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.

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.

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 trans-resveratrol in mouse liver by high performance liquid chromatography

To develop a sensitive high performance liquid chromatography （HPLC） assay for the determination of trans-resveratrol in mouse liver. The whole liver of a mouse was removed from the body, homogenated, and extracted by ethyl acetate. The organic layer was isolated and evaporated to dryness, the residue was reconstituted in 0.2 mL mobile phase for centrifugation, and 50 uL of the supernatant was injected into the/-IPLC instrument. The sample was separated on a Shimadzu ODS column （150 mm × 4.6 mm, 5 um） at 35 ℃ and detected by ultraviolet （UV） detector at the wavelength of 305 nm. The mobile phase consisted of methanol and 0.1 mol/L acetic acid （4：6, v/v） with the flow-rote at 1 mL/min. The limit of detection was 3.0 ng/g in liver homogenate with a signal/noise ratio of 3：1. The linear range of the calibration curve was 5.0-120.0 ng/g. The mean recoveries at the concentrations of 6, 10 and 80 ng/g were 102%, 96.0% and 91.5%, respectively. The RSDs for inter- and intra-day assays were less than 5%. Compared with other reported methods, this method was faster and more sensitive. It was also proved to be of good linearity, selectivity, accuracy and precision, and can be efficiently applied to the pharmacoldnetic study of trans-resveratrol in mouse liver.

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.

THYMINE BONDED-STATIONARY PHASE FOR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A new type of HPLC stationary phase containing thymine derivative was successfully prepared.It was found to give selective separation of nucleic acid bases and several purine derivatives,such as caffeine and theophylline.The retention behaviour and elution order of the solutes were interpreted in terms of molecular structure.

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.

Detection of 36 antibiotics in coastal waters using high performance liquid chromatography-tandem mass spectrometry

Among pharmaceuticals and personal care products released into the aquatic environment, antibiotics are of particular concern, because of their ubiquity and health effects. Although scientists have recently paid more attention to the threat of antibiotics to coastal ecosystems, researchers have often focused on relatively few antibiotics, because of the absence of suitable analytical methods. We have therefore developed a method for the rapid detection of 36 antibiotic residues in coastal waters, including tetracyclines （TCs）, sulfanilamides （SAs）, and quinolones （QLs）. The method consists of solid-phase extraction （SPE） and liquid chromatography-tandem mass spectrometry （LC-MS/MS） analysis, using electrospray ionization （ESI） in positive mode. The SPE was performed with Oasis HLB and Oasis MCX cartridges. Chromatographic separation on a Cr8 column was achieved using a binary eluent containing methanol and water with 0.1% formic acid. Typical recoveries of the analytes ranged from 67.4% to 109.3% at a fortification level of 100 ng/L. The precision of the method, calculated as relative standard deviation （RSD）, was below 14.6% for all the compounds. The limits of detection （LODs） varied from 0.45 pg to 7.97 pg. The method was applied to detemaine the target analytes in coastal waters of the Yellow Sea in Liaoning, China. Among the tested antibiotics, 31 were found in coastal ＇waters, with their concentrations between the LOD and 212.5 ng/L. These data indicate that this method is valid for analysis of antibiotics in coastal waters. The study first reports such a large number of antibiotics along the Yellow Sea coast of Liaoning, and should facilitate future comprehensive evaluation of antibiotics in coastal ecosystems

Comparison of protocatechuic aldchyde in Radix Salvia miitiorrhiza and corresponding pharmacological sera from normal and fibrotic rats by high performance liquid chromatography

AIM： To observe the effect of protocatechuic aldchyde on the proliferation of hepatic stellate cells （HSCs）. METHODS： Liver fibrosis was induced in rats by carbon tetrachloride （CCh）. Then normal and fibrotic drug sera were extracted from rats. The effects of protocatechuic aldchyde, raw Radix Salvia miltiorrhiza and drug sera of Salvia miltiorrhiza on HSC growth were determined by CCKoS. The protocatechuic aldchyde was separated by high performance liquid chromatography （HPLC） in a AIItima C18 column （250 mm × 4.6 mm, 5 μm） with a mobile phase of acetonitrile-4% glacial acetic acid solution （gradient elution） at the wavelength of 281 nm. RESULTS： Protocatechuic aldchyde, raw Radix Salvia miltiorrhiza and drug sera of Salvia miltiorrhiza were found to have inhibitory effects on proliferation of rat HSCs. Raw Radix Salvia miltiorrhiza had a stronger inhibitory effect than the drug sera. The fibrotic drug sera showed a higher suppressive effect than the normal drug sera （P 〈 0.05）. Protocatechuic aldchyde was found in crude materials of both Radix Salvia miltiorrhiza and its corresponding drug sera. The average recovery （n = 6） was 110.5% for raw Salvia miltiorrhiza Bge, 102% for normal drug sera and 105.2% for fibrotic drug sera. The relative standard devitation （RSD） was 0.37%, 1.96% and 1.51%, respectively （n=6）. The contents of protocatechuic aldchyde were 0.22%, 0.15% and 0.19%, respectively （n = 6） （P〈 0.05）. The RSD was 0.33%, 0.75% and 1.24% （n=6） for raw material of Radix Salvia miltiorrhiza, normal drug sera and fibrotic drug sera, respectively. The samples were stable for 6 d. CONCLUSION： Protocatechuic aldchyde can inhibit the growth of HSCs. HPLC is suitable for the determination of virtual bioactive components of Chinese herbal medicines in vitro.

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.

Determination of Organic Acids in Root Exudates by High Performance Liquid Chromatography: I. Development and Assessment of Chromatographic Conditions *1

Methods for determining nine low molecular weight organic acids in root exudates were developed by using reversed phase high performance liquid chromatography with UV (ultraviolet) detection at 214 nm. The mobile phase was 18 mmol L -1 kH 2PO 4 adjusted to pH 2.25 with phosphoric acid and the flow rate was 0.3 mL min -1 . The analytical column was a reversed phase silica based C 18 column (Shim pack CLC ODS). The root exudates were collected through submerging the whole root system into aerated deionized water for 2 hours. The filtered exudate solutions were concentrated to dryness by rotary evaporation at 40 °C, dissolved in 10 mL mobile phase. The chromatographic conditions of organic acid determination were analyzed. The results showed that there was a high selectivity and sensitivity in the organic acid determination by reversed phase high performance liquid chromatography. Coefficients of variation for organic acid determination were lower than 10% except lactic acid. The recoveries were consistently between 80.1% to 108.3%. Detection limits were approximately 0.05 to 4.5 mg L -1 for organic acids except succinic acid with the detection limit of 7.0 mg L -1 . Phosphorus deficiency may contribute to the release of organic acids in soybean root exudates especially malic, lactic and citric acids.

Determination of the Flavonoids from Ginkgo Biloba Extract by High Performance Liquid Chromatography

HPLC method for analysis of the flavonoids from ginkgo biloba extract (GBE) was studied. By suitable selection of columns. symmetrical chromatographic peaks were obtained without using acidic modifier in the mobile phase, which can eliminate the time for cleaning the chromatographic system and simplify the analystic method for GBE Experimental conditions: column: Hypersil BDS C-18, 5mumx4x250 mm: column temperature: 35degreesC; mobile phase: 46% methanol-54% water; flow rate: 0.7 mL/min; detection wavelength: 360nm.

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.

Fingerprint analysis of placenta polypeptide injection by high performance liquid chromatography

Objective: To develop the representative fingerprint for the quality control of placenta polypeptide injection. Methods: The chromatographic separation was performed using a Phenomenex Gemini C18 column (250 mm 4.6 mm, 5 mm) maintained at 30 1C. 0.1% aqueous trifiuoroacetic acid (Solvent A) and acetonitrile contained 0.1% TFA (Solvent B) were used as mobile phase with a gradient elution. Detection wavelength was 280 nm with the sample injection volume of 50 mL; the fiow rate was 1.0 mL/min. The fingerprints of different samples were investigated by similarity analysis. Results: Nine peaks were identified as the characteristic common peaks. The similarities of the fingerprints of the 10 batches of samples were above 0.992. Conclusion: This method showed high precision and good repeatability, and provided the basis for the improvement of the quality control of placenta polypeptide injection.

Determination of 17<i>α</i>-Methyltestosterone in Freshwater Samples of Tilapia Farming by High Performance Liquid Chromatography

17α-methyltestosterone is used to induce the sex reversal of Tilapia sp. to obtain cultures mono-sex to an economically viable. This practice may lead to environmental contamination and problems in human health. Therefore methods need to be developed to detect residues of 17α-methyltestosterone in aqueous matrices. A simple high-performance liquid chromatographic method using ultraviolet detection (245 nm) and testosterone as internal standard has been developed for the monitoring 17α-methyltestosterone in freshwater samples of tilapia aquaculture. The method described involves limited sample preparation as it includes a filtration followed by a single solid-phase extraction step using C18 cartridge. Validation data indicated that the HPLC-UV method for 17α-methyltestosterone determination in the concentration range of 50 - 2000 μg/L provided good linearity, sensitivity, accuracy and precision. Method performance was efficiently applied to monitoring the freshwater samples of fish ponds and the surrounding aquatic channels.

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.

Determination of Organic Acids in Root Exudates by High Performance Liquid Chromatography:Ⅱ.Influence of Several Testing Conditions

Effects of column temperature and flow rate on separation of organic acids were studied by determining nine low-molecular-weight organic acids on reversed- phase C18 column, using high performance liquid chromatography (HPLC) with a wavelength of UV (ultraviolet) 214 urn and a mobile phase of 18 mmol L-1 KH2PO4 buffer solution (pH 2.1). The thermal stability of organic acids was determined by comparing the recoveries of organic acids in different temperature treatments. The relationships between column temperature, flow rate or solvent pH and retention time were analyzed. At low solvent pH, separation efficiency of organic acids was increased by raising the flow rate of the solvent because of lowering the retention time of organic acids. High column temperature was unfavorable for the separation of organic acids. The separating effect can be enhanced through reducing column temperature in organic acid determination due to increasing retention time. High thermal stability of organic acids with low concentrations was observed at temperature of 40 ℃-45℃. Sensitivity and separation effect of organic acid determination by HPLC were clearly improved by a combination of raising flow rate and lowering column temperature at low solvent pH.

Determination of Trace Amount of Polycyclic Aromatic Hydrocarbons in Urban Sewage by Solid-phase Extraction Coupled with High Performance Liquid Chromatograph

[Method] This study aimed to determine trace amount of polycyclic aromatic hydrocarbons(PAHs) in urban sewage by using solid-phase extraction(SPE) coupled with high performance liquid chromatograph(HPLC).[Method] From the aspects of solid-phase extraction column,elution solvent,elution volume,elution speed and so forth,the test conditions of SPE-HPLC method were optimized,and trace amount of PAHs in urban sewage was determined.[Result] The optimized solid-phase extraction conditions were SUPELCLEAN LC-18 solid-phase extraction column,methylene dichloride as elution solvent,15 ml elution volume,2 ml/min elution speed,5 ml/min loading speed,1 000 ml water with 200 ml methanol loading volume.Under the optimized extraction conditions,the recovery was high,namely 76.3%-105.2%;relative standard deviation was 3.8%-6.0%,showing good precision;detection limit was low,only 0.000 8-0.048 0 μg/L.[Conclusion] This method is user-friendly,with high sensitivity and good precision,and suitable for continuous determination of a large volume of water samples.

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.

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.