrodin contents in fresh wild G. elata harvested in winter in Bomi County, artificially cultivated fresh G. elata harvested in winter in Bomi County, and nonlocal G. elata were determined by high-performance liquid chromatography （HPLC） under the following conditions., chromato- graphic column, AngilentTc-C18 （5 μm, 4.6 mm × 250.0 mm） ; mobile phase, acetonitrile-water （5：95, V/V） ; wavelength, 220 nm; flow rate, 1.0 ml/min; column temperature, 35℃; and injection volume, 10 μl. The gastrodin contents in G. elata respectively harvested in winter and summer were compared. [ Result] The gastrodin content was higher in the fresh wild G. elata harvested in winter in Bomi County （0.076%） than in the artificially cultivated fresh G. elata harvested in winter in Bomi County （0.045%） and nonlocal G. elata （0.025%）. Additionally, the gastrodin content was higher in G. elata harvested in winter than in those harvested in summer. [ Conclusion] G. elata from Bomi Country is an excellent variety and should be harvested in winter.

Development of a reversed dispersive based graphene functionalized with multiwalled carbon nanotubes for detection ofβ2-agonists in pork by high-performance liquid chromatography

A reliable and inexpensive pretreatment procedure in the determination ofβ2-agonists in pork was developped.The procedure used a nanocomposite of multiwalled carbon nanotubes(CNTs)functionalized with graphene(rGO)as the reversed dispersive sorbent.It was analyzed after purification by highperformance liquid chromatography,the extraction time and the properties of the nanocomposite were optimized.Under optimized conditions,present method has linear response over concentration range of 0.5–50 ng/mL in pork samples with a satisfactory detection limit close to 0.1 ng/mL.The precisions of the current method(coefficient of variation)are lower than 5%,while recoveries are more than 98.3%.The nanocomposite exhibited high adsorptivity,long-term storage stability,satisfactory anti-interfering activity and high selectivity toward2-agonists compared with those of rGO and CNTs.

The New Type of Liquid Chromatography—Colloidal High-Performance Liquid Chromatography (CHPLC): Further Applications

The recent paper by I. Brondz (2018), “One-Step Procedure for Direct Purification of Pediocin-Like Bacteriocins and Cationic Antimicrobial Peptides from Complex Culture Medium on an Analytical, Semipreparative, and Preparative Scale. A New Type of Liquid Chromatography—Colloidal High-Performance Liquid Chromatography (CHPLC)” (International Journal of Analytical Mass Spectrometry and Chromatography, 6, 41-49, https://doi.org/10.4236/ijamsc.2018.63004), described a new type of CHPLC. This technique allows colloidal liquids and suspensions to be chromatographed directly despite the presence in the liquids of material such as bacteria, fungi, and other soft and hard particles. The significance of this development lies in enabling the single-step cleanup and concentration of the target substance from a complex mixture of soluble molecules in the presence of insoluble particles by high performance liquid chromatography (HPLC). The technique also allows the use of viscose liquids (as described in Brondz (2018) that are not suitable for analysis by conventional HPLC. In the previous paper, emphasis was placed on describing the applications of the techniques for the preparation of target substances such as small peptides, bacteriocins, bacitracin, and lysosome. Normally, the industrial preparation of these substances requires multistep procedures, which are time- and labor-consuming, and typically results in significant loss of target material and specific activity. In the present paper, the application of CHPLC for the isolation of alkaloids from crude raw material such as opium cake is demonstrated. In the opium cake, large amounts of hard vegetable particles and even sand corns are present together with the target alkaloids. Despite this, isolation by CHPLC of the desired compound was achieved in a single step by using a water/ethanol-based liquid. Isolation of alkaloids from such raw material normally requires a multistep procedure that includes the preparation of insoluble tartrate or picrate complex and this process includes dissolving the substance in flammable organic liquid. The isolation described here was performed in a single step by using the water/ethanol-based liquid.

Fingerprint Analysis of PolygonumavculareL. Using High-Performance Liquid Chromatography

An HPLC method was established to control the quality of PolygonumavculareL. The active ingredients in PolygonumavculareL. samples were extracted with methanol, and then analyzed by high-performance liquid chromatography （HPLC）. The fingerprint of PolygonumavculareL. as well as its 11 typical peaks were established. The HPLC method demonstrated good precision, reproducibility, and stability, with the relative standard deviations of retention time and peak area less than 3%.

STUDY OF DERIVATIZATION OF PROTEIN WITH A EUROPIUM CHELATE AND APPLICATION TO HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

A europium chelate of 5-chlorosulfoyl-2- thenoyltrifluoroacetone(CTTA)was investigated for precolumn derivatization of protein for high performance liquid chromatography.This new label was highly fluorescent and suitable for time-resolved fluorometric detection.The detective limit of protein is less than 0.3ug in sample with a volume of 100ul.Theoretically,the new label can also be used in gel electrophoresis and protein blotting.

Cation-exchange high-performance liquid chromatography for variant hemoglobins and HbF/A2:What must hematopathologists know about methodology?

Cation-exchange high-performance liquid chromatography(CE-HPLC) is a widely used laboratory test to detect variant hemoglobins as well as quantify hemoglobins F and A2 for the diagnosis of thalassemia syndromes. It's versatility, speed, reproducibility and convenience have made CE-HPLC the method of choice to initially screen for hemoglobin disorders. Despite its popularity, several methodological aspects of the technology remain obscure to pathologists and this may have consequences in specific situations. This paper discusses the basic principles of the technique, the initial quality control steps and the interpretation of various controls and variables that are available on the instrument output. Subsequent sections are devoted to methodological considerations that arise during reporting of cases. For instance, common problems of misidentified peaks, totals crossing 100%, causes of total area being above or below acceptable limits and the importance of pre-integration region peaks are dealt with. Ultimately, CE-HPLC remains an investigation, the reporting of which combines in-depth knowledge of the biological basics with more than a working knowledge of the technological aspects of the technique.