Search

In recent years, more sensitive and less invasive methods for detecting organ transplantation rejection have been receiving increasing interest. Metabonomics is a recent development in the transplantation field. In this paper, a metabonomic investigation on plasma samples of liver transplanted rats was presented. Samples from control, syngeneic, and allogeneic transplanted groups were analyzed using GC-MS after derivatization. Thirty-six metabolites in the acquired GC-MS data, including amino acids, organic acids, carbohydrates, and some other metabolites were identified and matched by NIST spectral library. The scores plot of principal component analysis (PCA) showed that the three groups were unambiguously apart from each other; the syngeneic liver transplanted group and the control group were adjacent, indicating a high similarity between the two groups; the allogeneic liver transplanted group, however, was apparently separate from the other two groups, demonstrating the significant differences of meta...

The separation and determination of four important active components (imperatorin, isoimperatorin, phelloptorin and falcarindiol) from Angelica dahurica extract has been performed using capillary electrochromatography (CEC) with a methacrylate ester-based monolithic column. The effect of the porogen ratio on the column preparation was studied. The mobile phase composition, such as the concentration of organic solvent, the ionic strength and the pH of the buffer were also optimized. Under the optimized conditions (50% acetonitrile and 50% of a 20 mmol/L sodium dihydrogen phosphate electrolyte at pH 4.95, - 25 kV), a fast and baseline separation of the four analytes was achieved. The calibration curves showed a good linearity (r2 > 0.997) and the limits of detection were lower than 0.34 mg/L. The mean recoveries of the studied components ranged between 95.18% and 98.44%. The method developed is sensitive, reliable and suitable for the quality control. With this CEC system, the quality of Angelica dahurica extracts from 18 various regions was evaluated.

A rapid HPLC method using a monolithic column with fluorescence detection has been developed for determination of telmisartan in human plasma. Sample preparation was done by protein precipitation with acetonitrile and naproxen was used as IS. The compounds were detected by fluorescence detection, using an excitation wavelength of 300 nm and emission wavelength of 385 nm. Calibration curves of telmisartan were linear in the range of 1-200 ng/mL. The assay was high throughput, sensitive and precise, and it was successfully applied to a bioequivalence study of two formulations of telmisartan.

This paper describes a sensitive, specific and rapid high-performance liquid chromatography (HPLC) method for the determination of curcumin in rat plasma. After a simple step of protein precipitation in 96-well format using acetonitrile containing the internal standard (IS), emodin, plasma samples were analyzed by reverse-phase HPLC. Curcumin and the IS emodin were separated on a Diamonsil C(18) analytical column (4.6 x 100 mm, 5 microm) using acetonitrile-5% acetic acid (75:25, v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was sensitive with a lower limit of quantitation of 1 ng/mL, with good linearity (r(2) >or= 0.999) over the linear range 1-500 ng/mL. All the validation data, such as accuracy and precision, were within the required limits. A run time of 3.0 min for each sample made high-throughput bioanalysis possible. The assay method was successfully applied to the study of the pharmacokinetics of curcumin liposome in rats.

A two-dimensional column-switching system without sample loop trapping, where two columns were operated via a six-port switching valve, was employed in the isolation and purification of five isoflavonoids from Rhizoma Belamcandae: tectoridin, iridin, tectorigenin, irigenin and irisflorentin. The introduction of the six-port switching value, instead of a sample loop, assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension were not restricted. Two configurations were tested in this study. In the first mode, only one column was used in the second dimension and two ‘heart-cutting’ fractions were transported to the same second-dimensional column. In the second mode, two parallel columns were used in the second dimension and two fractions were transported to the two columns. Between the two configurations, the one with two second dimensional columns had double sample size, better resolution and one more purified compound. Both two-dimensional configurations consumed less solvent with even greater efficiency and shorter cycle time compared with conventional gradient methods. All of the isoflavonoids were isolated at high purities of greater than 95% with yields of above 82%. Copyright © 2009 John Wiley & Sons, Ltd.

Three flavonoids including 4″′-O-rhamnosylrutin, 2″-O-rhamnosylvitexin, and 4″-O-glucosylvitexin were isolated and purified from the hawthorn leaves by high speed countercurrent chromatography (HSCCC), combined together with isocratic preparative reversed-phase high performance liquid chromatography. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of n-butanol-water (1:1, v/v). After two useful fractions were obtained by HSCCC using the optimal solvent system, one of them, fraction II, was further purified by reversed-phase semipreparative high performance liquid chromatography. From 500 mg partially purified extract of the hawthorn leaves, 188.4 mg 2″-O-rhamnosylvitexin, 28.5 mg 4″′-O-rhamnosylrutin, and 55.3 mg 4″-O-glucosylvitexin with purities of over 97%, respectively, were obtained. The structure identification of all pure fractions was carried out by UV, MS, 1H NMR and 13C NMR.

This study presents an efficient strategy based on microwave-assisted extraction (MAE), HPLC-DAD-MS/MS and high-speed counter-current chromatography (HSCCC) for the rapid extraction, identification, separation and purification of active components from the traditional Chinese medicine Fructus Aurantii Immaturus. An LC-DAD-MS/MS method was applied for the screening and structural identification of main components in crude extract, and five components were preliminarily identified as neoeriocitrin, narirutin, naringin, hesperidin and neohesperidin according to their UV and mass spectra. An efficient MAE method for the extraction of the three most abundant components (narirutin, naringin and neohesperidin) was optimized by the combination of univariate and multivariate approaches. The crude extract was then separated and purified by HSCCC and a total of 61.6 mg of narirutin, 207.3 mg of naringin and 159.5 mg of neohesperidin at high purities of 98.1, 97.2 and 99.5%, respectively, were obtained from 1.42 g of crude extract. The recoveries of these compounds were 86, 93 and 89%, respectively.

An optimized temperature-programmed gas chromatography–mass spectrometry system combined with chemometric methods was firstly applied to analyze the volatile components of M. biondii Pamp. A total of 65 components were identified using similarity searches between mass spectra and MS database. This number was extended to 80 components with the help of chemometric techniques. The peak purity of two-way data was controlled by fixed size moving window evolving factor analysis, two dimensional-evolving latent projection graph and three dimensional-evolving latent projection graph. Then the overlapped peak clusters were resolved using heuristic evolving latent projection. The results prove that the reported approach is powerful for the analysis of complex herbal samples.

A tandem liquid chromatographic–time-of-flight mass spectrometric (LC–TOFMS) method has been developed for rapid separation and identification of diarylheptanoids and gingerol-related compounds in aqueous extracts of dried ginger. Total-ion-current chromatograms and mass spectra were acquired. A formula database of known compounds was established, against which components of dried ginger could be rapidly identified by matching their exact masses with theoretical masses of compounds calculated from their empirical formulae. Identification of 20 compounds was accomplished with error of 4 ppm, and further confirmation of elemental composition was obtained from the abundance of the isotope peaks. LC–TOFMS has been shown to be a useful tool for rapid identification of compounds in aqueous extracts of dried ginger.

A rapid, simple, sensitive, and accurate high performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of 6-gingerol, 8-gingerol, and 10-gingerol in three medicinal gingers. The chromatographic separation of three gingerols was achieved within 4 min on a sub-2 µm particles column. Gradient elution was performed using acetonitrile-water as the mobile phase at a flow rate of 1.0 mL/min. The injection volume was 2 µL, and the detection wavelength was set at 280 nm. Validation studies revealed that the method was reliable and reproducible for the simultaneous quantitation of the three gingerols in three medicinal gingers.

Carbon tetrachloride (CCl 4 ) is a well-known model compound for producing chemical hepatic injury. Cytochrome P450 is an important monooxygenase in biology. We investigated the CYP450 protein expression in the in vivo hepatotoxicity of rats induced by CCl 4 . In this experiment, CCl 4 were administered to male rats, and their livers at 24 h post-dosing were applied to the proteomic analysis. Blood biochemistry and histopathology were examined to identify specific changes. At the same time, a novel acetylation stable isotopic labeling method coupled with LTQ-FTICR mass spectrometry was applied to disclose the changes of cytochrome P450 expression amounts. The quantitative proteomics method demonstrated its correlation coefficient was 0.9998 in a 100-fold dynamic range and the average ratio of the labeled peptides was 1.04, which was very close to the theoretical ratio of 1.00 and the standard deviation (S.D.) of 0.21. With this approach, 17 cytochrome P450 proteins were identified and quantified with high confidence. Among them, the expression amount of 2C11, 3A2, and 2 E1 were down-regulated, while that of 2C6, 2B2, and 2B1 were up-regulated.

A new drug, quick-acting anti-motion capsule (QAAMC) composed of d-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion-sickness use. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of d-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 microL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm x 3.0 mm, 3.5 microm) with a mobile phase composed of methanol-water-formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0-->91.0 (D-amphetamine), 256.0-->167.0 (diphenhydramine) and 166.1-->148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for d-amphetamine and 1 ng/mL for diphenhydramine, with good linearity in the range 0.5-200 ng/mL for D-amphetamine and 1-500 ng/mL for diphenhydramine (r(2)> or =0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs.

A cheap, simple and rapid sample preparation method has been developed for quantification of ulifloxacin, the active metabolite of prulifloxacin in human plasma, by HPLC with fluorescence detection using lemefloxacin as the internal standard. One-step protein precipitation with 10% perchloric acid (2:1, v/v) on a 200 μL sample was used. The separation was performed at 30 °C on a C18 column using an eluent of acetonitrile-0.5% triethylamine buffer. The compounds were monitored at λex of 280 nm, λem of 425 nm. The calibration curve for ulifloxacin in human plasma was linear over the range 0.01–1.00 μg mL−1. The lower limit of quantification is 0.01 μg mL−1. The intra- and inter-day precision ranged from 3.0 to 6.7%, respectively. The method had been used for clinical pharmacokinetic studies of prulifloxacin formulation product after oral administration to healthy volunteers.

Developing a simple and fast method to analyze possibly adulterated synthetic drugs in suspected herbal medicines (HM) is both methodologically and commercially significant. This paper constructs a new approach named local straight-line screening (LSLS), to the solution of the problem, after carefully observing the characteristics of the spectral line shapes. LSLS can be applied to both the qualitative and quantitative analysis of suspected HM, based solely on infrared spectrum of the possible synthetic drug and the suspected HM without any sample pretreatment. The concept and the application of the method are exemplified by analysis of sibutramine hydrochloride (SH), an anti-obesity medicine, adulterated in some commercially available HM-based diet pills, and compared with MSFDSS, and SNICA methods. The results show that, despite highly overlapping spectra and unresolved data, it is possible with LSLS to obtain an accurate discrimination and determination of SH in the HM-based diet pills. This allows the method to be potentially used in the primary screening of other synthetic drugs suspiciously adulterated in HM, with high rapidity, accuracy and cost-effectiveness.

The quick separation and simultaneous determination of d-amphetamine and diphenhydramine in the quick-acting anti-motion capsules was investigated by capillary zone electrophoresis. The influence of different parameters (internal standard, injection modes, pH, concentration of the running buffer and applied voltage) was systematically studied. The two compounds could be well separated within 2.0 min in a 40.2 cm fused-silica capillary at a separation voltage of 20 kV in a 50 mM phosphate–12.5 mM borate buffer adjusted to pH 5.5. Correlation coefficients for calibration curves in the range 0.50–1.50 μg mL−1 for d-amphetamine and 2.75–8.25 μg mL−1 for diphenhydramine were higher than 0.999. The limits of detection of d-amphetamine and diphenhydramine were 10.0 and 5.5 ng mL−1 and the recoveries of the compounds in the QAAMC were 99.80 and 99.85%, respectively.

High-speed counter-current chromatography (HSCCC) was applied to the isolation and purification of geniposide from Gardenia jasminoides Ellis. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of ethyl acetate– n- butanol–water (2:1:3, v/v/v). According to the above solvent system, preparative HSCCC was successfully performed with the optimal solvent system composed of ethyl acetate– n- butanol–water (2:1.5:3, v/v/v) yielding 389 mg of geniposide at over 98% purity from 1 g of the partially purified extract with 38.9% recovery in a one-step separation.

A selective chiral high performance liquid chromatographic (HPLC) method coupled with achiral column was developed and validated to separate and quantify tetrahydropalmatine (THP) enantiomers in dog plasma. Chromatography was accomplished by two steps: (1) racemic THP was separated from biological matrix and collected on a Kromasil C18 column (150 mmx4.6 mm, 5 microm) with the mobile phase acetonitrile-0.1% phosphoric acid solution, adjusted with triethylamine to pH 6.15 (47:53); (2) enantiomeric separation was performed on a Chiralcel OJ-H column (250 mmx4.6 mm, 5 microm) with the mobile phase anhydrous ethanol. The detection wavelength was set at 230 nm. (+)-THP and (-)-THP were separated with a resolution factor (Rs) of at least 1.6 and a separation factor (alpha) greater than 1.29. Linear calibration curves were obtained over the range of 0.025-4 microg/ml in plasma for each of (+)-THP and (-)-THP (R2>0.999) with a limit of detection (LOD) of 0.005 microg/ml and the recovery was greater than 88% for each enantiomer. The relative standard deviation (R.S.D.) and relative error values were less than 10% at upper and lower concentrations. The method was used to determine the pharmacokinetics of THP enantiomers after oral administration of racemic THP. The results presented herein showed the stereoselective disposition kinetics of THP in dogs and were a further contribution to the understanding of the kinetic behavior of THP analogues.

Three flavonoid glycosides including orientin, vitexin, quercetin-3-O-neohesperidoside and one unknown compound were isolated and purified by high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC from Trollius ledebouri Reichb., a traditional Chinese medicine. Preparative HSCCC with a two-phase solvent system composed of ethyl acetate–n-butanol–water (2:1:3, v/v/v) was successfully performed by increasing the flow-rate of the mobile phase from 1.5 to 2.5 ml/min after 190 min. Consequently, 95.8 mg orientin, 11.6 mg vitexin, 9.3 mg unknown compound with purities of over 97% and one partially purified peak fraction (contained quercetin-3-O-neohesperidoside at 85.1% purity) were obtained from 500 mg of the crude extract. Then the partially purified fraction was further purified by reversed-phase semi-preparative high-performance liquid chromatography. The structure identification of all pure fractions was carried out by UV, MS, 1H NMR and 13C NMR.

Following an initial cleaning-up step on the D101 macroporous resin, a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (1:0.9:0.9:1, v/v) was used to isolate and separate schizandrin and gomisin A from Schisandra chinensis. A total of 107 mg schizandrin and 36 mg gomisin A with purities of 99.5% and 99.1% were obtained from 400 mg crude extract in one-step elution and less than 3 h, and the structure identification was performed by UV, IR, MS, 1 H NMR and 13 C NMR.

A preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of ethyl acetate-n-butanol-water (2:7:9, v/v/v) was successfully performed to isolate and separate clemastanin B and indigoticoside A from the plant of Radix Isatidis, a traditional Chinese medicine. A total of 59.2 mg clemastanin B and 66.1 mg indigoticoside A with purities of 94.6% and 99.0% determined by high performance liquid chromatography (HPLC) were obtained in one-step elution from 250 mg crude extract, which contained clemastanin B 24.8% and indigoticoside A 28.4%, and the recoveries of clemastanin B and indigoticoside A were 90.3% and 92.2%, respectively. The chemical structure was identified by IR, MS, 1H NMR and 13C NMR.

Extracts of herbs with promising antidiabetic capacity, used in traditional medicine, have been analyzed by high-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD). Optimization of the chromatographic conditions is discussed. Two kinds of xanthone glycoside were successfully separated and quantified in herbs from seven regions. The neomangiferin and mangiferin content of herbs from different regions varied greatly. The sensitivity, selectivity, accuracy, precision, and ruggedness of the proposed method were excellent, and the method can be used for quality control of Anemarrhena asphodeloides rhizome raw material.

High-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v) was used to isolate and separate two C-glycosylflavones from Patrinia villosa Juss, a traditional Chinese medicine. The separation produced 42.9 mg isovitexin and 20.1 mg isoorientin with purities of 99.3% and 98.5%, respectively as determined by high-performance liquid chromatography (HPLC) in one step elution from 250 mg crude extract, and identification was performed by MS, 1H NMR and 13C NMR. It is the first report of discovering isovitexin and isoorientin from the plant of Patrinia genus.

A rapid and sensitive CEC method with methacrylate ester-based monolithic column has been developed for separation and determination of five coumarins (byakangelicin, oxypeucedanin hydrate, xanthotoxol, 5-hydroxy-8-methoxypsoralen and bergapten) in Angelica dahurica extract. Surfactant sodium desoxycholate (SDC) was introduced into the mobile phase as the pseudostationary to dynamically increase the selectivity of analytes instead of increasing the hydrophobicity of stationary phase. In addition, other factors, pH of phosphate buffer, ACN content and applied voltage, for instance, have also an obvious effect on the resolution but little on the retention time. Satisfactory separation of these five coumarins was achieved within 6 min under a 30:70 v/v ACN-buffer containing 20 mM sodium dihydrogen phosphate (NaH(2) PO(4) ) and 0.25 mM SDC at pH 2.51. The RSDs of intraday and interday for relative peak areas were less than 3.0% and 4.7%, respectively; and the recoveries were between 87.5% and 95.0%. The LODs were lower than 0.15 μg/mL and the LOQs were lower than 0.30 μg/mL, respectively, while calibration curves showed a good linearity (r(2) > 0.9979). Finally, five target coumarins from the crude extracts of A. dahurica were separated, purified, and concentrated by D-101 macroporous resin, and were successfully separated and quantitatively determined within 6 min.

A comprehensive two-dimensional (2D) separation is one that employs two separation dimensions (columns) and draws on all of the available resolving power from each of the dimensions of separate the components in a sample. In this study, a comprehensive 2D chromatography approach was developed for the separation and identification of membrane permeable compounds in a famous traditional Chinese medicine of Schisandra chinensis. The first dimensional column was the immobilized liposome chromatography (ILC) column, which mimics the biological membranes and can be used to study drug-membrane interactions in liquid chromatography. Using an automatic ten-port switching valve equipped with two sample loops, the section of the first-dimension was introduced in the second-dimension consist of a silica monolithic column. More than 40 components in Schisandra chinensis were resolved by using the developed separation system and among them 14 compounds were identified interacting with the ILC column based on their retention action, UV and mass data. With this comprehensive 2D-HPLC system, the three-dimensional chromatographic fingerprints of Schisandra chinensis were preliminarily established and processed by using principal component analysis and hierarchical clustering analysis. The obtained information can distinguish the unacceptable samples of the quality control. The result demonstrated that the 2D biochromatography system has been demonstrated to have more advantages of finding strong binding bioactive components, providing an enhanced peak capacity, good sensitivity and powerful resolution biological fingerprinting analysis of complex TCMs, which was a useful means to control the quality of and to clarify the membrane permeability of the compounds in Schisandra chinensis.

Introduction – Further studies of active coumarin components in Radix Angelicae Dahuricae (AE) are absolutely essential to provide data on pharmacology, toxicology and quality for innovative drug candidates. Thus, the preparation of active component standards and the administration of coumarin monomers should be carried out. The isolation of the low-level active components from complex Traditional Chinese Medicine (TCM) samples necessitates the development of rapid, simple and economical modern extraction, separation, identification and purification methods. Objective – To develop an efficient strategy for the rapid extraction, separation, identification and purification of coumarins from AE. Methodology – First, active coumarins in AE were extracted with microwave-assisted extraction (MAE) after the extraction conditions were optimised. Second, gradient extraction methods with MAE were used to partially purify AE. Third, a high-performance liquid chromatography–diode array detection-electrospray ionisation tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) method was applied for the preliminary on-line identification and screening of the main coumarins in AE extract. Finally, a two-dimensional preparative high-performance liquid chromatography–diode array detection (2D-prep-HPLC-DAD) system was developed for further preparative separation of those target components. Results – Altogether 10 coumarins have been identified and five of them including xanthotoxol, osthenol, oxypeucedanin hydrate, byakangelicin and imperatorin were deemed as target components for the preparative isolation. All of the five isolated coumarins were at high purities of over 99% and the production rate was much higher than the traditional methods. Conclusion – The present paper demonstrates that these consecutive approaches are very useful for to isolate chemical constituents from TCM.

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, followed by a 96-well protein precipitation, has been developed and fully validated for the determination of Phakellistatin 13 (PK13), a new cyclic heptapeptide isolated from the sponge Phakellia fusca Thiele, in rat plasma. After protein precipitation of the plasma samples (50 microL) in a 96-well plate by methanol (200 microL) containing the internal standard Pseudostellarin B (20 ng/mL), the plate was vortex mixed for 3 min. Following filtration for 5 min, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on an XB-C18 analytical column (5 microm, 50 mm x 4.6 mm i.d.) using an eluent of methanol-water (85:15, v/v) and detected by electrospray ionization mass spectrometry in the negative multiple reaction monitoring mode with a chromatographic run time of 5.0 min. The method was sensitive with a lower limit of quantification (LLOQ) of 0.1 ng/mL, with good linearity (r0.999) over the quantitation range of 0.1-5 ng/mL. The validation results demonstrated that this method was significantly specific, accurate, precise, and was successfully applied in measuring levels of PK13 in rat plasma following intravenous administration of 20, 50, and 100 microg/kg of peptide in rats, respectively, which was suitable for the preclinical pharmacokinetic studies on PK13.

A non-aqueous capillary electrophoretic method was established for the determination of berberine in Rhizoma coptidis and its preparations. The effects of organic solvent and the concentrations of sodium acetate were studied, which showed that berberine in extracts of traditional Chinese medicine can be separated successfully in a buffer solution of 75 mmol/L of sodium acetate in methanol containing 1 mol/L of acetic acid. The simple and rapid method was linear in the range 25-200 microgram/mL of berberine and had a good reproducibility, with the relative standard deviation below 2%. Non-aqueous capillary electrophoresis is a satisfactory system for the analysis of alkaloids in traditional Chinese medicine.

A capillary zone electrophoresis method was set up for the separation and determination of glycyrrhizin in Chinese medicinal preparations. Concentrations of Na(2)B(4)O(7) were optimized, which showed that glycyrrhizin in the sample could be separated from interference in the running buffer of 30 mmol/L Na(2)B(4)O(7). Using declofenac as internal standard, the simple method was linear in the range 25-300 microg/mL of glycyrrhizin, and good reproducibility was obtained. The extracts of Radix glycyrrhizae and its preparations could be injected directly for analysis without any pretreatment.

Tetrahydropalmatine in Rhizoma corydalis and its preparations were separated and determined with no pretreatment in the buffer solution of 50 mmol/L of sodium acetate in methanol containing 2 mol/L acetic acid.

A 96-well protein precipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated for the determination of fudosteine in human plasma. After protein precipitation of the plasma samples (50 microL) by the methanol (150 microL) containing the internal standard (IS), erdosteine, the 96-well plate was vortexed for 5 min and centrifuged for 15 min. The 100 microL supernatant and 100 microL mobile phase were added to another plate and mixed and then the mixture was directly injected into the LC-MS/MS system in the negative ionization mode. The separation was performed on a XB-CN column for 3.0 min per sample using an eluent of methanol-water (60:40, v/v) containing 0.005% formic acid. Multiple reaction monitoring (MRM) using the precursor-product ion transitions m/z 178--91 and m/z 284--91 was performed to quantify fudosteine and erdosteine, respectively. The method was sensitive with a lower limit of quantification (LLOQ) of 0.02 microg mL(-1), with good linearity (r0.999) over the linear range of 0.02-10 microg mL(-1). The within- and between-run precision was less than 5.5% and accuracy ranged from 94.2 to 106.7% for quality control (QC) samples at three concentrations of 0.05, 1 and 8 microg mL(-1). The method was employed in the clinical pharmacokinetic study of fudosteine formulation product after oral administration to healthy volunteers.

A two-dimensional column-switching system without sample loop trapping, where two columns were switched directly via a six-port two-position switching valve, was successfully applied for the first time to the isolation and purification of six iridoid glycosides including geniposide, gardenoside, shanzhiside, scandoside methyl ester, deacetyl-asperulosidic acid methyl ester and genipin-1-β- d -gentiobioside from Gardenia jasminoides Ellis, a plant used in the traditional Chinese medicine. The introduction of the six-port switching valve instead of sample loop assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension could reach 20 ml. In this mode of operation, the sample size of the two-dimensional approach was more than 1.3 times that of conventional gradient methods with even less solvent consumption. And the simultaneous operations of the two dimensions allowed the cycle time to be less than 19 min, compared with that (90 min) in the gradient elution single-dimension mode of operation. All of the six isolated iridoid glycosides were isolated at high purities of over 99% with approximately 96% recoveries.

Bestatin is a low molecular weight aminopeptidase inhibitor originally isolated from culture filtrates of Streptomyces olivoreticuli. We have developed a sensitive, specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the quantitative determination of bestatin in rat plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 50 μL plasma samples by solid phase extraction (SPE). Reverse-phase HPLC separation was accomplished on a Lichrospher C18 column (4.6 mm × 50 mm, 5 μm) with a mobile phase composed of methanol–water–formic acid (70:30:0.5, v/v/v) at a flow rate of 0.8 mL/min. The method had a chromatographic total run time of 3 min. A Varian 1200L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 309.2 → 120.0 (bestatin) and 313.4 → 138.0 (granisetron) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 5 ng/mL, with good linearity (r2 ≥ 0.999) over the linear range of 5–2000 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of bestatin in rats.

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of a potent 5-HT(1B/1D) receptor agonist, rizatriptan in human plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 100 microL plasma samples by liquid-liquid extraction (LLE) and chromatographed on a Lichrospher C18 column (4.6mm x 50mm, 5 microm) with a mobile phase consisting of acetonitrile-10mM aqueous ammonium acetate-acetic acid (50:50:0.5, v/v/v) pumped at 1.0 mL/min. The method had a chromatographic total run time of 2 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 270-->201 (rizatriptan) and 313.4-->138 (granisetron) used for quantitation. The assay was validated over the concentration range of 0.05-50 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 98%. The intra-day accuracy of the assay was within 12% of nominal and intra-day precision was better than 13% C.V. Following a 10mg dose of the compound administered to human subjects, mean concentrations of rizatriptan ranged from 0.2 to 70.6 ng/mL in plasma samples collected up to 24h after dosing. Inter-day accuracy and precision results for quality control samples run over a 5-day period alongside clinical samples showed mean accuracies of within 12% of nominal and precision better than 9.5% C.V.

Following preparative isolation and purification by high-speed counter-current chromatography (HSCCC), the collected fractions were generally analyzed by high-performance liquid chromatography (HPLC) to determine the relative purities of each fraction. Our paper reports for the first time a preparative isolation-purity detection hyphenated system: online coupling of HSCCC with high-performance liquid chromatography-diode array detection (HSCCC-HPLC-DAD). The introduction of online purity analysis in HSCCC has dramatically improved the efficiency of this technique by overcoming the drawbacks of post analysis in HSCCC isolation. The effluent from the outlet of HSCCC was splitted into two parts: one was collected, while the other was introduced directly into an HPLC-DAD system for purity analysis through a switch valve. Therefore, the purities of the obtained fractions from HSCCC were monitored, and fractions with high purities were collected. This strategy has been successfully demonstrated with the preparative isolation and purification of hyperoside from Hypericum perforatum (St. Jone's Wort); a model of TBE-300A HSCCC was used to isolate and separate hyperoside from H. perforatum with a two-phase solvent system composed of ethyl acetate-ethanol-water at the volume ratio of 5:1:5 (v/v) using online detection technique. The isolation was done in less than 3.5 h, and a total of 83.0-mg hyperoside at over 99.0% purity was yielded from 300 mg of the partially purified extract. This new strategy possesses general utility in the preparation of bioactive compounds from traditional Chinese medicine (TCM).

A preparative counter-current chromatography (CCC) was used to isolate and separate chemical constituents from the leaf of Patrinia villosa, a famous traditional Chinese medicinal herb. Six flavonoids including two known and four novel compounds were successfully simultaneous purified by CCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10: 13: 13: 10, v/v) by increasing the flow rate of the mobile phase from 1.0 ml/min to 2.0 ml/min after 110 min to bring out the late eluters. The separation produced total of 44.9 mg fraction I with 99.1% purity, 35.5 mg fraction II with 98.8% purity, 79.8 mg fraction III with 99.3% purity, 45.8 mg fraction IV with 98.8% purity, 39.8 mg fraction V with 98.6% purity and 9.6 mg fraction VI with 97.5% purity from 400 mg crude extract in one single isolation procedure and less than 10 h, and the obtained fractions were all analyzed by high performance liquid chromatography (HPLC). Their chemical structures were elucidated as (2S)-5,7,2',6'-tetrahydroxy-6,8-di (gamma,gamma-dimethylallyl) flavanone (1), (2S)-5,7,2',6'-tetrahydroxy-6-lavandulylated flavanone (2), (2S)-5,7,2',6'-tetrahydroxy-4'-lavandulylated flavanone (3), (2S)-5,2',6'-trihydroxy-2'',2''-dimethylpyrano [5'', 6'': 6, 7] flavanone (4), (2S, 3''S)-5,2',6'-trihydroxy-3''-gamma,gamma-dimethylallyl-2'',2''-dimethyl-3'',4''-dihydropyrano [5'', 6'': 6, 7] flavanone (5) and licoagrochalcone B (6), respectively, by spectrum methods including UV, IR, high resolution (HR)-electrospray ionization (ESI)-MS, 1-dimension (1D) and 2-dimension (2D) NMR techniques. Among them, compounds 2, 3, 4, and 5 were new compounds and discovered from nature for the first time. The recoveries of the six compounds were 91.2%, 91.4%, 92.1%, 90.5%, 90.3% and 89.7%, respectively, in CCC step. Subsequently, their anticancer activities were also examined to inhibit human cancer cells' growth including A549, BEL-7402, SGC-7901, MCF-7, HT-29, K562 and A498 cell lines by MTT method in vitro. The results indicated that the compounds 1, 2 and 3 exhibited high anticancer activities (IC50 < 7 microg/ml), especially to K562 cancer cell (IC50 < 3.1 microg/ml), and the compounds 4, 5 and 6 exhibited weaker inhibition effect (IC50 < 30 microg/ml).

Supercritical fluid extraction (SFE) of aurentiamide acetate from Patrinia villosa Juss was performed. The optimization of parameters was carried out using an analytical-scale supercritical fluid extraction (SFE) system. Then the extraction was scaled up by 100 times using a preparative SFE system under the optimized conditions of 55 °C, 35 MPa and modified CO 2 with 10% methanol. Then, the crude extract I obtained by SFE was chromatographed on silica gel and the solvent system composed of petroleum ether–ethyl acetate (5:1, v/v) was used to produce the crude extract II, which was further isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n -hexane–ethyl acetate–methanol–water (1:1.2:1.2:1, v/v/v/v). One hundred fifty-five milligrams of aurentiamide acetate was obtained from 400 mg crude extract II (contained 42% target) with a purity of 99.3% determined by HPLC and 92.3% recovery in one-step elution, and identification was performed by UV, MS, 1 H NMR and 13 C NMR. As far as we know, this is the first report of discovering aurentiamide acetate from the plant of Patrinia genius .

Supercritical fluid extraction (SFE) of orotinin, orotinin-5-methyl ether and licoagrochalcone B from Patrinia villosa was performed. The optimization of parameters including pressure, temperature, modifier and sample particle size on yield was carried out using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa, 45 degrees C, a sample particle size 40-60 mesh and modified CO2 with 20% methanol. The yield of the preparative SFE was 2.82% (crude extract I) and the combined yield of orotinin, orotinin-5-methyl ether and licoagrochalcone B was 0.82 mg/g of dry sample mass. Then the crude extract I was re-dissolved in methanol and methanol soluble fraction (crude extract II, 0.17%) was obtained, which was successfully isolated and separated by a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:6:6:6, v/v/v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 3 h. The target compounds isolated and purified by HSCCC were analyzed by high performance liquid chromatography. The separation produced total of 38.2 mg of orotinin at 99.2% purity, 19.8 mg of orotinin-5-methyl ether at 98.5% purity and 21.5 mg of licoagrochalcone B at 97.6% purity from 400 mg of the crude extract in a one-step separation. The recoveries of orotinin, orotinin-5-methyl ether and licoagrochalcone B were 91.1, 91.6 and 90.3%, respectively, and the chemical structure identification was carried out by UV, IR, MS, 1H NMR and 13C NMR.

Preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and separation three flavonoids including bolusanthol B, a novel compound named 5,7,2',6'-tetrahydroxy-6,8-di(gamma,gamma-dimethylallyl) flavanone and tetrapterol I from Patrinia villosa Juss using two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water at the volume ratio of 10:11:11:8 (v/v). A total of 25.4 mg bolusanthol B, 52.5 mg 5,7,2',6'-tetrahydroxy-6,8-di(gamma,gamma-dimethylallyl) flavanone and 50.2 mg tetrapterol I were obtained from 250 mg crude extract with purities of 96.8%, 99.2% and 99.3%, respectively determined by HPLC in one single operation and less than 5 h. The structure identification was performed by UV, IR, MS, 1H NMR, 13C NMR and 2D NMR. Among then, bolusanthol B and tetrapterol I were obtained from the plant of Patrinia genius for the first time, and 5,7,2',6'-tetrahydroxy-6,8-di(gamma,gamma-dimethylallyl) flavanone was a novel prenylated flavonoid and discovered from nature for the first time.

Icariin in Chinese traditional medicine was determined by an accurate and simple capillary zone electrophoresis method which used 30 mmol/L of borate solution containing 10% of acetonitrile as running buffer and needed no pretreatment. Copyright © 1999 John Wiley & Sons, Ltd.

A single syringe pump is employed in this report to add modifier (methanol) through a parallel connection device, which can maintain a constant and lasting (at least 6 h) ratio of methanol without any contamination of the pump. The amount of methanol in supercritical CO2 is measured by gas chromatography. Psoralen and isopsoralen are applied as examples to illustrate the elution effect. With a fixed modifier line, the longer the CO2 line, the higher the concentration. Variations in pressure and/or temperature also have some influence upon the methanol ratio. The single-pump parallel device can conveniently alter the property and ratio of the modifier, and is a probable substitute for gradient programming performed by a dual-pump system.