衍生化GC法测定重组乙型肝炎疫苗中游离甲醛的含量

目的建立衍生化毛细管气相色谱法测定重组乙型肝炎疫苗[酿酒酵母或中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)]中游离甲醛含量。方法应用2,4-二硝基苯肼(2,4-dinitrophenylhydrazine,2,4-DNPH)衍生重组乙型肝炎疫苗中的游离甲醛后,环己烷萃取衍生物,HP-5毛细管色谱柱分离,电子捕获检测器测定,外标法定量。色谱条件:初始温度为150℃,保持1 min,再以20℃·min-1的速率升温至250℃,保持10 min;检测器温度为350℃,尾吹60 m L·min-1;进样口温度为300℃,分流比为50∶1;载气为N2;进样量为1μL。结果游离甲醛质量浓度在1.01~30.3 mg·L-1内,与峰面积呈良好的线性关系,回归方程为A=3 968.5ρ-284.1(r=0.999 6)。低、中、高3个浓度的平均加样回收率为99.1%,RSD值为2.87%。结论衍生化毛细管气相色谱法可用于重组乙型肝炎疫苗中游离甲醛含量的质量控制。     

柱前衍生化HPLC/UV法测定黄杨宁片中环维黄杨星D的含量

目的建立柱前衍生化HPLC/UV法测定黄杨宁片中环维黄杨星D的含量。方法异氰酸苯酯为柱前衍生化试剂,流动相：甲醇-水（83∶17）;ZORBAX SB-C18（250 mm×4.6 mm,3.5μm）柱分离;检测波长242 nm;流速：1 mL.min-1;柱温：30℃;进样量：10μL。结果环维黄杨星D在2～400μg.mL-1与峰面积呈良好线性关系,回归方程为：Y=2 616X＋198.2（r=0.999 9）;环维黄杨星D平均回收率为102.6%,RSD为0.4%（n=6）;结论该法简单、准确度高、灵敏度好,为黄杨宁片质量控制提供了依据。     

聚乙二醇400分子量测定方法比较研究

目的：建立高效凝胶渗透色谱法（HPGPC）测定聚乙二醇400（PEG400）分子量及其分布,并与《中国药典》所用的端基分析滴定法比较。方法：采用Shodex OHpak SB-802.5HQ与Shodex OHpakSB-803HQ色谱柱串联分离,以0.1 mol·L^-1硝酸钠（0.02%叠氮钠）为流动相,流速0.5 mL·min^-1,柱温30℃,样品浓度2 mg·mL^-1,采用示差折光检测器进行测定。结果：HPGPC测定的聚乙二醇400的分子量结果与《中国药典》2015年版所用的端基分析法测得的平均分子量结果接近,采用不同厂家色谱柱、对照品可影响PEG400的分子量测定结果,选用Shodex OHpak SB色谱柱串联测定聚乙二醇400重均分子量的范围为407~440,分子量分布宽度1.0~1.1,该方法的精密性RSD为0.2%,重复性为2.89%,24 h内样品稳定性RSD为0.66%。结论：经方法学验证,HPGPC法准确、重复性好,可用于聚乙二醇400分子量及其分布测定。     

聚乙二醇化重组人干扰素β-1a注射液中游离聚乙二醇残留量的测定

目的对聚乙二醇化重组人干扰素β-1a（PEG-rhIFN-β-1a）注射液中的残留聚乙二醇丁醛（PEG）进行方法学研究。方法采用Phenomenex JupiterC4色谱柱,流动相：A相,0．1％三氟乙酸．水溶液;B相,0．1％三氟乙酸一乙腈溶液,梯度洗脱;流速：1.0mL·min^-1,柱温为25℃,蒸发光检测器,漂移管温度为113．0℃,栽气流速为3．1L．min。结果PEG在0.6～2.0μg·mL^-1内线性良好,相关系数为0．9984,检出限为0.20μg,定量限为0．40gg,精密度、稳定性和重复性试验的RSD均＜2．O％,平均回收率为96％～100％。结论运用该方法对PEG-rhIFN-β-1a注射液中游离PEG残留量测定,残留量＜1.0％。     

柱前衍生液相色谱法与柱后衍生液相色谱法测定疫苗中游离甲醛残留量的对比研究

目的：建立柱前衍生液相色谱法与柱后衍生液相色谱法测定疫苗中游离甲醛残留量,并考察2种方法测定结果的一致性程度。方法：柱前衍生液相色谱法色谱条件：岛津LC-20AT液相色谱仪（SPD-20A紫外检测器）,采用Kromasil 100-5-C18（250mm×4.6mm）色谱柱,流动相为60%乙腈溶液,流速0.8mL·min-1,柱温40℃,测定波长360 nm。柱后衍生液相色谱法色谱条件：岛津LC-20AT液相色谱仪（SPD-M20A二极管阵列检测器和vector衍生装置）,采用纳谱AQ-C18（250 mm×4.6 mm）色谱柱,流动相为0.2%（V/V）磷酸溶液,流速1.0 mL·min-1,柱温25℃,检测波长412 nm;衍生溶液为醋酸缓冲液,流速0.5 mL·min-1,温度100℃。分别对2种方法的精密度、重复性、加样回收率等项目进行考察,并对测定结果进行显著性检验（F检验和t检验）。结果：柱前衍生液相色谱法线性范围为0.025~100 μg·mL-1（R=0.999 9,n=12）,精密度RSD为0.06%,重复性RSD为0.3%~1.4%,平均加样回收率为97.3%~104.8%（RSD为0.7~2.9%）,定量限为0.02μg·mL-1,检出限为0.01 μg·mL-1。柱后衍生液相色谱法线性范围为0.025~100 μg·mL-1（R=0.9999,n=12）,精密度RSD为0.02%,重复性RSD为0.07%~3.5%,平均加样回收率为105.6%~114.6%（RSD为0.3~1.9%）,定量限为0.02μg·mL-1,检出限为0.006 μg·mL-1。对21批样品测定结果的F检验和t检验结果表明两者无显著性差异。结论：2种方法操作简便,专属性强,灵敏度高,结果准确,可用于疫苗中游离甲醛残留量测定。     

甲醛、乙醛衍生化条件的探讨

目的建立通过HPLC方法同时控制甲醛量、乙醛量的衍生化条件。方法甲醛、乙醛与2,4-二硝基苯肼溶液在60℃水浴中加热60 min衍生化反应,采用Agilent TC-C18色谱柱(4.6 mm×250 mm,5μm),以水-乙腈(45∶55)为流动相,流速1.0 mL·min 1,检测波长350 nm;柱温为40℃;进样量20μL。结果甲醛、乙醛衍生化物峰的分离度为7.2,甲醛的线性范围为0.028~9.23μg.mL 1(r=0.999 4),平均回收率(n=9)为102.6%(RSD=0.9%),定量限为3.7×10 6μg、检测限为1.8×10 6μg;乙醛的线性范围为0.063~20.90μg.mL 1(r=0.999 5),平均回收率(n=9)为102.0%(RSD=1.2%),定量限为1.1×10 4μg、检测限为5.5×10 5μg。结论本法准确、灵敏,为甲醛、乙醛的安全监测提供检测依据。     

多巴对映异构体的柱前衍生化HPLC法手性拆分

目的：建立多巴对映异构体的柱前衍生化拆分分析方法。方法：采用柱前衍生化反相高效液相色谱法,以氯甲酸乙酯为酰化剂,L-丙氨酸-β-萘胺（L-Ala-β-NA）为柱前衍生化试剂,在室温反应20 min 后,依利特 Hypersil ODS2（4.6 mm ×250 mm,5μm）为色谱柱;乙腈-0.05%三氟乙酸为流动相,梯度洗脱;流速为1.0 mL·min-1;柱温为25℃;检测波长为280 nm。结果：多巴对映体衍生物获得了基线分离,并确认了R（-）-和S（+）-两个衍生物的色谱峰。结论：该方法经济可行,操作较简便,为多巴的药理学研究和光学异构体纯度测定提供了参考。     

柱前衍生高效液相色谱法测定盐酸四环素原料药中微量甲醛含量

目的建立测定盐酸四环素中微量甲醛含量的柱前衍生化-高效液相色谱法。方法衍生化试剂为2,4-二硝基苯肼乙腈溶液乙酸钠/冰乙酸缓冲液(p H=5)等体积混合,色谱柱为Phenomenex Hyperclone BDS C18柱(250 mm×4.6 mm,5μm),流动相为乙腈-水(45∶55,V/V)等度洗脱,柱温为35℃,检测波长为355 nm,流速为1.0 m L/min,进样量为20μL。结果甲醛质量浓度在0.012 13~0.485 20μg/m L范围内与峰面积线性关系良好(r=0.999 9);柱前衍生化反应获得的衍生物较稳定,建立的方法精密度高、重复性好,检测限(LOD)和定量限(LOQ)分别为0.012 1μg/m L和0.036 4μg/m L;甲醛的平均加样回收率为96.57%,RSD为4.18%;两个不同厂家盐酸四环素原料药中游离甲醛相对百分含量分别为(4.75±0.036)×10-6%和(4.78±0.031)×10-6%。结论该方法简便、可行,适用于盐酸四环素原料药中游离甲醛的含量测定及质量评价。     

毛细管气相色谱法测定盐酸多塞平中有机溶剂的残留量

目的建立测定盐酸多塞平中有机残留溶剂的测定方法。方法采用100%聚二甲基硅氧烷毛细管柱（30.0m×320μm×0.4μm）;氢火焰离子化检测器（FID）;柱温采用程序升温：初温30℃（保持14min）,以30℃.min-1升温至85℃（保持2min）,以50℃.min-1升温至180℃（保持4min）;载气为N2,流速：起始为0.7mL.min-1（保持14min）,以1mL.min-1升到2mL.min-1（保持7.7min）;进样口温度：220℃;检测器温度：250℃。结果 7种残留溶剂达到完全分离,且均呈现良好的线性关系和回收率,3批样品检测残留溶剂均未超标。结论本测定方法简单,准确,可以用于盐酸多塞平原料药中残留溶剂的测定。     

程序升温毛细管气相色谱法测定盐酸氨溴索的溶剂残留量

目的建立测定盐酸氨溴索中残留溶剂的测定方法。方法采用聚乙二醇毛细管柱（30.0m×530μm×3.00μm）,氢火焰离子化检测器（FID）,色谱条件：初温60℃（保持5min）,以30℃/min升温至180℃（保持5min）,进样口温度：180℃,检测器温度：200℃。结果4种残留溶剂达到完全分离,且均呈现良好的线性关系,3批样品检测残留溶剂均未超标。结论本测定方法简单,准确,可以有效用于盐酸氨溴索原料药中残留溶剂的测定。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.