用HPLC法测定发酵液中的生物素含量

采用高效液相色谱法测定发酵液中生物素的含量。色谱柱为Symmetry C18,流动相为V(甲醇):V(pH 2.5磷酸缓冲溶液)=14:86,检测波长为210 nm,柱温为35℃。结果表明,样品进样量在5-150μg/mL范围内线性关系良好,加样回收率在95%以上,相对标准偏差RSD为6.6%,最低检测限为0.01μg/mL。该方法简便、快捷、准确、重现性好,可用于发酵液中生物素的含量测定。     

HPLC法测定葛花中鸢尾苷的含量

目的：测定8个产地葛花中鸢尾苷的含量,建立葛花中鸢尾苷含量测定的HPLC方法。方法：采用GRACEC18（250mm×4.6mm,5μm）色谱柱,流动相为乙腈-水梯度洗脱,流速为0.8mL/min,紫外检测波长为265nm;柱温为室温。结果：鸢尾苷的峰面积（Y）与浓度（X）在11.8～236.4μg/mL范围内具有良好线性关系,Y=34920X-1156.5,r=0.9995（n=7）;平均加样回收率为103.66%,RSD〈2%（n=9）;测得8批不同产地的葛花药材中鸢尾苷含量在37.00～113.1mg/g。结论：建立了高效液相色谱法测定葛花中鸢尾苷含量的方法,该法准确、可靠,可用于葛花中主要成分鸢尾苷的含量测定;不同产地葛花中均检测到鸢尾苷,但其含量有一定区别。     

高效液相色谱法测定甜菊糖苷中甜菊醇的含量

建立高效液相色谱法测定甜菊糖苷中甜菊醇的含量。色谱条件:色谱柱为Phenomenex C18(4.6 mm×250 mm,5μm);流动相为乙腈-水(50∶50);流速1.0 mL/min;检测波长213 nm;柱温30℃;进样量10μL。线性范围为1.046μg/mL~52.3μg/mL(r=0.9997),加标平均回收率为96.60%,RSD为0.51%(n=6)。本方法准确度高、精密度高、重复性好、简捷易操作,可以作为甜菊糖苷中甜菊醇含量的测定方法。     

HPLC检测三叶青块根中白藜芦醇和白藜芦醇苷含量的研究

目的：建立三叶青地下部分白藜芦醇、白藜芦醇苷以及总黄酮的提取分离和含量测定方法。方法：白藜芦醇提取分离选取溶剂为70%乙醇，白藜芦醇苷的提取分离选取溶剂为40%乙醇。含量测定采用高效液相色谱法，色谱柱选择Agilent ZORBAX SB-C18色谱柱(250 mm×4.6 mm,5μm)，流动相为甲醇-水梯度洗脱，流速为0.8 mL/min，检测波长为304 nm，柱温25℃。结果：白藜芦醇的线性关系方程为Y=113.836X+0.911 5，在0.02～2μg/mL范围内的峰面积与进样浓度线性关系良好(R²=0.997 6)。采用70%乙醇提取三叶青根茎中白藜芦醇的平均含量为0.59μg/g。白藜芦醇苷的线性关系方程为Y=52.515X-0.497 7，在0.02～2μg/mL范围内的峰面积与进样浓度线性关系良好(R²=0.994 4)。采用40%乙醇提取三叶青根茎中白藜芦醇的平均含量为1.76μg/g。结论：建立了三叶青地下部分白藜芦醇、白藜芦醇苷以及总黄酮的提取分离和含量测定方法。建立的高效液相色谱法方法简便，准确，重复性好，可应用于...     

反相高效液相色谱法测定双唑泰软膏中三组分的含量

以C_(18) ODS色谱柱为分析柱,甲醇-水-三乙胺(体积比70:30:0.3,含庚烷磺酸钠10mmol/L,用磷酸调pH 4.0)为流动相,260 nm为检测波长,用反相高效液相色谱法测定了双唑泰软膏中甲硝唑、克霉唑和醋酸氯己定3个组分的含量。测定结果表明:甲硝唑、醋酸氯己定和克霉唑分别在3.92～39.2μg/mL,3.32～33.2μg/mL和1.64～16.4μg/mL质量浓度范围内,与峰面积呈良好的相关性(r=0.999 9、r=0.999 4和r=0.999 1),平均回收率分别为99.95%、101.14%和99.47%(n=9)。该法快速、方便,分离度好,辅料无干扰,适用于该复方制剂中3种成分的同时测定。     

高效液相色谱法测定氟伐他汀钠缓释片的含量

建立高效液相色谱法测定含量和含量均匀度的方法。采用SHIMADZU CLC-ODS色谱柱,以乙睛-水-冰醋酸(380∶120∶0.2)为流动相,234nm波长处检测。氟伐他汀钠在41μg/mL~328μg/mL浓度范围内线性关系良好(r=0.9998),平均回收率为100.5%,相对标准偏差(relative standaral deviation,RSD)0.5%(n=9)。本方法具有简单,柱效高,经济等优点。     

生物检材中佐匹克隆的GC/μECD测定

目的：建立液-液萃取、GC/μECD气相色谱测定生物检材中佐匹克隆的方法。方法：以家兔为模型动物,500 mg佐匹克隆标准品灌胃家兔,6.5小时后处死,取其胃、肝、血、脑、心脏、尿,采用乙醚液-液萃取提取,Rtx-5毛细管柱分离,电子捕获检测器检测,标准曲线法定量。结果：该方法灵敏度达0.02μg/mL,在0.05-50μg/mL范围内线性关系良好;低、中、高3种浓度的加样回收率分别为91.7%、99.5%、94.4%,相对标准偏差（RSD）均小于5%;佐匹克隆在家兔脏器中的浓度分别为7.31μg/mL（血液）、7.63μg/g（肝脏）、7.96μg/g（脑）、14.67μg/g（心脏）、32.18μg/g（胃组织）、42.65μg/mL（尿液）,在各脏器中呈特异性分布。结论：该法操作简单、灵敏度高,结果可靠,完全满足临床检测及公安机关在办理佐匹克隆麻醉案件中的需要。     

气相色谱法检测水痘减毒活疫苗原液中二甲基亚砜残留量

目的 建立测定水痘减毒活疫苗原液中二甲基亚砜(dimethyl sulfoxide, DMSO)残留量的气相色谱法,并进行方法学的验证和初步应用。方法 采用气相色谱法检测,对色谱柱、柱温、分流比以及供试品预处理方法进行了优化,并对优化后的方法进行系统适用性和专属性、线性和范围、准确度和精密度、定量限和检测限、耐用性的方法学验证。结果 色谱柱使用DB-624(30 m×0.53 mm, 3.00μm)毛细管柱,以氮气为载气,进样口温度设定为250℃,FID检测器温度设定为270℃,柱温为120℃,分流比为5∶1。供试品在检测前采用乙腈沉淀蛋白的预处理方式(体积比1∶9～1∶19)以去除其中的干扰杂质。方法学验证结果显示,系统适用性和专属性良好,DMSO色谱峰与杂质分离良好,在50～1 000μg/mL范围内线性关系良好(R²=0.999 9)。检测限为20μg/mL,定量限为50μg/mL;准确度结果均在90%～110%之间,精密度的RSD均<10%。水痘减毒活疫苗原液中的DMSO残留量均未检出。结论 建立的气相色谱法能稳定、准确、快速地检测水痘减毒活疫苗原液...     

高效液相色谱法测定川射干鸢尾苷元磺酸钠及其制剂泰克吉宁注射液含量和有关物质

建立高效液相色谱法测定鸢尾苷元磺酸钠(4′,5,7-三羟基-6-甲氧基异黄酮-5′-磺酸钠)及其制剂泰克吉宁注射液的含量及有关物质。采用C18色谱柱(150×4.6mm,5μm),流动相为甲醇-5%醋酸水(80:20),流速为1.0mL/min,检测波长为263nm。鸢尾苷元磺酸钠在11～100μg/mL范围内线性关系良好,回归方程Y=43.3609X-1.5973(r=0.99998),平均回收率为99.77%(n=9)。最低检测限为0.052μg/mL,与有关物质分离良好。本法快速、简便、准确,专属性强。     

异丙威在稻田土壤中的残留检测方法

建立稻田土壤中异丙威的气相色谱测定方法。稻田土中的异丙威经二氯甲烷液液分配提取后,用气相色谱氮磷检测器(GC-NPD)测定,以保留时间定性,外标法定量。方法的线性范围为0.20-40μg/mL,r=0.9994,回收率为86.86-92.91%,RSD为0.70-2.27%。结果表明该方法灵敏、准确,并能很好地排除干扰,可满足稻田土中痕量异丙威的测定。     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (-43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2-8°C for more than 3 months. IC(50) value of Ambisome (0.18 μg/mL) was comparatively similar to F-1a (0.17 μg/mL) and F-2a (0.16 μg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.     

肉桂抑制α-葡萄糖苷酶活性成分研究

为寻找肉桂中具有抑制α-葡萄糖苷酶活性的化学成分,采用高效液相色谱结合体外抑制α-葡萄糖苷酶活性筛选模型的方法,进行活性成分的跟踪分离,并对活性化合物进行酶抑制动力学研究.结果显示,肉桂石油醚提取物(IC50=350.37 μg/mL)的活性明显高于阳性对照阿卡波糖(IC50=1028.99 μg/mL),从中分离出2个活性成分,分别鉴定为桂皮醛( IC50 =277.89 μg/mL)和肉桂酸(IC50=286.22 μg/mL).酶抑制动力学结果表明它们对α-葡萄糖苷酶的抑制类型均为非竞争性抑制,Ki值分别为178.07 μg/mL和229.43 μg/mL.     

黄连素和黄芩苷不同配伍对肠道细菌体外生长的影响

研究不同配伍的黄连素和黄芩苷在模拟肠道厌氧环境下对肠道内有害菌、条件致病菌和益生菌体外生长的影响。

采用微量肉汤稀释法测定黄连素和黄芩苷不同配伍对肠道内金黄色葡萄球菌、沙门菌、肠炎链球菌、大肠埃希菌、乳杆菌及嗜黏蛋白阿克曼菌体外生长24 h后吸光度的影响。

对于金黄色葡萄球菌,抑制作用最强的是黄连素∶黄芩苷 = 10∶1（组合六）125 μg/mL（t = –39.350,P＜0.001）,抑制率为99.91%;对于沙门菌,抑制作用最强的是黄连素∶黄芩苷 = 100∶1（组合七）250 μg/mL（t = –73.788,P＜0.001）,抑制率为80.47%;对于肠炎链球菌,抑制作用最强的是黄连素∶黄芩苷 = 1∶0（组合一）250 μg/mL（t = –57.549,P＜0.001）;对于大肠埃希菌,抑制作用最强的是黄连素∶黄芩苷 = 1∶0（组合一）125 μg/mL（t = –29.085,P＜0.001）;对于乳杆菌,增殖作用最强的是黄连素∶黄芩苷 = 100∶1（组合七）31.25 μg/mL（t = 76.280,P＜0.001）;对于嗜黏蛋白阿克曼菌,增殖作用最强的是黄连素∶黄芩苷 = 100∶1（组合七）62.5 μg/mL（t = 8.357,P＜0.001）。

黄连素∶黄芩苷 = 10∶1（组合六）、黄连素∶黄芩苷 = 100∶1（组合七）对有害菌的抑制作用更强,黄连素∶黄芩苷 = 100∶1（组合七）对益生菌的增殖作用更强,而黄连素和黄芩苷联合使用对条件致病菌的抑制作用较弱。

     

Comparison of the Activity of Meropenem and Imipenem Against Anaerobic Bacteria in Bulgaria

The in vitro activities of meropenem and imipenem were compared against 154 clinical isolates of anaerobic bacteria representing 23 species of 10 genera. The NCCLS-recommended agar dilution method with Brucella agar from the Wadsworth Anaerobic Laboratory was used. Meropenem proved to be more active than imipenem with an MIC range of ≤0.125–4 μg/mL, MIC₅₀=0.25 μg/mL, MIC₉₀=1 μg/mL with 100% of strains susceptible at the breakpoint=4 μg/mL. Imipenem showed a lower activity with an MIC range of ≤0.125 to 16 μg/mL, MIC₅₀of 2 μg/mL and MIC₉₀of 4 μg/mL with 10% of the strains not inhibited at this concentration. Ninety-six per cent were susceptible at 8 μg/mL and 100% at 16 μg/mL. The MIC of both antibiotics (especially of imipenem) were higher for the Bacteroides fragilis group than for the rest of the Gram-negative organism higher still than the Gram-positive anaerobes.     

Simultaneous determination of serum lathosterol and cholesterol by semi-micro high-performance liquid chromatography with electrochemical detection

A simple method has been developed for the simultaneous determination of lathosterol and cholesterol by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Lathosterol was found to be electrochemically oxidized and its current peak height was linearly related to the amount of lathosterol injected, ranging from 0.15 μmol/L to 300 μmol/L (r=0.995). Similar results were obtained with cholesterol from 15 μmol/L to 600 μmol/L (r=0.995). The separation was carried out with an ODS column, acetonitrile containing 30 mmol/L lithium perchlorate as a mobile phase, and an applied potential at +2.8 V vs. Ag/AgCl. The detection limit (S/N=3) of lathosterol as well as cholesterol was 0.03 μmol/L (0.15 pmol). Total lathosterol in control human and rat serum was determined by the present method with a recovery of more than 95.8% and an RSD (n=5) of less than 7.3%. The present method was applied to an experiment with rats to examine the effect of lathosterol feeding. There were no significant changes in serum lathosterol or cholesterol levels in rats fed with a high-lathosterol diet for six days. Therefore, we found this method to be both simple and useful for the simultaneous determination of lathosterol and cholesterol in serum.     

凹叶厚朴抑制α-糖苷酶与抗氧化活性研究

首次采用96微孔板法检测贵州和河南产凹叶厚朴抑制α-葡萄糖苷酶活性;并采用DPPH、ABTS和FRAP三种方法测定其抗氧化活性.贵州产凹叶厚朴乙酸乙酯(IC50 =7.22 μg,/mL)和正丁醇提取部位(IC50=36.59 μg/mL),河南产凹叶厚朴石油醚(IC50=107.04 μg/mL)和乙酸乙酯提取部位(IC50=17.17μg/mL),它们的活性都远高于于阳性对照Acarhose( IC50=1081.27 μg/mL).贵州产凹叶厚朴乙酸乙酯提取部位清除ABTS自由基的能力最强(IC50=8.81 μg/mL),强于阳性对照BHT(IC50=11.94 μg/mL);其次为河南产凹叶厚朴乙酸乙酯提取部位(IC50=12.73 μg/mL).研究结果表明,贵州产凹叶厚朴乙酸乙酯提取部位抑制α-葡萄糖苷酶和抗氧化活性最好.     

新型柱前衍生试剂分析草甘膦的高效液相色谱研究

以2,5-二甲氧基苯磺酰氯(DMOSC)为柱前衍生化试剂,建立了柱前衍生草甘膦的紫外检测反相高效液相色谱法,并优化了衍生化条件,得最佳条件:衍生温度35℃,时间15 min,pH 10.0,草甘膦与DMOSC的摩尔比为1∶6。HPLC分析条件:采用Kromasil C18柱,流速1.0 mL/min,柱温30℃,检测波长220 nm,流动相为甲醇-乙腈-磷酸盐缓冲溶液(0.02 mol/L、pH 5.5),三者的体积比为15∶5∶80。结果表明:草甘膦质量浓度在5~100μg/mL范围内线性关系良好,相关系数为0.996 2,检测限为0.067μg/mL。实验表明该方法反应条件温和,灵敏度高,衍生产物稳定。     

Semi-solid state fermentation of food waste for production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> biopesticide

In this study, Bacillus thuringiensis (Bt) biopesticide was produced through different fermentation processes using food waste with different water contents. The semi-solid state sample with 75% water content presented the highest δ-endotoxin efficiency of 862 μg/mL. δ-endotoxin efficiency increased by 30.2% from that of the solid-state sample (50% water content) and 73.8% from that of the submerged sample (99% water content). Results confirmed that semi-solid state fermentation presents considerable advantages compared with other fermentation types (solid-state and submerged). The timing adjustment of pH and recycling fermentation effectively counteracted the inhibition of pH and product, thereby increasing δ- endotoxin efficiency to 1,648 and 2,478 μg/mL (accumulated value of all four fermentation loops), respectively. A δ- endotoxin slow-release formulation using polylactic acid as the carrier was also developed to effectively promote the stability of Bt biopesticide.     

提高展青霉(Penicillium patulum)产灰黄霉素效价的诱变育种研究

为进一步筛选高产灰黄霉素的工业生产菌株,分别对前期采用紫外线-氯化锂（UV-LiCl）、半导体激光（LD laser）及CO2激光（CO2laser）对展青霉FS80-1复合诱变获得三株高产菌株进行液体发酵和固体培养比较。结果表明,通过UV-LiCl复合诱变获得突变菌株GM120-43的液体发酵产灰黄霉素效价11 982μg/mL,比出发菌株提高37.52%,固体培养效价为89 496μg/g（干重）,比出发菌株提高80.04%。;半导体激光诱变获得突变株LD100-1的液体发酵效价9 440μg/mL,固体培养效价119 766μg/g干重,比出发菌株FS80-1提高了140%;两个突变株的生物学特性均发生不同程度的变化,突菌株GM120-43适合于液体发酵生产,突变株LD100-1适合于固体发酵培养。