二维液相色谱法在线检测哈茨木霉发酵液中2460A含量

山广志等
摘 要 建立了在线检测哈茨木霉发酵液中微量2460A的二维液相色谱方法。利用Ultimate 3000双三元液相色谱仪,采用阀切换二维色谱技术,组合3根色谱柱实现2460A的在线净化、富集和含量检测。净化柱采用资生堂MF C
1 引 言
2460A是从植物病原菌拮抗菌哈茨木霉(Trichoderma hazianum)的发酵产物中分离获得的一种具有抗肿瘤活性的新结构化合物,结构见图1插图。2460A具有白细胞介素6受体(IL6R)的配基活性,同时对骨髓癌CM126细胞和结肠癌HT29细胞具有抑制作用[ 1]。
2460A水溶性较差(水中溶解度<1 mg/L),采用常规分析型HPLC方法在进样量100 μL条件下仅能达到定量限水平。哈茨木霉发酵液基质较为复杂,严重干扰目标物的分离。为了研究和优化2460A的发酵工艺,需要对不同发酵条件下哈茨木霉发酵液中的2460A含量进行测定。采用传统液液萃取方法进行样品预处理时,需要发酵液样品量较大,操作步骤繁琐,耗时较长,难以满足发酵工艺优化所需的快速、准确的检测要求。
近年来,由普通一维液相色谱发展起来的多维液相色谱技术有力地推动了复杂体系成分的分离[ 2,3]。Gidding等[ 4,5]指出,多维色谱分离系统比一维色谱具有更好的分辨率和更高的峰容量,更适合分离复杂体系。本研究利用Ultimate 3000双三元液相色谱仪,采用六通阀切换技术(见图2)[ 6~9]、大体积进样器和3根色谱柱,综合利用“中心切割”、在线富集等二维色谱技术,实现了复杂发酵液的在线预处理,减轻了实验人员操作强度,节约了样品处理时间,提高了检测的自动化程度和通量,方法重现性较手动提取得到较大提高。通过大体积进样和在线富集,提高了方法灵敏度,解决了微量组分的检测难题。建立的方法准确、简便、灵敏、通用性强,适用于2460A发酵工艺优化过程中的大量检测研究工作,同时为复杂基质中微量代谢产物的测定提供了方法依据和借鉴。
2 实验部分
2.1 仪器与试剂
Determination of 2460A in Trichoderma Hazianum Fermentation
Liquor by Online Twodimensional Liquid Chromatographic Method
SHAN GuangZhi*, ZHOU Jie, ZUO LiMin, JIANG Wei, LIU GuiXia, ZHANG Yang, LI Yuan, JIANG Rong*
(Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing 100050, China)
Abstract An online two dimensional liquid chromatographic (2DLC) method was established by using an ultimate dual gradient liquid chromatography, three chromatographic columns and valveswitching technology to detect 2460A in trichoderma hazianum fermentation liquor. MF C8 column (10 mm×4.6 mm, 5.0 μm) was used as purification column and MG C18 column (20 mm×4.6 mm, 5.0 μm) was used as enriching column. Methanol and water were used as mobile phase with a gradient elution at a flow rate of 2.0 mL/min. The sample was separated on the Thermo Hypersil GOLD C18 column (250 mm×4.6 mm, 5.0 μm) maintained at 40 ℃ using methanol and water. The flow rate was 1.0 mL/min and 1.0 mL sample was injected into the 2DLC system. The detection wavelength was 424 nm. The whole analytical time was less than 60 min. The standard curve was linear over the 2460A concentration range of 0.0025-10.0 mg/L(r=0.9981, n=8). The limit of detection was calculated to be 1.2 μg/L (S/N=3) and the limit of quantification was calculated to be 2.5 μg/L (S/N=10). The average recoveries varied from 88.0% to 104.4%.
Keywords Twodimensional liquid chromatography; Trichoderma hazianum; Fermentation liquor; 2460A
(Received 29 August 2014; accepted 20 October 2014)
This work was supported by the National S & T Major Special Project on Major New Drug Innovation (No. 2012zx09301002001019)
摘 要 建立了在线检测哈茨木霉发酵液中微量2460A的二维液相色谱方法。利用Ultimate 3000双三元液相色谱仪,采用阀切换二维色谱技术,组合3根色谱柱实现2460A的在线净化、富集和含量检测。净化柱采用资生堂MF C
1 引 言
2460A是从植物病原菌拮抗菌哈茨木霉(Trichoderma hazianum)的发酵产物中分离获得的一种具有抗肿瘤活性的新结构化合物,结构见图1插图。2460A具有白细胞介素6受体(IL6R)的配基活性,同时对骨髓癌CM126细胞和结肠癌HT29细胞具有抑制作用[ 1]。
2460A水溶性较差(水中溶解度<1 mg/L),采用常规分析型HPLC方法在进样量100 μL条件下仅能达到定量限水平。哈茨木霉发酵液基质较为复杂,严重干扰目标物的分离。为了研究和优化2460A的发酵工艺,需要对不同发酵条件下哈茨木霉发酵液中的2460A含量进行测定。采用传统液液萃取方法进行样品预处理时,需要发酵液样品量较大,操作步骤繁琐,耗时较长,难以满足发酵工艺优化所需的快速、准确的检测要求。
近年来,由普通一维液相色谱发展起来的多维液相色谱技术有力地推动了复杂体系成分的分离[ 2,3]。Gidding等[ 4,5]指出,多维色谱分离系统比一维色谱具有更好的分辨率和更高的峰容量,更适合分离复杂体系。本研究利用Ultimate 3000双三元液相色谱仪,采用六通阀切换技术(见图2)[ 6~9]、大体积进样器和3根色谱柱,综合利用“中心切割”、在线富集等二维色谱技术,实现了复杂发酵液的在线预处理,减轻了实验人员操作强度,节约了样品处理时间,提高了检测的自动化程度和通量,方法重现性较手动提取得到较大提高。通过大体积进样和在线富集,提高了方法灵敏度,解决了微量组分的检测难题。建立的方法准确、简便、灵敏、通用性强,适用于2460A发酵工艺优化过程中的大量检测研究工作,同时为复杂基质中微量代谢产物的测定提供了方法依据和借鉴。
2 实验部分
2.1 仪器与试剂
Determination of 2460A in Trichoderma Hazianum Fermentation
Liquor by Online Twodimensional Liquid Chromatographic Method
SHAN GuangZhi*, ZHOU Jie, ZUO LiMin, JIANG Wei, LIU GuiXia, ZHANG Yang, LI Yuan, JIANG Rong*
(Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing 100050, China)
Abstract An online two dimensional liquid chromatographic (2DLC) method was established by using an ultimate dual gradient liquid chromatography, three chromatographic columns and valveswitching technology to detect 2460A in trichoderma hazianum fermentation liquor. MF C8 column (10 mm×4.6 mm, 5.0 μm) was used as purification column and MG C18 column (20 mm×4.6 mm, 5.0 μm) was used as enriching column. Methanol and water were used as mobile phase with a gradient elution at a flow rate of 2.0 mL/min. The sample was separated on the Thermo Hypersil GOLD C18 column (250 mm×4.6 mm, 5.0 μm) maintained at 40 ℃ using methanol and water. The flow rate was 1.0 mL/min and 1.0 mL sample was injected into the 2DLC system. The detection wavelength was 424 nm. The whole analytical time was less than 60 min. The standard curve was linear over the 2460A concentration range of 0.0025-10.0 mg/L(r=0.9981, n=8). The limit of detection was calculated to be 1.2 μg/L (S/N=3) and the limit of quantification was calculated to be 2.5 μg/L (S/N=10). The average recoveries varied from 88.0% to 104.4%.
Keywords Twodimensional liquid chromatography; Trichoderma hazianum; Fermentation liquor; 2460A
(Received 29 August 2014; accepted 20 October 2014)
This work was supported by the National S & T Major Special Project on Major New Drug Innovation (No. 2012zx09301002001019)
摘 要 建立了在线检测哈茨木霉发酵液中微量2460A的二维液相色谱方法。利用Ultimate 3000双三元液相色谱仪,采用阀切换二维色谱技术,组合3根色谱柱实现2460A的在线净化、富集和含量检测。净化柱采用资生堂MF C
1 引 言
2460A是从植物病原菌拮抗菌哈茨木霉(Trichoderma hazianum)的发酵产物中分离获得的一种具有抗肿瘤活性的新结构化合物,结构见图1插图。2460A具有白细胞介素6受体(IL6R)的配基活性,同时对骨髓癌CM126细胞和结肠癌HT29细胞具有抑制作用[ 1]。
2460A水溶性较差(水中溶解度<1 mg/L),采用常规分析型HPLC方法在进样量100 μL条件下仅能达到定量限水平。哈茨木霉发酵液基质较为复杂,严重干扰目标物的分离。为了研究和优化2460A的发酵工艺,需要对不同发酵条件下哈茨木霉发酵液中的2460A含量进行测定。采用传统液液萃取方法进行样品预处理时,需要发酵液样品量较大,操作步骤繁琐,耗时较长,难以满足发酵工艺优化所需的快速、准确的检测要求。
近年来,由普通一维液相色谱发展起来的多维液相色谱技术有力地推动了复杂体系成分的分离[ 2,3]。Gidding等[ 4,5]指出,多维色谱分离系统比一维色谱具有更好的分辨率和更高的峰容量,更适合分离复杂体系。本研究利用Ultimate 3000双三元液相色谱仪,采用六通阀切换技术(见图2)[ 6~9]、大体积进样器和3根色谱柱,综合利用“中心切割”、在线富集等二维色谱技术,实现了复杂发酵液的在线预处理,减轻了实验人员操作强度,节约了样品处理时间,提高了检测的自动化程度和通量,方法重现性较手动提取得到较大提高。通过大体积进样和在线富集,提高了方法灵敏度,解决了微量组分的检测难题。建立的方法准确、简便、灵敏、通用性强,适用于2460A发酵工艺优化过程中的大量检测研究工作,同时为复杂基质中微量代谢产物的测定提供了方法依据和借鉴。
2 实验部分
2.1 仪器与试剂
Determination of 2460A in Trichoderma Hazianum Fermentation
Liquor by Online Twodimensional Liquid Chromatographic Method
SHAN GuangZhi*, ZHOU Jie, ZUO LiMin, JIANG Wei, LIU GuiXia, ZHANG Yang, LI Yuan, JIANG Rong*
(Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing 100050, China)
Abstract An online two dimensional liquid chromatographic (2DLC) method was established by using an ultimate dual gradient liquid chromatography, three chromatographic columns and valveswitching technology to detect 2460A in trichoderma hazianum fermentation liquor. MF C8 column (10 mm×4.6 mm, 5.0 μm) was used as purification column and MG C18 column (20 mm×4.6 mm, 5.0 μm) was used as enriching column. Methanol and water were used as mobile phase with a gradient elution at a flow rate of 2.0 mL/min. The sample was separated on the Thermo Hypersil GOLD C18 column (250 mm×4.6 mm, 5.0 μm) maintained at 40 ℃ using methanol and water. The flow rate was 1.0 mL/min and 1.0 mL sample was injected into the 2DLC system. The detection wavelength was 424 nm. The whole analytical time was less than 60 min. The standard curve was linear over the 2460A concentration range of 0.0025-10.0 mg/L(r=0.9981, n=8). The limit of detection was calculated to be 1.2 μg/L (S/N=3) and the limit of quantification was calculated to be 2.5 μg/L (S/N=10). The average recoveries varied from 88.0% to 104.4%.
Keywords Twodimensional liquid chromatography; Trichoderma hazianum; Fermentation liquor; 2460A
(Received 29 August 2014; accepted 20 October 2014)
This work was supported by the National S & T Major Special Project on Major New Drug Innovation (No. 2012zx09301002001019)
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