HPLC determination of the related substances in erdosteine / 药学学报

An HPLC method was established for the determination of the related substance in erdosteine. Waters ODS-SunFire (250 mm x 4.6 mm ID, 5 microm) column was used, the mobile phase was composed of methanol-acetonitrile-0.01 mol x L(-1) citric acid (20:4:76, the pH value was adjusted by triethylamine to 2.5). The flow rate was 1 mL x min(-1). The detection wavelength was 254 nm. The related substances in the sample of erdosteine taken were calculated by self control with or without the response factor of impurity relative to that of erdosteine. Under the chromatographic condition developed, the impurities in erdosteine were isolated well. The detection limit was 0.2 microg x mL(-1) (signal/noise = 3) by principal component calculated. The method can be adopted to control the related substances in erdosteine.

Determination of residual solvents in 7-amino-3-chloro cephalosporanic acid by gas chromatography / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a gas chromatography method for determination of residual solvents in 7-amino-3-chloro cephalosporanic acid (7-ACCA).</p><p><b>METHODS</b>The residual levels of acetone, methanol, dichloromethane, ethyl acetate, isobutanol, pyridine and toluene in 7-ACCA were measured by gas chromatography using Agilent INNOWAX capillary column (30 m × 0.32 mm,0.5 μm). The initial column temperature was 70° maintained for 6 min and then raised (10°C/min) to 160°C for 1 min. Nitrogen gas was used as carrier and FID as detector. The flow of carrier was 1.0 ml/min, the temperature of injection port and detector was 200°C and 250°C, respectively.</p><p><b>RESULT</b>The limits of detection for acetone, methanol, dichloromethane, ethyl acetate, isobutanol, pyridine, toluene in 7-ACCA were 2.5 μg/ml, 1.5 μg/ml, 15 μg/ml, 2.5 μg/ml, 2.5 μg/ml, 2.5 μg/ml and 11 μg/ml, respectively. Only acetone was detected in the sample, and was less than the limits of Ch.P.</p><p><b>CONCLUSION</b>The method can effectively detect the residual solvents in 7-ACCA.</p>

Absorption and distribution of extracts of Elsholtzia blanda in rats / 药学学报

The study is aimed to establish a RP-HPLC method for determination of luteolin from the extracts of Elsholtzia blanda (EEB) in rats' biological specimen. A RP-HPLC method was established for determination of free and total luteolin in SD rats' plasma and gastrointestinal tract and total luteolin in SD rats' heart, liver, lung and kidney at 0.17, 0.5, 1, 2, 4 and 6 h after administration of EEB to 24 male SD rats (4 rats per one time spot). Luteolin glycoside was hydrolyzed to aglycone luteolin in intestinal tract, and then luteolin was absorbed. The main form of luteolin existed in gastrointestinal tract after administration of EEB is aglycone. The content of luteolin in liver and kidney were higher than that in heart and lung. The content of luteolin in kidney, heart and lung were showed max at 1 h. Its peak time was similar to that in blood. However, in liver, the drug was distributed quickly, and showed max at 0.17 h. And because of the sensitivity of the method, luteolin was not be detected in other tissues. The method is sensitive, specific, accurate, and is appropriate for determination of luteolin in vivo.

Pharmacokinetics and plasma protein binding of rutin deca (H-) sulfate sodium / 药学学报

Rutin deca (H-) sulfate sodium (RDS) possesses very good activity as an inhibitor of the complement system of warm-blooded animals and HIV. An ion-pair coupled with solid-phase extraction technique (IP-SPE) was developed to extract RDS from rat plasma, urine, bile and protein solution samples. The assay was applied to pharmacokinetics of RDS, including plasma pharmacokinetics, excretion and protein binding studies. After i.v. 5, 20 and 100 mg x kg(-1) RDS via tail vein in rats, the plasma concentration-time profiles were fitted using 3P97 software. The average terminal half-life (t(1/2)) was 3.432 +/- 0.185 2 h. The relationship of dose and AUC of RDS was linear within the dosage range. This suggested that the disposition of RDS in rats belong to linear kinetics and the pharmacokinetic parameters of RDS were dose independent. After iv RDS 20 mg x kg(-1) in rats, the biliary excretion amount of parent drug amount was only 0.3181% +/- 0.2087% of given dosage, and the urinary excretion was 86.0% +/- 6.1% in 36 h. Ultrafiltration techniques were applied to determine the protein binding of RDS in plasma (from SD rat, Beagle dog and human), human serum albumin (HSA) and human alpha1-acid glycoprotein (AGP). The mean protein binding rate in plasma of SD rat, Beagle dog and human plasma of RDS were 80%-90%, in which the range of concentration of RDS was 5 to 100 microg x mL(-1). The protein binding to HSA was 85.7% +/- 1.3% and 14.0% +/- 3.2% to AGP.

Expression of human CYP2E1 in insect cells using bac-to-bac expression system / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To obtain recombinant human CYP2E1 and to determine its activity by using the specific probe substrate.</p><p><b>METHODS</b>CYP2E1 cDNA was obtained by RT-PCR using human liver RNA as template. The cloned CYP2E1 cDNA was ligated with pFastBac vector to generate recombinant pFastBac-CYP2E1, which was then transformed into E. coli DH 10 Bac. Recombinant Bacmid-CYP2E1 was generated by transposition. Then Spodoptera frugiperda (Sf9) insect cells was infected with Bacmid-CYP2E1 to generate recombinant baculoviruses carrying human CYP2E1 cDNA. Finally, Sf9 insect cells were triinfected with recombinant baculoviruses carrying human CYP2E1, CYPOR and CYPb5. The activity of the recombinant enzymes was determined using chlorzoxazone as the substrate.</p><p><b>RESULT</b>The Kmand Vmaxof recombinant CYP2E1 to chlorzoxazone was (72.4 +/-8.7) micromol. L(-1) and (2.41 +/-0.10) micromol.min(-1)?g(-1)protein, respectively.</p><p><b>CONCLUSION</b>Active recombinant CYP2E1 has been obtained by bac-to-bac expression system and its activity is similar to previous reports.</p>

Luteolin excretion after oral administration of Elsholtzia blanda benth extracts in rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To observe the excretion of luteolin after oral administration of Elsholtzia blanda benth extracts in rats.</p><p><b>METHODS</b>Samples of urine, feces and bile were collected after oral administration of Elsholtzia blanda benth extracts in rate. After deconjugation with beta-glucuronidase/sulfatase, the levels of luteolin in urine, feces and bile were measured by RP-HPLC.</p><p><b>RESULT</b>The recovery rate of luteolin was 98.0 %-106.0 % and the extract recoveries were 85.0 %-108.0%. Relative standard deviation (RSD) of intra-and inter-day assay was less than 10.0 %. The total accumulative excretion was 37 % (11 % in urine, 26 % in feces and bile).</p><p><b>CONCLUSION</b>The established RP-HPLC method is sensitive, specific, accurate, and is applicable for determination of luteolin in rat urine,feces and bile.</p>

Metabolism-based interaction of diphenytriazol and flavone compounds / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To observe the metabolism-based interaction of diphenytriazol and flavone compounds.</p><p><b>METHODS</b>Flavone compounds kaempferol, isoharmnten and Elsholtzia blanda benth extract were chosen as the substrate of glucuronidation in the phase II metabolism. The metabolism was investigated in different rat liver microsome incubates pretreated with beta-naphthoflavone (BNF), diphenytriazol and tea oil (control). The concentrations of residual substrate were determined by HPLC. Quercetin and kaempferol were coincubated with diphenytriazol in control microsome to evaluate the inhibition for phase I metabolism. The concentration of diphenytriazol was determined by HPLC.</p><p><b>RESULT</b>The phase II metabolic activity of kaempferol, isoharmnten and Elsholtzia blanda benth extract in diphenytriazol-treated microsome was more potent than that in BNF-treated microsome (P<0.01). The phase I metabolism of diphenytriazol was markedly inhibited by quercetin and kaempferol, with the inhibition constants (Ki) (12.41 +/-0.26)microg . ml(-1) and (7.97 +/-0.08)microg . ml(-1), respectively.</p><p><b>CONCLUSION</b>Diphenytriazol demonstrates metabolism-based interaction with flavone compounds in vitro.</p>

Determination of dimethyl sulphate residual in granisetron hydrochloride by headspace gas chromatography / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a headspace gas chromatography method for determining dimethyl sulphate residual in granisetron hydrochloride.</p><p><b>METHODS</b>An Angilent INNOWAX capillary column with nitrogen gas as carrier and FID as detector was applied in this study. Dimethyl sulphate was tested under a constant column temperature.</p><p><b>RESULT</b>Dimethyl sulphate had different retention time from other organic solvents such as alcohol,acetoacetate, isopropanol, dichlormethane and chloroform, which might exist in granisetron hydrochloride. The detection limit of dimethyl sulphate;s was 0.0016%.</p><p><b>CONCLUSION</b>The method can be used for the determination of dimethyl sulphate residual in granisetron hydrochloride.</p>

Research progress on interactions between luteolin (glucosides) and drug-metabolizing enzyme / 药学学报

The paper summarizes the interactions between luteolin (glucosides) and drug-metabolizing enzyme from the literature of recent years and our research work. The metabolism of luteolin is chiefly mediated by phase II metabolic enzyme. Its glucosides are firstly hydrolyzed into aglycone in intestinal tract, and then absorbed and metabolized. Luteolin has the effect on the induction of CYP3A, and on the inhibition of CYPIA, 1B and 2E. Also, luteolin is an effective inhibitor of CYP2B6, CYP2C9 and CYP2D6. Luteolin can induce and inhibit UGTs and SULTs. It can also inhibit multi ABC transport proteins. Understanding the interactions between luteolin (glucosides) and drug-metabolizing enzyme has an important significance in guiding clinical use of the drug.

Pharmacokinetics of luteolin from Elsholtzia blanda extracts in rats / 药学学报

An RP-HPLC method for determination of luteolin from Elsholtzia blanda Benth. extracts in rats' plasma was established and the pharmacokinetics of luteolin in rats was studied. Drug blood samples from caudal vein were gotten after oral administration of luteolin. Plasma samples were determined by RP-HPLC after being deproteinized with trichloroacetic acid and extracted with ethyl acetate. The calibration curve was linear in the range of 0.37-47.27 microg x mL(-1). The limit of quantification was 0.37 microg x mL(-1). The method recovery of luteolin was 93%-99%. The extract recovery was 75%-85%. RSDs of intra-and inter-day precisions were less than 5%. The concentration-time curve of luteolin after oral administration of Elsholtzia blanda Benth. extracts was fitted to two compartment open model. Two factors analysis of variance were adopted in the evaluation of gender and time spots for collection of blood. The result suggested that the gender-based difference in blood-drug concentrations had statistical significance. The metabolite in blood was identified as galcuronide. The method is sensitive, specific, accurate, and is appropriate for determination of luteolin in vivo.

Determination of residual organic solvents in Panax notoginseng extracts by capillary gas chromatography with headspace sampling / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a headspace gas chromatography method for the determination of residual organic solvents in Panax notoginseng extracts.</p><p><b>METHODS</b>The samples were injected into HP-INNOWAX capillary column by headspace sampler and analyzed with FID detector using standard addition method.</p><p><b>RESULTS</b>There was a good linearity in the experimental concentration (r=0.9932-0.9999). The rate of recovery was in the rang of 81.74%-111.2%. The numbers of theoretical plates were more than 15000 and the resolutions between the adjacent peaks were more than 2. The RSD of precision and accuracy were both less than 10 %.</p><p><b>CONCLUSION</b>The method is simple,accurate and sensitive with good reproducibility, which can be used for the determination of the residual organic solvents in Panax notoginseng extracts.</p>

Interaction between (E)-2-(4-(diethylamino methyl) benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one and P-glycoprotein / 药学学报

Cell lines of Bcap37 and Bcap37/MDR1 (the high P-glycoprotein (P-gp) expressing cell line) were used as model to investigate the different accumulations of (E)-2-(4-(diethylamino methyl) benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one (BYZX) in the two kinds of cells. It was authenticated that whether BYZX was the substrate of P-gp. Meanwhile, the inhibitive effects of BYZX on the P-gp were investigated by determining the fluorescence intensity of rhodamine 123 in the model cells, with and without BYZX. A reversed-phase high-performance liquid chromatography (RP-HPLC) method was used to determine the accumulations of BYZX in the two cells. The results showed that the amount of BYZX accumulation in Bcap37/MDR1 cells were as many as those in Bcap37 cells (P > 0.05), and the concentrations of BYZX accumulated in the Bcap37/MDR1 cells did not increase when co-incubated with P-gp inhibitor verapamil. Furthermore, different concentrations of BYZX also had no effects on the efflux of rhodamine 123 (P > 0.05). These results indicated that there were no interactions between BYZX and P-gp. BYZX will not be pumped out of the cells, and it also not inhibited the P-gp. It was the useful advantage for its absorption.

In vitro metabolic interaction between diphenytriazol and steroid hormone drugs / 药学学报

<p><b>AIM</b>To observe the metabolic interaction between diphenytriazol and steroid hormone drugs, and provide some useful information for clinical medication.</p><p><b>METHODS</b>The steroid hormone drugs which may be co-administrated with diphenytriazol were selected, such as mifepriston, estradiol, medroxyprogesterone acetate, progesterone, norethisterone and so on. Diphenytriazol was incubated with each drug in rat liver microsome. The residual concentration of diphenytriazol or steroid hormone drugs in the microsomal incubates was determined by reversed-phase high-performance liquid chromatography, separately. The inhibition constants (K(i)) for each of them were calculated.</p><p><b>RESULTS</b>The inhibition constant K(is) of diphenytriazol for the metabolism of mifepristone, estradiol, medroxyprogesterone acetate, progesterone and norethisterone were (201.3 +/- 1.0), (94 +/- 4), (128.7 +/- 2.2), (64 +/- 5) and (80 +/- 4) micromol x L(-1), respectively. The inhibition constants K(i) of steroid hormone drugs for the metabolism of diphenytriazol was (66.9 +/- 2.2) micromol x L(-1) for estradiol, (60.0 +/- 2.3) micromol x L(-1) for medroxyprogesterone acetate, (163 +/- 10) micromol x L(-1) for progesterone and (88 +/- 5) micromol x L(-1) for norethisterone, respectively.</p><p><b>CONCLUSION</b>Diphenytriazol shows metabolism interaction with steroid hormone drugs such as estradiol, medroxyprogesterone acetate, progesterone and norethisterone.</p>

Cultivation of Students' Comprehensive Abilities in Independence Experiment and Practice / 中华医学教育探索杂志

This article introduces the independent experiment and social investigation activities in the course of medication analy- sis set up for strengthening students' comprehensive abilities.These activities create a good study atmosphere for enhancing stu- dents' ability to do research and their humanistic qualities.

Glucuronidation and in vitro interaction of Ginkgo flavonoids with other drugs / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To obtain the information on the glucuronidation of Ginkgo flavonoid and the interaction profile of Ginkgo flavones with other drugs in vitro.</p><p><b>METHODS</b>Ginkgo flavonoids (quercetin, isorhamnetin and keampferol) and other drugs were co-incubated with rat hepatic microsome at 25 degree; the residual concentrations of flavonoids were determined by HPLC. The enzymatic parameters of quercetin, isorhamnetin and keampferol metabolism were assessed. The interactions between flavonoids and these drugs on glucuronidation were observed.</p><p><b>RESULT</b>The K(m) values were ( 24+/-0.05), (148+/-0.09) and (110+/-0.03) micromol/L and the V(max) values were (60+/-0.21), (48+/-0.02) and (34+/-0.02) micromol x g(-1) x min(-1) for quercetin, isorhamnetin and kaempferol, respectively. The IC(50) of nifedipine propafenone ipriflavone and diphenytriazol on flavonoids metabolism were 54 - 70, 69 - 122, 85 - 98 and 210 - 362 micromol, respectively. The inhibition constants (Ki) of diphenytriazol propafenone and ipriflavone on quercetin, isorhamnetin and keampferol metabolism were (57.6, 50.5, 33.1) (33.6, 59.5, 45.2) and(13.7,24.0,15.7) microg/ml respectively. The ratio [I]/[Ki] of the plasma concentration and inhibition constant for propafenone was 0.002 - 0.003.</p><p><b>CONCLUSION</b>The metabolic level of quercetin is the strongest among three Ginkgo flavonoids. Nifedipine propafenone and ipriflavone inhibit the metabolism of quercetin, isorhamnetin and keampferol at different levels. Because of the interaction between Ginkgo flavonoids with nifedipine, caution must be taken when two drugs are used together clinically.</p>

Interaction between ipriflavone and other drugs in metabolism in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the metabolism-based interaction between ipriflavone(IP) and other drugs in vitro.</p><p><b>METHODS</b>IP and propafenone or estradiol, or other drugs were co-incubated with rat liver microsome induced by beta-naphthoflavone. The concentrations of residual substrate in microsomal incubates were determined by HPLC.</p><p><b>RESULT</b>The metabolism of propafenone (10 microg/ml) incubated with 2 microg/ml or 50 microg/ml IP was markedly inhibited compared with control group(P<0.01). While the metabolism of 10 microg/ml of estradiol incubated with 10 microg/ml and 100 microg/ml IP showed no marked inhibition. When IP (20 microg/ml) was incubated with 0.5 microg/ml propranolol, 0.5 microg/ml propafenone or 5.0 microg/ml estradiol respectively the metabolic activity of IP was markedly inhibited compared with control group (P<0.05 P<0.05 P<0.02 respectively).</p><p><b>CONCLUSION</b>The results indicate that there is an interaction between ipriflavone and propafenone during in vitro metabolism, and there might also be clinical significance.</p>

HPLC fingerprint of the tablets of Ginkgo biloba L / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish the method for determination of the fingerprint of tablets of Ginkgo biloba L.</p><p><b>METHODS</b>HPLC-DAD was used to determine the constituents in tablets. Diamonsil C18(200 mm x 4.6 mm, 5 microm) was used as analysis column and acetonitrile/KH(2)PO(4) as mobile phase with gradient elution. The column temperature was at 24 degree. The profile of chemical constituents in control sample and tablets obtained from the chromatograms were analyzed by similarity software.</p><p><b>RESULT</b>The method developed for components analysis of the standard extracts was linear within certain concentration (r>0.999). There was no difference between the fingerprints of 3 batches of products. The fingerprints of tablets and the extract showed a good similarity(>0.965).</p><p><b>CONCLUSION</b>This method is accurate simple and can be used for the quality control of Ginkgo biloba L. preparations.</p>

Determination of three formulations of pilocarpine in rabbit ocular aqueous by RP-HPLC / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop an RP-HPLC method for assay of pilocarpine in rabbit ocular aqueous humor.</p><p><b>METHODS</b>The RP-HPLC method was performed on a column of ODS-C(18) with the mobile phase consisting of 0.5% of triethylamine (TEA) of phosphate solutions (10 mmol/L, pH 2.5) and acetonitrile (98/2,v/v). The detection wavelength was 215 nm and flow rate was 1.0 ml/min. Ninety albino rabbits were divided into 3 groups (30 in each):group 1 received 50 microl of eye drops containing 1% generic pilocarpine, group 21% mixture pilocarpine solution consisting of aqueous sample and liposome and group 31% liposome pilocarpine, respectively. The aqueous humor was withdrawn at 5, 10, 30, 40, 60, 90, 120, 180, 240 and 360 min. Pilocarpine was extracted from aqueous humor with dichloromethane.</p><p><b>RESULT</b>The linear calibration curve was obtained in the concentration range of 0.1 - 20 microg/ml. The average recovery was (68.1+/-2.7)% (n=9). Inter-day and intra-day RSD were 4.33% and 2.87%, respectively. In three formations 1% liposome pilocarpine was the best for the areas under curve and measurable amounts.</p><p><b>CONCLUSION</b>The RP-HPLC method is simple and reliable for pilocarpine measurement in ocular aqueous. Liposome formulation can significantly increase the bioavailability of pilocarpine in ocular aqueous.</p>

HPLC determination of zolmitriptan and its related substances / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop an analytical method and quality control for determination of zolmitriptan and related substances.</p><p><b>METHODS</b>Zolmitriptan and related substances were separated and determined on a shimadzu CLC-C(8) column (150 mm x 6 mm, 10 microm) with a mobile phase of acetonitrile-10 mmol/L phosphate buffer (25:75 pH 7.5) and a flow-rate of 1 ml/min; the UV-VIS detector was operated at 229 nm.</p><p><b>RESULT</b>The limit of detection for the related substances was 0.5 ng on the zolmitriptan basis (S/N >3). Linear calibration curve was gene rated from 4 - 40 microg/ml with a correlation coefficient of 0.9999. The recovery rate of zolmitriptan was 99.1% with a standard deviation of 0.2%. The results of HPLC method were consistent with those of nonaqueous titration method.</p><p><b>CONCLUSION</b>HPLC method is a rapid sensitive and accurate method for the determination of zolmitriptan and its related substances.</p>

Ginkgo flavones in in vitro metabolism and its clinical application / 药学学报

<p><b>AIM</b>To develop a method for assaying Ginkgo flavones in rat hepatical microsome.</p><p><b>METHODS</b>Quercetin, isorhamnetin and keampferol were added to microsome incubate and incubated for a given time then extracted with ether-acetone. After evaporated, the residue was reconstituted with 100 microL of phosphate buffer solution (pH 2.0)-tetrahydrofuran-methanol-isopropanol (60:15:10:20). An aliquot of 20 microL was injected into the HPLC system. According to the result of estimate by means of HPLC, the results of metabolism of Ginkgo flavones in different conditions was compared.</p><p><b>RESULTS</b>The assay was linear over the rang of 0.2-8 mg.L-1 for Ginkgo flavones. The limit of quantification was 0.1 mg.L-1 (n = 3). The recoveries of three components of Ginkgo flavones were 99.9%-113.8% for quercetin (RSD < 0.8%), 100.8%-117.3% for isorhamnetin (RSD < 1.9%) and 100.7%-116.5% for keampferol (RSD < 1.03%, n = 5).</p><p><b>CONCLUSION</b>The method is simple, fast and accurate. It can be used for investigation of the metabolism of Ginkgo flavones.</p>