高效毛细管电泳法在食品和药品检测中的应用
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摘要
毛细管电泳技术是分析科学领域继液相色谱之后又一重大的进展,在分析和测定复杂样品的过程中发挥着巨大的作用。本论文开展了毛细管电泳技术在食品、中药、化妆品和生物样品中的应用研究,旨在完善毛细管电泳技术和拓展其应用范围,重点集中在以下四个方面:
1.首次采用毛细管电泳脉冲电导法同时测定了市售饮品中的葡萄糖、果糖和蔗糖,该法分析速度快、灵敏度高。
2.毛细管电泳结合电化学和间接紫外检测方法,快速分离和测定了中药枸杞和枸杞胶囊中的单糖和寡糖,方法分析时间短,结果准确可靠。
3.首次采用胶束电动毛细管色谱测定了八种不同化妆品中的糖皮质激素类物质地塞米松,为有效地监测化妆品的行业安全提供了一个快速、有效的检测方法。
4.首次应用胶束电动毛细管色谱测定了人血样和尿液中的地塞米松和倍他米松对映体,为运动员是否滥用糖皮质激素的常规分析提供了一个新途径。
High performance capillary electrophoresis has many advantages such as high efficiency, short analysis time, small sample size and inexpensive instrumentation, which can be useful in the rapid and efficient determination of active compounds in complex samples. In this paper, four developed methods for the analysis of active compounds in food and medicine were presented.
1. Carbohydrates are the focus of new analytical techniques because of their importance in several areas ranging from medicine to food science.
In this study, a method for simultaneous determination of mono–and disaccharides by capillary zone electrophoresis with pulsed conductivity detection at Au microelectrode was developed. The effects of running buffer, separation voltage and injection time were studied and optimized. Common mono– and disaccharides, including fructose, glucose and sucrose, were baseline separated in 10 mmol/L sodium hydroxide–10mmol/L sodium chloride–0.5mmol/L cetryltrimetylammonium bromide within 9 min at the separation voltage of–15kV. Under the optimized conditions, the linear relationship between peak area and concentration almost existed in the rangs of 2.0×10~(–4) - 1.0×10~(–2)mol/L with the detection limits were 2.0×10~(–5)mol/L, 3.0×10~(–5)mol/L, 1.0×10~(–5)mol/L for fructose, glucose and sucrose, respectively. The method was successfully applied to the separation and determination of mono–and disaccharides in three soft drinks with satisfactory results.
2. Lycium fruit is a kind of popular Chinese herb and food supplement. The content of sugars in Lycium fruit is important because it decides the quality of Lycium fruit and affects the contents of other activities in Lycium fruit.
In this part, a method of rapid separation and determination of monosaccharides and oligosaccharides in lycium fruit by capillary electrophoresis with electrochemical detection and indirect UV detection was presented. The effects of detection potential, injection time, buffer concentration and separation voltage were all investigated. Under the optimum condition, seven sugars including xylose, fructose, glucose, galatose, maltotetraose, maltohexoaose and sucrose were well separated within 8 min. This method has linear ranges of 1.0×10~(–4) - 5.0×10~(–3) mol/L for CE–ECD and 5×10~(–4) - 7×10~(–3) mol/L for CE–UV for all the analysts with the detection limits of 1×10~(–6) - 2.5×10~(–6 )mol/L for CE–ECD and 1×10~(–4) - 3×10~(–4) mol/L for CE–UV, which shows that the direct ECD is more sensitive and selective than indirect UV detection. The method were first applied to the rapid determination of sugars in Lycium fruit and Lycium fruit capsule with satisfied results.
3. In the hygienic standardization of cosmetics in the People’s Republic of China and the directive of cosmetics in the European Economic and Monetary Union, the glucocorticoids are forbidden. The reason for this is that prolonged use of cosmetics containing glucocorticoids leads to severe side effects on the skin. However, glucocorticoids are still added illegally since their initial results are favorable. Hence, a rapid and reliable method to monitor the content of glucocorticoids in cosmetics is urgently required. Up to now, the assay of glucocorticoids in cosmetics has always been based on LC despite the fact that its separation efficiency needs to be further improved.
In this section, a rapid and reliable method based on micellar electrokinetic capillary chromatography was developed for the determination of dexamethasone in cosmetics. Effects of the buffer composition, concentration and pH, and detection wavelength, separation voltage, injection time were systematically investigated to optimize the method. The optimum conditions were: 30 mmol/L borax buffer containing 20 mmol/L sodium dodecyl sulfate at pH 9.0, detection wavelength 254 nm, injection time 10 s at a height of 10 cm, and separation voltage at 15 kV, under which the analysis of dexamethasone in cosmetics was carried out within 6 min. The proposed method was validated through stability, precision, linearity and accuracy experiments. Excellent linearity was obtained in the range of 50ˇ1000μg/mL with a correlation coefficient of 0.9997, and acceptable precision, in intra–day and inter–day analysis, was also obtained with relative standard deviation in the range of 0.19ˇ0.86% and 2.50ˇ4.90% for migration time and peak area ratio, respectively. Additionally, the developed method was applied to assay eight different cosmetics from local market with the recoveries ranging from 93.3 to 97.6%.
4. Betamethasone (BM) and dexamethasone (DM) are epimeric synthetic glucocorticoids with different configuration of the methyl group on C–16 and frequently employed as ant–inflammatory agents in clinic to treat a wide range of diseases. However, in clinic, prolonged and systemic administration of these drugs is often accompanied with significant side effects, such as skin fragility, abnormal fat, poor wound healing, suppression of growth, etc. Simultaneity, BM and DM, in sport medicine, are used by reason of their anti–inflammatory and analgesic properties, but systemic administration of them is forbidden by the International Olympic Committee (IOC) because of their potential positive effects on sport performance and associated toxicological risks. Therefore, in order to monitor the systemic glucocorticoids administration, a simple, rapid and reliable method for measurement of BM and DM in human urine and serum is urgently required. Up to now, it is still a challenge for analysts to determine BM and DM in the body fluids of athletes owing to not only their very similar structures but also, in many cases, their low concentrations found in biofluids.
In this study, simultaneous determination of betamethasone and its epimer dexamethasone in human urine and serum by a simple and reliable micellar electrokinetic capillary chromatography method has been presented. A three level full factorial experimental design was employed to search for the optimum conditions. Rapid and baseline separation of BM and DM was obtained within 7 min with the optimum conditions of 30 mmol/L borax buffer, 30 mmol/L sodium dodecyl sulfate at pH 10.0, separation voltage at 18 kV, injection time 15 s at a height of 10 cm, using sodium sorbate as internal standard. The proposed method was validated with respect to stability, precision, linearity and accuracy. Good relationship between peak area ratio and analyte concentration was linear over 30ˇ1000μg/mL for BM and DM with correlation coefficients≥0.9993. Relative standard deviations of the method were all less than 4.50% in the intra–day and inter–day analysis. The developed method was applied to assay spiked human urine and serum samples containing both compounds with recoveries in the range of 97.5ˇ100.5%.
1.首次采用毛细管电泳脉冲电导法同时测定了市售饮品中的葡萄糖、果糖和蔗糖,该法分析速度快、灵敏度高。
2.毛细管电泳结合电化学和间接紫外检测方法,快速分离和测定了中药枸杞和枸杞胶囊中的单糖和寡糖,方法分析时间短,结果准确可靠。
3.首次采用胶束电动毛细管色谱测定了八种不同化妆品中的糖皮质激素类物质地塞米松,为有效地监测化妆品的行业安全提供了一个快速、有效的检测方法。
4.首次应用胶束电动毛细管色谱测定了人血样和尿液中的地塞米松和倍他米松对映体,为运动员是否滥用糖皮质激素的常规分析提供了一个新途径。
High performance capillary electrophoresis has many advantages such as high efficiency, short analysis time, small sample size and inexpensive instrumentation, which can be useful in the rapid and efficient determination of active compounds in complex samples. In this paper, four developed methods for the analysis of active compounds in food and medicine were presented.
1. Carbohydrates are the focus of new analytical techniques because of their importance in several areas ranging from medicine to food science.
In this study, a method for simultaneous determination of mono–and disaccharides by capillary zone electrophoresis with pulsed conductivity detection at Au microelectrode was developed. The effects of running buffer, separation voltage and injection time were studied and optimized. Common mono– and disaccharides, including fructose, glucose and sucrose, were baseline separated in 10 mmol/L sodium hydroxide–10mmol/L sodium chloride–0.5mmol/L cetryltrimetylammonium bromide within 9 min at the separation voltage of–15kV. Under the optimized conditions, the linear relationship between peak area and concentration almost existed in the rangs of 2.0×10~(–4) - 1.0×10~(–2)mol/L with the detection limits were 2.0×10~(–5)mol/L, 3.0×10~(–5)mol/L, 1.0×10~(–5)mol/L for fructose, glucose and sucrose, respectively. The method was successfully applied to the separation and determination of mono–and disaccharides in three soft drinks with satisfactory results.
2. Lycium fruit is a kind of popular Chinese herb and food supplement. The content of sugars in Lycium fruit is important because it decides the quality of Lycium fruit and affects the contents of other activities in Lycium fruit.
In this part, a method of rapid separation and determination of monosaccharides and oligosaccharides in lycium fruit by capillary electrophoresis with electrochemical detection and indirect UV detection was presented. The effects of detection potential, injection time, buffer concentration and separation voltage were all investigated. Under the optimum condition, seven sugars including xylose, fructose, glucose, galatose, maltotetraose, maltohexoaose and sucrose were well separated within 8 min. This method has linear ranges of 1.0×10~(–4) - 5.0×10~(–3) mol/L for CE–ECD and 5×10~(–4) - 7×10~(–3) mol/L for CE–UV for all the analysts with the detection limits of 1×10~(–6) - 2.5×10~(–6 )mol/L for CE–ECD and 1×10~(–4) - 3×10~(–4) mol/L for CE–UV, which shows that the direct ECD is more sensitive and selective than indirect UV detection. The method were first applied to the rapid determination of sugars in Lycium fruit and Lycium fruit capsule with satisfied results.
3. In the hygienic standardization of cosmetics in the People’s Republic of China and the directive of cosmetics in the European Economic and Monetary Union, the glucocorticoids are forbidden. The reason for this is that prolonged use of cosmetics containing glucocorticoids leads to severe side effects on the skin. However, glucocorticoids are still added illegally since their initial results are favorable. Hence, a rapid and reliable method to monitor the content of glucocorticoids in cosmetics is urgently required. Up to now, the assay of glucocorticoids in cosmetics has always been based on LC despite the fact that its separation efficiency needs to be further improved.
In this section, a rapid and reliable method based on micellar electrokinetic capillary chromatography was developed for the determination of dexamethasone in cosmetics. Effects of the buffer composition, concentration and pH, and detection wavelength, separation voltage, injection time were systematically investigated to optimize the method. The optimum conditions were: 30 mmol/L borax buffer containing 20 mmol/L sodium dodecyl sulfate at pH 9.0, detection wavelength 254 nm, injection time 10 s at a height of 10 cm, and separation voltage at 15 kV, under which the analysis of dexamethasone in cosmetics was carried out within 6 min. The proposed method was validated through stability, precision, linearity and accuracy experiments. Excellent linearity was obtained in the range of 50ˇ1000μg/mL with a correlation coefficient of 0.9997, and acceptable precision, in intra–day and inter–day analysis, was also obtained with relative standard deviation in the range of 0.19ˇ0.86% and 2.50ˇ4.90% for migration time and peak area ratio, respectively. Additionally, the developed method was applied to assay eight different cosmetics from local market with the recoveries ranging from 93.3 to 97.6%.
4. Betamethasone (BM) and dexamethasone (DM) are epimeric synthetic glucocorticoids with different configuration of the methyl group on C–16 and frequently employed as ant–inflammatory agents in clinic to treat a wide range of diseases. However, in clinic, prolonged and systemic administration of these drugs is often accompanied with significant side effects, such as skin fragility, abnormal fat, poor wound healing, suppression of growth, etc. Simultaneity, BM and DM, in sport medicine, are used by reason of their anti–inflammatory and analgesic properties, but systemic administration of them is forbidden by the International Olympic Committee (IOC) because of their potential positive effects on sport performance and associated toxicological risks. Therefore, in order to monitor the systemic glucocorticoids administration, a simple, rapid and reliable method for measurement of BM and DM in human urine and serum is urgently required. Up to now, it is still a challenge for analysts to determine BM and DM in the body fluids of athletes owing to not only their very similar structures but also, in many cases, their low concentrations found in biofluids.
In this study, simultaneous determination of betamethasone and its epimer dexamethasone in human urine and serum by a simple and reliable micellar electrokinetic capillary chromatography method has been presented. A three level full factorial experimental design was employed to search for the optimum conditions. Rapid and baseline separation of BM and DM was obtained within 7 min with the optimum conditions of 30 mmol/L borax buffer, 30 mmol/L sodium dodecyl sulfate at pH 10.0, separation voltage at 18 kV, injection time 15 s at a height of 10 cm, using sodium sorbate as internal standard. The proposed method was validated with respect to stability, precision, linearity and accuracy. Good relationship between peak area ratio and analyte concentration was linear over 30ˇ1000μg/mL for BM and DM with correlation coefficients≥0.9993. Relative standard deviations of the method were all less than 4.50% in the intra–day and inter–day analysis. The developed method was applied to assay spiked human urine and serum samples containing both compounds with recoveries in the range of 97.5ˇ100.5%.
引文
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