流动注射化学发光法测定吩噻嗪类药物

目的确定以鲁米诺-高碘酸钾发光体系测定吩噻嗪类药物的化学发光分析方法。方法在碱性介质中,盐酸氯丙嗪和盐酸异丙嗪对鲁米诺-高碘酸钾发光体系有明显的增敏作用,且增敏效果与其浓度呈良好的线性关系。基于此,建立了盐酸氯丙嗪和盐酸异丙嗪的流动注射化学发光分析方法。结果在优化的实验条件下,盐酸氯丙嗪和盐酸异丙嗪的线性范围分别为3.0×10-9-1.0×10-6g/mL和3.0×10-8-7.0×10-5g/mL,检测限分别为5.0×10-10g/mL和7.3×10-9g/mL。结论本方法简便、快速、准确、灵敏度高、线形范围宽,应用于相应注射剂和片剂分析,并与药典方法进行对照,结果令人满意。     

流动注射-化学发光法测定氢化可的松

目的确定以高锰酸钾-亚硫酸钠体系测定氢化可的松的流动注射-化学发光分析方法。方法在酸性条件下,氢化可的松对高锰酸钾-亚硫酸钠体系发光反应具有明显的增敏作用。据此,建立了流动注射化学发光测定氢化可的松的分析方法。结果在优化的实验条件下,氢化可的松质量浓度在1.0×10-9-1.0×10-6g/mL范围内与发光强度呈良好的线性关系,检出限(3R)为4.0×10-10g/mL,对氢化可的松进行11次平行测定,其相对标准偏差为2.2%。结论本方法应用于注射液中氢化可的松含量的测定,快速、准确、简便,灵敏度高、线性范围宽。     

奋乃静药物的流动注射化学发光测定方法的建立

目的建立快速测定奋乃静的流动注射化学发光新方法。方法在硝酸介质中,奋乃静能被硫酸铈氧化生成发光物质奋乃静砜,从而产生化学发光。基于此,建立了奋乃静的流动注射化学发光分析方法。结果在优化的实验条件下,不用任何发光增敏剂,奋乃静在1.0×10-7-7.0×10-5g/mL范围内与化学发光强度呈良好的线性关系,检出限(3σ)为8.0×10-8g/mL,对1.0×10-6g/mL的奋乃静进行了11次平行测定,其相对标准偏差为1.8%。结论本方法简便、快速、准确、灵敏度高、线形范围宽,应用于奋乃静片剂分析,并与药典方法进行对照,结果满意。     

Luminol-potassium permanganate chemiluminescence system for the determination of three anthracycline antibiotics

Objective To establish a flow-injection chemiluminescence method for the determination of doxorubicin, epirubicin and mitoxantrone and study its reaction mechanism. Methods In alkaline medium, chemiluminescence of luminol-potassium permanganate system could be inhibited obviously by anthracycline antibiotics. Combined with flow-injection technique, a new chemiluminescence method for determining the anthracycline antibiotics was set up. The chemiluminescence mechanism of the luminol-potassium permanganate system was also discussed. Results Under optimal conditions, the good linear ranges of doxorubicin, epirubicin and mitoxantrone were 5.0×10-9-1.0×10-6g/mL, 1.0×10-9-1.0×10-5g/mL and 3×10-9-1.0×10-6g/mL, respectively. The detection limits of doxorubicin, epirubicin and mitoxantrone were 3.0×10-9g/mL, 5.0×10-8g/mL and 2.0×10-9g/mL, respectively. During eleven repeated inter-day and intra-day precision tests of 1.0×10-6g/mL samples, the relative standard deviations corresponded to reference values of 3.0%, 2.8% and 2.1%. Conclusion The developed method is sensitive, accurate, rapid and of low cost. It can be applied to determine doxorubicin hydrochloride, epirubicin hydrochloride and mitoxantrone hydrochloride in injection preparations.     

Flow-injection-enhanced chemiluminescence method for the determination of three β-blockers

Objective To develop a rapid, simple and sensitive chemiluminescence method for the determination of three β-blockers (bisoprolol, atenolol and propranolol). Methods The chemiluminescence of cerium (Ⅳ)-sulfite system was obviously sensitized by adding anyone of three β-blockers in acid media. A new chemiluminescence method was set up by combining with flow-injection technique and used to determine the three β-blockers. Results Good linear ranges were obtained at the concentrations of 2.0×10-7g/mL-4.0×10-5g/mL, 1.0×10-7g/mL-3.0×10-5g/mL and 7.0×10-7g/mL-1.0×10-5g/mL, respectively, with the detection limits of 5.0×10-8g/mL, 7.0×10-8g/mL and 5.0×10-8g/mL (S/N=3), respectively, and the relative standard deviations for 11 times consecutive injections of 1.0×10-6g/mL bisoprolol, atenolol and propranolol were 3.57%, 2.21% and 2.26%, respectively. Conclusion The developed method is sensitive, accurate, rapid and of low cost. And it can be applied to determine bisoprolol, atenolol and propranolol in pharmaceutical preparations.     

Capillary electrophoresis coupled with electrochemiluminescence for determination of cloperastine hydrochloride

Objective To investigate the electrochemiluminescence (ECL) behavior of cloperustine hydrochloride.Methods ECL intensity of tris (2,2'-bipyridyl) rutheniumo(Ⅱ) was enhanced, the method for the determination of dupernstine hydrochloride was established using capillary electrophoresis (CE) coupled with electrochemilumolinescence (ECL) detection. Results Under the optimum conditions, ECL intensity varied linearly with cloperastine hydrochloride concentration from 7.0×10-6 g/mL to 1.0×10-4 g/mL. The detection limit (S/N=3) was 8.05×10-7g/mL. he relative standard deviation of the ECL intensity and the migration time for 11 consecutive injections of 1.0 ×10-5g/mL cloperastine hydrochloride was 2.9% and 1.5%, respectively. This method was successfully applied to eloperastine hydrochloride tablet determination. Conclusion The method has been established, validated and applied for determination of cloperastine hydrochloride.     

治带片中苦参碱及氧化苦参碱的HPLC法测定

目的建立治带片中苦参碱及氧化苦参碱的高效液相色谱(HPLC)测定方法。方法色谱柱:Lichrospher-NH2(4.6 mm×250 mm,5μm),C18保护柱;流动相:乙腈-无水乙醇-0.5 mol/L磷酸水溶液(80∶10∶10);检测波长212 nm;流速1.0 mL/min;柱温:室温;进样量20μL。结果苦参碱和氧化苦参碱线性范围均为1.0-10.0μg/mL,回归方程苦参碱为:C=1.201×10-4A+0.161,r=0.9992;氧化苦参碱为:C=1.366×10-4A+0.221,r=0.9996,平均回收率分别为99.9%和99.4%,RSD分别为1.48%和4.33%。结论本法简便快捷,结果准确,可用于该制剂的质量控制。     

抗坏血酸对CH2（COOH）2-BrO3^--Mn^2＋-H2SO4体系化学振荡反应的影响

研究了抗坏血酸对CH2（COOH）2-BrO3^--Mn^2＋-H2SO4体系化学振荡反应的影响,考察了该体系中各反应物的初始浓度范围及主要影响因素．结果表明：在5．7×10^-4～8．5×10^-2mol／L的范围内,抗坏血酸对振荡反应的诱导期有较大影响,且抗坏血酸的浓度与振幅及抗坏血酸浓度的对数与诱导期倒数的对数均存在良好线形关系．诱导期和周期的表观活化参数分别为56．63kJ／mol,73．81kJ／mol;同时还对抗坏血酸参与下可能振荡反应机理进行了探讨．     

尾加压素Ⅱ促进乳鼠心肌成纤维细胞分泌胶原及增殖的细胞内信号转导机制

目的探讨尾加压素Ⅱ(UⅡ)促进乳鼠心肌成纤维细胞(CFs)分泌胶原及增殖的细胞内信号转导机制。方法体外培养CFs,采用免疫组织化学染色法及羟脯氨酸测定法分别观察不同浓度UⅡ作用下,CFs中磷酸化ERK1/2细胞灰度和CFs培养上清中胶原含量的变化;蛋白激酶C(PKC)抑制剂chelerythrine chloride(Che)、ERK1/2抑制剂PD98059和钙调神经磷酸酶(CaN)抑制剂cyclosporin A(CsA)各自对UⅡ诱导的细胞增殖的影响。结果在1×10-10、1×10-9、1×10-8mol/L UⅡ作用下,CFs培养上清中胶原含量均较对照组明显增加,而CFs中磷酸化ERK1/2细胞灰度均较对照组有显著降低(P<0.01);在1×10-7mol/L UⅡ作用下,上述各参数与对照组比较无统计学意义(P>0.05)。1×10-6mol/L Che+1×10-8mol/L UⅡ组、1×10-5mol/L PD98059+1×10-8mol/L UⅡ组和5μg/mLCsA+1×10-8mol/L UⅡ组的胶原含量均高于对照组而低于1×10-8mol/L UⅡ组(P<0.05)。1×10-6mol/L Che+1×10-8mol/L UⅡ组、1×10-5mol/L PD98059+1×10-8mol/L UⅡ组和5μg/mL CsA+1×10-8mol/L UⅡ组的p-ERK1/2的灰度低于对照组(P<0.01);1×10-6mol/L Che+1×10-8mol/L UⅡ组和5μg/mL CsA+1×10-8mol/LUⅡ组的p-ERK1/2的灰度高于1×10-8mol/L UⅡ组(P<0.01),而1×10-5mol/L PD98059+1×10-8mol/L UⅡ组与之比较则无统计学意义(P>0.05)。结论UⅡ具有促进CFs分泌胶原及增殖的作用,其作用可能是通过PKC/MAPK/CaN途径实现的。     

Analysis and determination of diterpenoids in unprocessed and processed Euphorbia lathyris seeds by HPLC-ESI-MS

Euphorbia lathyris （Caper spurge） is a toxic and potent Chinese materia medica （T/PCMM）. This study sought a method for identifying five diterpenoids （Euphorbia factors LI-L3, L7a, and Ls） with the spectra of UV and mass, quantifying three diterpenoids L1, L2, and L8 in crude extracts of unprocessed and processed E. lathyris seeds by liquid chromatography/ electrospray ionization mass spectrometry （LC-ESI-MS）. The analysis was achieved on an Agilent Eclipse XDB-C18 column （4.6 mm× 150mm i.d., 5 μm） with an isocratic elution with a mobile phase consisting of water and acetonitrile at a flow rate of 0.25 mL/min at column temperature of 30 ℃ and UV detection was set at 272 nm. An ESI source was used with a positive ionization mode. The calibration curve was linear in the ranges of 9.9-79 μg/mL for Euphorbia factor Lb 3.8-30.5μg/mL for Euphorbia factor L2, and 1.0-20.6 μg/mL for Euphorbia factor LB. The average recoveries （n=6） of three diterpenoids were 98.39%, 91.10% and 96.94%, respectively, with RSD of 2.5%, 2.4% and 2.1%, respectively. The contents of the three diterpenoids in processed E. lathyris seeds were 3.435, 1.367 and 0.286 mg/g, respectively, which decreased more sharply than those in unprocessed E. lathyris seeds which were 4.915, 1.944 and 0.425 mg/g, respectively. The method is simple, accurate, reliable and reproducible, and it can be applied to control the quality of unprocessed and processed E. lathyris seeds.     

流动注射-光度法测定亚硝酸盐

为快速测定微量亚硝酸盐,建立了一种新的测定亚硝酸盐的方法--流动注射-催化光度法.该方法结合流动注射技术,以亚硝酸盐对氯酸钠与亚甲基蓝发生的褪色反应有催化作用的研究结果为基础.最优试验条件下,在5～150 μg/L和150～1 000 μg/L范围内吸光强度与NO_2~-的质量浓度呈良好的线性关系,该方法的检出限为1 μg/L.对质量浓度为10和100 μg/L的NO_2~-分别进行了11次连续测定,相对标准偏差分别为3.1%和4.6%.用此方法检测河水和地下水水样中的亚硝酸盐,标准偏差为1.1%～3.4%,加标回收率为97.6%～100.3%.     

健脾补血片中阿魏酸的质量浓度测定

目的建立健脾补血片中阿魏酸的高效液相色谱(HPLC)测定方法。方法采用HPLC法。色谱柱Lichrospher C18(4.6 mm×250 mm,5μm),C18保护柱;流动相:乙腈-0.378 mol/L冰醋酸水溶液(30∶70);检测波长321 nm;流速0.8 mL/min;柱温:室温;进样量10μL。结果阿魏酸质量浓度在1.0-10.0 mg/L范围内,与峰面积呈良好的线性关系(r=0.9996),平均回收率为99.8%,RSD为0.26%。结论HPLC可用于健脾补血片中阿魏酸的质量浓度测定。     

Simultaneous determination of 12, 13-dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture by high performance liquid chromatography

A reversed phase high performance liquid chromatography （HPLC） method was established for the simultaneous determination of 12, 13-dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture. A Grace Apollo Cl8 column （250 mm × 4.6 mm, 5 μm） was used as the stationary phase and the mobile phase was composed of acetonitrile and aqueous phosphoric acid （0.2%, v/v）. Gradient elution was carried out at the flow rate of 1.0 mL/min and the column temperature was 30 ℃. An ultraviolet （UV） detector was used with a selected wavelength of 240 nm. Calibration curves were linear within the concentration range of 4.6-45.75 μg/mL for 12, 13-dihydroxyeuparin （r〉0.9999） and 106.9-1068.9μg/mL for glycyrrhizic acid （r〉0.9999）, respectively. Recoveries were 102.18% for 12, 13-dihydroxyeuparin and 101.17% for glycyrrhizic acid. The method developed could be applied to the simultaneous determination of 12, 13- dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture.     

Analysis of ganciclovir and its related substances using high performance liquid chromatography and liquid chromatography-mass spectrometry methods

Objective High performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS) methods were developed for the determination of ganciclovir and its related substances. Methods A Hypersil ODS2 column (4.6mm×250mm, 5μm) was used with a mobile phase of 0.02M potassium 1.0mL/min, and UV detector set at 254nm was used for monitoring the eluents. Results The method was simple, rapid, selective and capable of separating all related substances at trace level with a detection limit of 0.04μg/mL. It has been validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The linearity range was 10.2-153.0μg/mL with r=0.9998. The percentage recoveries ranged from 96.7% to 101.6%, and RSD was 1.24%-1.96% (n=5). Conclusion The method was found to be suitable not only for monitoring the reactions during the process development but also for quality control of ganciclovir. For identification of related substances, LC/MS was used. The mainly related substances of ganciclovir active pharmaceutical ingredients (API) were determined as guanine, (1, 3-dioxolan-4-yl) methyl acetate, and diacetyl guanine.     

尼古丁损伤大鼠肠系膜动脉内皮功能及卡托普利的干预作用

目的观察尼古丁对大鼠离体肠系膜动脉内皮依赖性舒张反应的影响以及卡托普利的干预作用。方法应用器官培养技术和离体血管环张力描记法,研究不同浓度的尼古丁(10-5、10-4、10-3mol/L)在有或无卡托普利(0.01、0.030、.1 mmol/L)的情况下,对培养24 h的成年SD大鼠肠系膜动脉环(1 mm)内皮依赖性舒张反应的影响。结果尼古丁呈浓度依赖性损伤乙酰胆碱(ACh)诱导的内皮依赖性血管舒张反应(P<0.01,与空白对照组比较)。卡托普利呈浓度依赖性改善尼古丁对血管内皮依赖性舒张反应的损害。低浓度时对尼古丁损伤没有明显影响,高浓度具有显著的保护作用(P<0.05,与尼古丁10-4mol/L组比较)。结论卡托普利对尼古丁所引起的血管内皮依赖性舒张功能的损伤具有明显的保护作用。     

熊果酸对大鼠子宫平滑肌收缩的影响

目的观察熊果酸对离体大鼠子宫活动的影响。方法将离体子宫肌条在1 g的前负荷下温育,加入熊果酸1.0×10-8、2.0×10-8、3.0×10-8、4.0×10-8g/L,以等容量生理盐水作为自身对照,记录30 min;分别使用不同的受体拮抗剂5 min后,再加入熊果酸3.0×10-8g/L。用Biolap 410生物机能仪记录熊果酸对离体子宫平滑肌运动的影响。结果熊果酸可增强离体子宫平滑肌的收缩振幅、频率和持续时间,熊果酸剂量与子宫肌兴奋作用存在剂量-反应关系。并使用受体阻断剂苯海拉明(2×10-6mol/L)、酚妥拉明(2×10-6mol/L)、异博定(2×10-7mol/L)及消炎痛(2×10-5mol/L)观察其改变。该增强作用可被消炎痛阻断,而异博定、酚妥拉明、苯海拉明未能阻断熊果酸的作用。结论熊果酸增强作用与前列腺素酶的合成与释放有关,不通过L型电压依赖性钙通道、α受体以及H1受体发挥作用。     

RP-HPLC method for the quantitation of β-Sitosterol in Elaeagnus Gon yanthes Benth

Objective To establish an RP-HPLC method for the determination of β-Sitosterol in Elaeagnus Gonyanthes Benth. Methods The separation was performed on a luna C8 (2) (150 mm×4.6 mm, 5μm) column with the mobile phase of methanol-water (88∶12, v/v) at a flow rate of 1.0 mL/min, the detection wavelength was set at 210 nm, and the temperature of the column was maintained at 35 ℃. Results The calibration curve of β-Sitosterol was linear over the concentration range of 0.075-0.375 mg/mL (r=0.9999) and the average recovery of β-Sitosterol was 96.30% with RSD of 3.60%(n=3). Conclusion The method is simple, rapid, and accurate, and can be used for the quality control of Elaeagnus Gonyanthes Benth.     

Simultaneous determination of berberine, matrine and oxymatrine in traditional Chinese medicines by using nonaqueous capillary electrophoresis

A rapid method for the simultaneous determination of berberine (BBR), matrine (MT) and oxymatrine (OMT) by nonaqueous capillary electrophoresis (NACE) was developed. Optimum separation of the analytes was obtained on a 50cm×50μm i.d. fused-silica capillary using a non-aqueous buffer system of 70mM ammonium acetate, 7.0% acetic acid and 10% acetonitrile at 25kV and 20℃. The relative standard deviations (R.S.D.) of the migration times and peak areas of the three active components were 0.06%-0.20% and 0.12%-3.41% for berberine, 0.11%-0.60% and 0.74%-1.63% for matrine, 0.15% and 0.45% for oxymatrine, respectively. Detection limits of berberine, matrine and oxymtrine were 0.18μg/mL, 4.08μg/mL and 4.16μg/mL, respectively. In the tested concentration range, good linear relationships (0.9992 for berberine, 0.9988 for matrine and 0.9988 for oxymatrine) were observed. The linear calibration ranges were 0.45-360.0μg/mL for berberine, 8.16-408.0μg/mL for matrine and 20.8-416.0μg/mL for oxymatrine. This method has been successfully applied to the phytochemical analysis of alkaloids extracts from two commonly used traditional Chinese herbal drugs: Sophora flavescens Ait. (Kushen) and Cortex phellodendri chinensis (Huangbai) and their medicinal preparations.     

引阳素胶囊中淫羊藿苷的含量测定

目的建立引阳素胶囊中淫羊藿苷的高效液相色谱(HPLC)含量测定方法。方法采用HPLC法。色谱柱Li-chrospher C18(4.6 mm×250 mm,5μm),C18保护柱;流动相为乙腈-20 g/L冰醋酸水溶液(30∶70);检测波长270 nm;流速1.0 mL/min;柱温为室温;进样量10μL。样品加50%(体积分数)乙醇超声提取,浸提液过0.45μm滤膜后测定。结果淫羊藿苷线性范围为10.0-60.0 mg/L,回归方程为C=2.042×10-5A+0.554,r=0.9993,平均回收率为99.8%,RSD为0.65%。结论本法用于引阳素胶囊中淫羊藿苷的含量测定时简便快捷、准确。     

Preparation of recombinant firefly luciferase by a simple and rapid expression and purification method and its application in bacterial detection

A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase, equipped with a polyhistidine affinity tag, was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108, and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein, the ATP content of bacteria was 9.48×10-16mol/mL, and was identical to the bacteria counts (4500CFU/mL) in order of magnitude. Taken together, our results provided a simple and efficacious method of the preparation of recombinant luciferase, which could be applied in the determination of bacteria via ATP bioluminescence.