GC法测定法罗培南钠有机溶剂残留量

目的:建立法罗培南钠原料药中有机溶剂残留量测定方法。方法:采用气相色谱法测定,色谱柱为HP-5弹性石英毛细管柱(30 m×0.53 mm,1.5 μm,以5％苯基-95％甲基聚硅氧烷为固定液),进样口温度:200℃,氢火焰离子化检测器(FID),检测器温度:250℃;柱温:程序升温,初始温度35℃,保持5 min,以每分钟10℃的升温速率升温至120℃,保持2 min,再以每分钟35℃的升温速率升温至250 ℃,保持5 min,载气:氮气,流速:2 mL/min,分流比:1:1,尾吹流速:30mL/min,以N-甲基吡咯烷酮为溶剂。结果:待测物均能得到很好的分离,峰面积与浓度呈良好的线性关系,精密度良好。结论:该法可用于法罗培南钠原料药中有机溶剂残留量的检测。     

毛细管气相色谱法测定奥沙利铂原料药中有机溶剂的残留量

目的建立以毛细管气相色谱法检测奥沙利铂原料中2种有机溶剂乙醚、乙醇残留量的方法。方法采用聚乙二醇毛细管柱(30.0m×530μm×3μm);氢火焰离子化检测器(FID);柱温采用程序升温:初温60℃(保持4min),以30℃/min升温至180℃(保持5min);载气为N2;进样口温度:220℃;检测器温度:250℃;丙酮为内标进行测定。结果奥沙利铂原料药中残留溶剂乙醚和乙醇达到完全分离,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,适用于奥沙利铂原料药中残留溶剂的测定。     

顶空气相色谱法测定尼伐地平中的残留溶剂

目的建立顶空气相色谱法测定尼伐地平原料药中5种残留有机溶剂的方法。方法色谱柱为DB-624（30 m×0.53 mm,3μm）,氢火焰离子化检测器（FID）,进样口温度为200℃,检测器温度为250℃,顶空平衡温度为80℃,顶空平衡时间为30 min,程序升温40℃保持8 min,以10℃min-1的速度升至180℃并保持5 min,N,N-二甲基甲酰胺（DMF）为溶剂。结果 5种有机溶剂和DMF的峰完全分离,且线性关系良好,平均回收率为乙醇102.1%、丙酮99.4%、异丙醇97.9%、乙酸乙酯98.9%和甲苯100.2%。5种残留溶剂的检出量均符合2010版中国药典规定（乙醇、丙酮、异丙醇和乙酸乙酯限度均为0.5%,甲苯为0.089%）。结论该方法操作简单,结果可靠,可用于尼伐地平原料药中的残留有机溶剂的测定。     

顶空毛细管气相色谱法测定乌拉地尔原料药的溶剂残留量

目的:建立乌拉地尔原料药中5种溶剂残留量的毛细管气相色谱测定方法。方法:采用Agilent DB-5毛细管气相色谱柱(30m×320μm×0.25μm),FID检测器,进样口温度200℃,检测器温度230℃;载气为高纯氮气,流速3.0mL.min-1,柱温为程序升温:40℃保持10min,10℃.min-1升温至220℃,保持5min。顶空进样,顶空瓶平衡温度80℃,平衡时间30min。结果:在上述条件下,5种残留溶剂色谱峰的理论板数均大于10 000,相邻峰的分离度均大于2;甲醇、乙醇、丙酮、二氯甲烷、1,2-二氯乙烷的线性关系良好(r分别为0.999 2,0.999 3,0.999 9,0.999 8和0.999 8);最低检出浓度分别为:0.46,0.46,0.23,0.31,0.090μg.mL-1;平均回收率均在90%以上。结论:本方法简单、准确,灵敏度高、重复性好,适用于乌拉地尔原料药中有机溶剂残留量的测定。     

盐酸替罗非班中残留溶剂的测定

目的建立盐酸替罗非班中残留溶剂的分离测定方法。方法采用气相色谱法,Agilent DB-624柱（30m×0．32mm,1．80μm）,氢火焰离子化检测器,以氮气为载气,进样口温度为200,检测器温度为300℃;起始温度为40℃,保持20min,以每分钟20℃的速率升温至220℃,保持10min。结果被测溶剂均能得到良好的分离,每个溶剂的峰面积与浓度在一定的范围内能够呈现良好的线性关系。结论该方法能够准确测定盐酸替罗非班中的残留溶剂,可用于盐酸替罗非班原料药有机溶剂的检测。     

艾拉莫德原料药中残留溶剂检测方法的研究

目的:检查艾拉莫德原料药中的有机溶剂残留量。方法:采用气相色谱法测定,色谱柱为HP-5弹性石英毛细管柱(以5％苯基-95％甲基聚硅氧烷为固定液,1.5μm,30 m×0.53 mm);进样口温度:200℃;氢火焰离子化检测器(FID),检测器温度:300℃;柱温:程序升温,初始温度30℃,保持13 min,以5℃·min-1升温至35℃,保持7 min,再以10℃·min-1升温至230℃,保持5 min;载气:氮气;流速:1.5 mL·min-1;以N-甲基吡咯烷酮为溶剂,正丙醇为内标物。结果:被测物均能得到很好的分离,峰面积与浓度呈良好的线性关系,精密度良好。结论:该法可用于艾拉莫德原料药中有机溶剂残留量检测。     

环孢素原料药中残留溶剂检测方法的研究

目的检查环孢素原料药中的有机溶剂残留量。方法采用气相色谱法,色谱柱为弹性石英毛细管柱SE-54(聚甲基苯基乙烯基硅氧烷为固定液,30 m×0.53 mm×2.5μm),进样口温度200℃;氢火焰离子化检测器(FID)温度:250℃;柱温:程序升温,初始温度55℃,维持1 min,以10℃/min的速率升温至145℃,再以55℃/min的速率升温至200℃,维持2 min;载气:氮气,进样方式:分流进样,分流比:8∶1;流速2.3 ml/min。以二甲基乙酰胺为溶剂,外标法测定。结果被测物均能得到很好的分离,峰面积与浓度呈良好的线性关系,精密度良好。结论该法可用于环孢素原料药中有机溶剂残留量检测。     

气相色谱法测定高乌甲素传递体中有机溶剂残留量

目的:建立气相色谱法测定高乌甲素传递体中有机溶剂戏留量的方法方法:采用FID检测器,色谱枉为DB-624（30m×0.32 mm,1.8μm）,进样口及检测器温度均为250℃,载气为氮气,柱温采用程序升温:初始温度为40℃,保持10min,以8℃·min^-1升至120℃,保持4 min,再以40℃·min^-1升至200℃,保持2 min;流速为1.0 ml·min^-1。顶空瓶平衡温度120℃,平衡时间15 min,分流比为10:1,以二甲基亚砜为溶剂结果:在甲醇、乙醇、三氮甲烷三种溶剂均能得到良好的分离,空白对照无干扰;各自检测浓度在所考察的范围内与峰面积具有良好的线性;平均回收率为96.6%～102.7%,精密度试验及准确度试验的RSD均小于2.0%;甲醇、乙醇、三氯甲烷的最低检测限分别为20,18,9.8μg·ml^-1。结论:本方法简便、准确,灵敏度高,适用于高乌甲素传递体原料药中有机溶剂残留量的检测。     

毛细管气相色谱法测定洛索洛芬钠的溶剂残留量

目的建立测定洛索洛芬钠中残留溶剂的测定方法。方法色谱柱：PEG-20M（交联）毛细管柱（30.0m×0.53mm×5.0μm）,检测器：FID;色谱条件：初始温度40℃（保持5min）,以每分钟20℃升温速率升温至180℃（保持3min）,进样口温度：150℃;检测器：200℃。结果被测3种有机溶剂均能完全的分离,且均呈良好的线性关系,精密度和回收率均能符合要求。结论本方法简单,准确,可以有效用于洛索洛芬钠原料药中残留溶剂的检测。     

顶空气相色谱法测定托拉塞米原料药中残留溶剂

目的:检查托拉塞米原料药中甲醇、二氯甲烷、甲苯、二氧六环等有机溶剂残留量。方法:采用气相色谱(GC)法测定,色谱柱为HP-5弹性石英毛细管柱(5%-苯基甲基聚硅氧烷为固定液,30 m×0.53 mm,1μm),进样口温度220℃;氢火焰离子化检测器(FID)温度:240℃;柱温:程序升温,初始温度40℃,保持10 min,以40℃·min-1升温至200℃,保持3 min;载气:N2;流速:5 mL·min-1;以二甲亚砜为溶剂,丙酮为内标物。结果:被测物均能得到很好的分离,峰面积与浓度呈良好的线性关系,精密度良好。结论:该法可用于托拉塞米原料药中有机溶剂残留量检测。     

Metabolism and hepatotoxicity of N,N-dimethylformamide,N-hydroxymethyl-N-methylformamide,and N-methylformamide in the rat

The metabolism and hepatotoxicity ofN,N-dimethylformamide (DMF) and two of its metabolites,N-hydroxymethyl-N-methylformamide (HMMF) andN-methylformamide (NMF) were evaluated over a 4-day period in rats. DMF toxicity was dose dependent and delayed toxicity after the administration of a high DMF dose (13.7 mmol/kg) in comparison to a lower dose (4.1 mmol/kg) was observed. Treatment of rats with 13.7 mmol/kg DMF, HMMF, or NMF showed i) that DMF is more toxic than HMMF or NMR, and ii) that hepatotoxicity occurs later for DMF than for HMMF or NMF. Analysis of serum and urine samples demonstrated that DMF is first metabolized to HMMF, which is then partially converted to NMF. After HMMF administration, NMF was found both in serum and in urine. The time course of DMF and HMMF toxicity in relation to NMF formation fitted the hypothesis that the hepatotoxicity of DMF and HMMF is mediated via NMF. The degree of hepatotoxicity after HMMF and NMF treatment is similar. However, the degree of DMF hepatotoxicity is much higher than in the case of NMF or HMMF. The role of NMF as an obligatory intermediate in DMF and HMMF hepatotoxicity is discussed.     

Varying the Unsaturation in N 4,N 9-Dioctadecanoyl Spermines: Nonviral Lipopolyamine Vectors for More Efficient Plasmid DNA Formulation

Purpose The aim of the study is to analyze the effect of varying the degree of unsaturation in synthesized N⁴,N⁹-dioctadecanoyl spermines on DNA condensation and then to compare their transfection efficiency in cell culture. Methods The N⁴,N⁹-di-C18 lipopolyamines—saturated (stearoyl), C9-cis- (oleoyl), and C9,12-di-cis- (linoleoyl)—were synthesized from the naturally occurring polyamine spermine. The ability of these novel compounds to condense DNA and form nanoparticles was studied using ethidium bromide fluorescence quenching and nanoparticle characterization techniques. Transfection efficiency was studied in several primary skin cells (FEK4, FCP4, FCP5, FCP7, and FCP8) and in an immortalized cancer cell line (HtTA) and was compared with the commercially available nonliposomal transfection formulation Transfectam^? (dioctadecylamidoglycyl spermine), which also contains two saturated C18 lipid chains. Results N⁴,N⁹-Dilinoleoyl spermine (C18, di-cis-9,12) is efficient at circular plasmid DNA (pEGFP) condensation and gives the most effective transfection in a series of primary skin cells and cancer cell lines at low charge ratios of 5.5 (± ammonium/phosphate). Conclusions The dienoic fatty acyl spermine conjugate N⁴,N⁹-dilinoleoyl spermine efficiently condenses DNA and achieves the highest transfection levels among the studied lipopolyamines in cultured cells.     

Lipopolyamine-Mediated Single Nanoparticle Formation of Calf Thymus DNA Analyzed by Fluorescence Correlation Spectroscopy

Purpose The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N⁴,N⁹-dioleoylspermine and N¹-cholesteryl spermine carbamate. Methods Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen (PG) to allow fluorescence intensity fluctuation measurement and analysis. Results N⁴,N⁹-Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 × 10⁻¹² m²/s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio=1.0–1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N¹-Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5–2.5) with D = 1.3 × 10⁻¹² m²/s and PN value of 5.2. N⁴,N⁹-Dioleoylspermine is a more efficient DNA-condensing agent than N¹-cholesteryl spermine carbamate. Conclusions FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.     

Diphenyl diselenide supplementation delays the development of <Emphasis Type="Italic">N</Emphasis>-nitroso-<Emphasis Type="Italic">N</Emphasis>-methylurea-induced mammary tumors

The effect of dietary diphenyl diselenide (1 ppm) on N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis was examined in female Wistar rats. Beginning at 5 weeks of age, the animals were fed with either control or diphenyl-diselenide-supplied diets until the end of the study (210 days). At 50 days of age, mammary tumor was induced by the administration of three doses of NMU (50 mg/kg body wt, intraperitoneally) once a week for 3 weeks. In experimental trials, latency to tumor onset was extended in rats fed with diet supplemented with diphenyl diselenide (P < 0.05). The incidence and frequency of tumors were significantly small in animals supplemented with diphenyl diselenide. However, the multiplicity of tumors was not altered by dietary diphenyl diselenide. Diphenyl diselenide supplementation also restored superoxide dismutase (SOD) activity and vitamin C levels altered in the NMU group (P < 0.05). Our results suggest that diphenyl diselenide can be considered a chemopreventive agent, even when supplemented at a relatively low concentration.     

Dodecyl N,N-Dimethylamino Acetate and Azone Enhance Drug Penetration Across Human,Snake, and Rabbit Skin

The effectiveness of the penetration enhancers, dodecyl N, N-dimethylamino acetate (DDAA) and Azone, on pretreated human epidermis for the permeation of model drugs, indomethacin, 5-fluorouracil, and propranolol-HCl, was studied in in vitro diffusion cells. Snakeskin (Elaphe obsoleta) and rabbit pinna skin were compared as possible models for human skin. The drug concentrations were analyzed by HPLC. With all skins and all model drugs, DDAA increased drug permeability at least as well as Azone, and in most cases it was a more effective permeation enhancer. The relative permeation improvements in human skin, snakeskin, and rabbit skin were 10- to 20-, 5- to 50-, and 20- to 120-fold, respectively. Tritiated water served as an indicator of skin condition. Its penetration in the skin samples was independent of the drugs used, and both penetration enhancers significantly increased the flux of tritiated water through all skins. Thus, DDAA and Azone significantly increased the permeation of lipophilic and hydrophilic model compounds. Rabbit pinna skin was a poor model for human skin in vitro, while snakeskin was much closer to human skin in terms of transdermal permeability. In most cases drug permeability decreased in the order rabbit human > or < snake.     

Tryptophan–NAD+ pathway metabolites as putative biomarkers and predictors of peroxisome proliferation

The present study was designed to provide further information about the relevance of raised urinary levels of N-methylnicotinamide (NMN), and/or its metabolites N-methyl-4-pyridone-3-carboxamide (4PY) and N-methyl-2-pyridone-3-carboxamide (2PY), to peroxisome proliferation by dosing rats with known peroxisome proliferator-activated receptor (PPAR) ligands [fenofibrate, diethylhexylphthalate (DEHP) and long-chain fatty acids (LCFA)] and other compounds believed to modulate lipid metabolism via PPAR-independent mechanisms (simvastatin, hydrazine and chlorpromazine). Urinary NMN was correlated with standard markers of peroxisome proliferation and serum lipid parameters with the aim of establishing whether urinary NMN could be used as a biomarker for peroxisome proliferation in the rat. Data from this study were also used to validate a previously constructed multivariate statistical model of peroxisome proliferation (PP) in the rat. The predictive model, based on ¹H nuclear magnetic resonance (NMR) spectroscopy of urine, uses spectral patterns of NMN, 4PY and other endogenous metabolites to predict hepatocellular peroxisome count. Each treatment induced pharmacological (serum lipid) effects characteristic of their class, but only fenofibrate, DEHP and simvastatin increased peroxisome number and raised urinary NMN, 2PY and 4PY, with simvastatin having only a transient effect on the latter. These compounds also reduced mRNA expression for aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase, EC 4.1.1.45), the enzyme believed to be involved in modulating the flux of tryptophan through this pathway, with decreasing order of potency, fenofibrate (–10.39-fold) >DEHP (–3.09-fold) >simvastatin (–1.84-fold). Of the other treatments, only LCFA influenced mRNA expression of ACMSDase (–3.62-fold reduction) and quinolinate phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) (–2.42-fold) without any change in urinary NMN excretion. Although there were no correlations between urinary NMN concentration and serum lipid parameters, NMN did correlate with peroxisome count (r²=0.63) and acyl-CoA oxidase activity (r²=0.61). These correlations were biased by the large response to fenofibrate compared to the other treatments; nevertheless the data do indicate a relationship between the tryptophan–NAD⁺ pathway and PPAR-dependent pathways, making this metabolite a potentially useful biomarker to detect PP. In order to strengthen the observed link between the metabolites associated with the tryptophan–NAD⁺ pathway and more accurately predict PP, other urinary metabolites were included in a predictive statistical model. This statistical model was found to predict the observed PP in 26/27 instances using a pre-determined threshold of 2-fold mean control peroxisome count. The model also predicted a time-dependent increase in peroxisome count for the fenofibrate group, which is important when considering the use of such modelling to predict the onset and progression of PP prior to its observation in samples taken at autopsy.     

A novel compound N,N-(Z)-N-(E)-tri-p-coumaroylspermidine isolated from Carthamus tinctorius L. and acting by serotonin transporter inhibition

Safflower, the dry flower of Carthamus tinctorius L., has long been applied for empirically treating cerebral ischemia and depression in traditional Chinese medicine. Pathogenesis of major depression involves monoaminergic transmission. The present study assessed whether safflower or its isolate would be effective in functionally regulating monoamine transporter using in vitro screening cell lines. We discovered that safflower insoluble fraction significantly inhibited serotonin uptake in Chinese hamster ovary cells stably expressing serotonin transporter (i.e. S6 cells). This fraction went through an activity-guided isolation and an active ingredient was obtained, which was subsequently elucidated as a novel coumaroylspermidine analog N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine using NMR techniques. Pharmacologically, this compound potently and selectively inhibited serotonin uptake in S6 cells or in synaptosomes, with IC₅₀ of 0.74 ± 0.15 µM for S6 cells or 1.07 ± 0.23 µM for synaptosomes and with a reversible competitive property for the 5HT-uptake inhibition. The potency of it for 5HT uptake was weaker than that of fluoxetine whereas efficacy generally similar for both. Animals treated with this testing compound showed a significant decrease in synaptosomal 5HT uptake capacity. Thus, N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine is a novel serotonin transporter inhibitor, which could improve neuropsychological disorders through regulating serotoninergic transmission.     

Metabolic activation of aromatic and heterocyclicN-hydroxyarylamines by wild-type and mutant recombinant human NAT1 and NAT2 acetyltransferases

Recombinant human NAT1 and polymorphic NAT2 wild-type and mutantN-acetyltransferases (encoded byNAT2 alleles with mutations at 282/857, 191, 282/590, 341/803, 341/481/803, and 341/481) were expressed inEscherichia coli strains XA90 and/or JM105, and tested for their capacity to catalyze the metabolic activation (viaO-acetylation) of theN-hydroxy (N-OH) derivatives of 2-aminofluorene (AF), and the heterocyclic arylamine mutagens 2-amino-3-methylimidazo [4,5-f]quinoline (IQ), 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Both NAT1 and NAT2 (including all mutant human NAT2s tested) catalyzed the metabolic activation of each of theN-hydroxyarylamines to products that bound to DNA. Metabolic activation of N-OH-AF was greater than that of the heterocyclicN-hydroxyarylamines. The relative capacity of NAT1 versus NAT2 to catalyze activation varied withN-hydroxyarylamine substrate. N-OH-MeIQx and N-OH-PhIP exhibited a relative specificity for NAT2. These results provide mechanistic support for a role of the genetic acetylation polymorphism in the metabolic activation of heterocyclic amine mutagens and carcinogens.     

温敏性吲哚美辛/β-环糊精包合物的制备及体外评价

目的合成兼具温敏性及药物包合能力的新型药物载体聚(N-异丙基丙烯酰胺)-β-环糊精(PNIPA-β-CD)，以吲哚美辛为模型药物，考察该载体的释药行为。方法末端带羧基的PNIPA与改性后的环糊精衍生物在1-(3-二甲氨基丙基)-3-乙基-碳二亚胺(EDC)的作用下缩合，得到PNIPA-β-CD，采用冻干法制备吲哚美辛/PNIPA-β-CD包合物。红外、¹H NMR和DSC表征载体的结构及包合物的形成；用分光光度法测定载体材料的LCST，并进行包合物体外释药研究。结果PNIPA-β-CD在35 ℃发生相转变，吲哚美辛/PNIPA-β-CD包合物的载药量为5.8％，药物与载体摩尔比为0.97∶1。体外释放研究表明吲哚美辛/PNIPA-β-CD包合物在37 ℃的释药比其在25 ℃的释放要慢，在LCST以上具有一定的缓释作用。结论该载体既具有温敏性，又具有药物包合作用，并且在体温条件下具有缓释作用，是一种新型的温敏性药物载体。