CN101266231A - A method for identifying paederin - Google Patents
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- CN101266231A CN101266231A CNA2008100362454A CN200810036245A CN101266231A CN 101266231 A CN101266231 A CN 101266231A CN A2008100362454 A CNA2008100362454 A CN A2008100362454A CN 200810036245 A CN200810036245 A CN 200810036245A CN 101266231 A CN101266231 A CN 101266231A
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Abstract
本发明公开了一种毒隐翅虫素的鉴别方法,其包括样品处理及数据获取过程,所述样品处理是将毒隐翅虫提取物以50%HPLC级甲醇稀释100倍;所述数据获取是首先利用液相色谱-质谱联用仪在负离子模式和正离子模式下分别扫描毒隐翅虫提取物稀释液,以获取母离子峰数据;然后采用微量进样泵柱后注射方式进行碰撞诱导解离和扫描,以获取子离子峰数据。本发明提供的毒隐翅虫素的鉴别方法,由于采用了液相色谱-质谱联用系统,使得该鉴别方法具有优良的灵敏度,鉴别结果准确、有效及可靠,且操作方便,为毒隐翅虫提取物在临床上的研究提供了方便。
The invention discloses a method for identifying paederin, which includes sample processing and data acquisition processes. The sample processing is to dilute the extract of paederin with 50% HPLC grade methanol 100 times; the data acquisition First, use liquid chromatography-mass spectrometry to scan the diluent of Paederus venomosa extract in negative ion mode and positive ion mode to obtain the parent ion peak data; ionization and scanning to obtain product ion peak data. The identification method of the poisonous paederin provided by the present invention adopts the liquid chromatography-mass spectrometry system, so that the identification method has excellent sensitivity, the identification result is accurate, effective and reliable, and the operation is convenient. The clinical research of worm extract provides convenience.
Description
技术领域 technical field
本发明涉及一种毒隐翅虫素的鉴别方法,具体说,是涉及一种利用液相色谱-质谱联用法鉴别毒隐翅虫素的方法。The invention relates to a method for identifying paederin, in particular to a method for identifying paederin by means of liquid chromatography-mass spectrometry.
背景技术 Background technique
毒隐翅虫是隐翅虫科中的一个属,已知种类超过250种,产自除南极洲以外的世界各地,中国已报道的毒隐翅虫有21种。毒隐翅虫主要生活在潮湿的农田、菜地、杂草丛中,是山沟、农田常见的小型昆虫。我国常见的毒隐翅虫有青翅蚁形隐翅虫(又称梭形毒隐翅虫、黄足蚁形隐翅虫),黑足蚁形隐翅虫,多毛隐翅虫,巾形毒隐翅虫,小红毒隐翅虫,蚁态毒隐翅虫,西藏毒隐翅虫等7种,其中以青翅蚁形隐翅虫分布最广,遍及各省及台湾等地区。Poisonous paederus is a genus of Paederidae, with more than 250 known species, which are produced in all parts of the world except Antarctica. There are 21 species of poisonous paederus reported in China. Poisonous paederus mainly lives in wet farmland, vegetable fields, and weeds, and is a common small insect in ravines and farmland. Common poisonous paederus in my country include green winged paederus (also known as fusiform poisonous paederus, yellow-footed paederus), black-footed paederus, hairy paederus, towel-shaped paederus There are 7 kinds of paederus, paederus small red, paederus ant, and paederus tibetan. Among them, paederus blue-winged ant is the most widely distributed, covering all provinces and Taiwan and other regions.
毒隐翅虫的卵、幼虫、蛹和成虫(除翅以外的各部分)都有毒素存在,成虫的毒素主要来源于血淋巴,雌、雄虫体内都含有毒素。毒隐翅虫的毒素是虫体用来抵御外来侵袭的化学物质。毒隐翅虫的毒液化学成分复杂,含有醛类、醌类、萜类等,有报道其毒素主要为3种,分别是毒隐翅虫素、拟毒隐翅虫素和毒隐翅虫酮。其中,毒隐翅虫素的含量最高,分子量为503.65,溶于有机溶剂乙醇、乙醚、氯仿及苯,不溶于水,结晶的毒隐翅虫素在112℃熔化,弱酸性,水中煮沸1h不被破坏。Eggs, larvae, pupae and adults (all parts except wings) of poisonous paederus have toxins, and the toxins of adults mainly come from hemolymph, and both female and male worms contain toxins. The toxins of poisonous paederus are chemicals that the insect body uses to defend against foreign invasion. The chemical composition of the venom of the poisonous paederus is complex, containing aldehydes, quinones, terpenes, etc. It has been reported that there are mainly three types of toxins, namely, the poisonous paederin, the poisonous paederin and the poisonous paederin . Among them, paederin has the highest content, with a molecular weight of 503.65. It is soluble in organic solvents such as ethanol, ether, chloroform and benzene, but insoluble in water. The crystallized paederin melts at 112°C. It is weakly acidic and can be boiled in water for 1 hour. destroyed.
本发明人研究发现毒隐翅虫提取物具有促进糖尿病皮肤溃疡愈合、抑制HL-60人白血病细胞株生长、抑制BEL-7402人肝癌细胞株生长及促进毛发生长的药用功效,在医学上具有广泛的应用前景,可望用于制备治疗糖尿病皮肤顽固性溃疡、治疗某些类型的人白血病和人肝癌及促进毛发生长的药物制剂。但目前尚未有毒隐翅虫素的鉴别方法,使毒隐翅虫提取物在临床上的研究存在很大困难。The present inventors have found that the extract of Paederus venomosa has the medicinal effects of promoting the healing of diabetic skin ulcers, inhibiting the growth of HL-60 human leukemia cell lines, inhibiting the growth of BEL-7402 human liver cancer cell lines, and promoting hair growth. It has wide application prospects and is expected to be used for preparing pharmaceutical preparations for treating refractory diabetic skin ulcers, treating certain types of human leukemia and human liver cancer, and promoting hair growth. However, there is no identification method for toxic paederin, which makes the clinical research of poisonous paederin extract very difficult.
发明内容Contents of the invention
本发明的目的是提供一种毒隐翅虫素的鉴别方法,为毒隐翅虫提取物在临床上的研究提供便捷。The purpose of the present invention is to provide a method for identifying paederin to provide convenience for the clinical research of the extract of paederin.
为解决上述技术问题,本发明的技术方案如下:In order to solve the problems of the technologies described above, the technical solution of the present invention is as follows:
本发明的毒隐翅虫素的鉴别方法,包括样品处理及数据获取过程,其特征在于,所述样品处理是将毒隐翅虫提取物以50%HPLC级甲醇稀释100倍;所述数据获取是首先利用液相色谱-质谱联用仪在负离子模式和正离子模式下分别扫描毒隐翅虫提取物稀释液,以获取母离子峰数据;然后采用微量进样泵柱后注射方式进行碰撞诱导解离和扫描,以获取子离子峰数据。The identification method of paederin of the present invention comprises sample processing and data acquisition process, is characterized in that, described sample processing is that the extract of paederus is diluted 100 times with 50% HPLC grade methanol; The data acquisition First, use liquid chromatography-mass spectrometry to scan the diluent of Paederus venomosa extract in negative ion mode and positive ion mode to obtain the parent ion peak data; ionization and scanning to obtain product ion peak data.
所述液相色谱条件为:色谱柱是Shiseido C18/100×2.1mm/5u、流动相是体积比为70∶30的甲醇一水及含1mM甲酸铵的溶液体系、流速是0.2ml·min-1、柱温是30℃、进样量为10μl;所述质谱条件为:Turbo Ionspray离子源、三重四极杆质量检测器、喷雾气13L/min、气帘气12L/min、喷雾电压5KV、聚焦电压400V、入口电压10V、出口电压10V、辅助加热器温度450℃及扫描质量范围为400~800amu。The liquid chromatography conditions are as follows: the chromatographic column is Shiseido C18/100×2.1mm/5u, the mobile phase is a solution system of methanol-water with a volume ratio of 70:30 and 1mM ammonium formate, and the flow rate is 0.2ml min- 1. The column temperature is 30°C, the injection volume is 10 μl; the mass spectrometry conditions are: Turbo Ionspray ion source, triple quadrupole mass detector, spray gas 13L/min, curtain gas 12L/min, spray voltage 5KV, focusing The voltage is 400V, the inlet voltage is 10V, the outlet voltage is 10V, the auxiliary heater temperature is 450°C and the scanning mass range is 400-800amu.
所述母离子峰数据为:色谱峰保留时间tR=4.0min,准分子离子峰[M-H]-为m/z=502.4,与甲酸根的加合离子峰[M+HCOO]-为m/z=548.4,与钠离子的加合离子峰[M+Na]+为m/z=526.4;所述子离子峰数据为:m/z 470、m/z 438,m/z502.2、m/z 470、m/z 438,m/z 494.2、m/z 462.2、m/z 340。The parent ion peak data are: chromatographic peak retention time t R =4.0min, quasi-molecular ion peak [MH] -is m/z=502.4, and formate ion peak [M+HCOO ] -is m/ z=548.4, the adducted ion peak [M+Na] + with sodium ion is m/z=526.4; the product ion peak data are: m/z 470, m/z 438, m/z 502.2, m /z 470, m/z 438, m/z 494.2, m/z 462.2, m/
本发明提供的毒隐翅虫素的鉴别方法,由于采用了液相色谱-质谱联用系统,使得该鉴别方法具有优良的灵敏度,鉴别结果准确、有效及可靠,且操作方便,为毒隐翅虫提取物在临床上的研究提供了方便。The identification method of the poisonous paederin provided by the present invention adopts the liquid chromatography-mass spectrometry system, so that the identification method has excellent sensitivity, the identification result is accurate, effective and reliable, and the operation is convenient. The clinical research of worm extract provides convenience.
附图说明 Description of drawings
图1为毒隐翅虫提取物稀释液在负离子模式下扫描的0~8min色谱图;Fig. 1 is the 0~8min chromatogram of the poisonous paederus extract diluent scanned in negative ion mode;
图2为毒隐翅虫提取物稀释液在正离子模式下扫描的0~8min色谱图;Fig. 2 is the 0~8min chromatogram of the poisonous paederus extract diluent scanned in positive ion mode;
图3为母离子峰(tR=4.0min)在负离子模式下的质谱图;Fig. 3 is the mass spectrogram of parent ion peak (t R =4.0min) in negative ion mode;
图4为母离子峰(tR=4.0min)在正离子模式下的质谱图;Fig. 4 is the mass spectrogram of parent ion peak (t R =4.0min) in positive ion mode;
图5为m/z=526.4离子的子离子质谱图;Fig. 5 is the product ion mass spectrum of m/z=526.4 ion;
图6为m/z=548.4离子的子离子质谱图;Fig. 6 is the product ion mass spectrum of m/z=548.4 ion;
图7为m/z=502.2离子的子离子质谱图;Fig. 7 is the product ion mass spectrum of m/z=502.2 ion;
图8为毒隐翅虫素化合物的化学分子结构式。Fig. 8 is the chemical molecular structural formula of the compound of paederin.
具体实施方式 Detailed ways
下面通过实施例对本发明作进一步说明,其目的仅在于更好理解本发明的内容而非限制本发明的保护范围。The present invention will be further described below by embodiment, and its purpose is only to understand content of the present invention better but not limit the protection scope of the present invention.
实施例Example
1、样品处理1. Sample processing
将毒隐翅虫提取物以50%HPLC级甲醇稀释100倍。The Paederus venom extract was diluted 100-fold with 50% HPLC grade methanol.
2、数据获取2. Data acquisition
a)母离子峰数据的获取a) Acquisition of parent ion peak data
液相色谱条件为:色谱柱是Shiseido C18/100×2.1mm/5u、流动相是体积比为70∶30的甲醇一水及含1mM甲酸铵的溶液体系、流速是0.2ml·min-1、柱温是30℃、进样量为10μl;质谱条件为:Turbo Ionspray离子源、三重四极杆质量检测器、喷雾气13L/min、气帘气12L/min、喷雾电压5KV、聚焦电压400V、入口电压10V、出口电压10V及辅助加热器温度450℃;质谱工作方式设定为Q1扫描,扫描质量范围为400~800amu,分别在负离子模式和正离子模式下扫描毒隐翅虫提取物稀释液,得到在不同电离模式下扫描的0~8min色谱图(见图1和图2所示),可见两图在tR=4.0min处有一个共有峰。The liquid chromatographic conditions are as follows: the chromatographic column is Shiseido C18/100×2.1mm/5u, the mobile phase is a solution system of methanol-water with a volume ratio of 70:30 and 1mM ammonium formate, and the flow rate is 0.2ml·min -1 , The column temperature is 30°C, the injection volume is 10 μl; the mass spectrometry conditions are: Turbo Ionspray ion source, triple quadrupole mass detector, spray gas 13L/min, curtain gas 12L/min, spray voltage 5KV, focusing voltage 400V, inlet The voltage is 10V, the outlet voltage is 10V, and the temperature of the auxiliary heater is 450°C; the mass spectrometry work mode is set to Q1 scanning, and the scanning mass range is 400-800 amu. The diluent of Paederus venomosa extract is scanned in negative ion mode and positive ion mode respectively, and the obtained The 0-8min chromatograms scanned under different ionization modes (see Fig. 1 and Fig. 2), it can be seen that there is a common peak at t R = 4.0min in the two diagrams.
由该共有峰(tR=4.0min)在负离子模式和正离子模式下的质谱图(见图3和图4所示)可见:m/z=502.4的离子为毒隐翅虫素化合物分子的准分子离子,可表示为[M-H]-;m/z=548.4的离子为毒隐翅虫素化合物分子与甲酸根的加合离子,可表示为[M+HCOO]-;m/z=526.4离子为毒隐翅虫素化合物分子与钠离子的加合离子,可表示为[M+Na]+。From the mass spectrograms (shown in Figure 3 and Figure 4) of the common peak (t R =4.0min) in negative ion mode and positive ion mode: it can be seen that the ion of m/z=502.4 is the quasi The molecular ion can be expressed as [MH] - ; the ion of m/z=548.4 is the adduct ion of the compound molecule of paederin compound and formate, which can be expressed as [M+HCOO] - ; the ion of m/z=526.4 It is the adduct ion of paederin compound molecule and sodium ion, which can be expressed as [M+Na] + .
b)子离子峰数据的获取b) Acquisition of product ion peak data
采用微量进样泵柱后注射方式,对m/z=526.4、548.4、502.2进行子离子扫描,毒隐翅虫提取物稀释液进样速度为10ul/min,碰撞气为7L/min。The post-column injection method of the micro-injection pump was used to scan the product ion for m/z=526.4, 548.4, and 502.2. The injection speed of the diluent of Paederus venomosa extract was 10ul/min, and the collision gas was 7L/min.
由图5可见,m/z=526.4离子的子离子质谱有m/z=494.2、m/z=462.2及m/z=340,其中m/z=494.2、m/z=462.2为[M+Na]+丢失CH3OH的子离子,m/z=340为羰基α侧链断裂的形成的子离子。It can be seen from Fig. 5 that the product ion mass spectrum of m/z=526.4 ion has m/z=494.2, m/z=462.2 and m/z=340, wherein m/z=494.2, m/z=462.2 are [M+ Na] + loses the product ion of CH 3 OH, and m/z=340 is the product ion formed by the carbonyl α side chain breaking.
由图6可见,m/z=548.4离子的子离子质谱有m/z=502.2、m/z=470及m/z=438,其中m/z=502.2佐证了m/z=548.4([M+HCOO]-)和[M-H]-的关系,m/z=470、m/z=438为[M-H]-+丢失CH3OH的子离子。As can be seen from Figure 6, the product ion mass spectrum of m/z=548.4 ion has m/z=502.2, m/z=470 and m/z=438, wherein m/z=502.2 proves that m/z=548.4 ([M +HCOO] - ) and [MH] - relationship, m/z=470, m/z=438 is the product ion of [MH] -+ missing CH 3 OH.
由图7可见,m/z=502.2离子的子离子质谱有m/z=470和m/z=438,它们为[M-H]-+丢失CH3OH的子离子。It can be seen from Fig. 7 that the product ion mass spectrum of m/z=502.2 ion has m/z=470 and m/z=438, which are the product ions of [MH] -+ loss of CH 3 OH.
综合上述数据分析可见,tR=4.0min色谱峰对应的化合物分子结构中有多个CH3O基团,该化合物的ESI质谱易形成加合离子,分子量为503,与毒隐翅虫素化合物的化学分子结构特征完全吻合,由此可以说明本发明的毒隐翅虫素的鉴别方法可以准确鉴别出毒隐翅虫提取物中的毒隐翅虫素。Based on the analysis of the above data, it can be seen that there are multiple CH 3 O groups in the molecular structure of the compound corresponding to the t R =4.0min chromatographic peak, and the ESI mass spectrum of this compound is easy to form adducted ions, with a molecular weight of 503, which is similar to that of the compound Pederin. The chemical molecular structure characteristics of the two compounds are completely consistent, thus it can be explained that the identification method of paederin in the present invention can accurately identify the paederin in the extract of paederin.
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CN101773101B (en) * | 2010-03-03 | 2012-07-04 | 中国农业科学院植物保护研究所 | Method for artificially and massively feeding natural enemy staphylinid |
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