HPLC测定薄膜衣片剂中食用色淀的含量

目的 建立薄膜衣片剂中食用色淀的HPLC含量测定新方法.方法 高效液相色谱梯度洗脱法.色谱柱:Alltima-C18 (4.6 mm×250 mm,5 μm);流动相:甲醇(A)-0.05 mol·L-1醋酸铵水溶液(B)梯度洗脱;检测波长:254 nm;流速:1.0 mL· min-1;进样量为20 μL.结果 柠檬黄色素和日落黄色素分别在10.03～200.6 μg·mL-1和10.01～200.2 μg·mL-1范围内呈良好线性关系(r≥0.999 9),色淀总的平均回收率为96.7%,RSD=1.26%.结论 所建立的方法操作简便、灵敏度高、重复性好,为薄膜衣片剂中食用色淀含量提供有效的质量控制,建立了新的检测方法.     

山橿药材中主要有效成分含量的研究

目的:利用RP-HPLC梯度洗脱的方法对山橿药材中主要有效成分进行了研究,为科学评价与有效控制山橿药材质量提供了方法。方法:依利特Hypersil ODS柱(250 mm×4.6 mm,5μm),以甲醇-水(60%~100%)非线形梯度洗脱,体积流1.0 ml/min,柱温30℃,在296 nm波长下进行检测。测定10批次山橿药材中银松素、乔松素和球松素的含量。结果:采用非线性梯度洗脱的方法可以同时控制3种主要有效成分的含量,方法学考察表明该方法具有较好的精密度、重复性和稳定性。结论:采用的梯度洗脱方法测定有效成分的含量可以作为山橿质量评价的重要依据。     

高效液相色谱法测定万古霉素注射液中的含量

目的:探讨高效液相色谱法测定万古霉素注射液中的万古霉素含量。方法:应用高效液相色谱法同时测定3份万古霉素注射液中的样品中万古霉素的含量,并分析比较了测定条件。结果:万古霉素在214nm处有最大吸收,最佳色谱条件为:色谱柱:硅胶ALOTMCl8色谱柱;流动相:A相为15mmol/L乙酸铵;B相为乙腈;流速为1.0ml/min;梯度洗脱;柱温:30℃,进样量:20μl。结论:高效液相色谱法测定万古霉素注射液中的万古霉素,操作简便迅速,精密度和准确度均较理想,稳定性强,回收含量高,可为测定中万古霉素注射液中万古霉素物质提供方法依据。     

HPLC梯度洗脱法测定对乙酰氨基酚口服混悬液中有关物质

目的：采用高效液相色谱法（HPLC）梯度洗脱测定对乙酰氨基酚口服混悬液中有关物质含量。方法：用Inertsustain C18（250mm×4.6mm,5μm）为色谱柱;以0.05mol·L-1醋酸铵溶液和甲醇为流动相进行梯度洗脱;检测波长为245nm,流速为1.0mL·min-1,柱温为25℃。结果：各峰间能完全分离;对乙酰氨基酚和对氨基酚的最低检测浓度分别为0.027μg·mL-1和0.093μg·mL-1;对乙酰氨基酚在0.037～0.33μg·mL-1范围内峰面积与浓度呈良好的线性关系,对氨基酚在0.26～3.09μg·mL-1范围内峰面积与浓度呈良好的线性关系;中间精密度试验对氨基酚RSD为13.77%。结论：该方法专属性好、灵敏、结果准确,可用于对乙酰氨基酚口服混悬液的有关物质含量控制。     

HPLC法测定人血浆中比阿培南的浓度及其药动学研究

目的：建立人血浆中比阿培南的高效液相色谱检测方法。方法：色谱柱为Agilent ZORBAX Bonus-RP（4.6 mm×250 mm,5μm）;流动相为甲醇和0.03%醋酸水溶液,采用梯度洗脱法;流速为1.00 ml/min;检测波长为300 nm,血浆样品处理选用5-羟基吲哚乙酸为内标,采用硫酸锌沉淀蛋白。结果：比阿培南在0.2～50.0μg/ml范围内线性良好,HPLC测定定量下限为0.2μg/ml,比阿培南和内标在血浆的保留时间分别为3.9 min和9.7 min,出峰迅速。比阿培南浓度分别为0.5、5.0和40.0μg/ml,血浆样本回收率分别为101.8%、100.8%、100.4%;精密度试验日内和日间RSD均小于15%,稳定性试验结果表明血浆样品在样品处理过程中较稳定（RSD〈15%）。结论：本方法灵敏度高,操作简便,快速,重复性好,适用于比阿培南血浆浓度的测定。     

梯度洗脱联合波长切换HPLC法同时测定豨莶风湿丸中的豨莶苷、奇壬醇、豨莶精醇、防己诺林碱和粉防己碱

目的 建立梯度洗脱联合波长切换HPLC法同时测定稀莶风湿丸中稀莶苷、奇壬醇、稀莶精醇、防己诺林碱和粉防己碱.方法 采用Kromasil C18色谱柱(4.6 mm× 250 mm,5μm),以流动相A:乙腈-甲醇(2∶1)、流动相B:0.05％冰醋酸溶液进行梯度洗脱;流速为1.0 mL/min;样量为20 μL;豨莶苷、奇壬醇和稀莶精醇检测波长为215 nm,防己诺林碱和粉防己碱检测波长为280 nm.结果 豨莶苷、奇壬醇、稀莶精醇、防己诺林碱和粉防己碱在给定浓度范围内的线性范围分别为6.45～129.00 μg/mL(r=0.999 5)、5.61～112.20 μg/mL(r=-0.999 9)、4.25～85.00 μg/mL(r=0.999 3)、9.19～183.80 μg/mL(r=0.999 8)和11.05～221.00 μg/mL(r=0.999 7),线性关系良好;平均加样回收率和相对标准偏差(RSD)值分别为98.73％(1.43％)、97.63％(1.28％)、99.44％(1.29％)、98.33％(1.38％)、97.36％ (1.37％).结论 建立的梯度洗脱联合波长切换HPLC法能同时测定豨莶风湿丸中稀莶苷、奇壬醇、稀莶精醇、防己诺林碱和粉防己碱含量,方法简便、准确、重现性好,为稀莶风湿丸质量控制提供了可靠方法.     

EDTA alkaline elution characteristics and measurement of DNA damage in unlabeled DNA using Hoechst 33258 fluorescence

Hoechst 33258 fluorescence of single stranded DNA has been used to perform alkaline elution with unlabeled DNA. The high background fluorescence of "standard" elution solutions has prompted others to use EDTA but the elution characteristics of DNA in EDTA-containing solutions and the comparability of results with those using "standard" tetrapropyl ammonium hydroxide solutions have not previously been examined. We report here the elution characteristics of DNA in EDTA and the relevant parameters for the successful use of EDTA as an elution solution. An increase in elution pH to 12.4 is required but elution solutions of higher pH cause alkaline hydrolysis of undamaged DNA. Drug-treated DNA from which DNA-protein crosslinks have been removed can be completely removed from the filters at the end of the elution by a Pronase filter digestion. The simplest and most efficient removal of DNA-protein crosslinks is through the inclusion of proteinase-K in an SDS containing lysis solution. EDTA elution can measure interstrand crosslinks and single strand breaks as easily as is performed using radiolabeled DNA under "standard" elution conditions and requires only 1.5-2 x 10(6) cells per elution filter. DNA-protein crosslinking measurements were unsatisfactory, however, since even the Pronase digestion failed to completely remove protein-crosslinked DNA from the elution filters.     

CYP1A1 activation of aminoflavone leads to DNA damage in human tumor cell lines

Purpose: Aminoflavone (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one; AF; NSC 686288) is a novel anticancer agent with a unique pattern of growth inhibitory activity in the National Cancer Institute (NCI) 60 tumor cell line screen. Phase I clinical trials with AF will begin soon. We previously demonstrated extensive metabolism of AF by cytochrome P450 (CYP) 1A1 and CYP1A2, metabolic activation, formation of irreversible protein and DNA adducts and p53 stabilization in sensitive, but not resistant, human tumor cell lines treated with AF [9]. The present studies focus on the effects of AF on cellular DNA and cellular responses to DNA damage. Methods: Phosphorylation of H2AX in MCF7 cells treated with AF was determined with immunofluorescence. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) assays were used to determine the effect of cotreatment with caffeine or wortmannin, inhibitors of ataxia-telangiectasia-mutated protein (ATM) and ATR (ATM- and rad3-related protein), on the AF IC₅₀ values for MCF7 cells. DNA damage in MCF7 cells treated with AF was determined by alkaline elution. DNA-topoisomerase complex stabilization was ascertained by the ICE (in vitro complex of enzyme) assay. Results: Treatment of sensitive cells with AF resulted in phosphorylation of H2AX, a histone 2A variant that is phosphorylated in response to DNA damage. AF IC₅₀ values for MCF7 cells were lowered by cotreatment with caffeine or wortmannin, further implicating DNA damage in AF cytotoxicity. There was no evidence of DNA–DNA cross-linking in sensitive cells, but protein-associated single-strand breaks were observed after AF treatment. Although this pattern of DNA damage is commonly associated with topoisomerase poisons, there was no evidence for AF-induced stabilization of either topoisomerase I- or II-DNA complexes. Conclusions: These studies further implicate DNA damage in the cytotoxicity of AF and identify biochemical features of that damage including formation of protein-associated single-strand breaks not involving topoisomerase I or II.     

Elution of antitransglutaminase antibodies from duodenal biopsies: a novel approach in the diagnosis of celiac disease

Celiac disease (CeD) is a disease more prevalent and multisymptomatic than was earlier recognized. Whereas prompt initiation of gluten-free diet (GFD) is beneficial in relieving the symptoms, an accurate CeD diagnosis is necessary also to avoid years of restricted diet on uncertain grounds. We propose a new diagnostic method, based on elution of deposited antibodies against transglutaminase (anti-tTG) from duodenal biopsies in patients with symptoms and screening serology analyses suggestive of CeD. The eluates were analyzed in a Phadia 250 fluoroimmunoassay, demonstrating elevated concentrations of anti-tTG in CeD patients, corresponding to serology and histopathology findings. In one case histology was inconclusive, displaying only unspecific inflammation, but eluted anti-tTG was positive. This patient has clinically improved following GFD. We conclude that our novel method represents a new tool in the diagnostic work up in CeD. The detection of deposited anti-tTG at the site of inflammation appears to provide a high sensitivity and specificity using a technique that is quick, simple and reliable. Further studies are needed for optimization and elucidation of suitable applications for this elution method.     

梯度淋洗离子色谱法测定大气颗粒物PM2.5中四种水溶性阴离子

目的:建立梯度淋洗离子色谱法测定大气颗粒物PM2.5中的F-、Cl-、SO42-和NO3-。方法:以IonPacAS18阴离子分离柱分离,KOH自动淋洗装置设定梯度淋洗程序,流速为1.0 ml/min,电导检测器检测。结果:方法线性关系良好,相关系数均大于0.9995,检出限分别为:F-0.005 mg/L、Cl-0.005 mg/L、SO24-0.010 mg/L、NO3-0.003 mg/L。F-的相对标准偏差小于10%,其他三种离子相对标准偏差均小于5%。结论:该方法线性范围宽,重现性好,快速准确,适用于大气颗粒物中F-、Cl-、SO42-和NO3-的检测。