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.

Determination of Benzo[a]pyrene in Edible Oil by High Performance Liquid Chromatography-Fluorescence Detector (HPLC-FL)

In this study, an optimized high performance liquid chromatography-fluorescence detector (HPLC-FL) method for the determination of benzo[a]pyrene in edible oil was established. HPLC was performed with Thermo Fisher Scientific C18 column (250 mm×4.6 mm, 5 μm) as the chromatographic column and acetonitrile and water as the mobile phase, and the excitation wavelength and emission wavelength of fluorescence detector were 286 and 430 nm, respectively. The response was high, and the linear range was 0.5-10.0 ng/ml. The lowest limit of detection was 0.11 ng/ml, and the average recovery was 92.5%. This method is suitable for quantitative analysis of benzo[a]pyrene content in edible oil.

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.

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.

Novel method for the determination of five carbamate pesticides in water samples by dispersive liquid-liquid microextraction combined with high performance liquid chromatography

A novel method for the determination of five carbamate pesticides （metolcarb, carbofuran, carbaryl, isoprocard and diethofencard） in water samples was developed by dispersive liquid-liquid microextraction （DLLME） coupled with high performance liquid chromatography-diode array detector （HPLC-DAD）. Some experimental parameters that influence the extraction efficiency were studied and optimized to obtain the best extraction results. Under the optimum conditions for the method, the calibration curve was linear in the concentration range from 5 to 1000 ng mL^-1 for all the five carbamate pesticides, with the correlation coefficients （r^2） varying from 0.9984 to 0.9994. Good enrichment factors were achieved ranging from 80 to 177- fold, depending on the compound. The limits of detection （LODs） （S/N = 3） were ranged from 0.1 to 0.5 ng mL^-1. The method has been successfully applied to the analysis of the pesticide residues in environmental water samples.

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.

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.

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.

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.

Research Advances in Detection Techniques of High Performance Liquid Chromatography-Refractive Index Detector

As a highly sensitive and stable detector,refractive index detector is usually used for quantitative detection of substances such as polymer,sugar and organic acid. The research reviewed the application of HPLC-RID in the fields of quantitative determination of medicine and food,in order to lay a foundation for wider use of RID.

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.

Optimization of Ultrasonic Extraction and Clean-up Protocol for the Determination of Polycyclic Aromatic Hydrocarbons in Marine Sediments by High-performance Liquid Chroma-tography Coupled with Fluorescence Detection

The procedures of ultrasonic extraction and clean-up were optimized for the determination of polycyclic aromatic hydrocarbons (PAHs) in marine sediments. Samples were ultrasonically extracted, and the extracts were purified with a miniaturized silica gel chromatographic column and analyzed with high performance liquid chromatography (HPLC) with a fluorescence detector. Ultrasonication with methanol-dichloromethane (2:1, v/v) mixture gave higher extraction efficiency than that with dichloromethane. Among the three elution solvents used in clean-up step, dichloromethane-hexane (2:3, v/v) mixture was the most satisfactory. Under the optimized conditions, the recoveries in the range of 54.82% to 94.70% with RSDs of 3.02% to 23.22% for a spiked blank, and in the range of 61.20% to 127.08% with RSDs of 7.61% to 26.93% for a spiked matrix, were obtained for the 15 PAHs studied, while the recoveries for a NIST standard reference SRM 1941b were in the range of 50.79% to 83.78% with RSDs of 5.24% to 21.38%. The detection limits were between 0.75 ng L-1 and 10.99 ng L-1for different PAHs. A sample from the Jiaozhou Bay area was examined to test the established methods.

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.

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 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.

Determination of neomycin in water samples by high performance anion chromatography with pulsed amperometric detection

A simple, fast and reliable method, using high performance anion chromatography with pulsed amperometric detection, had been developed for the analysis of neomycin in water samples. The elution and separation were carried out with an isocratic mobile phase, containing 10 mmol/L NaOH. The influence of the concentration and pH of the mobile phase on the separation and detection was investigated. A quadruple-potential waveform used for the detection was optimized. The detection limit of neomycin was down to 0.027 μg/mL. The linearity of neomycin calibration curve ranged from 0.050 to 0.505 μg/mL with correlation coefficient of 0.9997. R.S.D. (n = 11) was 4.0%.

Study on the Flow Injection Micro-Column Pre-Separation System Coupled With High Performance Liquid Chromatography for the Determination of Ecdysterone in Traditional Chinese Medicine

A flow injection (FI) micro-column system coupled with high performance liquid chromatography (HPLC) was proposed for the pre-separation and determination of active organic component (ecdysterone) in traditional Chinese medicine, Loulu. The factors influencing separation performance were investigated and optimized. Under the optimal conditions, the contents of ecdysterone in Loulu were determined by HPLC system using MeOH-H_2O (40: 60,V/V) as the mobile phase at a flow rate of 1. 0 mL/min. The calibration curve was linear in the range of 0. 5~ 100 mg/L of ecdysterone concentrations. The detection limit of the analyte was 0. 11mol/L(3) with a precision of 0. 38% RSD (n=7 f c= 10. 0 mg/L). The average recovery of the method was 98. 7%. The proposed method has been applied to determine ecdysterone in practical samples, and the determined values by both external standard method and standard addition method were in good agreement. Compared to the traditional solid extraction method, the system proposed has the advantages of simple procedure, good reproducibility, minimum volume requirement, reduction of matrix interference and low contamination risk.

Analysis of microsatellite instability in stool DNA of patients with colorectal cancer using denaturing high performance liquid chromatography

AIM： To evaluate the usefulness of denaturing high performance liquid chromatography （DHPLC） for analyzing microsatellite instability （MSI） status in stool DNA of patients with colorectal cancer. METHODS： A total of 80 cancer tissues from patients with primary sporadic colorectal tumor （proximal cancer： 27, distal cancer： 53） and matched stool （which were employed for comparison with the tissues） were analyzed for MSI status in BAT 26. DNA samples extracted from stool were evaluated by nested polymerase chain reaction （PCR） and DHPLC for MSI analysis. RESULTS： Six cases （7.5%） of MSI were identified in BAT 26 from 80 cancer tissues. All the stool DNA samples from patients whose cancer tissue showed IVlSI also displayed MSI in BAT 26. CONCLUSION： As MSI is one of the established fecal DNA markers to screen colorectal cancer, we propose to use DHPLC for the IVlSI analysis in fecal DNA.

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.