A High-Performance Liquid Chromatography Method for the Simultaneous Determination of Five Index Components in Danhong Injection

The purpose of this study was to establish a high-performance liquid chromatography (HPLC) method for the simultaneous determination of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid in Danhong injection. The chromatographic method employed was as follows: the column was a Welch Ultimate XB-C18 column (250 mm × 4.6 mm, 10 μm), the mobile phase was a gradient elution of 0.4% formic acid aqueous solution (A) and acetonitrile (B), the detection wavelengths were 280 nm for sodium danshensu, protocatechuic aldehyde, and salvianolic acid B and 326 nm for 4-coumaric acid and rosmarinic acid, the sample volume was 10 μL, the flow rate was 1.0 mL/min, and the column temperature was 35°C. This method can realize the separation and determination of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid within 50 minutes. The linear relationships of the five peak areas and their concentrations are good (R2> 0.9997). The precision RSD values are all less than 1.0%. The reproducibility RSD values are all less than 1.3%. The stability RSD values are all less than 2.2%. The recovery values ranged from 92.4% to 99.4%. This method is simple, accurate, and reproducible. It can be used for the determination of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid in Danhong injection.

Analysis of Pesticide Raid^(█) in Feed of Wistar Rat by High-Pressure Liquid Chromatography (HPLC)

The distribution of pesticide by-product in tissues of wistar rats were analyzed using high pressure liquid chromatography. The limit of detection of the HPLC was 0.1 μg. Results show bioaccumulation factor of pesticide “Raid?” in lipid, up to three times that of the feed at the first concentration and gradually decreased as the concentration increased in the muscle > (0.7), brain > (0.5) and liver > (0.3) as indicated in the text. At higher concentration of 961 μg/g, bioaccumulation factor decreased in the lipid to 1.2 and 0.6 in the muscle, 0.03 in the brain and 0.08 in the liver respectively. High Pressure Liquid Chromatography (HPLC) analysis of raid extract suggests the presence of micprothrin and palethrin. The implications are numerous, but simply put that accidental ingestion of chlorinated hydrocarbon as in “Raid?” may involve convulsions, collapse and coma after only brief excitation and ataxia at the onset.

On-line enrichment and determination of polycyclic aromatic hydrocarbons in atmospheric particulates using high performance liquid chromatography with fluorescence as detector

Seven polycyclic aromatic hydrocarbons （PAHs） in atmospheric particulates were determinated by high performance liquid chromatography （HPLC） with fluorescence detector using direction injection and an on-line enrichment trap column. The method simplified the sample pretreatment, saved time and increased the efficiency. With the on-line trap column, PAHs were separated availably even underground injecting 1.0 ml sample with relatively high column efficiency. The recoveries of the seven PAHs were from 85% to 120% for spiked atmospheric particulate sample. The limit of detection was 15.3-39.6 ng/L （S/N=3.3）. There were good linear correlations between the peak areas and concentrations of the seven kinds of PAHs in the range of 1-50 ng/ml with the correlation coefficients over 0.9970. Furthermore, it also indicated that the method is available to determine PAHs in atmospheric particulates well.

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.

Bio-analytical method development and validation of Rasagiline by high performance liquid chromatography tandem mass spectrometry detection and its application to pharmacokinetic study

The most suitable bio-analytical method based on liquid liquid extraction has been developed and validated for quantification of Rasagiline in human plasma. Rasagiline-13C3 mesylate was used as an internal standard for Rasagiline. Zorbax Eclipse Plus C18 （2.1 mm × 50 mm, 3.5 um） column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involved simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode using an API-4000 system. The lotal run time was 3.0 min. The proposed method has been validated with the linear range of 5 12000 pg/mL for Rasagiline. The intra-run and inter-run precision values were within 1.3% 2.9% and 1.6% 2.2% respectively for Rasagiline. The overall recovery for Rasagiline and Rasagiline-13C3 mesylate analog was 96.9% and 96.7% respectively. This validated method was successfully applied to the bioequivalence and pharmacokinetic study of human volunteers under fasting condition.

Analysis of ganciclovir and its related substances using high performance liquid chromatography and liquid chromatography-mass spectrometry methods

Objective High performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC/MS)methods were developed for the determination of ganciclovir and its related substances.Methods A Hypersil ODS2 column(4.6 mm×250 mm,5 μm)was used with a mobile phase of 0.02 M potassium dihydrogen phosphate buffer(pH 6.0)-methanol(92∶8)at a flow rate of 1.0 mL/min,and UV detector set at 254 nm was used for monitoring the eluents.Results The method was simple,rapid,selective and capable of separating all related substances at trace level with a detection limit of 0.04 μg/mL.It has been validated with respect to accuracy,precision,linearity,and limits of detection and quantification.The linearity range was 10.2-153.0 μg/mL with r=0.9998.The percentage recoveries ranged from 96.7% to 101.6%,and RSD was 1.24%-1.96%(n=5).Conclusion The method was found to be suitable not only for monitoring the reactions during the process development but also for quality control of ganciclovir.For identification of related substances,LC/MS was used.The mainly related substances of ganciclovir active pharmaceutical ingredients(API)were determined as guanine,(1,3-dioxolan-4-yl)methyl acetate,and diacetyl guanine.

Isolation and Purification of Unstable Iridoid Glucosides from Traditional Chinese Medicine by Preparative High Performance Liquid Chromatography Coupled with Solid-phase Extraction

An efficient preparative method was successfully developed for isolation and purification of unstable components from medicinal plant extracts, using a combined method of preparative high performance liquid chro-matography(HPLC) and solid-phase extraction(SPE). The aim of this study was to obtain an effective method with high preparative efficiency and importantly to avoid the transformation of unstable compounds. The preparative HPLC system was based on an LC/MS controlled four-channel autopurification system. The SPE method was performed with a C18 packing material to trap the target compounds and to remove the acidic additive derived from the mobile phase. Using this method, the unstable iridoid glucosides(IGs) as model compounds were successfully isolated and purified from the extract of Hedyotis diffusa Willd. Six IGs(including one new minor IG) and one nucleotide compound were simultaneously obtained, each with a purity of 91% as determined by HPLC. The structures of the isolated compounds were identified by UPLC/Q-TOF MS, UV, 1D and/or 2D NMR. It was demonstrated that the combination of preparative HPLC with SPE is a versatile tool for preparative purification of unstable compounds from complex natural products.

Rapid Determination of Dopamine and Its Metabolites During in vivo Cerebral Microdialysis by Routine High Performance Liquid Chromatography With Electrochemical Detection

To determine dopamine and its metabolites during in vivo cerebral microdialysis by routine high performance liquid chromatography with electrochemical detection. Methods Microdialysis probes were placed into the right striatum of Wistar rat brains and perfused with Ringer＇s solution at a rate of 1.5 pL/min. A reverse phase HPLC with electrochemistry was used to assay DA, DOPAC, and HVA after cerebral microdialysates were collected every 20 minutes from awake and freely moving rats. In order to identify the reliability of this method, its selectivity, linear range, precision and accuracy were tested and the contents of DA, DOPAC, and HVA in rat microdialysates were determined. Results The standard curve was in good linear at the concentration ranging from 74 nmol/L to 1.5 pmol/L for DOPAC （r^2= 0.9996）, from 66 nmol/L to 1.3 gmol/L for DA （r^2=l.0000） and from 69 nmol/L to 1.4 pmol/L for HVA （r^2=0.9992）. The recovery of DOPAC （0.30, 0.77, 1.49 gmol/L）, DA （0,26, 0.69, 1.32 gmol/L）, and HVA （0.27, 0.71, 1.37 gmol/L） was 82.00±1.70%, 104.00±4.00%, 98.70±3.10%; 92.30± 1.50%, 105.30±2.30%, 108.00±2.00%; 80.00±7.80%, 107.69±8.00%, and 108.66±3.10%, respectively at each concentration. Their intra-day RSD was 3.3%, 3.4%, and 2.5%, and inter-day RSD was 4.2%, 2.3%, and 5.6%, respectively. The mean extracellular concentrations of DOPAC, DA, and HVA in rat brain microdialysates were 10.7, 2.4, and 9.2 gmol/L （n=6）, respectively. Conclusion The findings of our study suggested that the simple, accurate and stable method can be applied to basic researches of diseases related to monoamines neurotransmitters by cerebral microdialysis in rats.

High Performance Liquid Chromatography Analysis of Sugars and Acid Components in ’Xintai Tianhong’ Hawthorn Fruit

[Objectives]This study was conducted to provide basic information for the utilization,quality control and deep processing of‘Xintai Tianhong’hawthorn fruit resources.[Methods]The contents of sugar and acid components in‘Xintai Tianhong’hawthorn fruit were determined by high performance liquid chromatography(HPLC).[Results]‘Xintai Tianhong’hawthorn fruit contained such four kinds of sugar components as fructose,sorbitol,glucose and sucrose,and such six kinds of organic acid components as oxalic acid,tartaric acid,malic acid,acetic acid,citric acid and succinic acid.The fruit had the highest sucrose content,accounting for 70.53%of the total sugar content,followed by fructose,glucose and sorbitol.For the organic acid components,the content of succinic acid was the highest,accounting for 47.32%of the total acid content,followed by citric acid,malic acid,oxalic acid and tartaric acid,and acetic acid had the lowest content.[Conclusions]‘Xintai Tianhong’hawthorn should be a succinic acid-type hawthorn variety,which can be used as a parent in genetic research and breeding practice.

Metabolomic Studies Using High Performance Liquid Chromatography and Tandem Mass Spectrometry to Discover Quality Markers for Oriental Beauty (Dongfang Meiren) Tea

In this study, a high-performance liquid chromatograph and an electrospray ionization (ESI) triple quadrupole mass spectrometry (TQ MS) detector were used to scan Oriental Beauty tea of different grades and prices. Principle component analysis (PCA) of the profiling data was performed for pattern recognition, clearly showing that the proposed MS profiling method was able to classify Oriental Beauty tea into different grades. The component mass ions primarily responsible for the separation were selected with high loading strength in the PCA for subsequent identification with tandem mass (MS/MS). Caffeine, citrate and salicylate were verified, whereas certain other compounds remained ambiguous. Regression analysis considering caffeine, citrate and salicylate showed a linear relationship between the prices of the Oriental Beauty tea with an adjusted R2 of 0.84. If all the selected marker ions (in addition to caffeine, citrate and salicylate) could have been identified and incorporated into regression analysis, a stronger relationship could have been confirmed. These results suggest that metabolomics can facilitate the determination of real markers in the quality control of Oriental Beauty tea, and may lead to the further application of metabolomics in other food quality controls.

Determination of AF and AFG in Red Ginseng by High Performance Liquid Chromatography with Evaporative Light Scattering Detector (HPLC-ELSD)

[Objective]The paper was to establish a method for determining AF and AFG in red ginseng.[Method]A new simple,rapid and sensitive method for simultaneous determination of two amadori compounds,arginyl-fructose(AF)and arginyl-fructosyl-glucose(AFG),in extracts of three kinds of ginseng preparations was developed and validated using high performance liquid chromatography with evaporative light scattering detector(HPLC-ELSD).Two target analytes were efficiently separated by Prevail CTM18 column(4.6 mm×250 mm,5μm)at the flow rate of 0.8 mL/min within 15 min of single chromatographic run.[Result]Under optimized conditions,the detection limits were 0.015 and 0.02 mg/mL for AF and AFG,respectively.Calibration curves of peak area for two analytes were linear over three orders of magnitude with the correlation coefficients greater than 0.999.The average recoveries,precision,reproducibility and stability for two analytes(AF and AFG)were 99.5% and 100.9%,0.43% and 0.47%,0.46% and 0.43%,0.41% and 0.49%,respectively.[Conclusion]This method was successfully applied for quantifying AF and AFG in red ginseng and the method was efficient,sensitive and accurate.

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.

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.

Determination of Residual Acrylamide in Medical Polyacrylamide Hydrogel by High Performance Liquid Chromatography tandem Mass Spectroscopy

Objective To determine residual acrylamide in medical polyacrylamide hydrogel by high performance liquid chromatography tandem mass spectroscopy （HPLC-MS）. Methods After 13C3 labeled acrylamide was added, the sample was extracted with water and then cleaned up with ExtrelutTM 20. The polyacrylamide hydrogel sample and 20 clinical cases were analyzed by HPLC-MS/MS and isotope dilution quantifying technique in selected reaction monitoring （SRM） mode. Results Acrylamide was separated from polyacrylamide hydrogel. The concentration of acrylamide in polyacrylamide hydrogel ranged from 3.9×10^-9 to 3.1×10^- 8g/L in the 20 clinical cases. The peak area was favorable linear and the range was up to 3 000 μg/L. The recovery rate was 103.1% with a relative standard deviation （RSD） of 6.20%, when the mark level was 50 lag/L. Conelusion HPLC-MS is a rapid, accurate, and sensitive method for the determination of residual acrylamide in medical polyacrylamide hydrogel.

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.

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.

Modernization of Chinese herbal compound and the high performance liquid chromatography tandem mass spectrometry (HPLC-MS)

Chinese herbal compound is playing an important role on curing human diseases.And it has been a trend that Chinese herbal compound is being used all over the world in 21 century.However,our Chinese herbal compound is facing serious challenge for the lack of canonical system of quality criterion for Chinese herbal compound so it has been a urgent problem to set up the quality control standards and reveal therapeutic basis of Chinese herbal compound.In order to give full play to the advantages of Chinese herbal compound,modern scientific and technological is used to research of Chinese herbal compound,especially the high performance liquid chromatography tandem mass spectrometry(HPLC-MS),because it is high sensitive,rapid,and obtain more information.It is very necessary that HPLC-MS is uesed to elucidate the effective components of basic substances of Chinese Herbal Compound,and endow traditional Chinese medicine with modern scientific connotation.

Quantitative Analysis of Components in OC-CS Sprays by High Performance Liquid Chromatography with Double Wavelength UV Detection

The method of reversed-phase high performance liquid chromatography was established for the determination of effective components in OC-CS spray using double wavelength UV detection.The method was utilized under following conditions:a column of Kromasil C18(250 mm × 4.6 mm,5 μm),mobile phase consisting of methanol/water(80 /20) at a flow rate of 0.8 mL/min,column temperature of 25 ℃,and the UV detection at 227 nm and 300 nm.Three key components in OC-CS spray could be distinguished clearly,including o-chlorobenzalmalononitrile(CS),oleoresin capsicum(OC) and dihydrocapsaicin(DC).This method has the advantages of fast,simple and satisfactory linear relationship between UV absorption and concentration.It may be considered to turn into a standard method for detection of related components in the spray.

Sensitive determination of 4-O-methylhonokiol in rabbit plasma by high performance liquid chromatography and application to its pharmacokinetic investigation

A novel high performance liquid chromatographic method was developed for the determination of 4-O- methylhonokiol in rabbit plasma and was applied to its pharmacokinetic investigation. Plasma samples were treated by one-fold volume of methanol and acetonitrile to remove the interference proteins. A reverse phase column of SHIM- PACK VP-ODS （150 mm × 4.6 mm, 5.0 μm） was used to separate 4-O-methylhonokiol in the plasma samples. The detection limit of 4-O-methylhonokiol was 0.2 μg/L and the linear range was 0. 012 - 1. 536 mg/L. The good extraction recoveries were obtained for the spiked samples （84.7%, 89.3% and 87.7% for low, middle and high concentrations of added standards, respectively）. The relative standard deviation of intra-day and inter-day precisions was in the range from 0.6% to 13.5%. The pharmacokinetic study of 4-O-methylhonokiol was made and the results from the plasmaconcentration curve of 4-0-methylhonokiol showed a two-apartment open model. This work developed a sensitive, stable and rapid HPLC method for the determination of 4-O-methylhonokiol and the developed method has been successfully applied to a pharmacokinetic study of 4-O-methylhonokiol.

High Performance Liquid Chromatography Separation and Simultaneous Fluorometric Detection of Trace Amount of Polyamines in Cell Culture Media

A highly sensitive and precise method for the determination of the polyamines (including putrescine, spermidine and spermine) in cell culture media is described. The samples were concentrated by C18 column and the polyamines were converted to fluorometric derivatives with DNS-Cl. The polyamine derivatives were then completely separated by HPLC and determined by simultaneous fluorometric detection. The CV of intragroup and intergroup were 2. 49 %-4. 26% and 4. 29 %-5. 16 %, respectively. The rate of recovery was 103%- 99%. There was trace amount of polyamines detected by this method in the media of F12, 8900, RPMI1640, DMEM and M199 even without incubation with cells. So this method can be used for detecting the changes of polyamines in a medium before and after incubation with cells. It is helpful for the researches on the regulation of cell proliferation by polyamines.