常规和小型化毛细管电泳—电化学检测技术在食品药品和生物样品分析中的应用研究
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摘要
毛细管电泳(Capillary Electrophoresis,CE)是近二十年来发展较快的分析分离技术之一,具有分离效率高、分析速度快、所需样品少等特点,应用范围包括无机离子、有机分子、生物大分子和对映体等,在分析化学、生物化学、分子生物学、药物化学、食品化学、环境化学等许多领域有着广阔的应用前景。在绪论中回顾了毛细管电泳发展的历史、现状以及发展趋势,对毛细管电泳的基本原理和特点进行了简单介绍,并详细介绍了毛细管电泳电化学检测技术及其在食品、药品分析中的应用。
当前毛细管电泳技术已经得到了广泛应用,本文使用常规毛细管电泳电化学检测技术测定了药品和食品中的生物活性成分,以及使用小型化的毛细管电泳装置测定了生物样品中的成分,主要内容如下:1.毛细管电泳电化学检测法测定洋葱中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了洋葱中芥子酸、槲皮素和原儿茶酸的含量。在优化条件下,以直径300 um的碳圆盘电极为工作电极,电极电位为+950 mV(vs.SCE),在pH9.0、40 mmol/L的硼酸运行缓冲液中,上述三组分在25 min内可实现基线分离。芥子酸、槲皮素和原儿茶酸的浓度分别在2.0×10-7-1.0×10-4 g/ml、2.0×10-7-5.0×10-5 g/ml和5.0×10-7-5.0×10-5g/ml的范围内与峰电流呈良好线性关系,检测下限分别为1.5×10-7 g/ml、1.6×10-7g/ml、3.6×10-7g/ml,该法简单可靠,已经成功地用于洋葱中活性成分的测定,样品无需预富集,检测结果令人满意。2.毛细管电泳电化学检测法测定山楂中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了山楂中表儿茶素、芦丁、金丝桃甙、绿原酸和槲皮素。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.95 V(vs.SCE),在40 mmol/L硼酸盐(pH:8.4)的运行缓冲液中,上述五组分在16 min内实现较好分离。被测物浓度与峰电流在两个数量级范围内呈良好线性关系,检测限在6.00×10-8g/ml到3.75×10-7g/ml之间,该法简单可靠,已经成功地用于山楂和山楂片中有效成分的测定,样品无需预先富集,检测结果令人满意。3.毛细管电泳电化学检测法测定莲子芯中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了莲子芯中儿茶素、芦丁、金丝桃甙和山奈酚。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.8 V(vs.SCE),在60 mmol/L(pH:8.0)硼酸盐和20 mmol/L十二烷基硫酸钠(SDS)的混合运行缓冲液中,上述四组分在20min内实现较好分离。被测物浓度与峰电流在两个数量级范围内呈良好线性关系,检测限在6.90×10-8g/mL到1.30×10-7g/mL之间,该法简单可靠,已经成功地用于莲子芯中活性成分的测定,样品无需预先富集,检测结果令人满意。4.毛细管电泳电化学检测法测定柿子叶中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了柿子叶中芦丁、维生素C、金丝桃甙、丁香酸、水杨酸和槲皮素。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.9 V(vs.SCE),在80 mmol/L硼酸盐(pH:9.0)的运行缓冲液中,上述六组分在20 min内实现较好分离。除维生素C外,其它被测物浓度与峰电流在三个数量级范围内呈良好线性关系,最低检测限达到2.50×10-8g/ml,该法简单可靠,已经成功地用于柿子叶中活性成分的测定,样品无需预先富集,检测结果令人满意。5.毛细管电泳电化学检测法测定三白草中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了三白草中芦丁、金丝桃甙、槲皮素、柯里拉京和没食子酸。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.95 V(vs SCE),在60 mmol/L硼酸盐(pH:8.7)的运行缓冲液中,上述五组分在20 min内实现较好分离。被测物浓度与峰电流在两个数量级范围内呈良好线性关系,检测限在3.05×10-8 g/ml到9.75×10-8 g/ml之间,该法简单可靠,已经成功地用于三白草中活性成分的测定,样品无需预先富集,检测结果令人满意。
6.毛细管电泳电化学检测法测定墓头回中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了墓头回中东莨菪素、金丝桃甙、绿原酸和槲皮素。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.95 V(vs.SCE),在40 mmol/L硼酸盐(pH:8.7)的运行缓冲液中,上述四组分在20 min内实现较好分离。被测物浓度与峰电流在两个数量级范围内呈良好线性关系,检测限在2.70×10-8 g/ml到1.30×10-7 g/ml之间,该法简单可靠,已经成功地用于墓头回中活性成分的测定,样品无需预先富集,检测结果令人满意。
7.小型化毛细管电泳电化学检测法测定猪尿中β-兴奋剂
本实验使用小型化毛细管电泳电化学检测法同时测定了猪尿中盐酸克伦特罗、莱克多巴胺和沙丁胺醇。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.95 V(vs.Ag/AgCl),在100 mmol/L硼酸盐(pH:9.15)的运行缓冲液中,上述三组分在7 min内实现较好分离。被测物浓度与峰电流在三个数量级范围内呈良好线性关系,检测限在1.20×10-7 g/ml到2.06×10-7 g/ml之间,该法简单可靠,已经成功地用于实际样品猪尿中β-兴奋剂的测定,样品无需预先富集,检测结果令人满意。
Capillary electrophoresis (CE), one of the most important separation techniques, has been greatly developed and improved in recent twenty years. The history, current situation and future trend of capillary electrophoresis is reviewed in this thesis. Capillary electrophoresis is becoming increasingly recognized as an important analytical separation technique due to its speed, efficiency, reproducibility, ultra-small sample volume and ease of clearing up the contaminants. Now it is widely applied in analytical chemistry, biological chemistry, environmental chemistry, and so on. In combination with electrochemical detection (ED), it offers high sensitivity and good selectivity for electroactive species. The present dissertation described the studies of capillary electrophoresis with electrochemical detection in the analyses of foods, traditional Chinese medicines and biological samples. The major content is described as follows:
1. Determination of sinapic acid, quercetin and protocatechuic acid in allium cepa linn by capillary zone electrophoresis with electrochemical detection
Simultaneous determination of sinapic acid, quercetin and protocatechuic acid in Allium cepa Linn by capillary zone electrophoresis with electrochemical detection was reported. The parameters affecting separation and detection such as detection potential, separation voltage and running buffer acidity were evaluated. Under the optimum conditions, the analytes were baseline separated within 25 min in a borax buffer (pH = 9.0). Good linear relationship was established between peak current and concentration of analytes in the range of 2.0 ×10~(-7)~ 1.0 ×10~(-4) g/mL for sinapic acid, 2.0 ×10~(-7)~ 5.0 ×10~(-5) g/mL for quercetin, and 5.0 ×10~(-7)~ 5.0 ×10~(-5) g/mL for protocatechuic acid. The detection limit (S/N = 3) is 1.5 ×10~(-7) g/mL, 1.6 ×10~(-5) g/mL, and 3.6 ×10~(-5) g/mL respectively for these three analytes. This method has been used for the determination of active ingredients in allium cepa linn with satisfactory results. 2. Determination of active ingredients in hawthorn and hawthorn piece by capillary electrophoresis with electrochemical detection
Capillary zone electrophoresis with electrochemical detection has been used for the separation and determination of (-)-epicatechin, rutin, hyperin, chlorogenic acid and quercetin. Effects of several important factors including running buffer acidity, separation voltage and working electrode potential, and so on, were evaluated to acquire the optimum conditions . The working electrode was a 300 um carbon disc electrode at a working potential of + 0.95 V ( vs. SCE ) . Under the optimum conditions , the analytes can be well separated within 16 min in a 75 cm length fused-silica capillary . The current response was linear over two orders of magnitude with detection limits ( S/N = 3 ) ranging from 6.00 × 10~(-8) g/ml to 3.75 × 10~(-7)g/ml for all analytes . This method was successfully used in the analysis of hawthorn and hawthorn piece and the assay results were satisfactory .
3. Determination of flavonoids in plumula nelumbini by micellar electrokinetic capillary electrophoresis with electrochemical detection
A simple and efficient method based on micellar electrokinetic capillary electrophoresis (MECC) with electrochemical detection, for the determination of catechin, rutin, hyperin and kaempferol in Plumula Nelumbini was described in this work. Under the optimum conditions, all analytes were well separated within 20 min at the separation voltage of 16 kV in a 60 mmol/L borate running buffer (pH 8.0) containing 20 mmol/L sodium dodecyl sulfate (SDS). The current response was linear over two orders of magnitude with detection limits (S/N = 3) ranging from 6.90 × 10~(-8) g/mL to 1.30 × 10~(-7) g/mL for all analytes. This work provides a useful method for the analysis of Plumula Nelumbini.
4. Determination of active ingredients in persimmon leaf by capillary electrophoresis with electrochemical detection
Capillary zone electrophoresis with electrochemical detection has been used for the separation and determination of rutin, Vc, hyperin, syringic acid, salicylic acid and quercetin. Effects of several important factors including running buffer acidity, separation voltage and working electrode potential, and so on, were evaluated to acquire the optimum conditions . The working electrode was a 300 um carbon disc electrode at a working potential of + 0.9 V ( vs. SCE ) . Under the optimum conditions , the analytes can be well separated within 20 min in a 75 cm length fused-silica capillary . The current response was linear over three orders of magnitude except that of Vc. The lowest detection limits ( S/N = 3 ) reached 2.50 × 10~(-8) g/ml. This method was successfully used in the analysis of Persimmon Leaf and the assay results were satisfactory.
5. Determination of active ingredients in saururus chinensis by capillary electrophoresis with electrochemical detection
Capillary zone electrophoresis with electrochemical detection has been used for the separation and determination of rutin , hyperin , quercetin , corilagin and gallic acid . Effects of several important factors including running buffer acidity , separation voltage and working potential , and so on , were evaluated to acquire the optimum conditions . The working electrode was a 300 um carbon disc electrode at a working potential of + 0.95 V ( vs. saturated calomel electrode ) . Under the optimum conditions , the analytes can be well separated within 20 min in a 75 cm length fused-silica capillary . The current response was linear over two orders of magnitude with detection limits ( S/N = 3 ) ranging from 3.05 × 10~(-8)g/ml to 9.75 × 10~(-8)g/ml for all analytes . This method was successfully used in the analysis of Saururus Chinensis and the assay results were satisfactory .
6. Determination of active ingredients in mutouhui by capillary electrophoresis with electrochemical detection
Capillary zone electrophoresis with electrochemical detection has been used for the separation and determination of scopoletin, hyperin, cholorigic acid and quercetin. Effects of several important factors including running buffer acidity, separation voltage and working electrode potential, and so on, were evaluated to acquire the optimum conditions . The working electrode was a 300 um carbon disc electrode at a working potential of + 0.95 V ( vs. SCE ) . Under the optimum conditions , the analytes can be well separated within 20 min in a 75 cm length fused-silica capillary . The current response was linear over two orders of magnitude with detection limits ( S/N = 3 ) ranging from 2.70 × 10~(-8) g/ml to 1.30 × 10~(-7)g/ml for all analytes . This method was successfully used in the analysis of Mutouhui and the assay results were satisfactory .
7. Method development for fast determination of β-agonists in pig urine by small-size capillary electrophoresis with electrochemical detection
Fast separation capability of a novel small-size capillary electrophoresis with electrochemical detection was demonstrated by determining clenbuterol hydrochloride, ractopamine and salbutamol in pig urine. Effects of several important factors including running buffer acidity, separation voltage and working electrode potential, and so on, were evaluated to acquire the optimum conditions. The working electrode was a carbon disc electrode at a working potential of + 0.95 V ( vs. Ag/AgCl). Under the optimum conditions , the analytes can be well separated within 7 min in a 14.5 cm length fused-silica capillary . The current response was linear over two orders of magnitude with detection limits ( S/N = 3 ) ranging from 1.20 × 10~(-7) g/ml to 2.06 × 10~(-7)g/ml for these analytes . This method was successfully used in the analysis of pig urine and the assay results were satisfactory .
当前毛细管电泳技术已经得到了广泛应用,本文使用常规毛细管电泳电化学检测技术测定了药品和食品中的生物活性成分,以及使用小型化的毛细管电泳装置测定了生物样品中的成分,主要内容如下:1.毛细管电泳电化学检测法测定洋葱中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了洋葱中芥子酸、槲皮素和原儿茶酸的含量。在优化条件下,以直径300 um的碳圆盘电极为工作电极,电极电位为+950 mV(vs.SCE),在pH9.0、40 mmol/L的硼酸运行缓冲液中,上述三组分在25 min内可实现基线分离。芥子酸、槲皮素和原儿茶酸的浓度分别在2.0×10-7-1.0×10-4 g/ml、2.0×10-7-5.0×10-5 g/ml和5.0×10-7-5.0×10-5g/ml的范围内与峰电流呈良好线性关系,检测下限分别为1.5×10-7 g/ml、1.6×10-7g/ml、3.6×10-7g/ml,该法简单可靠,已经成功地用于洋葱中活性成分的测定,样品无需预富集,检测结果令人满意。2.毛细管电泳电化学检测法测定山楂中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了山楂中表儿茶素、芦丁、金丝桃甙、绿原酸和槲皮素。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.95 V(vs.SCE),在40 mmol/L硼酸盐(pH:8.4)的运行缓冲液中,上述五组分在16 min内实现较好分离。被测物浓度与峰电流在两个数量级范围内呈良好线性关系,检测限在6.00×10-8g/ml到3.75×10-7g/ml之间,该法简单可靠,已经成功地用于山楂和山楂片中有效成分的测定,样品无需预先富集,检测结果令人满意。3.毛细管电泳电化学检测法测定莲子芯中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了莲子芯中儿茶素、芦丁、金丝桃甙和山奈酚。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.8 V(vs.SCE),在60 mmol/L(pH:8.0)硼酸盐和20 mmol/L十二烷基硫酸钠(SDS)的混合运行缓冲液中,上述四组分在20min内实现较好分离。被测物浓度与峰电流在两个数量级范围内呈良好线性关系,检测限在6.90×10-8g/mL到1.30×10-7g/mL之间,该法简单可靠,已经成功地用于莲子芯中活性成分的测定,样品无需预先富集,检测结果令人满意。4.毛细管电泳电化学检测法测定柿子叶中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了柿子叶中芦丁、维生素C、金丝桃甙、丁香酸、水杨酸和槲皮素。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.9 V(vs.SCE),在80 mmol/L硼酸盐(pH:9.0)的运行缓冲液中,上述六组分在20 min内实现较好分离。除维生素C外,其它被测物浓度与峰电流在三个数量级范围内呈良好线性关系,最低检测限达到2.50×10-8g/ml,该法简单可靠,已经成功地用于柿子叶中活性成分的测定,样品无需预先富集,检测结果令人满意。5.毛细管电泳电化学检测法测定三白草中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了三白草中芦丁、金丝桃甙、槲皮素、柯里拉京和没食子酸。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.95 V(vs SCE),在60 mmol/L硼酸盐(pH:8.7)的运行缓冲液中,上述五组分在20 min内实现较好分离。被测物浓度与峰电流在两个数量级范围内呈良好线性关系,检测限在3.05×10-8 g/ml到9.75×10-8 g/ml之间,该法简单可靠,已经成功地用于三白草中活性成分的测定,样品无需预先富集,检测结果令人满意。
6.毛细管电泳电化学检测法测定墓头回中的生物活性成分
本实验使用毛细管电泳电化学检测法同时测定了墓头回中东莨菪素、金丝桃甙、绿原酸和槲皮素。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.95 V(vs.SCE),在40 mmol/L硼酸盐(pH:8.7)的运行缓冲液中,上述四组分在20 min内实现较好分离。被测物浓度与峰电流在两个数量级范围内呈良好线性关系,检测限在2.70×10-8 g/ml到1.30×10-7 g/ml之间,该法简单可靠,已经成功地用于墓头回中活性成分的测定,样品无需预先富集,检测结果令人满意。
7.小型化毛细管电泳电化学检测法测定猪尿中β-兴奋剂
本实验使用小型化毛细管电泳电化学检测法同时测定了猪尿中盐酸克伦特罗、莱克多巴胺和沙丁胺醇。在优化条件下,以300 um直径的碳圆盘电极为检测电极,检测电位为+0.95 V(vs.Ag/AgCl),在100 mmol/L硼酸盐(pH:9.15)的运行缓冲液中,上述三组分在7 min内实现较好分离。被测物浓度与峰电流在三个数量级范围内呈良好线性关系,检测限在1.20×10-7 g/ml到2.06×10-7 g/ml之间,该法简单可靠,已经成功地用于实际样品猪尿中β-兴奋剂的测定,样品无需预先富集,检测结果令人满意。
Capillary electrophoresis (CE), one of the most important separation techniques, has been greatly developed and improved in recent twenty years. The history, current situation and future trend of capillary electrophoresis is reviewed in this thesis. Capillary electrophoresis is becoming increasingly recognized as an important analytical separation technique due to its speed, efficiency, reproducibility, ultra-small sample volume and ease of clearing up the contaminants. Now it is widely applied in analytical chemistry, biological chemistry, environmental chemistry, and so on. In combination with electrochemical detection (ED), it offers high sensitivity and good selectivity for electroactive species. The present dissertation described the studies of capillary electrophoresis with electrochemical detection in the analyses of foods, traditional Chinese medicines and biological samples. The major content is described as follows:
1. Determination of sinapic acid, quercetin and protocatechuic acid in allium cepa linn by capillary zone electrophoresis with electrochemical detection
Simultaneous determination of sinapic acid, quercetin and protocatechuic acid in Allium cepa Linn by capillary zone electrophoresis with electrochemical detection was reported. The parameters affecting separation and detection such as detection potential, separation voltage and running buffer acidity were evaluated. Under the optimum conditions, the analytes were baseline separated within 25 min in a borax buffer (pH = 9.0). Good linear relationship was established between peak current and concentration of analytes in the range of 2.0 ×10~(-7)~ 1.0 ×10~(-4) g/mL for sinapic acid, 2.0 ×10~(-7)~ 5.0 ×10~(-5) g/mL for quercetin, and 5.0 ×10~(-7)~ 5.0 ×10~(-5) g/mL for protocatechuic acid. The detection limit (S/N = 3) is 1.5 ×10~(-7) g/mL, 1.6 ×10~(-5) g/mL, and 3.6 ×10~(-5) g/mL respectively for these three analytes. This method has been used for the determination of active ingredients in allium cepa linn with satisfactory results. 2. Determination of active ingredients in hawthorn and hawthorn piece by capillary electrophoresis with electrochemical detection
Capillary zone electrophoresis with electrochemical detection has been used for the separation and determination of (-)-epicatechin, rutin, hyperin, chlorogenic acid and quercetin. Effects of several important factors including running buffer acidity, separation voltage and working electrode potential, and so on, were evaluated to acquire the optimum conditions . The working electrode was a 300 um carbon disc electrode at a working potential of + 0.95 V ( vs. SCE ) . Under the optimum conditions , the analytes can be well separated within 16 min in a 75 cm length fused-silica capillary . The current response was linear over two orders of magnitude with detection limits ( S/N = 3 ) ranging from 6.00 × 10~(-8) g/ml to 3.75 × 10~(-7)g/ml for all analytes . This method was successfully used in the analysis of hawthorn and hawthorn piece and the assay results were satisfactory .
3. Determination of flavonoids in plumula nelumbini by micellar electrokinetic capillary electrophoresis with electrochemical detection
A simple and efficient method based on micellar electrokinetic capillary electrophoresis (MECC) with electrochemical detection, for the determination of catechin, rutin, hyperin and kaempferol in Plumula Nelumbini was described in this work. Under the optimum conditions, all analytes were well separated within 20 min at the separation voltage of 16 kV in a 60 mmol/L borate running buffer (pH 8.0) containing 20 mmol/L sodium dodecyl sulfate (SDS). The current response was linear over two orders of magnitude with detection limits (S/N = 3) ranging from 6.90 × 10~(-8) g/mL to 1.30 × 10~(-7) g/mL for all analytes. This work provides a useful method for the analysis of Plumula Nelumbini.
4. Determination of active ingredients in persimmon leaf by capillary electrophoresis with electrochemical detection
Capillary zone electrophoresis with electrochemical detection has been used for the separation and determination of rutin, Vc, hyperin, syringic acid, salicylic acid and quercetin. Effects of several important factors including running buffer acidity, separation voltage and working electrode potential, and so on, were evaluated to acquire the optimum conditions . The working electrode was a 300 um carbon disc electrode at a working potential of + 0.9 V ( vs. SCE ) . Under the optimum conditions , the analytes can be well separated within 20 min in a 75 cm length fused-silica capillary . The current response was linear over three orders of magnitude except that of Vc. The lowest detection limits ( S/N = 3 ) reached 2.50 × 10~(-8) g/ml. This method was successfully used in the analysis of Persimmon Leaf and the assay results were satisfactory.
5. Determination of active ingredients in saururus chinensis by capillary electrophoresis with electrochemical detection
Capillary zone electrophoresis with electrochemical detection has been used for the separation and determination of rutin , hyperin , quercetin , corilagin and gallic acid . Effects of several important factors including running buffer acidity , separation voltage and working potential , and so on , were evaluated to acquire the optimum conditions . The working electrode was a 300 um carbon disc electrode at a working potential of + 0.95 V ( vs. saturated calomel electrode ) . Under the optimum conditions , the analytes can be well separated within 20 min in a 75 cm length fused-silica capillary . The current response was linear over two orders of magnitude with detection limits ( S/N = 3 ) ranging from 3.05 × 10~(-8)g/ml to 9.75 × 10~(-8)g/ml for all analytes . This method was successfully used in the analysis of Saururus Chinensis and the assay results were satisfactory .
6. Determination of active ingredients in mutouhui by capillary electrophoresis with electrochemical detection
Capillary zone electrophoresis with electrochemical detection has been used for the separation and determination of scopoletin, hyperin, cholorigic acid and quercetin. Effects of several important factors including running buffer acidity, separation voltage and working electrode potential, and so on, were evaluated to acquire the optimum conditions . The working electrode was a 300 um carbon disc electrode at a working potential of + 0.95 V ( vs. SCE ) . Under the optimum conditions , the analytes can be well separated within 20 min in a 75 cm length fused-silica capillary . The current response was linear over two orders of magnitude with detection limits ( S/N = 3 ) ranging from 2.70 × 10~(-8) g/ml to 1.30 × 10~(-7)g/ml for all analytes . This method was successfully used in the analysis of Mutouhui and the assay results were satisfactory .
7. Method development for fast determination of β-agonists in pig urine by small-size capillary electrophoresis with electrochemical detection
Fast separation capability of a novel small-size capillary electrophoresis with electrochemical detection was demonstrated by determining clenbuterol hydrochloride, ractopamine and salbutamol in pig urine. Effects of several important factors including running buffer acidity, separation voltage and working electrode potential, and so on, were evaluated to acquire the optimum conditions. The working electrode was a carbon disc electrode at a working potential of + 0.95 V ( vs. Ag/AgCl). Under the optimum conditions , the analytes can be well separated within 7 min in a 14.5 cm length fused-silica capillary . The current response was linear over two orders of magnitude with detection limits ( S/N = 3 ) ranging from 1.20 × 10~(-7) g/ml to 2.06 × 10~(-7)g/ml for these analytes . This method was successfully used in the analysis of pig urine and the assay results were satisfactory .
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