高效液相色谱-荧光检测法测定血浆中五聚赖氨酸-β-羰基酞菁锌

目的:建立高效液相色谱-荧光检测法测定小鼠血浆中五聚赖氨酸-β-羰基酞菁锌[ZnPc-(Lys)5]的浓度。方法:血浆样品经预处理后采用CNW C18色谱柱(250 mm×4.6 mm,5μm),流动相为甲醇-水(分别含有0.05%三氟乙酸),流速为1.0 mL.min-1,荧光检测器激发波长为610 nm,发射波长为690 nm,柱温为25℃。结果:在2～500 nmol.L-1检测浓度范围内线性良好(r=0.999 0),检出限为0.5 nmol.L-1,提取回收率为86.99%～88.80%,相对回收率为96.20%～101.76%,日内和日间RSD均低于10%。结论:该方法简便、快速、准确、重复性好,可用于血浆中ZnPc-(Lys)5浓度的测定。     

顺序注射氢化物发生-原子荧光光谱法同时测定中药材中砷和镉

目的:建立一种以顺序注射氢化物发生-原子荧光光谱法同时测定中药材中砷和镉含量的方法。方法:通过考察光电倍增管负高压、砷和镉灯电流、原子化器高度、载气流量、屏蔽气流量、增敏剂等因素对测定结果的的影响。在载流盐酸的浓度为0.6 mol·L-1,KBH4浓度为20 g.L-1,增敏剂硫脲和钴离子的浓度分别为80 g.L-1和70 mg.L-1时,同时测定砷和镉的效果最佳。结果:在最佳实验条件下,砷和镉的检出限分别为0.014μg.L-1和0.18μg.L-1,加标回收率为92.9%～103.5%,相对标准偏差小于3.2%,被测试样中共存的离子对砷和镉的测定没有干扰。结论:本方法操作方便、快速,用于中药材中砷和镉的同时测定,具有很好的可行性和实用性。     

奥美沙坦酯中4-氯甲基-5-甲基-1,3-二氧杂环戊烯-2-酮和4,5-二氯甲基-1,3-二氧杂环戊烯-2-酮的HPLC法测定

目的：建立高效液相色谱法（HPLC）测定奥美沙坦酯中氯代烷基结构类潜在基因毒性杂质（4-氯甲基-5-甲基-1,3-二氧杂环戊烯-2-酮（杂质1）和4,5二氯甲基-1,3-二氧杂环戊烯-2-酮（杂质2））。方法：采用色谱柱为Kromasil Eternity 5-PhenylHexyl 柱 250×4.6 mm;检测波长：215 nm,流动相A：乙腈：2.04 g?L-1磷酸二氢钾溶液（用1.73 g/L磷酸溶液调节pH值至3.4）（20：80）;流动相B：2.04 g?L-1磷酸二氢钾溶液（用1.73 g?L-1磷酸溶液调节pH值至3.4）：乙腈（20：80）,进行梯度洗脱,流速为1.0 mL·min-1,柱温40 ℃,进样量10 μL。结果：杂质1和杂质2与主峰分离良好,在浓度为0.1066~0.7104 μg·mL-1和0.1235~0.6174 μg·mL-1范围内杂质1和杂质2的线性关系良好（相关系数分别为1.0000和0.9971）;杂质1和杂质2的平均回收率分别为98.09%和114.85%,RSD（n=9）分别为7%和7%。结论：经方法学验证,本法准确性好、灵敏度高,适用于奥美沙坦酯中的杂质1和杂质2的定量控制。     

固相萃取-液相色谱-串联质谱法测定人血浆中莫沙必利的浓度

目的建立液相色谱-串联质谱(LC-MS/MS)法测定人血浆样本中莫沙必利的浓度。方法血浆样本经固相小柱萃取处理后,以甲醇-0.5%甲酸溶液(70:30)为流动相,采用Agilent TC-C18(4.6 mm×150mm,5μm)色谱柱进行分离,流速为0.5 m L·min-1,柱温25℃;采用ESI正离子模式检测,离子监测方式为MRM,用于定量分析的离子反应分别为m/z 422.1→m/z 198(莫沙必利)和m/z 466.1→m/z 184(西沙必利)。结果莫沙必利在0.512~125μg·L-1与峰面积线性关系良好(r2=0.999 3),定量下限为0.512μg·L-1,日内及日间RSD均<10%,莫沙必利的提取回收率和基质效应分别在95.8%~101.6%和101.6%~113.0%。结论本方法特异性强,灵敏度高,测定结果可靠,样本检测时间为3 min,适用于血浆样本中莫沙必利的浓度测定及其药物代谢动力学研究。     

光敏剂五聚赖氨酸-β-羰基酞菁锌对宫颈癌Hela细胞的光动力学治疗研究

目的 研究光敏剂五聚赖氨酸-β-羰基酞菁锌”[ZnPc-(Lys)5]光动力诱导宫颈癌Hela细胞死亡的方式.方法 测量Hela细胞对光敏剂ZnPc-(Lys)5的摄取、光毒性和暗毒性,利用流式细胞仪分析光动力疗法对Hela细胞的影响,包括其细胞凋亡和细胞坏死的比例;并通过透射电镜观察细胞超微结构的改变情况.结果 Hela细胞对该光敏剂的摄取随着ZnPc-(Lys)5浓度的升高而增强,相关光动力学实验结果显示:光敏剂ZnPc-(Lys)5对宫颈癌Hela细胞的光动力疗效作用理想,无暗毒性.其介导的光动力治疗是通过诱导细胞凋亡途径而达到消灭肿瘤细胞的作用.ZnPc-(Lys)5对Hela细胞光动力作用后随作用的不同时间,其凋亡的比率有显著性差异:0.5h的早期凋亡率为(50.36±1.02)％,1h为(40.80±0.72)％,2h为(30.87±2.80)％,4h为(26.87±0.81)％,各组比较差异均有统计学意义(P＜0.05).晚期凋亡率0.5h为(34.23±0.55)％,1h为(33.97±1.40)％,2h为(45.57±1.12)％,4h为(45.57±1.22)％,第0.5h、1h与第2h、4h比较,差异有统计学意义(P＜0.05).Hela细胞在光动力作用下1h超微结构主要表现为早期凋亡,2h后超微结构改变为晚期凋亡和胀亡的特征改变.结论 光敏剂ZnPc-(Lys)5介导的光动力治疗主要通过凋亡途径特异性诱导宫颈癌Hela细胞的死亡.     

HPLC法测定多维铁口服溶液中盐酸赖氨酸的含量

目的：建立HPLC法测定多维铁口服溶液中盐酸赖氨酸的含量。方法：采用2,4-二硝基氟苯为衍生化试剂,使盐酸赖氨酸衍生化。采用Gemini C18柱（4.6mm×250mm,5μm）;以0.05 mol?L-1醋酸钠溶液（用冰醋酸调pH至6.4）-甲醇（45∶55）为流动相,检测波长为354nm。结果：盐酸赖氨酸在1.076～21.52μg?mL-1浓度范围内线性关系良好,平均加样回收率为99.1%（RSD=0.6%,n=9）。4个厂家样品（共12批）中盐酸赖氨酸的标示百分含量分别为91.0%~92.2%、81.5%~84.3%、101.9%~103.1%、98.9%~100.2%。结论：本法简便准确,稳定性好,可用于测定多维铁口服溶液中盐酸赖氨酸的含量。     

增敏荧光法测定血浆中亚甲蓝含量及其残留量检测

目的 建立检测血浆中微量亚甲蓝的增敏荧光分光光度法.方法 利用β-环糊精对亚甲蓝荧光信号的增敏作用,以665nm为激发波长,680nm为发射波长,用荧光分光光度法直接测定血浆中的微量亚甲蓝.结果 用β-环糊精增敏时,亚甲蓝浓度在0.012~2.283μmol/L范围内呈现良好的线性关系（r=0.9994）,高中低浓度的日内变异系数（CV）分别为0.19%、0.41%和 1.14%,日间CV分别为1.76%、2.24%和2.97%,标准曲线法测得回收率为99.39~102.30%,对照品比较法测得回收率为96.93%~104.02%.结论 本研究体现出了简单,准确、快捷的特点,实现了无需进行分离、富集达到直接测定血浆中亚甲蓝浓度的目的.     

高效液相色谱法测定复方伪麻敏芬胶囊中3组分的含量

（1. ［摘要］目的建立高效液相色谱法测定复方伪麻敏芬胶囊中3组分的含量的方法。方法Zorbax C18柱（4.6 mm×250 mm，5 μm）；流动相为甲醇 6 mmol&#8226;L-1十二烷基硫酸钠溶液 0.025 mol&#8226;L-1磷酸二氢钾溶液 三乙胺（75：20：20：0.25，用磷酸调pH值至3.25）；流速：1 mL&#8226; min 1；检测波长：盐酸伪麻黄碱、马来酸氯苯那敏为259 nm，布洛芬为264 nm. 结果布洛芬浓度在100.6～704.2 μg&#8226;mL-1范围内，盐酸伪麻黄碱50.0～600.0 μg&#8226;mL-1范围内，马来酸氯苯那敏5.02～50.20 μg&#8226;mL-1范围内，其浓度与吸收之间线性关系均良好，平均回收率分别为99.7%，99.1%，99.1%，RSD分别为0.71%，0.73%，0.71%（n＝5）。结论该方法准确，灵敏度高，重现性好，可用于测定复方伪麻敏芬胶囊中布洛芬、盐酸伪麻黄碱、马来酸氯苯那敏的含量。     

液相色谱-串联质谱法测定晚期实体癌患者静脉滴注紫杉醇胶束后的药动学

目的建立测定人血浆中紫杉醇浓度的液相色谱-串联质谱(LC-MS/MS)法。方法 8例晚期实体癌患者,进行单次静脉滴注注射用紫杉醇胶束(300 mg·m-2),采集血浆样本并测定其中紫杉醇的浓度。血浆样本以氘5-紫杉醇为内标,血浆经直接沉淀后进样分析,选用CAPCELL PAK C18 MGIII(100 mm×2.0 mm,5μm)为分析柱,以0.2%甲酸的水溶液-乙腈溶液=40∶60(V/V)为流动相,流速为0.4 m L·min-1。选用三重四级杆串联质谱仪的多重反应监测(MRM)扫描方式进行监测,电喷雾离子化源,正离子方式。并采用Phoenix Win Nonlin 6.2软件对数据进行处理,计算药动学参数。结果血液中紫杉醇的线性范围10~20 000μg·L-1。日内、日间RSD均小于8%,平均提取回收率在89.5%~97.7%范围内。内标校正基质因子为0.888 7~1.033,RSD<4%。应用此法,检测8例晚期实体癌患者静脉滴注注射用紫杉醇胶束(300 mg·m-2)所得主要药动学参数:ρmax为(3 872±1 062)μg·L-1,AUC0-∞为(14 603±3 390)μg·h·L-1,t1/2为(18.3±6.4)h,CL为(22.0±5.0)L·h-1·m-2。结论建立的LC-MS/MS测定人血浆中紫杉醇浓度的方法灵敏度高、专一性好、操作简单。     

6-[4-(4′-吡啶)氨基苯]-4,5-二氢-3(2H)哒嗪酮对大鼠胸主动脉环收缩功能的影响

目的研究新型钙增敏强心剂6-[4-(4′-吡啶)氨基苯]-4,5-二氢-3(2H)哒嗪酮(MCI-154)的扩血管作用机制。方法采用生物张力换能器及生理记录仪测定大鼠离体胸主动脉环和蜕膜胸主动脉环的收缩张力。结果MCI-154可浓度依赖性抑制1nmol.L-1~10μmol.L-1去甲肾上腺素(pD2′为4.21±0.23)和80mmol.L-1KCl(IC50为7μmol.L-1)引起的血管环收缩,提示其可通过抑制血管平滑肌细胞膜上受体操纵性和电压依赖性钙通道而减少胞外钙内流。在无Ca2+K-H液中,MCI-154预处理可浓度依赖性降低3μmol.L-1苯肾上腺素(IC50为5μmol.L-1)及20mmol.L-1咖啡因(IC50为16μmol.L-1)引起的血管环收缩张力,提示其可抑制血管平滑肌细胞胞内钙释放。在1μmol.L-1Ca2+溶液中,MCI-154可显著降低蜕膜血管环收缩张力(IC50为10μmol.L-1),提示其可降低血管平滑肌对Ca2+的敏感性。结论MCI-154可通过抑制血管平滑肌胞外钙内流、胞内钙释放和降低其对Ca2+敏感性来降低血管平滑肌收缩张力,体外具有扩血管效应。     

Photophysicals and photochemicals studies of zinc(II) phthalocyanine in long time circulation micelles for photodynamic therapy use.

Long time circulation systems, such as polymeric micelles, represent a growing area in biomedical research. These microparticles can be used in many biological systems to provide appropriate drug levels with a specific biodistribution. Long time circulation micelles (LTCM) were routinely prepared using PEG-5000-DSPE (polyethyleneglycol-5000-distearoil-phosphatidyl-ethanolamine) and zinc(II) phthalocyanine (ZnPc) as a photosensitizer and fluorescent probe. This compound belongs to a second generation of photoactive agents, mainly used in photodynamic therapy (PDT) of neoplasic tissues. Their high selectivity for tumoral target tissues as well as high phototoxicity based on singlet oxygen generation renders the utilization of these compounds feasible as an alternative therapy for cancer treatment. LTCM were characterized by classical spectroscopic techniques. Absorbance measurements indicated that the drug was s completely loaded into LTCM (epsilon = 2.41 x 10(5) cm(-1)). This was also verified by steady state and time-resolved fluorescence measurements. The lifetime profiles of ZnPc decay curves were fitted according to biexponential function (tau1 = 3.9 ns and tau2 = 15.5 ns) indicating different locations for ZnPc into LTCM. The time-resolved spectroscopy measurements for ZnPc triplet excited state lifetimes (tauT) were calculated from the kinetic analysis of transient decays at the absorption maximum (480 nm), by using laser flash photolysis technique. All the spectroscopy measurements performed allowed us to conclude that, ZnPc in LTCM is a promising drug delivery system (DDS) for PDT.     

Topical delivery system of ophthalmic drugs by periocular injection with viscous solution.

The purpose of this study is to evaluate periocular injections with viscous solution as a topical delivery system of ophthalmic drugs. Tilisolol and carboxymethylcellulose (CMC) were used as a model beta-blocker and a viscous polymer, respectively. After intracapsular, retrobulbar and palpebral conjunctival injections (50 microl) of tilisolol with 3% CMC into rabbits, drug concentrations in the tear fluid, blood, aqueous humor and vitreous body were determined by HPLC. Periocular injection (50 microl) of tilisolol with 3% CMC showed slight leakage of the drug in the tear fluid from the injection site. The viscous vehicle decreased the absorption rate constant of the drug from the injection site to systemic circulation compared with the buffer solution. It suggests that the viscous solution improved the retention of drug at both the injection site and in periocular tissues. Although the periocular injections with viscous vehicle (3% CMC) showed lower AUC in the aqueous humor than that observed in instillation, they showed comparable AUC in the vitreous humor. Compared to the results after the periocular injections with buffer solution, CMC increased the AUCs in the vitreous body 3.1-fold with retrobulbar injection and 1.4-fold with palpebral conjunctival injection, respectively. As a result, periocular injections with 3% CMC showed higher delivery of tilisolol to the vitreous body against the aqueous humor than the instillation and periocular injections with buffer solution.     

The absorption of warfarin from the rat small intestine in situ.

The in situ absorption from the rat small intestine of the weakly acidic drug, warfarin (pKa 5-05), at 200 mug ml-1 in the instilled fluid with initial pH levels of 3, 5, 7 or 8 has been examined. These initial pH's in the buffer changed rapidly towards neutrality. The buffers at pH's 3 and 5 probably caused different amounts of warfarin precipitation, which resulted in different rates of warfarin disappearance from the instilled fluid which paralleled the initial rates of accumulation of warfarin in (or on) the intestinal wall. Where greater drug precipitation had probably occurred the initial rates of absorption into the plasma were slower. At the initial pH of 3 and by solubilization of warfarin with propylene glycol, the rate of absorption was similar to that from a fluid of pH 7. Propylene glycol in 15% solution did not affect the system significantly. The relatively high transfer of warfarin into octanol from buffer solution at pH 7 might indicate that the small fraction of unionized drug (1 : 100) at pH 7 is enough for remarkable transfer of this highly lipid-soluble drug.     

The distribution of quinidine in human blood.

The uptake of quinidine by washed human red blood cells from isotonic buffer solution (pH 7.4) occurred rapidly and was proportional to the concentration of drug in buffer. A constant red cell/buffer partition ratio of 4.16+/-0.15 s.e. mean was found. 2. Uptake from buffer solution was not affected by temperature or ouabain or by gassing with nitrogen or carbon monoxide and there was no evidence of saturability. Drug in red blood cells was associated largely with the cell contents (94.4+/-1.5% s.e. mean) following partition. 3 Plasma reduced the uptake of quinidine so that a red cell/plasma partition ratio of 0.82+/-0.09 s.e. mean was found. 4 Alteration of plasma binding by dilution of plasma with buffer showed that uptake was proportional to free drug concentration. 5 The possibility of red cell uptake of drug should be included in any considerations concerning pharmacokinetic aspects of drug action in the body.     

Photodynamic ultradeformable liposomes: Design and characterization

Hydrophobic ([tetrakis(2,4-dimetil-3-pentyloxi)-phthalocyaninate]zinc(II)) (ZnPc) and hydrophilic ([tetrakis(N,N,N-trimethylammoniumetoxi)-phthalocyaninate]zinc(II) tetraiodide) (ZnPcMet) phthalocyanines were synthesized and loaded in ultradeformable liposomes (UDL) of soybean phosphatidylcholine and sodium cholate (6:1, w/w, ratio), resulting 100 nm mean size vesicles of negative Zeta potential, with encapsulation efficiencies of 85 and 53%, enthalpy of phase transition of 5.33 and 158 J/mmol for ZnPc and ZnPcMet, respectively, indicating their deep and moderate partition into UD matrices. Matrix elasticity of UDL-phthalocyanines resulted 28-fold greater than that of non-UDL, leaking only 25% of its inner aqueous content after passage through a nanoporous barrier versus 100% leakage for non-UDL. UDL-ZnPc made ZnPc soluble in aqueous buffer while kept the monomeric state, rendering singlet oxygen quantum yield (Φ_Δ) similar to that obtained in ethanol (0.61), whereas UDL-ZnPcMet had a four-fold higher Φ_Δ than that of free ZnPcMet (0.21). Free phthalocyanines were non-toxic at 1 and 10 μM, both in dark or upon irradiation at 15 J/cm² on Vero and J-774 cells (MTT assay). Only liposomal ZnPc at 10 μM was toxic for J-774 cells under both conditions. Aditionally, endo-lysosomal confinement of the HPTS dye was kept after irradiation at 15 J/cm² in the presence of UDL-phtalocyanines. This could lead to improve effects of singlet oxygen against intra-vesicular pathogen targets inside the endo-lysosomal system.     

Spectrofluorimetric and micelle-enhanced spectrofluorimetric methods for the determination of gemfibrozil in pharmaceutical preparations

A spectrofluorimetric method for the determination of antihyperlipoproteinemic gemfibrozil was developed based on its native fluorescence. This method allows the determination of 0.10-6 microg ml(-1) gemfibrozil in aqueous solution (without using any buffer solution) with excitation and emission wavelengths of 276 and 304 nm, respectively. Detection and quantification limits were 0.03 and 0.10 microg ml(-1), respectively. The fluorescence properties of gemfibrozil in micellar media were also studied. It was shown that in the presence of 0.4% Brij-35 surfactant (pH 4.0, acetic acid-acetate buffer) about 2.4-fold enhancement can be achieved in the fluorescence of this drug. Based on the obtained results, a micelle-enhanced fluorescence method was also developed that is more sensitive than aqueous fluorescence method and has lower detection limit (0.02 microg ml(-1)). Both methods were applied satisfactorily to the determination of gemfibrozil in a commercial pharmaceutical formulation.     

亚胺培南/西司他丁注射液无菌检查冲洗液选择的比较

选择适当的冲洗液,可减少亚胺培南/西司他丁注射液无菌检查的冲洗量.分别采用0.1%蛋白胨水溶液和pH 7.0氯化钠-蛋白胨缓冲液作为冲洗液,对亚胺培南/西司他丁注射液进行无菌检查方法验证,结果表明,采用pH 7.0氯化钠-蛋白胨缓冲液作为冲洗液可明显减少无菌检查中冲洗液的使用.pH7.0氯化钠-蛋白胨缓冲液作为亚胺培南/西司他丁注射液无菌检查中的冲洗液可提高无菌检查工作效率.     

Preparation and improvement of release behavior of chitosan microspheres containing insulin

Sophisticated delivery systems, such as nanoparticles, represent a growing area in biomedical research. Nanoparticles (Np) were prepared using a solvent emulsion evaporation method (SEEM) to load zinc(II) phthalocyanine (ZnPc). Np were obtained using poly (d,l latic-co-glycolic acid) (PLGA). ZnPc is a second generation of photoactive agents used in photodynamic therapy.

ZnPc loaded PLGA nanoparticles were prepared by SEEM, characterized and available in cellular culture. The process yield and encapsulation efficiency were 80 and 70%, respectively. The nanoparticles have a mean diameter of 285 nm, a narrow size distribution with polydispersive index of 0.12, smooth surface and spherical shape. ZnPc loaded nanoparticles maintains its photophysical behavior after encapsulation. Photosensitizer release from nanoparticles was sustained with a moderate and burst effect of 15% for 3 days. The photocytotoxicity of ZnPc loaded PLGA Np was evaluated on P388-D1 cells what were incubated with ZnPc loaded Np (5 μM) by 6 h and exposed to red light (675 nm) for 120 s, and light dose of 30 J/cm². After 24 h of incubation, the cellular viability was determined, obtaining 61% of cellular death. All the physical–chemical, photophysical and photobiological measurements performed allow us conclude that ZnPc loaded PLGA nanoparticles is a promising drug delivery system for photodynamic therapy.     

蝙蝠葛碱透皮扩散接收液的筛选

目的：筛选蝙蝠葛碱透皮吸收研究所用的接收液。方法：以改良Franz扩散池为实验装置,兔皮为透皮扩散皮肤,以Q—t方程和透皮速率常数为考察指标,从pH4．5醋酸-醋酸钠生理盐水、30％乙醇生理盐水、生理盐水、pH7．2磷酸盐生理盐水、pH5．8磷酸盐生理盐水、pH4．5醋酸-醋酸钠30％乙醇生理盐水、20％乙醇生理盐水中筛选最佳接收液。结果：综合比较Q—t方程和透皮速率常数,均以pH5．8磷酸盐生理盐水为最佳。结论：本方法简单易行,重复性好,pH5．8磷酸盐生理盐水可作为蝙蝠葛碱透皮吸收研究的接收液。     

Oral delivery of mono-PEGylated sCT (Lys18) in rats: regional difference in stability and hypocalcemic effect

In the in vitro experiment using a luminal, mucosal, and fecal fluid/extract from jejunum and colon of a rat, Lys18-residue modified mono-PEG(2k)-sCT (Lys18-PEG(2K)-sCT) exhibited a longer half-life than salmon calcitonin (sCT) in a colonic fluid and its extract. A physical adsorption study showed that Lys18-PEG(2K)-sCT had lower adsorption in the feces than sCT over an 8-hr period. An absorption study of the sCT and Lys18-PEG(2K)-sCT from the jejunum and colon using an in situ closed-loop technique in anesthetized rats showed a dose-dependent reduction in the plasma Ca2+ level but to a certain limit. Furthermore, the hypocalcemic response by intracolonic administration was significantly higher than the intrajejunal one, demonstrating that the colon had better absorption. In particular, Lys18-PEG(2K)-sCT (5 microg/rats) produced the most pronounced hypocalcemia after the intracolonic administration, which resulted in a sustained reduction in the serum calcium level over an 8-hr period, with a maximum reduction (% max(d)) of 38% after 4 hr. The overall reduction in the serum calcium levels, which was expressed as the net change in the AUC relative to the control over an 8-hr period, was 25.51 +/- 3.38 for Lys18-PEG(2K)-sCT. The relative pharmacological bioavailability of the intracolonically administered Lys18-PEG(2K)-sCT was 2.1-fold higher than sCT and the absolute pharmacological bioavailability was 73.59% of i.v.-injected sCT in an 8-hr period. Overall, this study highlights the feasibility of the oral delivery of Lys18-PEG(2K)-sCT in achieving a sustained calcium-lowering effect.