【作者】 李海霞；

【导师】 蒋晔；

【作者基本信息】 河北医科大学 ， 药物分析学， 2010， 硕士

【摘要】 “痕量组分”一般是指其样本含量小于万分之一的物质,如药物中的某些活性较强的成分、环境中药物或毒素污染物或者药物金属离子污染等,这些物质的浓度极低,但都有很强的生理活性,或者在体内后有很强的蓄积能力,进而产生对肌体的产生毒害作用,其一直是药物分析及分析化学家的研究的焦点。这些物质含量较低,采用常规的分析方法(如高效液相色谱法、紫外光谱法)检测困难。提高分析方法的灵敏度或开发高灵敏度的新的分析方法是解决这一问题的主要途径,然而,对于极其痕量的物质,尤其是某些pg级以下的物质,即使使用了高灵敏度的现代分析方法(如MS等),其检测分析仍然困难,因此,采用适当的样品前处理方法,提纯富集这些痕量样品,使其达到可分析的灵敏度水平,是现代分析技术解决这一问题的另一途径,也是现代分析技术的研究热点;这些新的样品前处理技术同时也为解决某些难监测物质(如样品MS检测离子化效率低)的分析检测提供了新的分析手段。本课题研究了磷钼杂多酸-罗丹明B络合物共振瑞利散射(RRS)体系,建立了更加灵敏的唑来膦酸分析方法,解决了唑来膦酸输注液中唑来膦酸检测困难的问题,考察了体系中硫酸浓度、过硫酸钾溶液浓度、钼酸铵溶液浓度、罗丹明B(RDB)溶液浓度、煮沸时间、反应温度及反应时间对RRS的影响,优化了分析条件,提高了检测灵敏度,其唑来膦酸的检测限达了1.55 ng/ml,成功应用于唑来膦酸输注液中唑来膦酸的测定。课题研究了石墨炉原子吸收光谱法分析药物注射剂中铝离子污染的检测中的背景和分析条件,通过塞曼背景校正和基底改进技术成功解决了样品背景的干扰问题,通过优化干燥、灰化、原子化温度和时间,提高了方法灵敏度,检出限达0.14 ng/ml,所建分析方法可不经样品消化处理直接进行分析,满足了唑来膦酸注射液铝离子污染的检测要求。课题采用新型的中空纤维液相微萃取技术,建立了环境水中痕量雌二醇的富集和纯化方法,解决了传统的液液萃取和固相萃取方式富集率低、有机溶剂使用量大、分析成本高以及交叉污染等难题,研究了萃取效率的影响因素,在优化的萃取条件下,环境水中痕量雌二醇的富集倍数可达300倍,采用HPLC-UV检测,其分析灵敏度可达0.1 ng/ml,该方法成功应用于环境水样中雌二醇的检测。一共振瑞利散射法测定唑来膦酸目的:利用共振瑞利散射技术(RRS),建立灵敏、快速、简便的唑来膦酸输注液中唑来膦酸的测定方法。方法:精密量取供试品1ml,加1%过硫酸钾煮沸破坏10min,在0.125 mol/L酸性条件下,依次与0.25 mg/ml钼酸铵和7×10-6 mol/L罗丹明B(RDB)反应,静置45 min。在λex=λem=370 nm,狭缝宽度5 nm的条件下,测定RRS强度,用同样的方法测定空白值强度,以共振散射信号的增加值(ΔI)计算样品浓度。结果:在优化的条件下,共振散射信号的增加值(ΔI)与唑来膦酸的浓度成正比。唑来膦酸在6.25~100 ng/ml的浓度范围内线性关系良好,最低检出限1.55 ng/ml,RSD小于2.1%,回收率为100~101%。磷酸盐、亚磷酸盐和其他常见共存物质(金属离子、非金属离子和无机酸根)等均不干扰测定。结论:所建立的共振瑞利散射光谱法分析测定唑来膦酸,方法简便快速,其灵敏度高达1.55 ng/ml,较传统钼蓝法提高了近500倍。该方法为唑来膦酸输注液提供新的分析手段,可用于唑来膦酸输注液的质量控制。二GF-AAS研究唑来膦酸注射剂中铝离子的污染目的:采用石墨炉原子吸收光谱法(GF-AAS),建立唑来膦酸注射剂中痕量铝离子的分析方法,用于考察唑来膦酸注射液中铝离子污染的来源和水平。方法:取样品溶液100μl,加入50μl 5 mg/ml基底改性剂硝酸镁,1%硝酸定稀释至2.5 ml,直接进样,注入石墨炉中。AAS分析采用热解涂层石墨管,铝空心阴极灯检测波长309.3 nm,灯电流10 mA,狭缝宽度0.4 nm,干燥温度80℃和220℃,灰化温度700℃和1200℃,原子化温度2600℃,采用塞曼偏振背景校正的原子吸收光谱法,标准曲线法测定唑来膦酸注射剂中铝的含量。结果:所建立的方法能有效降低唑来膦酸注射液基底效应。铝离子在4.00~20.0 ng/ml的浓度范围内线性关系良好,相关系数r为0.9995。检出限为0.14 ng/ml,加样回收率为99% ~104%,RSD为2.7% ~3.5%。唑来膦酸注射液中铝离子30天检测结果表明,随着放置时间的延长,铝离子浓度显著提高。结论:本文建立了塞曼-石墨炉原子吸收法分析唑来膦酸注射液中痕量铝离子,方法灵敏度高,其检出限达0.14 ng/ml,样品未经复杂的消化处理,直接进样,且加入基底改进剂能有效降低样品基底效应。唑来膦酸注射液稳定性试验结果表明,唑来膦酸注射液中铝离子主要来源于安瓿瓶。三中空纤维液相微萃取-高效液相色谱测定环境水样中雌二醇目的:以新型的中空纤维液相微萃取技术(HF-LPME)为富集和纯化手段,建立HPLC-UV法分析环境水中痕量雌二醇(E2)的方法。方法:采用聚偏氟乙烯中空纤维,以正辛醇为萃取溶剂,将正辛醇装入11 cm的中空纤维膜内,将中空纤维两端密封后首尾连接,放入140 ml水样中,于室温、搅拌速度为500 rpm条件下萃取60 min,萃取后,中空纤维用100μl甲醇冲洗中空纤维内壁,合并萃取物在90℃经氮气流吹干后用甲醇溶解,进样20μl,进行HPLC分析,采用KROMASIL C18色谱柱(150 mm×4.6 mm, 5μm),流动相为甲醇-水(75:25),检测波长280 nm,流速1 ml/min,柱温室温。结果:在优化的实验条件下,样品中雌二醇的富集因子可达300,雌二醇在1~1000 ng/ml的浓度范围内线性关系良好,相关系数r为0.9995,最低检出限为0.1 ng/ml。回收率为97.5% ~120.5%,RSD为4.7% ~7.3%。结论:本文建立的中空纤维液相微萃取方法较文献报道的液液萃取方法富集率高,达300倍,提高了分析灵敏度;与固相萃取、固相微萃取等相比,方法简单,结果准确可靠,分析成本低,有机溶剂使用量少,且不存在交叉污染,为痕量雌激素的分析提供了可靠的富集手段。更多还原

【Abstract】 "Trace Elements" generally refers to samples less than 0.01%, such as active ingredients in drugs, drugs or toxins pollutants in environment, metal ion contamination in drugs. The concentration of these substances is low, but the physiological activity is strong. In addition, high accumulation accured gives toxic effect to human body. These problems, people paid more and more attention, have become the focuses of their research. However, it is difficult to analyze trace substances with conventional analytical methods, such as high-performance liquid chromatography, UV spectroscopy. The main method to solve the problem is to improve the sensitivity of analytical methods or develop more sensitive methods. However, it is difficult to detect some of substances which are below pg level, even if sensitive method is performed (such as MS, etc.). Therefore, appropriate sample pre-treatment method to enrich trace substances is introduced to solve this problem. New sample pre-treatment techniques provide new analytical tools to analyze substances which are difficult to monitor (such as substances with low ionization efficiency by MS detection).In this project, a sensitive method has been developed to solve the difficulty in the detection of zoledronic acid. Phosphorusmolybdenum heteropoly acid-rhodamin B produced high RRS, and greatly improved the sensitivity. The detection limit was 1.55ng/ml. The parameters affecting the reaction such as sulfuric acid concentration, potassium persulfate concentration, ammonium molybdate concentration, Rhodamine B concentration, as well as the boiling time, reaction temperature, reaction time were investigated. The method was successfully applied for the determination of zoledronic acid infusion solution. A GF-AAS was established for the determination of aluminum in zoledronic acid injection, and the resource of aluminum in zoledronic acid injection was discussed. The background interference was eliminated by Zeeman background correction. The sensitivity of the method was improved by optimizing the drying, ashing, atomization temperature and time, as well as choice of base modifier. The detection limit was 0.14ng/ml. The method supports direct analysis without digestion and was successfully applied to explore the sources of aluminum in zoledronic acid injection. Another job established in our project is to show the application of Hollow fiber liquid-phase micro-extraction. Hollow fiber liquid-phase micro-extraction, a new enrichment and purified pre-treatment technique, has been applied for the analysis of trace substances in environmental water. Compared with the reported sample preparation techniques, the proposed method seems to be more preferred for its high enrichment, cost effectiveness, minimization of organic waste, and absence of memory effect. Meanwhile, it earned many merits such as extraction and concentration in one set, outstanding purification, no cross-contamination and suitable for various analytes in different matrix. Under optimizing conditions, the enrichment efficiency was 300 and the detection limit was 0.1ng/ml.PART 1 Determination of Trace Zoledronic Acid by Resonance Rayleigh Scattering MethodObjective: To establish a sensitive, fast and simple resonance Rayleigh scattering method for the determination of trace zoledronic acid.Methods: In acid medium, ammonium molybdate of 0.25 mg/ml and inorganic phosphorus which is produced from 1 ml sample solution reacting with KPs of 1% for 10 min, reacts with rhodamin B of 7×10-6 mol/L to form an associated complex of phosphorusmolybdenum heteropoly acid- rhodamin B. The RRS(I) of complex was detected atλex=λem=370 nm with slit width of 5 nm. The same procedure was carried out to measure the blank signal(I0). Sample concentration was calculated by the enhancement of RRS (ΔI= I﹣I0).Results: Under the optimized conditions, the enhancement of RRS (ΔI) is proportional to the concentration of zoledronic acid in solution. The linear relationship was obtained in the range of 6.25 to 100ng/ml. The limit of detection is 1.55 ng/ml with RSD 2.1% and the recovery was 100~101%. Phosphate, phosphate and other coexistence substance (metal ion and mineral acid radical ion) did not interfere the determination of zoledronic acid.Conclusion: A simple and rapid RRS method for the determination of zoledronic acid has been developed. Compared with traditional molybdenum blue spectrophotometry method, it is 500 times more sensitive with limit of detection at 1.55 ng/ml. It provides a new method for the determination of zoledronic acid. The method was successfully applied to the quality control of zoledronic acid infusion solution.PART 2 Pollution of Aluminium in Zoledronic Acid Injection by Graphite Furnace Atomic Absorption SpectrometryObjective: A graphite furnace atomic absorption spectrometry was established for the study of the source of aluminium and the pollution in zoledronic acid injection.Methods: Sample solution of 100μl, adding 50μl 5 mg/ml of magnesium nitrate, diluted to 2.5 ml with 1% nitric acid, and then injected directly into the graphite furnace. Pyrolysis coating graphite tube and aluminium hollow cathode lamp with wavelength of 309.3 nm and lamp current of 10 mA were employed. 80℃and 220℃for drying, 700℃and 1200℃for ashing, 2600℃for atomizing were optimized for temperature program. The aluminium ion in zoledronic acid injection was determined directly by graphite furnace atomic absorption spectrometry with polarized Zeeman background correction.Results: Matrix effect in zoledronic acid injection was cut down in this condition. There was a good linear relationship in the concentration range of 4.00~20.0 ng/ml (r = 0.9995). The detection limit was 0.14 ng/ml, the relative standard deviation were 99% ~104%, and the RSD were 2.7% ~3.5%. The result of zoledronic acid injection of 30 days showed that concentration of aluminium increased with increasing the staying time.Conclusion: A graphite furnace atomic absorption spectrometry was established for the determination of aluminium in zoledronic acid injection. The method was sensitive with the detection limit of 0.14ng/ml. The sample was injected directly instead of complex digestive process. Basal modifier added can reduce the effects of the sample substrate effectively. The result of zoledronic acid injection stability test showed that aluminum ions came from ampoule.PART 3 Enrichment and determination of trace estradiol in environmental water samples by hollow fiber liquid phase microextraction prior to high performance liquid chromatographyObjective: A novel and simple cleanup procedure, based on hollow-fiber liquid-phase microextraction (HF-LPME) is described here, and HPLC-UV was used for the determination of trace estradiol in environmental water samples.Methods: For HF-LPME, the polyvinylidene difluoride hollow fiber membrane was used. N-octyl alcohol was used as n-octyl alcohol. The n-octyl alcohol was injected into the fiber of 11 cm. Then, the two ends of the fiber were enveloped, and then were introduced into a 140ml glass bottle with screw cap. The extraction was performed by agitation at 500 rpm at 25℃for 60 min. After that, 100μl methanol was slowly flushed through the lumen. The whole elution solution was evaporated to dryness at 90℃under nitrogen gas. To the dried sample, 100μl methanol was added before HPLC analysis. The analysis was performed on a KROMASIL C18 column (150 mm×4.6 mm, 5μm), with the mobile phase of methanol-water (75:25) at 25℃and detected at 280nm using UV detector. The flow rate was 1 ml/min.Results: Under the optimized experimental conditions, the enrichment factor was 300. The proposed method was found to be linear in the concentration 1-1000ng/ml for estradiol and the limit of detection for an S/N = 3 was 0.1ng/ml. The recovery of esdradiol was 97.5% ~120.5% with RSD of 4.7% ~7.3%.Conclusion: A hollow-fiber liquid-phase microextraction has been established for the analysis of estradiol. It takes a serial of advantages over liquid-liquid extraction such as high sensitivity with high enrichment factor of 300. Compared with the reported sample preparation techniques, such as solid phase extraction and solid phase microextraction , the proposed method seems to be more preferred for its cost effectiveness, minimization of organic waste, absence of memory effect, accuraty and reliability. It provides a new reliable means for the analysis of estradiol.更多还原