广西肝癌高发区不同水源微囊藻毒素含量调查

目的 了解广西肝癌高发区扶绥县饮用水水体微囊藻毒素(microcystin,MC)污染状况。方法 采集扶绥县11个自然村水源水样及28个自然村的末梢水水样,同时记录采样时水温及pH值。采用酶联免疫吸附法测定水样中的MC含量。结果 (1)水源水和末梢水之间温度和pH值差异无统计学意义(P>0.05)。(2)水源水和末梢水MC平均浓度分别为(15.64±2.08) ng/L、(14.42±2.28) ng/L,两者差异无统计学意义(P>0.05)。水源水中河水MC浓度最高,为16.69 ng/L,其他依次为地下河水和井水,各种水源水间MC浓度差异无统计学意义(P>0.05)。末梢水中MC浓度从高到低依次为水库水、井水、地下河水和河水,来源不同的末梢水间MC浓度差异无统计学意义(P>0.05)。(3)经水厂处理和未经水厂处理的末梢水水样MC平均浓度分别为(13.90±2.62) ng/L、(14.71±2.09) ng/L,两者差异亦无统计学意义(P>0.05)。结论 广西肝癌高发区扶绥县饮用水MC浓度均未超过WHO推荐的安全限值,不同水源水间的MC浓度无明显差异。     

癌症患者尿多胺高效液相色谱法测定

1971年Russell等报道癌症患者尿中多胺排量增多。此后,许多作者作了类似报道并认为测定尿多胺可作为诊断和判断肿瘤治疗效果的化学指示。目前高效液相色谱(HPLC)仪已逐渐普及,国内曾报道用国产填料对多胺标准品得到满意分离结果。我们应用此法测定癌症患者的尿多胺有明显增多,可为临床诊断癌症和疗效观察提供一种工具。     

反相高效液相色谱法测定人体血浆中冬凌草甲素含量

目的建立血浆中冬凌草甲素的RP-HPLC测定方法,为深入研究冬凌草的人体药代动力学特征提供有效和可靠的检测手段。方法采用甲醇水作为流动相,C-18反相色谱柱,乙醚作为萃取剂,检测血浆中冬凌草甲素含量,并评价方法的稳定性和重现性。结果RP—HPLC法测定乙醚萃取的血浆中冬凌草甲索保留时问稳定,标准曲线线性良好,结果准确、可靠。结论采用RP—HPLC方法检测乙醚萃取的血浆中的冬凌草甲素,可作为研究冬凌草甲素人体药代动力学的可靠检测手段。     

反相高效液相色谱法测定人血浆中消癌平注射液的绿原酸浓度

 目的　建立人血浆中消癌平高效液相色谱（HPLC）测定法。方法　采用HPLC法，色谱柱为C18柱（250 mm×4.6 mm，5 μm），流动相为乙腈-0.4 %磷酸溶液（13∶87），流速为1.0 ml／min，柱温为室温，检测波长327 nm。结果　绿原酸在浓度2.5～60.0 mg／L范围内呈良好的线性关系（r＝0.9996），最低检测浓度为0.25 mg／L。方法回收率为100.1 %～102.6 %，提取回收率为78.6 %～80.3 %。日内、日间相对标准偏差值（RSD）均小于3.8 %。结论　反相高效液相色谱法快速、简便、准确，可用于人血浆中消癌平注射液中绿原酸含量测定。     

高效液相色谱法测定人造靶点导向的吡柔比星在肿瘤组织中的浓度

[目的]制备小鼠抗人IgG-葡聚糖-盐酸吡柔比星免疫靶向偶联物，在肿瘤组织中接种人IgG非特异性靶点，探讨人造靶点免疫导向治疗肿瘤的可行性。[方法]制备小鼠抗人IgG-葡聚糖-盐酸吡柔比星（THP）偶联物，建立荷瘤小鼠模型。采用EUSA法测定小鼠抗人IgG-葡聚糖-盐酸毗柔比星偶联物抗体活性；体外细胞毒性实验（四甲基偶氮唑蓝法）比较分析小鼠抗人IgG、游离盐酸吡柔比星、小鼠抗人IgG-葡聚糖-盐酸吡柔比星对S180细胞的毒性作用；采用高效液相法（HPLC）测定肿瘤组织中盐酸吡柔比星药物浓度。[结果]制备的偶联物中，小鼠抗人IgG、葡聚糖与盐酸毗柔比星物质的量比为1：2．5：54；偶联物保留了小鼠抗人IgG抗体活性；体外细胞毒性实验证明偶联物对S180细胞有体外杀伤作用。对荷S180肉瘤小鼠进行体内导向治疗试验，高效液相色谱法测定小鼠抗人IgG-葡聚糖-盐酸吡柔比星＋人IgG组中肿瘤组织含盐酸吡柔比星含量较盐酸吡柔比星组增加（P〈0．01）。[结论]通过在肿瘤组织中接种IgG非特异性靶点，用抗人IgG单克隆免疫导向吡柔比星治疗小鼠S180肉瘤具有一定的可行性。     

假尿嘧啶核苷的高效液相色谱法测定及其与原发性肝癌的关系

应用高效液相色谱法（ＨＰＬＣ）测定了８０例原发性肝癌、３１例肝良性占位病变、４２例肝硬化、３８例健康志愿者血、尿中假尿嘧啶核苷（ＰＤ）的含量。结果显示，假尿嘧啶核苷的含量在原发性肝癌组升高显著；原发性肝癌ＰＤ阳性率７１．３％（５７／８０）；肝良性占位病变和健康组均无阳性病例；肝硬化组ＰＤ阳性者２例。对于原发性肝癌的诊断及预后判断有较好的指导作用。     

反相高效液相色谱法测定肿瘤患者血清中的二氢尿嘧啶和尿嘧啶

目的 建立肿瘤患者血清中二氢尿嘧啶和尿嘧啶浓度测定方法.方法 应用反相高效液相色谱法,流动相为磷酸盐缓冲溶液(含0.48%三乙胺),流速0.7 mL/min,检测波长206 nm.结果 二氢尿嘧啶最低检测限8μg/L,尿嘧啶最低检测限为5μg/L.血清中二氢尿嘧啶和尿嘧啶质量浓度为15.63～500μg/L时,与相应值线性关系良好(r≥0.9959).血清中二氢尿嘧啶和尿嘧啶回收率分别为74.56%～88.67%和83.95%～69.54%.结论 该方法精密度高、简单、快捷.     

采用高效液相色谱法检测余甘子中的没食子酸

目的：采用高效液相色谱法测定余甘子中没食子酸的含量,优化色谱条件,比较3个来源余甘子中没食子酸的含量差异。方法：分别对来自云南大理（2个批号）和福建漳州的余甘子中没食子酸进行含量测定,色谱条件为十八烷基硅烷键合硅胶色谱柱（粒径25μm,柱直径4.6 mm,柱长250 mm）;流动相乙腈-0.2%磷酸水溶液（5：95）;等度洗脱;流速1.000 mL/min;检测波长273nm;柱温30℃,标准曲线外标法。结果：没食子酸在13.010～130.104μg/mL范围内与峰面积有明显线性关系（R²=0.9994）;精密度良好[相对标准偏差（RSD）=0.08%];稳定性良好（RSD=0.29%）;重现性一般（RSD=4.23%）。对3批来自不同产地批号的余甘子药材进行了没食子酸含量评价,结果显示3个批次（福建20150304、云南20150205、云南20150304）的余甘子中没食子酸含量分别为2.60%、3.38%和2.82%,3个批次的余甘子中没食子酸含量均高于《中国药典》国家标准1.2%。结论：本方法测定余甘子中的没食子酸含量专属性强、精密度好、结果准确可靠。     

Novel method for DNA methylation analysis using high‐performance liquid chromatography and its clinical application

The aim of this study was to develop a new methodology that is suitable for DNA methylation diagnostics and to demonstrate its clinical applicability. We developed a new anion‐exchange column for high‐performance liquid chromatography (HPLC) with electrostatic and hydrophobic properties. Both cytosine and thymine, corresponding to methylated and unmethylated cytosine after bisulfite modification, respectively, are captured by electrostatic interaction and then discriminated from each other by their hydrophobic interactions. The DNA methylation levels of synthetic DNA were quantified accurately and reproducibly within 10 minutes without time‐consuming pretreatment of PCR products, and the measured values were unaffected by the distribution of methylated CpG within the synthetic DNA fragments. When the DNA methylation status of the FAM150A gene, a marker of the CpG island methylator phenotype specific to clear cell renal cell carcinoma (ccRCC), was examined in 98 patients with ccRCC, bulk specimens of tumorous tissue including cancer cells showing DNA methylation of the FAM150A gene were easily identifiable by simply viewing the differentiated chromatograms, even when the cancer cell content was low. Sixteen ccRCC showing DNA methylation more frequently exhibited clinicopathological parameters reflecting tumor aggressiveness (ie, a larger diameter, higher histological grade, vascular involvement, renal vein tumor thrombi, infiltrating growth, tumor necrosis, renal pelvis invasion and higher pathological TNM stage), and had significantly lower recurrence‐free and overall survival rates. These data indicate that HPLC analysis using this newly developed anion‐exchange column could be a powerful tool for DNA methylation diagnostics, including prognostication of patients with cancers, in a clinical setting.     

Comparison and evaluation of a novel bioassay and high‐performance liquid chromatography for the clinical measurement of serum voriconazole concentrations

The aim of this study was to develop and validate a novel bioassay for determining serum voriconazole (VRC) concentrations and to compare its routine clinical performance with that of high‐performance liquid chromatography (HPLC). The biological activity of VRC was measured by a plate diffusion assay using a VRC‐hypersusceptible Candida kefyr strain. The bioassay’s utility was tested by measuring steady‐state VRC concentrations in 100 serum probes from VRC‐treated patients. The HPLC system used solvent extraction with hexane : dichloromethane followed by reversed‐phase HPLC with ultraviolet detection. The intra‐day and inter‐day accuracy of the bioassay was <5%, while that of HPLC was <1%. The precision (mean coefficient of variation, 3.5%) was equal for both the methods. The limit of quantification was lower for HPLC (0.2 mg l^?1) than for the bioassay (0.5 mg l^?1). The result of linear regression analysis was HPLC = 1.0178 (bioassay) + 0.328; R² = 0.88; n = 100. Results of the serum panel ranged from 0.5 to more than 8.0 mg l^?1 for the bioassay and from 0.26 to 10.1 mg l^?1 for HPLC. Especially in laboratories without access to HPLC, the bioassay may be a clinically useful tool for therapeutic drug monitoring.     

Rapid determination of methylated purines in DNA treated with N-methyl-N-nitrosourea using high-performance liquid chromatography.

A method for determining methylated purine bases in [3H]N-methyl-N-nitrosourea (MeNOUr) treated DNA is described. The method combines reversed-phase high performance liquid chromatography (HPLC) of methylated DNA after hydrolysis in dilute acid with the determination of radioactivity in the fractionated eluates. The peaks of the respective methylated purines were indentified by the use of internal standards. The method allows quantitative separation of 3-methyl-adenine (m3Ade), 7-methyl-adenine (m7Ade), 3-methyl-guanine (m3Gua), 7-methyl-guanine (m7Gua) and O6-methyl-guanine (m6Gua) within 20 min. Thus the total time required for determination of methylated purines is limited only by radioactivity measurements in the respective fractions.     

Epidermal growth factor receptor mutations detected by denaturing high‐performance liquid chromatography in nonsmall cell lung cancer

BACKGROUND:

Somatic mutations in the epidermal growth factor receptor (EGFR) kinase domain are associated with sensitivity to EGFR‐tyrosine kinase inhibitors (EGFR‐TKI) in patients with nonsmall cell lung cancer (NSCLC).

METHODS:

The authors tested the possibility that nucleotide sequencing may be poorly suited for detection of mutations in tumor samples and found that denaturing high‐performance liquid chromatography (dHPLC) was an efficient and more sensitive method for screening.

RESULTS:

These results suggested that some reports based on standard DNA sequencing techniques may have underestimated mutation rates. In the present report, the authors examined the relationship between the presence and type of EGFR mutations detected by dHPLC and various clinicopathologic features of NSCLC, including response to therapy with EGFR‐TKI. Among 251 patients with advanced disease, 100 individuals received EGFR‐TKI. Those whose tumors harbored a detectable EGFR kinase mutation were much more likely to have a partial response (PR) or stable disease (SD) with EGFR‐TKI therapy than patients whose tumor contained no mutation (80% vs 35%; P = .001). Among the individual genotype subgroups, the frequency of a PR or SD was significantly different between patients with an exon 19 deletion compared with those with no detectable mutation (86% vs 35%; P < .001). Furthermore, patients whose tumors expressed an exon 19 mutant EGFR isoform exhibited a trend toward better EGFR‐TKI response (86% vs 67%; P = .171) and improved survival compared with patients whose tumors expressed an exon 21 mutation.

CONCLUSIONS:

Our findings warrant confirmation in large prospective trials and exploration of the biological mechanisms of the differences between mutation types. Cancer 2010. © 2010 American Cancer Society.     

Measurements of glutathione and other thiols in cells and tissues: a simplified procedure based on the HPLC separation of monobromobimane derivatives of thiols

Although there is intense interest in the role of thiols in controlling the efficiency of radiosensitizers, and in developing thiols (or pro-drugs liberating thiols) as radioprotectors, there is little information regarding the concentration of specific thiols in cells, tumors and normal tissues. Details are presented of a modified procedure using the thiol binding agent monobromobimane with separation using paired-ion reverse phase high performance liquid chromatography (HPLC). This method has been extended to include measurements of the radiosensitizer misonidazole and its desmethylated metabolite Ro 05-9963 in tissues.     

变性高效液相色谱技术检测非小细胞肺癌患者表皮生长因子受体基因突变的研究

 目的　探讨采用变性高效液相色谱（DHPLC）技术检测表皮生长因子受体（EGFR）基因突变的优势。方法　应用DHPLC技术检测49例非小细胞肺癌（NSCLC）患者EGFR基因第19与21外显子突变情况,并应用DNA直接测序法验证DHPLC检测基因突变的准确性。结果　49例NSCLC患者中,应用DHPLC检测出13例EGFR基因突变;其中第19外显子缺失突变10例（76.92 %）;第21外显子替代突变3例（23.08 %）。DNA直接测序法突变检测结果与DHPLC一致,DHPLC检测EGFR基因突变灵敏度为100 %。结论　DHPLC技术可以快速、准确、大规模筛选EGFR基因突变。     

32P-postlabeling analysis of DNA adducts from non-alternant PAH using thin-layer and high performance liquid chromatography

DNA adduct formation in vivo in mouse skin following a single topical application of benzo[a]fluoranthene (BbF), benzo[j]fluoranthene (BjF), benzo[k]fluoranthene (BkF), or indeno[1,2,3-cd]pyrene (IP) was investigated in female CD-1 mice using 32P-postlabeling analysis. Distinct adduct profiles were detected for each of the non-alternant hydrocarbons examined. Two adducts, one major and one minor, were detected using polyethyleneiminecellulose (PEI-cellulose) thin-layer chromatography (TLC) for BbF and BjF while a single major adduct was detected for BkF and IP. The relative extent of binding to mouse skin DNA was in the order BbF greater than BjF greater than BkF greater than IP. 32P-Postlabeled DNA adducts separated by PEI-cellulose TLC were further analyzed by high performance liquid chromatography (HPLC). A single radioactive peak was detected for 32P-labeled DNA adducts of BjF and BkF. Three general areas of radioactivity were detected when 32P-labeled DNA adducts of BbF were separated on HPLC.