Effects of Vaccination with the C-Strain Vaccine on Immune Cells and Cytokines of Pigs Against Classical Swine Fever Virus.

The attenuated C-strain vaccine against classical swine fever virus (CSFV) is one of the safest and most effective attenuated vaccines. However, little is known of the host immune response after vaccination with the C-strain vaccine. Blood samples from vaccinated pigs were collected to evaluate the number of immune cells, the level of specific CSFV antibody, and related cytokines induced by the vaccination of C-strain vaccine. The C-strain nucleic acid was gradually removed and specific antibody to vaccine kept increasing; the amount of the lymphocyte, Tc cell, and Th cell increased; some inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor-α mainly showed downregulated trends, but IL-6 and IL-8 were upregulated greatly; IL-2, IL-4, IL-5, IL-12p40, IL-13, interferon (IFN)-I, and Toll-like receptors (TLRs) kept high expression level after 28 days postvaccination (dpv); IFN-γ was upregulated slightly at 5 and 9 dpv, respectively. These results suggest that the C-strain vaccine induces a Th2 cell response to produce the specific antibody. The vaccine virus replicates at very low level. C-strain vaccine burden has close relationship with the expression of TLRs. The overexpression of TLRs initiates the innate immune system to clear up the vaccine. Meanwhile, ILs expressed by immune system induce the differentiation of B cells and produce specific antibody.

[HPLC tandem-mass spectrometric analysis of the chemical components and decomposition products of allicin extract of garlic].

To analyze the chemical components and decomposition products in allicin extract of garlic, the chemical components screening and identification were made with HPLC-MS/MS method by full scan TIC MS, HPLC retention time, product MS spectra and chemical reference standards. The stability of the extract in water and alcoholic solutions was also investigated. There were five major components in allicin extract which were all identified as thiosulfinates. The extract was stable for at least 3 months when stored at -20 degrees C as water solution, but obvious decomposition was observed with the increase of alcoholic concentration. The decomposition products were also identified by HPLC-MS/MS.

[Metabonomic study on the effects of allicin on rats].

To investigate the effects of allicin on rats by NMR-based metabonomic method, the changes of endogenous metabolites in normal rat urine and the influences on metabolism were analyzed with bio-nuclear magnetic resonance (NMR) method and partial least-squares discriminant analysis (PLS-DA) after intraperitoneal administration of allicin solution. The identified biochemical effects associated with allicin dosing included elevated then gradually recovered urinary levels of Kreb's cycle intermediates, such as citrate, alpha-ketoglutarate and succinate and increased concentrations of ketones. Meanwhile, decreased urinary concentrations of glucose, lactate, alanine, hippurate and trimethylamine oxide were observed. The PLS-DA revealed that the metabonomic profiles of allicin treated groups were obviously different from those of the control group. Allicin may change metabolism significantly in normal rats. The study of the pharmacologic mechanism of allicin by metabonomic method is practicable and it could be explored further.

[Related substances in purified alliin determined by HPLC-MS/MS].

To study the related substances in purified alliin, HPLC-MS/MS method carried out on a Phenomenex NH2 column was used for screen and identification of the related substances with full scan MS spectra determination of their [M + H] + ions and then the analyses of the retention time, product MS spectra and/or chemical reference standards. The full scan HPLC-MS chromatogram showed that there were seven major related substances in the purified alliin and their m/z of the [M + H] + ions with increasing retention were 116, 133, 147, 152, 175, 178 and 178, separately. And they were identified as proline, asparagine, glutamine, methiin, arginine, isoalliin and cycloalliin (both were isomers of alliin), respectively. The major related substances in purified alliin are amino acids, homologen and the isomers of alliin.

[Pharmacokinetic interactions between the main components in the extracts of Salvia miltiorrhiza Bge. in rat].

The pharmacokinetics of the main components of protocatechualdehyde, salvianolic acid B, tanshinone II(A), cryptotanshinone, and the hydrophilic or lipophilic extracts of Salvia Miltiorrhiza Bge., in rat plasma were studied after oral administration separately to explore the interactions between them. Some components in the hydrophilic extract depress the absorption of the protocatechualdehyde, on the contrary, enhance the absorption of the salvianolic acid B and depress its elimination rate. The concomitant components in the lipophilic extract might enhance the absorption of cryptotanshinone and its distribution from the centre compartment to the peripheral compartment, and the metabolism to tanshinone II(A). The 'concomitant components' in the extract of Chinese material medica had significant effect on the pharmacokinetics of its 'marker components'. It can not only be rival, synergic, but also have the effects on metabolism. Therefore the traditional Chinese medicine was a complicated system, It should be taken a scientific and dialectic view in the research and development processes.

[Metabolites of 1-(1-(6-methoxyl-2-naphthyl)ethyl)-2-(4-nitrobenzyl)-6,7-dimethoxyl-1,2,3,4-tetrahydroisoquinoline hydrobromide in rat by LC-MS/MS method].

To investigate the principal metabolites of 1-(1-(6-methoxyl-2-naphthyl) ethyl)-2-(4-nitrobenzyl)-6,7-dimethoxyl-1,2,3,4-tetrahydroisoquinoline hydrobromide (code designation: P91024) in rats after ig administration by LC-MS/MS, the phase I metabolites were discovered by comparing the fullscan and SIM chromatograms of the test samples with the corresponding blanks. The structures of phase I metabolites were identified by ESI-MS spectra and the product spectra of the corresponding adduct ions. The phase II metabolites were identified in the test samples after the phase I metabolites were completely removed with solvent extraction and then treated with glucuronidase for enzymolysis of phase II glucuronide conjugates and the hydrolysates. Two phase I metabolites of P91024 were identified in rat feces, one phase I and five phase II in bile, one phase I and three phase II in urine, and four phase I and one phase II in plasma. Their structures were elucidated, separately. P91024 was extensively metabolized in rat. The metabolites can be easily screened and identified by LC-MS/MS method.

[Photo-degradation products of 1-[1-(6-methoxy-2-naphthyl)ethyl]-2-(4-nitrobenzyl)-6,7-dimethoxyl-1,2,3,4-tetrahydroisoquinoline hydrobromide].

To study the photo-degradation products of 1-[1-(6-methoxy-2-naphthyl) ethyl]-2-(4-nitrobenzyl)-6,7-dimethoxyl-1,2,3,4-tetrahydroisoquinoline hydrobromide (code designation: P91024). The chemical structures of the major photo-degradation products of P91024 were identified by HPLC-MS and spectroscopic methods, and their reference substances were also synthesized for confirmation. The three major photo-degradation products were identified to be N-(4-nitrobenzyl)-6,7-dimethoxyl-3, 4-dihydroisoquinoline bromide, 1-[1-(6-methoxyl-2-naphthyl) ethyl]-6, 7-dimethoxyl-1, 2, 3, 4-tetrahydroisoquinoline and 2-isopropyl-6-methoxyl-naphthalene, respectively.

[Excretion of (-)-clausenamide in rats].

AIM: To study the excretion of (-)-clausenamide in rats. METHODS: The urine, feces and bile were collected at predetermined time points after (-)-clausenamide was orally administrated to 6 rats (30 mg x kg(-1)). The concentrations of (-)-clausenamide and its metabolite 6-OH-(-)-clausnamide were determined by HPLC-MS/MS method using glipzide as the internal reference, and the accumulative excretion amount of (-)-clausenamide and 6-OH-(-)-clausenamide was calculated in the urine, feces and bile, separately. RESULTS: (-)-Clausenamide was recovered mostly (44%) from feces in 112 hours, 7.1% was found from urine in 120 hours and 0.013% was detected from bile in 24 hours. The accumulative excretions of 6-OH-(-)-clausenamide were 0.92% , 0.46% and 0.0003% of the administered dose from feces, urine and bile, respectively. CONCLUSION: The major amount of (-)-clausenamide was recovered from feces after (-)-clausenamide was orally administrated to rats (30 mg kg(-1)).

[Structures of leucogen and its related substances in solution state].

AIM: To study the four diastereomers of leucogen and structure of the related substances. METHODS: LC-DAD, LC-MS/MS and LC- 1H NMR were used. LC was carried out with a Phenomnex Luna C18 (250 mm x 4.60 mm ID, 5 microm) column and a mobile phase of water-acetonitrile-glacial acetic acid (58:42: 0.3). RESULTS: The structures of leucogen and its related substances were identified. Leucogen and the related substances were found to have four diastereomers in solution state separately. The stability and transformation of the four diastereomers were analyzed and 3R4S5R was found to be more stable than the others according to quantum calculations. CONCLUSION: Leucogen have four diastereomers in solution state and it can transform from one diastereomer to the others, and the 3R4S5R is more stable than the others.

[Separation and determination of donepezil hydrochloride enantiomers in plasma by capillary electrophoresis].

AIM: To establish chiral separation method for donepezil hydrochloride (E2020) enantiomers by capillary electrophoresis (CE) and determine the two enantiomers in plasma. METHODS: Alkalized plasma was extracted by isopropanol-n-hexane (3 : 97) and L-butefeina was used as the internal standard. Enantioresolution was achieved using 2.5% sulfated-beta-cyclodextrin as chiral selector in 25 mmol x L(-1) triethylammonium phosphate solution (pH 2.5) on the uncoated fused-silica capillary column (70 cm x 50 microm ID). The feasibility of the method to be used as quantitation of E2020 enantiomers in rabbit plasma was also investigated. RESULTS: E2020 enantiomers were separated at a baseline level under the above condition. The linearity of the response was evaluated in the concentration range from 0.1 to 5 mg x L(-1). The linear regression analysis obtained by plotting the peak area ratio (A(s)/A(i)) of the analyte to the internal standard versus the concentration (C) showed excellent correlation coefficient (r = 0.999 2 for R(-)-E2020, r = 0.999 7 for S(+)-E2020) and the equations were A(s)/A(i) = 0.024 2 + 0.289 2C and A(s)/A(i) = 0.010 8 + 0.273 7C, respectively. The low limit of detection was 0.05 mg x L(_1). The inter- and intra-day precision (RSD) were all less than 20% . CONCLUSION: Compared with CSP by HPLC, the CE method is simple, reliable, inexpensive and suitable for studying the stereoseletive pharmacokinetics in rabbits.

Study on a new precolumn derivatization method in the determination of metformin hydrochloride.

Metformin hydrochloride is successfully determined by reversed-phase high-performance liquid chromatography with a new precolumn derivatization method using 9,10-anthraquinone-2-sulfonyl chloride as the derivatization agent. Several derivatization systems are tried to optimize the derivatization conditions, and a new post-derivatization treatment method is established. The derivatization product is analyzed on a Lichrosper C18 column (6.0 mm x 150 mm, 5 microm) at 256 nm with methanol-water (70:30, v/v) as the mobile phase. The calibration curves of the derivatives for the UV detector (0.01-4 mg/L) are linear with respect to peak area. The detection limit (peak area) for the metformin hydrochloride is 0.01 mg/L for a signal-to-noise ratio of 3:1. In human plasma, the detection limit is 0.02 mg/L. This assay is rapid, sensitive, and highly reproducible.

[Pharmacokinetics of (-)-clausenamide and its major metabolite 6-hydroxyl-clausenamide in beagle dogs by HPLC/MS].

UNLABELLED: To establish a sensitive and accurate method to study the pharmacokinetics of (-)-clausenamide [(-)-clau] and its major metabolite 6-hydroxyl-clausenamide (6-OH-clau) in the plasma of the Beagle dog. METHODS: (-)-Clau was orally administered to six Beagle dogs at the dose of 30 mg x kg(-1), venous blood from front leg was sampled and plasma was separated for analysis. After extraction with ethyl acetate, the plasma samples were analyzed by HPLC/MS and the mobile phase was a mixture of methanol-water-acetic acid (60: 40: 0. 8) at the flow rate of 1.0 mL x min(-1). The API-ES positive ion SIM detection was carried out for the detection of both (-)-clau ([M + H] (+), m/z 298 ) and 6-OH-clau ([M + H - H2 O](+), m/z 296) with glipzide (glip) ([M + H](+), m/z 446) as internal standard. The pharmacokinetic parameters were calculated by 3P97 software. RESULTS: There was good linear relationship ( r > 0. 999) between the SIM responses and the concentrations for (-)-clau and 6-OH-clau at the range from 1.0 to 200 ng x mL(-1) and 0.2 to 40.0 ng x mL(-1), respectively. The absolute recovery was greater than 85%. The plasma concentration-time curves of (-)-clau and 6-OH-clau were both best fitted to a two-compartment model. The C(max) of (-)-clau and 6-OH-clau were (21 +/- 10) ng x mL(-1) and (3.9 +/- 2.2) ng x mL(-1), T(max) were (0.8 +/- 0.5) h and (1.3 +/- 0.5) h, T 1/2 alpha were (0.9 +/- 0.6) hand (1.4 +/- 0.6) h, T 1/2 beta were (19 +/- 23) hand (13 +/- 12) h, AUC(0-24 h) were (69 +/- 14) h x ng x mL(-1) and (12 +/- 7) h x ng x mL(-1) respectively. CONCLUSION: The established HPLC/MS method was sensitive and specific for the determination of (-)-clau. It was shown that the absorption and first phase elimination of (-)-clau were very quick in Beagle dogs, but the terminal elimination was very slow. The plasma concentration profile of its major metabolite 6-OH-clau was similar to (-)-clau and the AUC was relatively small in comparison with (-)-clau.

[Pharmacokinetics and bioequivalence of trimebutine dispersive tablet in healthy subjects].

AIM: To develop an HPLC-ESI-MS assay for determination of trimebutine in human plasma and to investigate the pharmacokinetics and bioequivalence of two trimebutine tablets in human. METHODS: After being made alkaline with saturated sodium bicarbonate, plasma was extracted by cyclohexane and separated by HPLC on a reversed-phase C18 column with a mobile phase of 10 mmol x L(-1) ammonium acetate buffer solution (pH 3.5)-methanol (18:82). HPLC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 388 for trimebutine and m/z 280 for the internal standard (sibutramine, IS). The fragmentor voltage was 50 V. A randomized cross-over design was performed in 20 healthy volunteers. In the two study periods, a single 100 mg dose of each tablet was administered to each volunteer. RESULTS: Calibration curve was linear over the range of 0.3 - 150 microg x L(-1). The main pharmacokinetic parameters of T1/2, Tmax and Cmax were (9.2 +/- 2.8) h, (1.0 +/- 0.3) h and (40 +/- 20) microg x L(-1) for the reference tablet; (9.2 +/- 2.3) h, (0.9 +/- 0.4) h and (41 +/- 20) microg x L(-1) for the test tablet. The relative bioavalability of the test tablet was (97 +/- 13)%. The results of variance analysis and two one-sided t-test showed that there was no significant difference between the two formulations in the AUC and Cmax. CONCLUSION: The assay was proved to be sensitive, accurate and convenient. The two formulations were bioequivalent.

[SERS spectra of vitamin A acid in silver solution].

Vitamin A acid (VAA) was firstly dissolved in chloroform, and then the organic solution containing the samples was mixed with silver sol at the desired concentration. After sufficient shaking, collect SERS spectra of VAA with silver sol layer solution. The attribution of the peaks was illustrated by comparing SERS spectra with NR (Normal Raman) spectra, and the mechanism and orientation of adsorption were also discussed according to the peaks which was absent in NR spectrum but observed in SERS spectrum. To verify the feasibility of the method for the quantitative analysis, SER spectra at different concentration of VAA were collected. The intensities measured with the band at 1583 cm(-1) were plotted as a function of concentration. It was found that an effective limit for the detection of VAA could be as low as to 1.0 x 10(-7) mol x L(-1), the calibration curve was linear in the range from 1.0 x 10(-6)-5 x 10(-5) mol x L(-1). The proposed method has the potential to quantitative assay of the drugs having SERS effects but undissolvable in water.

[Determination of finasteride in human plasma by HPLC-MS].

AIM: To develop an HPLC-MS assay for determination of finasteride in human plasma and to investigate the bioequivalence in healthy volunteers. METHODS: After alkalization with sodium hydroxide, plasma was extracted with ethyl acetate and separated using a C18 column with a mobile phase of methanol-water (85:15). LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 395 for finasteride and m/z 407 for the IS. The fragmentor voltage was 120 V. A randomized crossover design was performed in 20 healthy volunteers. In the two study periods, a single 10 mg dose of each tablet was administered to each volunteer. RESULTS: Calibration curves were linear over the range 1-200 micrograms.L-1 (r = 0.9986). The limit of determination for finasteride in plasma was 0.05 microgram.L-1. The recovery of finasteride from plasma was in the range of 85.9%-98.7%. The results of variance analysis and two one-side t-test showed that there was no significant difference between the two formulations in the AUC and Cmax. CONCLUSION: The assay was proved to be sensitive, accurate and convenient. The two formulations were bioequivalent.

[Determination of donepezil in human plasma by HPLC-MS].

AIM: To establish a sensitive and specific liquid chromatography-mass spectrometry (time-of-flight) [LC-MS (TOF)] method for the determination of donepezil in human plasma after an oral administration of 5 mg donepezil hydrochloride tablet. METHODS: Alkalized plasma was extracted with isopropanol-n-hexane (3:97) and loratadine was used as internal standard. Solutes were separated on a C18 column with a mobile phase of methanol-acetate buffer (pH 4.0) (80:20). Detection was performed on a time-of-flight mass spectrometry equipped with an ESI interface and operated in positive-ionization mode. Donepezil quantitation was realized by computing the peak area ratio (donepezil-loratadine) (donepezil m/z 380[M + H]+ and loratadine m/z 383[M + H]+) and comparing them with calibration curve (r = 0.9998). RESULTS: The linear calibration curve was obtained in the concentration range of 0.1-15 micrograms.L-1. The detection limit of donepezil was 0.1 microgram.L-1. The average recovery was more than 90%. The intra- and inter-run precision was measured to be below 15% of RSD. CONCLUSION: The method is sensitive, simple and rapid, so, it can meet the need of the studies on the pharmacokinetics and bioavailability of donepezil.

[Determination of cyclovirobuxine D by RP-HPLC with precolumn fluorescence derivatization].

AIM: To establish a RP-HPLC method for determination of cyclovirobuxine D. METHODS: Cyclovirobuxine D reacted with a derivative reagent 1-naphthyl isocyanate in chloroform to form fluorescence derivatives, stopped the reaction by adding the mobile phase and then directly injected the solution into the chromatograph to seperate it by RP-HPLC. The analysis was carried out on C18 column, the mobile phase is methanol-water (85:15), the excitation wavelength was set at 305 nm, emission at wavelength 385 nm, and the flow rate was 1 mL.min-1. The effect of several factors including the reaction medium, temperature, time and amount of 1-naphthyl isocyanate on the yield of the derivatization was also investigated systematically. RESULTS: A simple and rapid RP-HPLC method for the simultaneous isolation and analysis of cyclovirobuxine D and its related substances was developed, and the absence of interference between the derivative peak responses of cyclovirobuxine D and its related substances were verified by UV diode array detecter and MS. The linearity was obtained from 0.75 microgram.mL-1 to 2.5 micrograms.mL-1 of cyclovirobuxine D derivatives with a correlation coefficient of 0.9991. The detection limit of cyclovirobuxine D derivative was 1 ng.mL-1, the repeatability of derivatization was good with relative standard derivation no more than 1.2% and derivative was stable within 48 h. The method described conforms to the validation of China Pharmacopiea compendial methods used for pharmaceutical products in general. CONCLUSION: The established method is proved to be reliable quantitative method for the quality control of cyclovirobuxine D.

Evaluation of anthraquinone-2-sulfonyl chloride for determination of phenol in water by liquid chromatography using pre-column phase-transfer catalysed derivatization.

A new reagent, anthraquinone-2-sulfonyl chloride (ASC), has been used for the derivatization of phenols. Several compounds with different polarity were selected to evaluate the new reagent and derivatives of these phenols prepared via a facile pathway. The optimal conditions for analytical derivatization and mechanism of the derivatization reaction are discussed. The derivatization procedure involves an ion-pair extraction of the deprotonated phenols with a tetrabutylammonium counter ion to an organic phase. At the interface of two phases, the derivatization reaction occurs quantitatively at room temperature within 3 min. The derivatives are stable and readily amenable to analysis by normal-phase and reversed-phase high performance liquid chromatography (HPLC). Excellent linearity response was demonstrated over the concentration range of 0.2 mumol/L to 200 mumol/L at 320 nm for normal-phase HPLC, at 256 nm for reversed-phase HPLC. Combined with preconcentration using a Waters Sep-Pak Plus C18 cartridge, detection limits of phenols for water sample analysis were as low as 1 x 10(-9) mol/L (ca. 0.1 microgram/L).