Guorong Fan

Separation and determination of coumarins in Fructus cnidii extracts by pressurized capillary electrochromatography using a packed column with a monolithic outlet frit

The pressurized capillary electrochromatography (pCEC) was utilized for the separation and determination of coumarins in Fructus cnidii extracts from 12 different regions. After a thorough study of analytical parameters such as acetonitrile content of the mobile phase, the concentration and pH of the buffer, and the applied voltage, a methodology was proposed to separate and determine six coumarins of F. cnidii extracts in less than 15 min. The experiments were performed in an in-house packed column with a monolithic outlet frit under the optimal conditions: pH 4.0 ammonium acetate buffer at 10 mM containing 50% acetonitrile at -6kV applied voltage. The calibration curves were linear in the range of 10.0-100.0 microg/mL for bergapten, 20.0-200.0 microg/mL for imperatorin, 5.0-400.0 microg/mL for osthole, 10.0-100.0 microg/mL for 2-acetylangelicin, 10.0-200.0 microg/mL for oroselone, and 10.0-200.0 microg/mL for O-acetylcolumbianetin. The correlation coefficients were between 0.9967 and 0.9995. With this pCEC system, fingerprints of F. cnidii extracts were preliminarily established to distinguish three types of coumarins by characteristic peaks, and the quality of various sources of raw materials was evaluated by determining the contents of six coumarins.

Rapid determination of telmisartan in human plasma by HPLC using a monolithic column with fluorescence detection and its application to a bioequivalence study.

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.

Chemical composition and antioxidant activity of volatiles from Patrinia Villosa Juss obtained by optimized supercritical fluid extraction

Supercritical CO(2) fluid extraction (SFE-CO(2)) was used to extract volatiles from Patrinia Villosa Juss. An orthogonal test L(9) (3)(4) including pressure, temperature, dynamic extraction time and modifier was performed to get the optimal conditions. Extract 1 was obtained by the optimal extraction condition 1: pressure=35 MPa, T=45 degrees C, dynamic extraction time=2.0 h and V(modifier (MeOH))=0% as guided by the index 1: the free radical scavenging activities in vitro against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) diammonium salt (ABTS). Extract 2 obtained by the optimal extraction condition 2: pressure=25 MPa, T=55 degrees C, dynamic time=2.5h and V(modifier (MeOH))=20% was guided by the index 2: the yield of the volatiles. The results showed that extract 1 possessed stronger antioxidant activity (EC(50)=32.01 microg/ml to DPPH and 50.90 microg/ml to ABTS(+)) than the extract 2 (EC(50)=95.62 microg/ml to DPPH and 99.78 microg/ml to ABTS(+)). Subsequently, the chemical compositions of the two extracts were identified by gas chromatography-mass spectrometry. Forty-six compounds were identified from extract 1, and the total volatile consisted of hydrocarbon (49.65%), aldehyde (16.66%), fatty acid (22.38%), terpene (9.04%) and little alcoholic. From extract 2, 32 compounds were identified, in which hydrocarbon, aldehyde, fatty acid and terpene possessed 58.21%, 5.97%, 13.19% and 21.79%, respectively. This is the first report of using SFE to extract the volatiles from P. Villosa Juss (a wild vegetable and medicine plant) and first time to systematically evaluate the volatiles antioxidant activity and chemical composition.

Simultaneous determination of rupatadine and its metabolite desloratadine in human plasma by a sensitive LC–MS/MS method: Application to the pharmacokinetic study in healthy Chinese volunteers

A sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of rupatadine and its metabolite desloratadine in human plasma. After the addition of diphenhydramine, the internal standard (IS), plasma samples were extracted with a mixture of methyl tert-butyl ether and n-hexane (1:1, v/v). The analysis was performed on a Ultimate AQ-C18 (4.6mm x 100mm, 5microm) column using a mobile phase consisting of a 80/20 mixture of methanol/water containing 0.0005% formic acid pumped at 0.3mlmin(-1). The analytes and the IS were detected in positive ionization mode and monitoring their precursor-->product ion combinations of m/z 416-->309, 311-->259, and 256-->167, respectively, in multiple reaction monitoring mode. The linear ranges of the assay were 0.1-50 and 0.1-20ngml(-1) for rupatadine and desloratadine, respectively. The lower limits of reliable quantification for both rupatadine and desloratadine were 0.1ngml(-1), which offered high sensitivity and selectivity. The within- and between-run precision was less than 7.2%. The accuracy ranged from -9.2% to +6.4% and -7.2% to +7.2% for rupatadine and desloratadine in quality control samples at three levels, respectively. The method has been successfully applied to a pharmacokinetic study of rupatadine and its major metabolite after oral administration of 10, 20 and 40mg rupatadine tablets to healthy Chinese volunteers.

High-throughput determination of fudosteine in human plasma by liquid chromatography–tandem mass spectrometry, following protein precipitation in the 96-well plate format

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 (r>0.999) 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 plasma metabonomic investigation into the intervention of volatile oil of Magnolia biondii Pamp on rat model of acute inflammation

ETHNOPHARMACOLOGICAL RELEVANCEnThe dried flower buds of Magnolia biondii Pamp (Magnoliaceae) possesses significant anti-inflammatory activities.nnnAIM OF THE STUDYnVolatile oil in Magnolia biondii Pamp (VOMbP) is considered to be important pharmacologically active individuals against acute inflammation, but its exact anti-inflammatory mechanism remains elusive. In this study, we aimed to investigate the intervention of VOMbP on rats with acute inflammation and explore the possible anti-inflammatory mechanisms of VOMbP with metabonomic strategy.nnnMATERIALS AND METHODSnAcute inflammation was induced by subcutaneously injection of carrageenan in the rats. Plasma was analyzed using gas chromatography-mass spectrometry (GC-MS), based on which the principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) models were established for metabonomic analysis.nnnRESULTSnIt was revealed that the pretreatment of VOMbP in acute inflammatory rats induces a substantial and characteristic change in their metabolic profiles. Some significantly changed metabolites, including hexadecanoic acid, linoleic acid, oleic acid, stearic acid, and cholesterol, were found to be reasonable in explaining the anti-inflammatory mechanism of VOMbP.nnnCONCLUSIONSnIn all, it is likely that VOMbP intervenes the metabolic process of inflammatory rats by affecting the fatty acid and cholesterol metabolism. Our work also indicated that the metabonomics method is a promising tool for performing intervention and mechanism research of traditional Chinese medicines.

Determination of ulifloxacin, the active metabolite of prulifloxacin, in human plasma by a 96-well format solid-phase extraction and capillary zone electrophoresis

This paper described a method for quantification of ulifloxacin, the active metabolite of prulifloxacin in human plasma by capillary zone electrophoresis using lomefloxacin as the internal standard. The separation was carried out at 25 degrees C in a 60.2 cm x 75 microm fused-silica capillary with an applied voltage of 20 kV using 200 mM borate buffer (pH 10.5). The detection wavelength was 275 nm. Clean-up and preconcentration of the samples were developed by 96-well format solid-phase extraction. 0.25 ml of plasma sample and 0.25 ml of IS were loaded onto the preconditioned wells, and the wells were washed using 1 ml of 20% methanol in acid water (1% phosphoric acid), and the analytes were eluted using 1 ml of 95/5 methanol/ammonia water. The method was suitably validated with respect to stability, specificity, linearity, lower limit of quantitation, accuracy, precision, extraction recovery and robustness. The calibration graph was linear for ulifloxacin from 0.02 to 2 microg/ml. The lower limit of quantification was 0.02 microg/ml. The intra- and inter-day precisions were within 4.0 and 8.2%, respectively. The method developed was successfully applied to the evaluation of clinical pharmacokinetic study of prulifloxacin formulation product after oral administration to healthy volunteers.

Development and validation of a rapid HPLC method for the determination of cefdinir in beagle dog plasma integrated with an automatic on-line solid-phase extraction following protein precipitation in the 96-well plate format

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining cefdinir in beagle dog plasma. After simple pretreatment for plasma with 6% perchloric acid, a volume of 100 μL upper layer of the plasma sample was injected into the self-made on-line SPE column. The analytes were retained on the trap column (Lichrospher C(18), 4.6 mm × 37 mm, 25 μm), and the biological matrix was washed out with the solvent (20mM KH(2)PO(4) adjusted pH 3.0) at flow rate of 2 mL/min. By rotation of the switching valve, the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (methanol-acetonitrile-20mM KH(2)PO(4) adjusted pH 3.0, 11.25:6.75:82, v/v/v) at flow rate of 1.5 mL/min, and then separated on the analytical column (Ultimate XB-C(18), 4.6 mm × 50 mm, 5 μm). The complete cycle of the on-line SPE preconcentration, purification and HPLC separation of the analytes was 4 min. The UV detection was performed at 286 nm. The calibration curves showed excellent linear relationship (R(2)=0.9995) over the concentration range of 0.05-50 μg/mL. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This method was successfully applied to quantify cefdinir in beagle dog plasma to support the pre-clinical pharmacokinetic trial.

Fast separation and determination of coumarins in Fructus cnidii extracts by CEC using poly(butyl methacrylate-co-ethylene dimethacrylate-co-[2-(methacryloyloxy)ethyl] trimethylammonium chloride) monolithic columns

A rapid CEC method with poly(butyl methacrylate-co-ethylene dimethacrylate-co-[2-(methacryloyloxy)ethyl] trimethylammonium chloride) monolithic column has been developed for separation and determination of four coumarins (isopimpinelline, bergapten, imperatorin, and osthole) in Fructus cnidii extracts. The effect of polymerization condition including the monomers ratio and the porogens ratio were studied. The mobile-phase composition, such as the composition of organic solvent, the concentration and pH of buffer, was also optimized. Under the same condition (50% ACN and 50% of a 10 mM sodium dihydrogen phosphate electrolyte at pH 4.95), in contrast to 25 min of analysis time in HPLC and 10 min of analysis time in pCEC, a fast separation of these analytes was achieved in less than 5 min in CEC. Method validation was developed in accordance with the analytical procedures. Intra- and interday precisions (RSD) for relative retention time and peak area were less than 1.69 and 4.63%, and LODs were lower than 0.5 microg/mL. Calibration curves of four compounds also showed good linearity (r(2)>0.995). The mean recoveries ranged between 93.91 and 98.65%. With this CEC system, the quality of F. cnidii extracts from various resources was evaluated by determining the contents of the four coumarins.

Sodium desoxycholate-assisted capillary electrochromatography with methacrylate ester-based monolithic column on fast separation and determination of coumarin analogs in Angelica dahurica extract

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 (NaH2PO4) 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 (r2 > 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.