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 Quinolone Antibiotics in Water Using Solid Phase Extraction-High Performance Liquid Chromatography-Fluorescence Method

[Objective] To develop a solid phase extraction-high performance liquid chromatography-fluorescence method for determination of quin- olone antibiotics in water. [ Metbod] The standard curves of four quinolones （norfloxacin, ciprofloxacin, Iomefloxacin and enrofloxacin） were pre- pared. The detection limit in water and recovery were determined. The water samples collected from different areas, river and tap water were trea- ted using solid-phese extraction method and analyzed by high performance liquid chromatography. Then the concentration of quinolones antibiotics was determined by fluorescence method. [ Result] The detection limit of quinolone antibiotics in water was 0.083 -0.248 μg/L, and their recovery was 63.7% -134.1%. The four quinolone antibiotics at different levels were detected in various water samples, and the total concentration of quin- olone antibiotics was 0.045 -3.969 μg/L. The total concentration of quinolone antibiotics was higher in the water samples collected from rivers in Shenzhen area than in the sewage samples. The four quinolone antibiotics could be detected in all tap water samples. [ CoaduLsion ] The solid phase extraction-high performance liquid chromatography-fluorescence method is feasible and effective to detect quinolones in water. In addition, this method needs low cost and can meet requirements of daily monitorina and analysis.

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.

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.

Molecularly Imprinted Micro-Solid-Phase Extraction for the Selective Determination of Phenolic Compounds in Environmental Water Samples with High Performance Liquid Chromatraphy

2,4,6-trichlorophenol molecularly imprinted suspension polymer has been prepared and applied to the molecularly imprinted micro-solid-phase extraction procedure for selective preconcentration of phenolic compounds from environmental water samples. The influence of functional monomer, cross-linker, polymerization condition, porogen, and the ratio of template molecule and functional monomer to cross-linker on the size of the obtained particles were investigated. It was found that methyacrylic acid as functional monomer, divinylbenzene as cross-linker, the molar ratio of template molecule and functional monomer to cross-linker was 1:4:20, the amount of AIBN was 100 mg, ultraviolet radiation at 365 nm were the optimal conditions, and at these conditions, the polymers had the best adsorption efficiency and had the monodispersity of 2 - 3 μm microsphere particles. The characteristics of the MIMSPE method were valid by high performance liquid chromatography. This MIMSPE-HPLC method has been successfully applied to the direct preconcentration and determination of phenolic compounds (phenol, 4-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol) in environmental water samples.

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

A highly sensitive SPE-liquid/liquid extraction RPLC method has been developed for the analysis of 6β-hydroxycortisol and cortisol in the urine of cancer patients. Methods： After SPE column purification and liquid-liquid extraction, the sample test solutions were analyzed with RPLC using a C18 analytical column. This improved analytical method has been validated for linearity, accuracy （recovery from urine）, repeatability （within-day and between-day precision）, specificity, sensitivity, and stability. This SPE-liquid/liquid extraction-RPLC is rapid, simple, accurate and reproducible. The technique is particularly useful for monitoring the CYP3A activity of cancer patients in clinical settings. The results are expressed as the ratio of 6β-hydroxycortisol to cortisol. Results： The CYP3A activity from a total of 153 samples was measured using this improved method. Considerable variation in the CYP3A activity of different cancer patients has been documented. Thus, personalized medical treatment based on the individual metabolic enzyme activity level is necessary. Conclusion： This new analytical method facilitates such individualized medical treatments.

Salting-Out Assisted Liquid-Liquid Extraction Combined with HPLC for Quantitative Extraction of Trace Multiclass Pesticide Residues from Environmental Waters

In this study, salting-out assisted liquid-liquid extraction combined with high performance liquid chromatography diode array detector (SALLE-HPLC-DAD) method was developed and validated for simultaneous analysis of carbaryl, atrazine, propazine, chlorothalonil, dimethametryn and terbutryn in environmental water samples. Parameters affecting the extraction efficiency such as type and volume of extraction solvent, sample volume, salt type and amount, centrifugation speed and time, and sample pH were optimized. Under the optimum extraction conditions the method was linear over the range of 10 - 100 μg/L (carbaryl), 8 - 100 μg/L (atarzine), 7 - 100 μg/L (propazine) and 9 - 100 μg/L (chlorothalonil, terbutryn and dimethametryn) with correlation coefficients (R2) between 0.99 and 0.999. Limits of detection and quantification ranged from 2.0 to 2.8 μg/L and 6.7 to 9.5 μg/L, respectively. The extraction recoveries obtained for ground, lake and river waters were in a range of 75.5% to 106.6%, with the intra-day and inter-day relative standard deviation lower than 3.4% for all the target analytes. All of the target analytes were not detected in these samples. Therefore, the proposed SALLE-HPLC-DAD method is simple, rapid, cheap and environmentally friendly for the determination of the aforementioned herbicides, insecticide and fungicide residues in environmental water samples.

Study on chromatographic fingerprint of sarcandra glabra (Thunb.) by microwave-assisted extraction coupled to HPLC/DAD

Microwave-assisted extraction(MAE)was used for extraction of effective components of sarcandra glabra(Thunb.),and then chromatographic fingerprint of sarcandra glabra(Thunb.)was studied by high performance liquid chromatography/diode array detector(HPLC/DAD).The conditions of MAE were optimized by an orthogonal experiment,and then the authentication and validation of the chromatographic fingerprint were conducted.Nine peaks were identified as common peaks in the fingerprint chromatograms,and isofraxidin was considered as a reference compound and quantified.Relative standard deviations of retention time and peak area of each component were less than 3% and 8%,respectively.Similarity and difference analysis were conducted by use of PCA and relation coefficient.Twenty batches of sarcandra glabra(Thunb.)samples from two different producing areas could be classified into two different groups in the PCA model.The results showed that MAE-HPLC/DAD method was simple,efficient and stable for the study of complex chromatographic fingerprint of sarcandra glabra(Thunb.),which could provide more reliable and precise information for quality evaluation.

Ionic Liquid-Based Ultrasound-Assisted Emulsification Microextraction Coupled with HPLC for Simultaneous Determination of Glucocorticoids and Sex Hormones in Cosmetics

An effective and environmentally friendly method based on ionic liquid-based ultrasound-assisted emulsification microextraction(IL-USAEME) and high performance liquid chromatography (HPLC) has been developed for the determination of nine hormones including three glucocorticoids, one androgen and five progestogens in cosmetics. Several factors that affect the extraction efficiency, such as the kinds and volume of ionic liquid, salt concentration, ultrasonic power and time, and centrifugation time were investigated and optimized. Under the optimum extraction condition, the recoveries of analytes ranged from 85.97% to 108.84% except prednisolone (62.30%). The intraday and interday precision was below 2.51% and 3.30%, respectively.

Ionic Liquid-based Microwave-assisted Liquid-liquid Microextraction and High Performance Liquid Chromatography Determination of Sulfonamides from Animal Oils

The authors performed ionic liquid-based microwave-assisted liquid-liquid microextraction（IL-based MALLME） coupled with high performance liquid chromatographic separation for the determination of 6 sulfonamides （SAs） from animal oils. The target analytes were extracted from animal oil samples with sodium hydroxide solution containing 1-butyl-3-methylimidazolium tetrafluoroborateand as the extraction solvent under microwave irradiation. The experimental parameters of the IL-based MALLME, including types of ILs, volume of IL, amount of ion-pairing agent（NHaPF6）, pH value of sample solution, and extraction temperature and time were evaluated. The limits of detection and quantification obtained were in a range of 0.4--0.5μg/kg and a range of 1.2--1.8μg/kg, respectively. The accuracy of the method was evaluated by analyzing five spiked animal oil samples at two fortified levels（5 and 50μg/kg）, and the recoveries of SAs varied from 81.4% to 114.5% with relative standard deviations ranging from 0.8% to 9.0%.

Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted,cloud-point extraction using AEO-9

This study aimed to establish a rapid analytical method to determine antioxidants in essence. A simple,efficient and practical, vortex-assisted, cloud-point extraction（VACPE） procedure is proposed for extracting and pre-concentrating four different of synthetic phenolic antioxidants（SPAs）, propyl gallate（PG）, tert-butylhydroquinone（TBHQ）, butylated hydroxyanisole（BHA）, butylated hydroxytoluene（BHT） in essence prior to high performance liquid chromatography（HPLC） analysis. The non-ionic surfactant, fatty alcohol polyoxyethylene ether-9（AEO-9）, was used as extractant and vortex-mixing was utilized to reduce extraction time and improve extraction efficiency. The effective parameters of the extraction process, such as volume of extraction solvent, pH, vortex-mixing time, equilibration temperature and time, were optimized. Under the optimum conditions, the linear range of PG, TBHQ,BHA and BHT was 8.0–800 ng/mL. All correlation coefficients of the calibration curves were higher than0.996 and relative standard deviations（RSD, n = 5） were 2.36%–5.46%. The proposed method was successfully applied to the extraction and determination of antioxidants in essence samples with satisfactory relative recoveries of 89.4%–103.5%. The results confirmed the SPAs of essence could be effectively monitored by this method and also established good reference criteria for essence.

Determination of Lignans in Schisandra sphenanthera and Schisandra chinensis Using Ionic Liquid-based Ultrasonic-assisted Extraction and High-performance Liquid Chromatography

A green, rapid and precise sample pretreatment technique, IL-based UAE（ionic liquid-based ultrasonic- assisted extraction）, was coupled with high-performance liquid chromatographic separation to identify the main effective components in Schisandra sphenanthera（S, sphenanthera） and Schisandra chinensis（S, ehinensis） including schisantherin A, schisandrin A, and deoxyschizandrin. Four different types of ionic liquids have been investigated, finally [C6MIM][BF4] was used as the extraction solvent. A powder form of S. sphenanthera and S. chinensis was mixed with the [C6MIM][BF4] to produce a suspension. This suspension was ultrasonically extracted in a water bath at room temperature. Several of the process parameters were optimized, including the type of ionic liquid used and its volume, the sample amount, the size of the sample particle, the extraction time, etc. HPLC calibration curves were estab-lished for all the analytes and proved to be linear（r〉0.9999）. The lowest detection level for schisandrin A was 0.12μg/mL, for schisantherin A was 0.08 μg/mL, and for deoxyschizandfin was 0.10μg/mL. The recoveries of the target compounds were from 74.19% to 109.33%. The standard deviations for detection were generally no more than 6.31%. In contrast to conventional extraction methods, the IL-based UAE did not involve volatile organic volatile solvents, and the analysis time, required sample and solvent vohtrnes were also lower than those of the conventional techniques.

A novel surface molecularly imprinted polymer as the solid-phase extraction adsorbent for the selective determination of ampicillin sodium in milk and blood samples

Surface molecularly imprinted polymers （SMIPs） for selective adsorption of ampicillin sodium were synthesized using surface molecular imprinting technique with silica gel as a support. The physical and morphological characteristics of the polymers were investigated by scanning electron microscope （SEM）, Fourier transform infrared spectroscopy （FT-IR）, thermogravimetric analysis （TGA）, elemental analysis and nitrogen adsorption-desorption test. The obtained results showed that the SMIPs displayed great adsorption capacity （13.5 lag/mg）, high recognition ability （the imprinted factor is 3.2） and good binding kinetics for ampicillin sodium. Finally, as solid phase extraction adsorbents, the SMIPs coupled with HPLC method were validated and applied for the enrichment, purification and determination of anapicillin sodium in real milk and blood samples. The averages of spiked accuracy ranged from 92.1% to 107.6%. The relative standard deviations of intra- and inter-day precisions were less than 4.6%. This study provides a new and promising method for enriching, extracting and determining ampicillin sodium in complex biological samples.

Extraction and RP-HPLC determination of taxol in rat plasma, cell culture and quality control samples

A rapid, sensitive, selective and validated reverse phase high-performance liquid chromatography （RP-HPLC） method for the estimation of paclitaxel in micro-sample of rat plasma and in culture of cancer cells was per- formed in this study. The mobile phase consisted of an （80： 20： 0.1, v/v/v）. Column elution at a flow rate of 1 optimized mixture of methanol：water： trifluroacetic acid mL/minute with UV detection at 225 nm at room tern- perature was used. The RP-HPLC method was successfully applied for the determination of paclitaxel in plasma samples and in culture of cancer cells with nano-quantity of estimation. The validation studies were performed in accordance with the International Conference on Harmonization （ICH） guidelines. The intra- and inter-day pre- cision showed that the coefficients of variation ranged from 1.07% to 4.27% at different levels of concentrations. To the best of our knowledge, this study also reported for the first time the optimization of different solvents for effective extraction of paclitaxel wherein tert.-butyl methyl ether （TBME）： diethyl ether （DEE） in 50：50 v/ v composition was found most efficient with extraction efficiency ranging between 77.99% and 91.74% and be- tween 76.14 and 93.66% in the plasma and cell culture, respectively. This proposed method was successfully ap- plied to study the pharmacokinetics of paclitaxel and the influence of verapamil and all-trans retinoic acid （atRA） on paclitaxel pharmacokinetics in rat models. This proposed method might emerge as a valuable aid in the labo- ratory monitoring of paclitaxel in a variety of in vitro as well as in vivo scenarios.

Matrix Solid-phase Dispersion Extraction of Alkaloids from the Roots of Aconitum kusnezoffii Reichb

Matrix solid-phase dispersion（MSPD） was developed for the extraction of four alkaloids, including aconitine, mesaconitine, hypaconitine and deoxyaconitine, from the roots ofAconitum kusnezoffii Reichb. The determination of the analyte was carried out by high performance liquid chromatography with UV detection. The alkaline alumina was used as sorbent. The mixture of acetonitrile and water was used as elution solvent. Several extraction parameters, such as type of sorbent, the ratio of sample to solid support material, type of the elution solvent and the volume of the elution solvent were tested. Mean recoveries ranged from 93.16% to 102.73%, with relative standard deviations from 0.27% to 4.17%. With the extraction efficiency and time expenditure taken into account, MSPD extraction should be a comparatively good method.

Comparison of Different Cartridges of Solid Phase Extraction for Determination of Polyphenols in Tobacco by UPLC/MS/MS and Multivariate Analysis

The comparison of solid phase extraction（SPE） for the preconcentration and isolation of polyphenols in tobacco samples was carried out by ultra-high performance liquid chromatography/tandem mass spectrometry （UPLC/MS/MS） and multivariate analysis.Several adsorbing materials of SPE（C18,NH2,SAX and OASIS） were investigated.It was found that the C18 and OASIS cartridges can not only speed up the purification process,but also simplify the SPE operation.A UPLC/MS/MS was used for the determination of polyphenols in tobacco samples after purification.All analytes were separated and determined in 2min.The limit of detection was 0.05 ng/mL.Cluster analysis（CA） and principal component analysis（PCA） were used for the analysis of 4 varieties（flue-cured tobacco,oriental tobacco,sun-cured tobacco and burley） in order to interpret the effect of planting and machining process on the concentration of polyphenols.The different types of tobacco samples could be easily clustered by CA.PCA on the chemical composition of tobacco resulted in two principal components（PCs） that take 84.2% of the total variance.The PCA and CA indicate that the polyphenols can be used for distinguishing tobacco types.

Extraction Process and Content Determination of Caffeic Acid in Zhuang Medicine Cryptolepis buchananii

[Objectives]To establish a method for determining the content of caffeic acid in Zhuang Medicine Cryptolepis buchananii.[Methods]The content of caffeic acid in C.buchananii leaves was determined by high performance liquid chromatography(HPLC).[Results]The results of HPLC determination showed that the RSD values were less than 3%,and the content of caffeic acid in 10 batches of C.buchananii leaves was in the range of 0.0965%-0.4772%.[Conclusions]This method is accurate and reliable,has good linear relationship and high precision,and can be used for quality evaluation of C.buchananii.

Optimization of the Extraction Process of Rutin from Flos Sophorae by Orthogonal Experiments

[Objectives]This study was conducted to optimize the extraction process of rutin in Flos Sophorae.[Methods]With the percentage content of rutin extracted from Flos Sophorae as the evaluation index and rutin as the reference substance,the content of rutin in Flos Sophorae extracted ultrasonically was determined by high performance liquid chromatography.L_(9)(3^(4))orthogonal experiments were carried out to optimize the three factors(solvent concentration,material-to-liquid ratio,extraction time)that affect the effect of ultrasonic extraction of rutin from Flos Sophorae.[Results]The extraction effect of the crude medicinal powder was best with the methanol concentration of 80%,the ratio of material to liquid at 0.1∶30,and the extraction time of 40 min.[Conclusions]This study provides a reference for optimizing the extraction process of rutin in Flos Sophorae.

全自动固相萃取—高效液相色谱法测定现制饮料中7种合成着色剂

目的:建立一种全自动固相萃取—高效液相色谱双波长法测定现制饮料中7种合成着色剂的方法。方法:样品经纯水提取2次,离心后,合并上清液,调节pH至3~4。利用全自动固相萃取装置活化Poly-sery色素专用固相萃取小柱,并上样、淋洗、洗脱。洗脱液氮吹浓缩至200μL,用50%流动相A+50%流动相B混合液定容至1.0 mL。色谱柱采用C 18柱,流动相为20 mmol/L乙酸铵—甲醇,梯度洗脱,流速1.0 mL/min,进样量10μL,检测波长254,628 nm。结果:各物质在0.5~50.0μg/mL范围内相关系数均大于0.9999,方法检出限为0.023~0.179 mg/kg,定量限为0.078~0.598 mg/kg,加标回收率为92.4%~96.8%,相对标准偏差(RSD)为1.2%~4.2%。结论:该方法试剂使用量小,操作步骤简便,自动化程度高,回收率高,重复性好,适合于批量样品测定。