光谱法在药物分析中应用与研究
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摘要
药物分析学是整个药学领域中的重要组成部分,药品质量的优劣,直接关系到人们的身体健康甚至生命安全,必须对药品质量进行全面控制,从而确保人们用药的安全、合理和有效。
目前,药物分析的趋势是要能够简便、快速地从组成复杂的样品中灵敏、可靠地检测痕量药物成分。适应这一要求,光谱法因其设备简单、操作方便、灵敏度高、准确度好以及适用范围广成了药物分析学科领域中最主要和最基本的研究手段和方法之一。
本文以光谱法作为检测手段,对药物的有效成分、药物中的重金属元素进行测定,探寻药物的更灵敏、更简单的检测方法,同时也对药物与蛋白质、氨基酸的相
互作用进行研究,主要研究内容包括:
1、混合微乳液增敏-流动注射法测定微量铅提出了混合微乳液增敏-流动注射法测定微量铅的新方法。在聚乙二醇辛基苯基醚(TritonX-100)/正庚烷/正丁醇/水—聚乙二醇-1000(PG-1000)混合微乳液介质中,Pb~(2+)、KI、结晶紫于流动注射体系中生成蓝色铅离子缔合物,于540nm测定该离子缔合物的吸光度。试验结果表明:在优化条件下,Pb 2+在0.0～800.0 ng/mL范围内符合比尔定律,检出限为13.2 ng/mL(S/N=3),检测速度为10/h,同时考察了共存离子的干扰并做了回收试验,本法用于测定中药葶苈子中的铅含量,结果满意。
2、流动注射—褪色光度法测定药物中Vc
建立了流动注射-褪色光度法测定药物中Vc含量的方法。在盐酸介质中维生素C (Vc)能使Fe~(3+)与SCN~-红色络合物褪色,且络合物吸光度减小与Vc用量成线性关系,于流动注射体系测定褪色前后络合物吸光度,测定波长为480 nm,流速为6.7mL/min。结果发现在优化条件下,Vc浓度在0.0- 6.4μg/mL范围内与吸光度符合朗伯-比耳定律,线性方程为?A= -0.00406+0.08347c(c:μg/mL),r=0.9995(S/N=3),检出限为0.18μg/mL,流通量为72/h。使用本法测定了Vc药片、注射液及银翘片中的Vc,结果与药典法一致。本法快速准确,可用于Vc的批量分析。
3、荧光探针法测药物及体液中加替沙星
提出了钇敏化荧光法测定药物中加替沙星(GFLX)含量的新方法,并对影响加替沙星荧光性质的各种因素进行了研究。在pH 7.0的缓冲溶液中,钇敏化的加替沙星溶液产生最强荧光,加替沙星浓度在4.0×10~(-8)~1.0×10~(-6)g/mL范围内与荧光强度呈良好的线性关系,线性方程为F = -1.26827+129.76556 C(μg/mL),r=0.9997,检出限为3.36×10~(-9) g/mL(S/N=3)。使用本法测定了加替沙星胶囊、注射液,并与高效液相色谱法比较,结果令人满意。
4、ICP-AES与GFAAS法测定中草药中铅、铜、镉、铬
采用石墨炉原子吸收分光光度法(GFAAS)与电感耦合等离子体原子发射光谱法(ICP-AES)测定了25种中药材中重金属铅、铜、镉、铬的含量,并对基体改进剂进行了探讨。实验结果表明两种测定方法结果基本一致,适用于中药材中这4种重金属含量的测定。
5、光谱法研究爱普列特与牛血清白蛋白的相互作用及应用
用光谱法研究了生理pH条件下爱普列特(EP)与牛血清白蛋白(BSA)结合反应的特征,发现爱普列特对BSA有较强的荧光猝灭作用。根据荧光猝灭数据获得了结合反应的结合常数、结合位点数和结合过程的基本热力学参数,并探讨了荧光猝灭机理。在实验条件下发现BSA的荧光强度猝灭程度与爱普列特的浓度在0.0-40.0μg/mL范围内有良好的关系,由此建立了测定爱普列特含量的荧光分析新方法。
6、荧光光谱法研究盐酸托烷司琼与L-色氨酸的相互作用及其应用用荧光光谱法研究了盐酸托烷司琼对L-色氨酸的荧光猝灭效应,结果发现在pH=9.0的氨-氯化铵介质中,盐酸托烷司琼对L-色氨酸的荧光猝灭作用最强,在此基础上建立了色氨酸荧光猝灭法测定盐酸托烷司琼含量的新方法并对荧光猝灭机理进行了探讨。实验发现当盐酸托烷司琼浓度在0.0-12.0μg/mL范围内时色氨酸荧光强度猝灭程度与盐酸托烷司琼浓度呈良好的线性关系,线性方程为ΔF=6.17+12.56C(μg/mL),r=0.997,检出限为0.04μg/mL(S/N=3)。使用本法测定了盐酸托烷司琼注射液,结果令人满意。
The analysis of druggery is one of the most important components in the whole pharmacology category. The quantity of the druggery directly affects people’s life health and safety. Therefore the whole control of druggery plays an important role in the clinic and medical treatment.
Nowdays, the trend of pharmaceutical analysis is to determine simply and reliably the trace medicine ingredient of complex samples. Therefore, Spectrophotometry has become one of the most essential study methods in the determination of pharmic products, due to its easy apparatus, simple procedure, highly sensitivity and selectivity.
The aim of this dissertation was to explore simpler, more sensitive determination methods of medication, with using spectrophotometry as research means. Meanwhile the interaction of drugs with serum albumin or with amino acid was also examined. The major contents are described as follows:
1. A Sensitive Method for trace Lead by Flow Injection Analysis with Mixed Microemulsion
In this paper, a new flow injection system in association with mixed microemulsion for the microanalysis of lead was proposed. In the mixed microemulsion system of TritonX-100/n-heptane/n-butanol/water-polyethyleneGlycol-1000, a blue ion association complex produced from the reaction between lead, potassium iodide and crystal violet in the flow stream, and the spectrophotometric detection was performed simultaneously at 540 nm. The results showed that under the optimum conditions, the linear range and detection limit was 0.0-800.0 ng/mL (r=0.9980) and 13.2 ng/mL respectively. The sample throughput was 10/h. Foreign substrates effection was also investigated. The proposed method has been successfully applied to the determination of lead in the lepidium apetalum willd.
2. Determination of Vc Using Discoloring Spectrophotometry in Flow Injection System
A method for determination of vitamine C (Vc) by FIA- discoloring spectrophotometry was established. In the medium of HCl, a new FIA-discoloring spectrophotometry for the determination of Vc was proposed, based on the discoloring effect of Vc on the reaction between Fe~(3+)and SCN~- and the absorbance drop of red complex of Fe~(3+)and SCN~- was proportional to the amount of Vc. The detection wavelength was 480 nm and flow rate was 6.7 mL/min The results showed that under the optimum conditions, the linear range and detection limit was 0.0-6.4μg/mL (r=0.9995) and 0.18μg/mL, respectively. The sample throughput was 72/h. The proposed method has been successfully applied to determine Vc in the Vc tablet, Vc injection and Cimetidine tablet. The method is simple, accurate and very suitable to determine Vc in batches.
3. Fluorescence Probe Enhanced spectrofluorimetry for the Determination of
Gatifloxacin in Pharmaceutical Formulations and Biological Fluids A spectrofluorimetry for the determination of gatifloxacin (GFLX) was developed, based on the strong fluorescence of gatifloxacin after adding fluorescence probe yttrium in buffer solution (pH=7.0). Various factors of influencing fluorescence have been researched. Under the optimum conditions, the liner range was 4.00×10~(-8) to 1.00×10~(-6) g/mL and the detection limit was 3.36×10~(-9) g/mL (correlation coefficient r=0.9997), respectively. The relative standard deviation was 1.1% for eleven measurements of 5.6×10~(-7) g/mL gatifloxacin standard solution. The mechanism of sensitizing effect of probe was discussed. The proposed method has been successfully applied to determine real samples and the obtained results were in good agreement with the results of HPLC.
4. Determination of Cd,Pb,Cu and Cr in Chinese Medicinal Herbs by ICP-AES and GFAAS
In this paper, a rapid method for the determination of Cd,Pb,Cu and Cr in 25 kinds of traditional Chinese medicinal herbs was developed by inductively coupled plasma atomic emission spectrometry (ICP-AES) and graphite furnace atomic absorption spectrometry(GFAAS). The influence of matrix modifier on determination was investigated. Under the optimal conditions, the recoveries of analyte were in the range of 95.0-108.0%. The results showed the two methods were in good agreement and the two methods could be used as routine techniques to analyze for these heavy metals.
5. Study of the Interaction between Epristeride and Bovine Serum Albumin by Spectroscopy and its Analytical Application
Under physiological condition (pH=7.4), the interaction between epristeride (EP) and bovine serum albumin (BSA) was studied by UV absorption and fluorescence spectroscopy. It was found that the fluorescence intensity of BSA was quenched strongly by EP through a static quenching procedure. The association constants, the number of binding sites and basic thermodynamic parameters were obtained based on fluorescence quenching data. On the basis of the relationship between the change of fluorescence intensity of BSA (?F) and the concentration EP, the proposed method had been successfully applied to determine EP in real samples and the obtained results were in good agreement with the results of official method, HPLC.
6. Study of the Interaction of Tropisetron Hydrochloride and L-Tryptophan by Spectrofluorimetry and its Analytical Application
A new method for the determination of tropisetron hydrochloride with L-Tryptophan in the medium pH 9.0 NH3-NH4Cl was studied, which based on the fluorescence quenching effect of tropisetron hydrochloride on L-Tryptophan. The fluorescence quenching mechanism and various factors of influencing fluorescence quenching were discussed. Under the optimum conditions, the liner range and detection limit was 0.03 to 12.0μg/mL and 0.04μg/mL (correlation coefficient r=0.997), respectively. The calibration curve equation was ?F=6.17+12.56 C (μg/mL). The proposed method had been successfully applied to determine tropisetron hydrochloride in real samples and the obtained results were in good agreement with the results of official method.
目前,药物分析的趋势是要能够简便、快速地从组成复杂的样品中灵敏、可靠地检测痕量药物成分。适应这一要求,光谱法因其设备简单、操作方便、灵敏度高、准确度好以及适用范围广成了药物分析学科领域中最主要和最基本的研究手段和方法之一。
本文以光谱法作为检测手段,对药物的有效成分、药物中的重金属元素进行测定,探寻药物的更灵敏、更简单的检测方法,同时也对药物与蛋白质、氨基酸的相
互作用进行研究,主要研究内容包括:
1、混合微乳液增敏-流动注射法测定微量铅提出了混合微乳液增敏-流动注射法测定微量铅的新方法。在聚乙二醇辛基苯基醚(TritonX-100)/正庚烷/正丁醇/水—聚乙二醇-1000(PG-1000)混合微乳液介质中,Pb~(2+)、KI、结晶紫于流动注射体系中生成蓝色铅离子缔合物,于540nm测定该离子缔合物的吸光度。试验结果表明:在优化条件下,Pb 2+在0.0～800.0 ng/mL范围内符合比尔定律,检出限为13.2 ng/mL(S/N=3),检测速度为10/h,同时考察了共存离子的干扰并做了回收试验,本法用于测定中药葶苈子中的铅含量,结果满意。
2、流动注射—褪色光度法测定药物中Vc
建立了流动注射-褪色光度法测定药物中Vc含量的方法。在盐酸介质中维生素C (Vc)能使Fe~(3+)与SCN~-红色络合物褪色,且络合物吸光度减小与Vc用量成线性关系,于流动注射体系测定褪色前后络合物吸光度,测定波长为480 nm,流速为6.7mL/min。结果发现在优化条件下,Vc浓度在0.0- 6.4μg/mL范围内与吸光度符合朗伯-比耳定律,线性方程为?A= -0.00406+0.08347c(c:μg/mL),r=0.9995(S/N=3),检出限为0.18μg/mL,流通量为72/h。使用本法测定了Vc药片、注射液及银翘片中的Vc,结果与药典法一致。本法快速准确,可用于Vc的批量分析。
3、荧光探针法测药物及体液中加替沙星
提出了钇敏化荧光法测定药物中加替沙星(GFLX)含量的新方法,并对影响加替沙星荧光性质的各种因素进行了研究。在pH 7.0的缓冲溶液中,钇敏化的加替沙星溶液产生最强荧光,加替沙星浓度在4.0×10~(-8)~1.0×10~(-6)g/mL范围内与荧光强度呈良好的线性关系,线性方程为F = -1.26827+129.76556 C(μg/mL),r=0.9997,检出限为3.36×10~(-9) g/mL(S/N=3)。使用本法测定了加替沙星胶囊、注射液,并与高效液相色谱法比较,结果令人满意。
4、ICP-AES与GFAAS法测定中草药中铅、铜、镉、铬
采用石墨炉原子吸收分光光度法(GFAAS)与电感耦合等离子体原子发射光谱法(ICP-AES)测定了25种中药材中重金属铅、铜、镉、铬的含量,并对基体改进剂进行了探讨。实验结果表明两种测定方法结果基本一致,适用于中药材中这4种重金属含量的测定。
5、光谱法研究爱普列特与牛血清白蛋白的相互作用及应用
用光谱法研究了生理pH条件下爱普列特(EP)与牛血清白蛋白(BSA)结合反应的特征,发现爱普列特对BSA有较强的荧光猝灭作用。根据荧光猝灭数据获得了结合反应的结合常数、结合位点数和结合过程的基本热力学参数,并探讨了荧光猝灭机理。在实验条件下发现BSA的荧光强度猝灭程度与爱普列特的浓度在0.0-40.0μg/mL范围内有良好的关系,由此建立了测定爱普列特含量的荧光分析新方法。
6、荧光光谱法研究盐酸托烷司琼与L-色氨酸的相互作用及其应用用荧光光谱法研究了盐酸托烷司琼对L-色氨酸的荧光猝灭效应,结果发现在pH=9.0的氨-氯化铵介质中,盐酸托烷司琼对L-色氨酸的荧光猝灭作用最强,在此基础上建立了色氨酸荧光猝灭法测定盐酸托烷司琼含量的新方法并对荧光猝灭机理进行了探讨。实验发现当盐酸托烷司琼浓度在0.0-12.0μg/mL范围内时色氨酸荧光强度猝灭程度与盐酸托烷司琼浓度呈良好的线性关系,线性方程为ΔF=6.17+12.56C(μg/mL),r=0.997,检出限为0.04μg/mL(S/N=3)。使用本法测定了盐酸托烷司琼注射液,结果令人满意。
The analysis of druggery is one of the most important components in the whole pharmacology category. The quantity of the druggery directly affects people’s life health and safety. Therefore the whole control of druggery plays an important role in the clinic and medical treatment.
Nowdays, the trend of pharmaceutical analysis is to determine simply and reliably the trace medicine ingredient of complex samples. Therefore, Spectrophotometry has become one of the most essential study methods in the determination of pharmic products, due to its easy apparatus, simple procedure, highly sensitivity and selectivity.
The aim of this dissertation was to explore simpler, more sensitive determination methods of medication, with using spectrophotometry as research means. Meanwhile the interaction of drugs with serum albumin or with amino acid was also examined. The major contents are described as follows:
1. A Sensitive Method for trace Lead by Flow Injection Analysis with Mixed Microemulsion
In this paper, a new flow injection system in association with mixed microemulsion for the microanalysis of lead was proposed. In the mixed microemulsion system of TritonX-100/n-heptane/n-butanol/water-polyethyleneGlycol-1000, a blue ion association complex produced from the reaction between lead, potassium iodide and crystal violet in the flow stream, and the spectrophotometric detection was performed simultaneously at 540 nm. The results showed that under the optimum conditions, the linear range and detection limit was 0.0-800.0 ng/mL (r=0.9980) and 13.2 ng/mL respectively. The sample throughput was 10/h. Foreign substrates effection was also investigated. The proposed method has been successfully applied to the determination of lead in the lepidium apetalum willd.
2. Determination of Vc Using Discoloring Spectrophotometry in Flow Injection System
A method for determination of vitamine C (Vc) by FIA- discoloring spectrophotometry was established. In the medium of HCl, a new FIA-discoloring spectrophotometry for the determination of Vc was proposed, based on the discoloring effect of Vc on the reaction between Fe~(3+)and SCN~- and the absorbance drop of red complex of Fe~(3+)and SCN~- was proportional to the amount of Vc. The detection wavelength was 480 nm and flow rate was 6.7 mL/min The results showed that under the optimum conditions, the linear range and detection limit was 0.0-6.4μg/mL (r=0.9995) and 0.18μg/mL, respectively. The sample throughput was 72/h. The proposed method has been successfully applied to determine Vc in the Vc tablet, Vc injection and Cimetidine tablet. The method is simple, accurate and very suitable to determine Vc in batches.
3. Fluorescence Probe Enhanced spectrofluorimetry for the Determination of
Gatifloxacin in Pharmaceutical Formulations and Biological Fluids A spectrofluorimetry for the determination of gatifloxacin (GFLX) was developed, based on the strong fluorescence of gatifloxacin after adding fluorescence probe yttrium in buffer solution (pH=7.0). Various factors of influencing fluorescence have been researched. Under the optimum conditions, the liner range was 4.00×10~(-8) to 1.00×10~(-6) g/mL and the detection limit was 3.36×10~(-9) g/mL (correlation coefficient r=0.9997), respectively. The relative standard deviation was 1.1% for eleven measurements of 5.6×10~(-7) g/mL gatifloxacin standard solution. The mechanism of sensitizing effect of probe was discussed. The proposed method has been successfully applied to determine real samples and the obtained results were in good agreement with the results of HPLC.
4. Determination of Cd,Pb,Cu and Cr in Chinese Medicinal Herbs by ICP-AES and GFAAS
In this paper, a rapid method for the determination of Cd,Pb,Cu and Cr in 25 kinds of traditional Chinese medicinal herbs was developed by inductively coupled plasma atomic emission spectrometry (ICP-AES) and graphite furnace atomic absorption spectrometry(GFAAS). The influence of matrix modifier on determination was investigated. Under the optimal conditions, the recoveries of analyte were in the range of 95.0-108.0%. The results showed the two methods were in good agreement and the two methods could be used as routine techniques to analyze for these heavy metals.
5. Study of the Interaction between Epristeride and Bovine Serum Albumin by Spectroscopy and its Analytical Application
Under physiological condition (pH=7.4), the interaction between epristeride (EP) and bovine serum albumin (BSA) was studied by UV absorption and fluorescence spectroscopy. It was found that the fluorescence intensity of BSA was quenched strongly by EP through a static quenching procedure. The association constants, the number of binding sites and basic thermodynamic parameters were obtained based on fluorescence quenching data. On the basis of the relationship between the change of fluorescence intensity of BSA (?F) and the concentration EP, the proposed method had been successfully applied to determine EP in real samples and the obtained results were in good agreement with the results of official method, HPLC.
6. Study of the Interaction of Tropisetron Hydrochloride and L-Tryptophan by Spectrofluorimetry and its Analytical Application
A new method for the determination of tropisetron hydrochloride with L-Tryptophan in the medium pH 9.0 NH3-NH4Cl was studied, which based on the fluorescence quenching effect of tropisetron hydrochloride on L-Tryptophan. The fluorescence quenching mechanism and various factors of influencing fluorescence quenching were discussed. Under the optimum conditions, the liner range and detection limit was 0.03 to 12.0μg/mL and 0.04μg/mL (correlation coefficient r=0.997), respectively. The calibration curve equation was ?F=6.17+12.56 C (μg/mL). The proposed method had been successfully applied to determine tropisetron hydrochloride in real samples and the obtained results were in good agreement with the results of official method.
引文
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