高效液相色谱—共振瑞利散射检测技术及其应用研究
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摘要
高效液相色谱法(High performance liquid chromatography,HPLC)以分离效率高,分析速度快,灵敏度高和应用广泛的特点,已成为分析科学的一个重要分支;新型高效通用的检测技术的研发和联用一直是高效液相色谱法(HPLC)的主要发展方向之一。共振瑞利散射检测技术以其灵敏度高、方法简便而引起广泛关注,但这种方法对复杂样品的测定略显不足,如能将液相色谱的分离功能与共振散射检测相结合,将是一个极具潜在应用前景的分析技术。
目前,蛋白质和氨基糖苷类抗生素的分析方法很多,但大多数需要衍生,或用短紫外波段来检测,准确度和灵敏度都较低,因此,对它们的分离与测定,在生物医学研究和质检等相关领域均具有重要意义。
本文建立了高效液相色谱-共振瑞利散射联用检测技术,并对样品进行了测定。主要研究内容有以下四方面:
1、研究并建立了高效液相色谱-共振瑞利散射联用新技术。用阴离子表面活性剂十二烷基硫酸钠(Sodium dodecyl sulfate,SDS)为光谱探针,商品荧光检测器检测,以牛胰岛素(Insulin,Ins)、细胞色素C(Cytochrome,Cyt-c)、溶菌酶(Lysozyme,Lys)和人血清白蛋白(Human serum albumin HSA)为分离分析目标物,考察高效液相色谱-共振瑞利散射联用技术的可行性,并进行方法学研究,通过柱后离子缔合模型的建立,确定了该技术的整体方案。结果表明,此新方法有较高的灵敏度,操作简便,重复性好,显示出在高效液相色谱检测方法中具有较广阔的应用前景,为实际样品的测定建立了可靠的分析方法。
2、基于建立的高效液相色谱-共振瑞利散射联用技术,以比布列西猩红为光谱探针,分离测定四种蛋白质。三氟乙酸-乙腈混合液为流动相,采用梯度淋洗技术,用Sinochrom ODS-AP色谱柱(150 mm×4.6 mm, 300à, 5μm)分离,Cyt-c、Lys、HSA、免疫球蛋白(γ-globulin,γ-Glo)四种蛋白质在12 min内完全分离,通过其与比布列西猩红相互结合,形成较各自体积更大的离子缔合物,用共振瑞利散射技术进行检测,检测波长为λEx=λEm=376 nm。Cyt-c,Lys,HSA,γ-Glo浓度分别在0.20～3.0μg·mL-1,0.25～2.5μg·mL-1,1.5～10μg·mL-1,2.0～15μg·mL-1时,与峰面积呈良好线性关系,线性相关系数都在0.99以上。当信噪比为3(S/N=3)时,四种蛋白质的检测限在0.2～1.0μg·mL-1范围内。并应用于对人血清中白蛋白和球蛋白的含量测定。
3、以滂胺天蓝作为光谱探针,采用高效液相色谱-共振瑞利散射联用技术,建立了测定妥布霉素的新方法。此方法用diamond ODS色谱柱分离,等度洗脱,在优化了分离条件后,研究了缓冲溶液的pH,反应管长,探针流速和探针的浓度等对灵敏度的影响。在选定的最佳分离条件和最优的结合条件下,妥布霉素在10 min内得到良好的峰形,将方法应用于妥布霉素滴眼液中妥布霉素含量的测定。结果表明:采用本法测定结果与商品标示值一致。本方法快速、简单、重现性好、精密度高、能检测无紫外荧光基团的物质,而且没有干扰,为生产、质检等部门提供了一种可靠的方法。
4、建立了分离检测庆大霉素组分的新方法。以滂胺天蓝为光谱探针,采用高效液相色谱-共振瑞利散射联用技术,对庆大霉素组分进行详细研究。用C18柱(150 mm×4.6 mm,5μm)分离,流动相为0.24%三氟乙酸水溶液-甲醇(96.5:3.5),流速为0.5 ml?min-1,等度洗脱。柱后探针溶液流速为0.08 ml?min-1,在λEx=λEm=356 nm检测。结果表明:庆大霉素中的4个主要组分C1、C1a、C2、C2a,在上述色谱条件下分离良好,分析时间仅为15 min。浓度分别在7.2～115.2、8.2～131.6、7.5～78.8、8.5～78.0μg·mL-1范围内具有良好线性关系,相关系数分别为0.9928、0.9973、0.9980、0.9981。方法检出限(S/N≥3)分别为7.2、6.0、6.0、6.8μg·mL-1。峰面积测定值的相对标准偏差(RSD%)分别为1.6%、2.2%、3.0%、3.0%。庆大霉素注射液采用本法测定结果与2005版的中国药典规定的范围一致。本法操作简单、快速、精密度、重现性能够很好的满足质量控制的要求。
High performance liquid chromatography (HPLC) has become an important branch of analytical science with the characteristics of high separation efficiency, rapid analysis, high sensitivity, and widely application, etc. The development of a new high efficient and general detection technique is the major development trend of HPLC. Resonance Rayleigh scattering(RRS)has aroused widespread concerned because of its simplicity and high sensitivity characteristics, but it is not enough for detecting complicated compound. Thus, there is great potential in the incorporation of HPLC and RRS and the incorporation will become a promising analysis technique.
At present, there are many proteins and aminoglycoside antibiotic analytic methods. Most of them need to be derivated or detected with short-ultraviolet, which could decrease the accuracy and sensitivity. So the separation and determination of them has great significance in the fields of medical study, quality supervision, biomedicine and other correlative fields. In this thesis, a novel detection technique for high performance liquid
chromatography-resonance Rayleigh scattering(HPLC-RRS) has been developed which can be applied to detect real samples. The research results are shown as follow:
PartⅠ.A new detection system for high performance liquid chromatography - resonance Rayleigh scattering(HPLC-RRS) has been explored. The feasibility of this technique of HPLC-RRS was studied by using SDS as the probe of Ins、Cyt-c、Lys, HSA,the methodology of HPLC-RRS technique has been researched as well. The comprehensive experimental scheme has been established by building the post-column ion-association model. The technique was applied to detect four proteins successfully, and a better linear relation、high reproducibility and high sensitivity were obtained and will have a perfect perspective.
PartⅡ. Proteins were separated and detected by using biebrich scarlet (BS) as the molecular recognition probes with the HPLC-RRS technique which has been established in PartⅠ. The experiment condition was performed on a sinochrom ODS-AP column(150 mm) with gradient elution, the mobile phase was trifluoroacetic acid- acetonitrile. The utility of present method was demonstrated by the separation and determination of four proteins involving Cyt-c, Lys, HSA,γ-Glo in 12 minutes. Combined with BS, the proteins formed larger ion-association complex, the RRS signal was detected atλEx =λEm=376 nm. A linear range was found between concentration and peak area at the range of 0.20～3.0μg·mL-1 for Cyt-c, 0.25～2.5μg·mL-1 for HSA, 1.5～10μg·mL-1for Lys, and 2.0～15μg·mL-1 forγ-Glo, with linear regression coefficients of all above 0.99. When the S/N = 3, the detection limit of the four kinds of proteins were within 2.0～10μg·mL-1. The novel method is first used to determine HSA andγ-Glo in human serum sample synchronously.
PartⅢ. A new method for detecting tobramycin (Tob) was established by using the pontamine sky blue as the spectroscopic probe by HPLC-RRS. This method required a separation condition performed on a diamond ODS with equivalent elution. The separation condition and the concentration of the probe have been optimized. The parameter of the experiment condition and post column-probe solution on the sensitivity have been optimized including the pH of the buffer solution, the length of the reaction tube, the probe flow rate. Under the optimal experiment condition, Tob could be separated in 10 minutes. This method had been applied to determine the Tob in tobramycin eye drops and the results were in good agreement with commodity marked. This method provided a reliable method for the production and the quality inspection department with rapidity, simpleness, high sensitivity, good repeatability and detected compound lack of chemical detectable characteristics.
PartⅣ.A HPLC-RRS method had been developed for the determination composition of gentamicin by using pontamine sky blue as spectroscopic probes. The separation condition was performed on a Platisil C18, the mobile phase consisted of 0.24 % trifluoroacetic acid-methanol (96.5:3.5, v/v) at a flow rate of 0.5 mL?min-1 and the probe flow rate was 0.08 mL?min-1. The RRS signal was detected atλEx =λEm=356 nm. The utility of presented method was demonstrated by the separation and determination of the composition in the gentamicin within 15 minutes. A detection limit of 6.0～7.2μg·mL-1 was reached and a linear range was found between peak area and concentration at the range of 7.2～115.2μg·mL-1 for C1, 8.2～131.6μg·mL-1 for C1a, 7.5～78.8μg·mL-1 for C2 and 8.5～78.0μg·mL-1 for C2a, with linear regression coefficients of all above 0.99. The correlation coefficients of calibration curves of C1, C1a, C2,C2a, were 0.9928,0.9973,0.9980 and 0.9981,respectively. The method was used to detect commercial sample and the results were in good agreement with specification of both China and European Pharmacopoeia (2005 version). This method can meet the requirement of quality controlling and it provides as a new method for the quality supervision station and medical real-time monitoring the antibiotics components with rapidity, simpleness, high sensitivity and good repeatability.
目前,蛋白质和氨基糖苷类抗生素的分析方法很多,但大多数需要衍生,或用短紫外波段来检测,准确度和灵敏度都较低,因此,对它们的分离与测定,在生物医学研究和质检等相关领域均具有重要意义。
本文建立了高效液相色谱-共振瑞利散射联用检测技术,并对样品进行了测定。主要研究内容有以下四方面:
1、研究并建立了高效液相色谱-共振瑞利散射联用新技术。用阴离子表面活性剂十二烷基硫酸钠(Sodium dodecyl sulfate,SDS)为光谱探针,商品荧光检测器检测,以牛胰岛素(Insulin,Ins)、细胞色素C(Cytochrome,Cyt-c)、溶菌酶(Lysozyme,Lys)和人血清白蛋白(Human serum albumin HSA)为分离分析目标物,考察高效液相色谱-共振瑞利散射联用技术的可行性,并进行方法学研究,通过柱后离子缔合模型的建立,确定了该技术的整体方案。结果表明,此新方法有较高的灵敏度,操作简便,重复性好,显示出在高效液相色谱检测方法中具有较广阔的应用前景,为实际样品的测定建立了可靠的分析方法。
2、基于建立的高效液相色谱-共振瑞利散射联用技术,以比布列西猩红为光谱探针,分离测定四种蛋白质。三氟乙酸-乙腈混合液为流动相,采用梯度淋洗技术,用Sinochrom ODS-AP色谱柱(150 mm×4.6 mm, 300à, 5μm)分离,Cyt-c、Lys、HSA、免疫球蛋白(γ-globulin,γ-Glo)四种蛋白质在12 min内完全分离,通过其与比布列西猩红相互结合,形成较各自体积更大的离子缔合物,用共振瑞利散射技术进行检测,检测波长为λEx=λEm=376 nm。Cyt-c,Lys,HSA,γ-Glo浓度分别在0.20～3.0μg·mL-1,0.25～2.5μg·mL-1,1.5～10μg·mL-1,2.0～15μg·mL-1时,与峰面积呈良好线性关系,线性相关系数都在0.99以上。当信噪比为3(S/N=3)时,四种蛋白质的检测限在0.2～1.0μg·mL-1范围内。并应用于对人血清中白蛋白和球蛋白的含量测定。
3、以滂胺天蓝作为光谱探针,采用高效液相色谱-共振瑞利散射联用技术,建立了测定妥布霉素的新方法。此方法用diamond ODS色谱柱分离,等度洗脱,在优化了分离条件后,研究了缓冲溶液的pH,反应管长,探针流速和探针的浓度等对灵敏度的影响。在选定的最佳分离条件和最优的结合条件下,妥布霉素在10 min内得到良好的峰形,将方法应用于妥布霉素滴眼液中妥布霉素含量的测定。结果表明:采用本法测定结果与商品标示值一致。本方法快速、简单、重现性好、精密度高、能检测无紫外荧光基团的物质,而且没有干扰,为生产、质检等部门提供了一种可靠的方法。
4、建立了分离检测庆大霉素组分的新方法。以滂胺天蓝为光谱探针,采用高效液相色谱-共振瑞利散射联用技术,对庆大霉素组分进行详细研究。用C18柱(150 mm×4.6 mm,5μm)分离,流动相为0.24%三氟乙酸水溶液-甲醇(96.5:3.5),流速为0.5 ml?min-1,等度洗脱。柱后探针溶液流速为0.08 ml?min-1,在λEx=λEm=356 nm检测。结果表明:庆大霉素中的4个主要组分C1、C1a、C2、C2a,在上述色谱条件下分离良好,分析时间仅为15 min。浓度分别在7.2～115.2、8.2～131.6、7.5～78.8、8.5～78.0μg·mL-1范围内具有良好线性关系,相关系数分别为0.9928、0.9973、0.9980、0.9981。方法检出限(S/N≥3)分别为7.2、6.0、6.0、6.8μg·mL-1。峰面积测定值的相对标准偏差(RSD%)分别为1.6%、2.2%、3.0%、3.0%。庆大霉素注射液采用本法测定结果与2005版的中国药典规定的范围一致。本法操作简单、快速、精密度、重现性能够很好的满足质量控制的要求。
High performance liquid chromatography (HPLC) has become an important branch of analytical science with the characteristics of high separation efficiency, rapid analysis, high sensitivity, and widely application, etc. The development of a new high efficient and general detection technique is the major development trend of HPLC. Resonance Rayleigh scattering(RRS)has aroused widespread concerned because of its simplicity and high sensitivity characteristics, but it is not enough for detecting complicated compound. Thus, there is great potential in the incorporation of HPLC and RRS and the incorporation will become a promising analysis technique.
At present, there are many proteins and aminoglycoside antibiotic analytic methods. Most of them need to be derivated or detected with short-ultraviolet, which could decrease the accuracy and sensitivity. So the separation and determination of them has great significance in the fields of medical study, quality supervision, biomedicine and other correlative fields. In this thesis, a novel detection technique for high performance liquid
chromatography-resonance Rayleigh scattering(HPLC-RRS) has been developed which can be applied to detect real samples. The research results are shown as follow:
PartⅠ.A new detection system for high performance liquid chromatography - resonance Rayleigh scattering(HPLC-RRS) has been explored. The feasibility of this technique of HPLC-RRS was studied by using SDS as the probe of Ins、Cyt-c、Lys, HSA,the methodology of HPLC-RRS technique has been researched as well. The comprehensive experimental scheme has been established by building the post-column ion-association model. The technique was applied to detect four proteins successfully, and a better linear relation、high reproducibility and high sensitivity were obtained and will have a perfect perspective.
PartⅡ. Proteins were separated and detected by using biebrich scarlet (BS) as the molecular recognition probes with the HPLC-RRS technique which has been established in PartⅠ. The experiment condition was performed on a sinochrom ODS-AP column(150 mm) with gradient elution, the mobile phase was trifluoroacetic acid- acetonitrile. The utility of present method was demonstrated by the separation and determination of four proteins involving Cyt-c, Lys, HSA,γ-Glo in 12 minutes. Combined with BS, the proteins formed larger ion-association complex, the RRS signal was detected atλEx =λEm=376 nm. A linear range was found between concentration and peak area at the range of 0.20～3.0μg·mL-1 for Cyt-c, 0.25～2.5μg·mL-1 for HSA, 1.5～10μg·mL-1for Lys, and 2.0～15μg·mL-1 forγ-Glo, with linear regression coefficients of all above 0.99. When the S/N = 3, the detection limit of the four kinds of proteins were within 2.0～10μg·mL-1. The novel method is first used to determine HSA andγ-Glo in human serum sample synchronously.
PartⅢ. A new method for detecting tobramycin (Tob) was established by using the pontamine sky blue as the spectroscopic probe by HPLC-RRS. This method required a separation condition performed on a diamond ODS with equivalent elution. The separation condition and the concentration of the probe have been optimized. The parameter of the experiment condition and post column-probe solution on the sensitivity have been optimized including the pH of the buffer solution, the length of the reaction tube, the probe flow rate. Under the optimal experiment condition, Tob could be separated in 10 minutes. This method had been applied to determine the Tob in tobramycin eye drops and the results were in good agreement with commodity marked. This method provided a reliable method for the production and the quality inspection department with rapidity, simpleness, high sensitivity, good repeatability and detected compound lack of chemical detectable characteristics.
PartⅣ.A HPLC-RRS method had been developed for the determination composition of gentamicin by using pontamine sky blue as spectroscopic probes. The separation condition was performed on a Platisil C18, the mobile phase consisted of 0.24 % trifluoroacetic acid-methanol (96.5:3.5, v/v) at a flow rate of 0.5 mL?min-1 and the probe flow rate was 0.08 mL?min-1. The RRS signal was detected atλEx =λEm=356 nm. The utility of presented method was demonstrated by the separation and determination of the composition in the gentamicin within 15 minutes. A detection limit of 6.0～7.2μg·mL-1 was reached and a linear range was found between peak area and concentration at the range of 7.2～115.2μg·mL-1 for C1, 8.2～131.6μg·mL-1 for C1a, 7.5～78.8μg·mL-1 for C2 and 8.5～78.0μg·mL-1 for C2a, with linear regression coefficients of all above 0.99. The correlation coefficients of calibration curves of C1, C1a, C2,C2a, were 0.9928,0.9973,0.9980 and 0.9981,respectively. The method was used to detect commercial sample and the results were in good agreement with specification of both China and European Pharmacopoeia (2005 version). This method can meet the requirement of quality controlling and it provides as a new method for the quality supervision station and medical real-time monitoring the antibiotics components with rapidity, simpleness, high sensitivity and good repeatability.
引文
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