HPLC法测定复方醋酸泼尼松氯霉素散中两组分的含量

目的建立高效液相色谱法同时测定复方醋酸泼尼松氯霉素散中醋酸泼尼松和氯霉素的含量。方法迪马ODSC18柱(4.6mm×250mm,5μm)色谱柱,以甲醇-水(60:40)为流动相,检测波长238nm,流速1.0ml·min-1,柱温30℃,进样量为20μl。结果醋酸泼尼松在2.5～15μg·ml-1范围内与吸收峰面积呈良好线性关系(r=0.9999,n=6),氯霉素在0.2～1.4mg·ml-1范围内与吸收峰面积呈良好线性关系(r=0.9999,n=6);平均回收率:氯霉素为99.5%,RSD=1.0%(n=9);醋酸泼尼松为1 0 0.9%,RSD=0.2%(n=9)。     

HPLC测定氯霉素滴眼液中氯霉素及二醇物的含量

目的采用RP-HPLC测定氯霉素滴眼液中氯霉素及二醇物的含量。方法采用Kromasil-C18柱(4.6 mm×150 mm,5μm),以乙腈-水-冰醋酸(40∶60∶0.5)为流动相,流速1.0 ml.min-1,检测波长272 nm。结果氯霉素的线性范围为8.0~128μg.ml-1(r=0.9998),二醇物的线性范围为2.5~40.0μg.ml-1(r=0.9998),平均回收率为98.88%,RSD=0.61%(n=6).结论该法分离效果好,灵敏度高、重复性好、结果准确可靠。     

美妮达搽剂的定量方法的研究

目的制定美妮达搽剂的质量标准,采用反相高效液相色谱法测定美妮达搽剂中的有效成分甲硝唑、氯霉素的含量,同时对氯霉素降解的二醇物进行含量控制。方法选用Inertsustain C_(18)柱(4.6mm×250 mm,5μm),以甲醇(A)-0.08%冰醋酸(B)系统梯度洗脱,洗脱条件为:0~13 min(25%A),13~18 min(25%A→80%A),18~25 min(80%A),25~30 min(80%A→25%A);检测波长为271nm,柱温35℃,流速:1.0 mL·min~(-1)。结果美妮达搽剂中甲硝唑、氯霉素以及氯霉素二醇物3种成分分离良好,甲硝唑、氯霉素检测浓度分别在38.1~304.8μg·mL~(-1)(r=1)、88.05~704.4μg·mL~(-1)(r=1)与峰面积呈良好的线性关系,甲硝唑低﹑中﹑高浓度的加样回收率在101.1%~102.4%(RSD<2.0%),氯霉素低、中﹑高浓度的加样回收率在98.6%~100.9%(RSD<2.0%);氯霉素二醇物浓度在3.065~24.52μg·mL~(-1)(r=0.9999)与峰面积呈良好的线性关系,低﹑中﹑高浓度的加样回收率在96.0%~96.8%(RSD<1.0%),检测限为0.25 ng。结论该法同时检测美妮达搽剂中甲硝唑、氯霉素以及氯霉素二醇物的含量,操作简单,准确可靠,可用于该制剂的质量控制。     

HPLC法测定复方氯霉素酊中氯霉素的含量

目的:建立复方氯霉素酊中氯霉素含量的测定方法。方法:采用Hypersil C18 ODS柱(200 mm×4.6 mm,5μm);流动相为:0.1%庚烷磺酸钠的溶液(取0.1%庚烷磺酸钠溶液500 ml与二甲基甲酰胺5 ml,冰醋酸0.5 ml,混匀)-乙腈(75:25);流速为1.0 ml·min-1;检测波长272 nm。结果:氯霉素在22.44～224.4μg·ml-1范围内线性关系良好,平均回收率为99.7%(n =6),RSD=0.5%。结论:该方法简便,准确快速,可测定复方氯霉素酊中氯霉素的含量。     

HPLC同时测定复方氯霉素乳膏中氯霉素和醋酸地塞米松的含量

目的:建立高效液相色谱法测定复方氯霉素乳膏(新复霜)中氯霉素和醋酸地塞米松的含量。方法:用十八烷基硅烷键合硅胶为填充剂[Symmetry C_(18)柱(4.6 mm×150 mm,5 μm)];以甲醇-水(64∶36)为流动相,流速为0.5 mL·min~(-1);检测波长为240 nm;进样量5 μL。结果:氯霉素及醋酸地塞米松在231.3～809.5μg·mL~(-1)(r=0.9999)和14.1～84.6μg·mL~(-1)(r=0.9999)范围内呈良好的线性关系;氯霉素和醋酸地塞米松精密度 RSD 分别为0.16%和0.22%;平均回收率(n=9)分别为100.1%和99.55%。结论:本方法快速、简便,重现性好,结果可靠,可作为复方氯霉素乳膏的质量控制标准。     

梯度洗脱HPLC同时测定鼻腔洗剂Ⅱ号中3种有效成分含量

目的建立高效液相色谱法同时测定鼻腔洗剂Ⅱ号中甲硝唑、氯霉素和氢化可的松含量.方法采用高效液相色谱法.Intersil C18分析色谱柱(4.6mm×250mm,5μm),流动相为8%乙腈-乙腈梯度洗脱,流速1.6mL·min-1,检测波长245nm.结果甲硝唑、氯霉素和氢化可的松的理论板数分别为16000,12000,5000;回归方程分别为Y=-7.769+3.734×10-6×(r=0.9995),Y=13.97+4.111×10-6×(r=0.9999),Y=8.433+1.046×10-6×(r=0.9999);线性范围分别为52.00～416.0μg·ml-1,131.8～659.0μg·ml-1,21.40～107.0μg·ml-1;平均回收率(±RSD)分别为99.2%(±2.6%),100.4(±1.1%),100.3%(±0.85%);最低检出浓度分别约为0.6,0.6,0.3μg·ml-1.结论用高效液相色谱法同时测定鼻腔洗剂Ⅱ号中甲硝唑、氯霉素和氢化可的松含量,操作简便,结果准确.     

HPLC法测定氯霉素地塞米松搽剂的含量

目的:建立HPLC法测定氯霉素地塞米松搽剂中氯霉素和醋酸地塞米松的含量。方法:色谱柱为C18柱(4.6mm×250mm,5μm),流动相为甲醇-水(68∶32),流速为1.0ml·min-1,检测波长为240nm。结果:氯霉素在400~1200μg·ml-1(r=1.000)范围内线性关系良好,平均回收率为100.1%(n=9),RSD为0.32%;醋酸地塞米松在10~30μg·ml-1(r=1.000)范围内线性关系良好,平均回收率为100.8%(n=9),RSD为0.71%。结论:该方法简便、准确、快速,可同时测定两组分的含量。     

HPLC法同时测定复方氯霉素硼酸酊中氯霉素及水杨酸的含量

目的建立高效液相色谱法（HPLC法）同时测定复方氯霉素硼酸酊中氯霉素及水杨酸含量的方法。方法采用Eclipse XDB-C18（5μm,4.6 mm×150 mm）色谱柱;甲醇-0.5%乙酸铵（60：40）为流动相;检测波长为300 nm;柱温：30℃。结果水杨酸在0.042-0.168 mg/ml浓度范围内,峰面积与浓度呈良好的线性关系,氯霉素在0.031-0.125 mg/ml浓度范围内,峰面积与浓度呈良好的线性关系,回归方程分别为：水杨酸：Y=13475 X-21.411（r=0.9997）;氯霉素：Y=8819.1 X-5.0643（r=0.9997）。结论此法专属性高、耐用性强、操作简便,适合作为测定复方氯霉素硼酸酊中氯霉素及水杨酸含量的分析方法。     

奥沙西泮原料及其片剂中有关物质的检查

目的:建立 HPLC 法同时测定奥沙西泮原料及片剂中有关物质的方法。方法:采用 C_(18)柱(150 mm×4.6 mm,5μm),以0.05 mol·L~(-1)磷酸二氢铵-甲醇(45:55,用三乙胺调 pH 8.0)为流动相,流速1.0 mL·min~(-1),检测波长为230 nm。结果:奥沙西泮峰及各杂质峰均能良好分离。杂质 A 浓度在0.092～9.2μg·mL~(-1)范围内与峰面积呈良好的线性关系,回归方程为 Y=67.9X 1.2,r=0.9999,最低检测限为0.13 ng,奥沙西泮中回收率为106.2%,RSD=2.1%(n=6),片剂中回收率为104.2%,RSD 为1.9%(n=6);杂质 B 浓度在0.106～10.6μg·mL~(-1)范围内与峰面积呈良好的线性关系,回归方程为 Y=51.4X-5.6,r=0.9999,最低检测限为0.31 ng,在奥沙西泮中回收率为97.9%,RSD=1.5%(n=6),片剂中回收率为101.5%,RSD 为1.2%(n=6);杂质 C 浓度在0.1～10.0μg·mL~(-1)范围内与峰面积呈良好的线性关系,回归方程为 Y=77.4X 0.28,r=0.9998,最低检测限为0.28 ng,奥沙西泮中回收率为113.1%,RSD=2.5%(n=6),片剂中回收率为110.5%,RSD为2.3%(n=6)。结论:该方法简便、灵敏、专属性好,可用于奥沙西泮原料和制剂中有关物质的检查。     

HPLC法测定复方滴眼液中氯霉素和盐酸丁卡因的含量

目的建立高效液相色谱法测定复方滴眼液中氯霉素和盐酸丁卡因含量的方法。方法色谱柱为C18柱(250mm×4.6mm;5μm),流动相为0.01mol·L-1醋酸铵(pH3.5)-甲醇(40:60),检测波长为290nm。结果氯霉素和盐酸丁卡因分别在0.075～0.225mg·ml-1和0.05～0.15mg·ml-1浓度范围内呈良好的线性关系,回归方程分别为C=47.6A+198.6,r=1.000和C=38.4A+232.8,r=1.000;平均回收率分别为100.4%(n=9);平均回收率为99.5%(n=9);精密度实验RSD均为0.1%,重现性试验RSD分别为0.31%和0.53%,结论本法简单、准确、重现性良好,可同时测定氯霉素盐酸丁卡因滴眼液中的氯霉素和盐酸丁卡因。     

Differential effects of the organochlorine pesticide DDT and its metabolite p,p'-DDE on p-glycoprotein activity and expression

1,1-Bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) is an organochlorine pesticide. Its metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethene (p,p'-DDE) is a persistent environmental contaminant and both compounds accumulate in animals. Because multidrug resistance transporters, such as p-glycoprotein, function as a defense against xenobiotic exposure, we analyzed the ability of DDT and p,p'-DDE to act as efflux modulators. Using a competitive intact cell assay based on the efflux of the fluorescent dye rhodamine 123, we found that DDT, but not p,p'-DDE, stimulated dye retention. Subsequent studies using verapamil as competitor suggested that DDT is a weak p-glycoprotein inhibitor. Further studies addressed the ability of DDT and p,p'-DDE to induce MDR1, the gene encoding p-glycoprotein. In HepG2 cells, we found that both compounds induced MDR1 by twofold to threefold. Similar results were observed in mouse liver after a single dose of p,p'-DDE, although some gender-specific induction differences were noted. By contrast, p,p'-DDE failed to induce MDR1 in HeLa cells, indicating some cell-specific effects for induction. Further expression studies demonstrated increased levels of the endoplasmic reticulum molecular chaperone, Bip, in response to DDT, but not p,p'-DDE. These results suggest that DDT, but not p,p'-DDE, induces an endoplasmic reticulum stress response.     

国产对乙酰氨基酚红光物质与国外样品的质量对比

本文论述了醋酐工艺、醋酸工艺、对氨基酚和活性炭,对对乙酰氨基酚质量的影响,由酰化反应动力学探讨了副产物的生成机理和对乙酰氨基酚带红光的来源,研究了控制产品中残留物、红光物质的测定方法,提高国产对乙酰氨基酚的内在质量标准,醋酸工艺的棕色粗品经脱红光专用炭精制后达到国外同类产品实样标准.     

Thenoyltrifluoroacetone, a potent inhibitor of carboxylesterase activity

Thenoyltrifluoroacetone (TTFA), a conventional mitochondrial complex II inhibitor, was found to inhibit purified porcine liver carboxylesterase non-competitively with a K(i) of 0.61x10(-6)M and an IC(50) of 0.54x10(-6)M. Both rat plasma and liver mitochondrial esterases were inhibited in a concentration-dependent fashion. Results indicate that TTFA is a potent inhibitor of carboxylesterase activity, in addition to its ability to inhibit mitochondrial complex II activity. Therefore, caution is warranted in using TTFA as a mitochondrial complex inhibitor in combination with esterase substrates, such as fluorescence probes or vitamin E esters.     

Inhibitory effects of halothane on the thermogenic pathway in brown adipocytes: localization to adenylyl cyclase and mitochondrial fatty acid oxidation

Volatile anesthetics such as halothane efficiently inhibit nonshivering thermogenesis as well as the cellular manifestation of that phenomenon: norepinephrine-induced respiration in brown adipocytes. To identify the molecular site(s) of action of such anesthetics, we have examined the effect of halothane on the sequential intracellular steps from the interaction of norepinephrine with isolated brown adipocytes to the stimulation of mitochondrial respiration (=thermogenesis). We did not identify an inhibition at the level of the adrenergic receptors, but a first site of inhibition was identified as the generation of cAMP by adenylyl cyclase; this led to inhibition of norepinephrine-induced expression of the uncoupling protein-1 (UCP1) gene and reduced norepinephrine-induced lipolysis as secondary effects. Although an inhibition of lipolysis in itself would inhibit thermogenesis, circumvention of this inhibition revealed that a second, postlipolytic, site of inhibition existed: halothane also inhibited the stimulatory effect of exogenous fatty acids on cellular respiration. This inhibition was independent of the presence of UCP1 in the mitochondria of the cells and was thus not directly on the thermogenic uncoupling mechanism. Since not only fatty acid oxidation but also pyruvate oxidation were inhibited by halothane in isolated mitochondria, whereas glycerol-3-phosphate oxidation was not, the second site of action of halothane, evident when cyclase/lipolytic inhibition was circumvented, was located to the respiratory chain, complex I. The results thus explain the inhibition of nonshivering thermogenesis by identifying two sites of action of halothane in brown adipocytes. In addition, the results may open for new formulations of the molecular background to anesthesia.     

Patulin causes DNA damage leading to cell cycle arrest and apoptosis through modulation of Bax, p and p proteins in skin of mice

Patulin (PAT), a mycotoxin found in apples, grapes, oranges, pear and peaches, is a potent genotoxic compound. WHO has highlighted the need for the study of cutaneous toxicity of PAT as manual labour is employed during pre and post harvest stages, thereby causing direct exposure to skin. In the present study cutaneous toxicity of PAT was evaluated following topical application to Swiss Albino mice. Dermal exposure of PAT, to mice for 4 h resulted in a dose (40-160 μg/animal) and time (up to 6 h) dependent enhancement of ornithine decarboxylase (ODC), a marker enzyme of cell proliferation. The ODC activity was found to be normal after 12 and 24 h treatment of patulin. Topical application of PAT (160 μg/100 μl acetone) for 24-72 h caused (a) DNA damage in skin cells showing significant increase (34-63%) in olive tail moment, a parameter of Comet assay (b) significant G 1 and S-phase arrest along with induction of apoptosis (2.8-10 folds) as shown by annexin V and PI staining assay through flow cytometer. Moreover PAT leads to over expression of p^21/WAF1 (3.6-3.9 fold), pro apoptotic protein Bax (1.3-2.6) and tumor suppressor wild type p⁵³ (2.8-3.9 fold) protein. It was also shown that PAT induced apoptosis was mediated through mitochondrial intrinsic pathway as revealed through the release of cytochrome C protein in cytosol leading to enhancement of caspase-3 activity in skin cells of mice. These results suggest that PAT has a potential to induce DNA damage leading to p⁵³ mediated cell cycle arrest along with intrinsic pathway mediated apoptosis that may also be correlated with enhanced polyamine production as evident by induction of ODC activity, which may have dermal toxicological implications.     

Elemental alteration, iron overloading and metallothionein induction in experimental hepatocarcinogenesis: a free radical-mediated process?

The azo-dye p-dimethylaminoazobenzene (p-DAB) is a potential tumor initiator in rodents, but the underlying mechanism is not clear. Following chronic feeding of the carcinogen for 3, 5 and 7 weeks, trace elemental status, free radical generation, oxidative damage, antioxidant profile were measured in male and female swiss albino mice. The feeding resulted in iron accumulation in male mice liver. No increase in iron level was observed in similarly exposed female mice. The results of this study suggest that p-DAB-induced iron accumulation in male mice with concomitant production of oxidative free radicals is an early event in the hepatocarcinogenic initiation. This occurs selectively in male mice and affects either directly or indirectly in development of chemically induced liver neoplasia. Again, that upregulation of metallothionein (MT) expression in association with increased free radical generation was demonstrated in male mice. Alteration of copper (Cu) and zinc (Zn) levels are described in the light of antioxidant profile in liver tissue. The current results thus provide evidence in support of iron accumulations producing oxidative damage, and enhanced metallothionein expression as possible contributors in the mode of action of p-DAB induced hepatocarcinogenesis.     

Cyp1a1(-/-) male mice: protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria

To study liver toxicity and uroporphyrin (URO) accumulation and urinary excretion, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent ligand for the aryl hydrocarbon receptor (AHR), is often used as the prototype. In this study, we asked the question how important is the role of CYP1A1 in causing TCDD toxicity. Using a single large intraperitoneal dose of TCDD (200 microg/kg) and following the response over an 8-week period, we found this dose: (a) was lethal in less than 4 weeks to Cyp1a1(+/+) males but not to Cyp1a1(-/-) males or to females of either genotype; (b) caused a wasting syndrome in Cyp1a1(+/+) but not Cyp1a1(-/-) mice; (c) resulted in thymic atrophy, regardless of gender or genotype; (d) decreased spleen size and caused leukocytopenia in males but not females of either genotype; (e) caused hepatocyte hypertrophy in Cyp1a1(+/+) more so than in Cyp1a1(-/-) mice; (f) increased intrahepatocyte lipids and total liver fat content in Cyp1a1(+/+) more than Cyp1a1(-/-) males and females; and (g) caused uroporphyria in Cyp1a1(+/+) males much more than Cyp1a1(+/+) females, or in Cyp1a1(-/-) mice. Contrary to Cyp1a2(-/-) knockout mice that exhibited 15 times less accumulation of TCDD in liver than Cyp1a1/1a2(+/+) wild-type mice, Cyp1a1(-/-) mice did not show this altered TCDD distribution-indicating that CYP1A2 but not CYP1A1 is the major hepatic TCDD-binding "sink". Our data demonstrate that CYP1A1 contributes to high-dose TCDD-induced toxicity, uroporphyria, and lethality.     

Restored expression and activity of organic ion transporters rOAT1, rOAT3 and rOCT2 after hyperuricemia in the rat kidney

We previously reported that in hyperuricemic rats, renal impairment occurred and organic ion transport activity decreased, accompanied with a specific decrease in the expression of rat organic anion transporters, rOAT1 and rOAT3, and organic cation transporter, rOCT2. In the present study, we investigated the reversibility of the organic ion transport activity and expression of organic ion transporters (slc22a) during recovery from hyperuricemia. Hyperuricemia was induced by the administration of a chow containing uric acid and oxonic acid, an inhibitor of uric acid metabolism. Four days after discontinuance of the chow, the plasma uric acid concentration returned to the normal level, and renal functions such as creatinine clearance and BUN levels were restored, although the recovery of tubulointerstitial injury was varied in sites of the kidney. Basolateral uptake of p-aminohippurate (PAH) and tetraethylammonium (TEA), and both protein and mRNA levels of rOAT1, rOAT3 and rOCT2 in the kidney gradually improved during 14 days of recovery from hyperuricemia. Basolateral PAH transport showed a higher correlation with the protein level of rOAT1 (r(2)=0.80) than rOAT3 (r(2)=0.34), whereas basolateral TEA transport showed a strong correlation with rOCT2 protein (r(2)=0.91). The plasma testosterone concentration, which is a dominant factor in the regulation of rOCT2, was gradually restored during the recovery from hyperuricemia, but the correlation between the plasma testosterone level and rOCT2 protein expression in the kidney was not significant. These results suggest that the regulation of organic ion transporters, rOAT1, rOAT3 and rOCT2, by hyperuricemia is reversible, and the organic ion transport activity restores according to the expression levels of these transporters.     

Time-dependent effect of p-aminophenol (PAP) toxicity in renal slices and development of oxidative stress

p-Aminophenol (PAP), a metabolite of acetaminophen, is nephrotoxic. This study investigated PAP-mediated changes as a function of time that occur prior to loss of membrane integrity. Experiments further evaluated the development of oxidative stress by PAP. Renal slices from male Fischer 344 (F344) rats (N = 4-6) were exposed to 0.1, 0.25, and 0.5 mM PAP for 15-120 min under oxygen and constant shaking at 37 degrees C. Pyruvate-stimulated gluconeogenesis, adenine nucleotide levels, and total glutathione (GSH) levels were diminished in a concentration- and time-dependent manner prior to detection of a rise in lactate dehydrogenase (LDH) leakage. Glutathione disulfide (GSSG) levels were increased by PAP suggesting the induction of oxidative stress. Western blot analysis confirmed a rise in 4-hydroxynonenal (4-HNE)-adducted proteins in tissues exposed to 0.1 and 0.25 mM PAP for 90 min. The appearance of 4-HNE-adducted proteins at the 0.1 mM concentration of PAP occurred prior to development of increased LDH leakage. Pretreatment with 1 mM glutathione (GSH) for 30 min only partially reduced PAP toxicity as LDH values were less severely depleted relative to tissues not pretreated with GSH. In contrast, pretreatment for 15 min with 2 mM ascorbic acid completely protected against PAP toxicity. Further studies showed that ascorbic acid pretreatment prevented PAP-mediated depletion of GSH. In summary, PAP rapidly depletes GSH and adenine nucleotides and inhibits gluconeogenesis prior to a rise in LDH leakage. PAP induces oxidative stress as indicated by an increase in GSSG and 4-HNE-adducted proteins. Ascorbic acid pretreatment prevents PAP toxicity by maintaining GSH status.     

Mechanism of action and determination of the best substrate for a thrombin-like enzyme from Lachesis muta muta venom by regression analysis of the kinetic parameters determined with peptidyl p-nitroanilide substrates.

The kinetic behavior of a thrombin-like enzyme from Lachesis muta muta venom has been studied with 13 tripeptidyl p-nitroanilide substrates. Eight substrates were unprotected at the N terminus and were used for the regression analysis of the experimentally determined kinetic parameters 1/Km, kcat and kcat/Km. The individual contribution of each amino acid side chain to the kinetic parameters was calculated. The amino acid sequence of the ideal substrate (D-Pro-Leu-Arg-pNA) was determined from a regression analysis for each kinetic parameter. This result was confirmed experimentally. The structural analysis of the tripeptides showed that the binding to the S3 sub-site had a small effect on Km. The binding of L-Leu to the S2 sub-site increased kcat without changing the value of Km. The analysis of the kinetic parameters revealed that, in the binding of L-Leu to the S2 sub-site, the enzyme bound the transition state configuration of the substrate/product transformation more tightly than that of the substrate.