CN101266231A

CN101266231A - Pederin discrimination method

Info

Publication number: CN101266231A
Application number: CNA2008100362454A
Authority: CN
Inventors: 万平; 李利珍; 赵梅君; 胡佳耀
Original assignee: 上海师范大学
Priority date: 2008-04-18
Filing date: 2008-04-18
Publication date: 2008-09-17
Also published as: CN101266231B

Abstract

The invention discloses a discrimination method of Paederus comprising a sample processing and data capturing process, wherein the Paederus extract product is diluted by 100 times using 50% of HPLC methanol and the data capturing process comprises the following steps: respectively scanning the Paederus extract product diluent in anion mode and cation mode using the liquid chromagraphy/mass spectrometry to acquire parent ion peak value; performing the collision induced dissociation and scanning using trace sampling pump post-column injection system to acquire daughter ion peak data. The discrimination method of Paederus uses liquid chromagraphy/mass spectrometry to obtain the features that: excellent sensitivity, accurate discrimination result, efficiency and reliability, and convenient operation and provides convenience for the clinical research on the Paederus extract product.

Description

A kind of discrimination method of malicious pederin

Technical field

The present invention relates to a kind of discrimination method of malicious pederin, specifically, relate to a kind of method of utilizing the liquid chromatograph mass spectrography method to differentiate malicious pederin.

Background technology

The poison rove beetle is a genus in the Staphylinidae, and known kind surpasses 250 kinds, originates from except that the Antarctica all over the world, and the Chinese malicious rove beetle of having reported has 21 kinds.The poison rove beetle mainly lives in moist farmland, vegetable plot, the weeds clump, is the common small insects in gully, farmland.The common malicious rove beetle of China has blue or green wing ant shape rove beetle (claiming fusiformis poison rove beetle, yellow sufficient ant shape rove beetle again), black sufficient ant shape rove beetle, the crinosity rove beetle, towel shape poison rove beetle, little red malicious rove beetle, ant attitude poison rove beetle, 7 kinds of Tibet poison rove beetles etc., it is the widest wherein to distribute with blue or green wing ant shape rove beetle, spreads all over areas such as each province and Taiwan.

Ovum, larva, pupa and the adult (each several part except that wing) of poison rove beetle all have toxin existence, and the toxin of adult is mainly derived from hemolymph, all contain toxin in female, the male worm body.The toxin of poison rove beetle is the chemical substance that polypide is used for resisting external invasion and attack.The venom complex chemical composition of poison rove beetle contains aldehydes, quinones, terpene etc., has its toxin of report to be mainly 3 kinds, is respectively malicious pederin, plan poison pederin and malicious rove beetle ketone.Wherein, the content of malicious pederin is the highest, and molecular weight is 503.65, is dissolved in organic solvent ethanol, ether, chloroform and benzene, and is water insoluble, and the malicious pederin of crystallization is 112 ℃ of fusings, and faintly acid is boiled 1h and is not destroyed in the water.

The inventor discovers that malicious rove beetle extract has the healing of the diabetes skin ulcer of promotion, suppresses the growth of HL-60 human leukemia cell line, suppresses growth of BEL-7402 human hepatoma cell strain and trichogenous medicinal efficacy, medically be with a wide range of applications, be expected to be used to prepare treatment diabetic skin telephium, the human leukemia for the treatment of some type and people's liver cancer and trichogenous pharmaceutical preparation.But at present the discrimination method of poisonous pederin not as yet makes the research clinically of malicious rove beetle extract have very big difficulty.

Summary of the invention

The discrimination method that the purpose of this invention is to provide a kind of malicious pederin is for the research clinically of malicious rove beetle extract provides convenient.

For solving the problems of the technologies described above, technical scheme of the present invention is as follows:

The discrimination method of malicious pederin of the present invention comprises sample preparation and data acquisition, it is characterized in that, described sample preparation is with 100 times of 50%HPLC level methyl alcohol dilutions with malicious rove beetle extract; It is at first to utilize liquid chromatograph-mass spectrometer to scan malicious rove beetle extract dilution respectively under negative ion mode and positive ion mode that described data are obtained, to obtain the parent ion peak data; Injection system carries out collision induced dissociation and scanning after adopting the micro-sampling pump plunger shaft then, to obtain the daughter ion peak data.

Described liquid phase chromatogram condition is: chromatographic column is that Shiseido C18/100 * 2.1mm/5u, moving phase are that volume ratio is that 70: 30 methyl alcohol one water and solution system, the flow velocity that contains the 1mM ammonium formate are 0.2mlmin ^-1, column temperature is that 30 ℃, sample size are 10 μ l; Described mass spectrum condition is: Turbo Ionspray ion gun, triple quadrupole bar mass detector, spraying gas 13L/min, gas curtain gas 12L/min, spray voltage 5KV, focus voltage 400V, inlet voltage 10V, outlet voltage 10V, 450 ℃ of auxiliary heating actuator temperatures and quality of scanning scope are 400～800amu.

Described parent ion peak data are: chromatographic peak retention time t _R=4.0min, quasi-molecular ion peak [M-H] ^-Be m/z=502.4, with the adduct ion peak [M+HCOO] of formate ^-Be m/z=548.4, with the adduct ion peak [M+Na] of sodion ⁺Be m/z=526.4; Described daughter ion peak data is: 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 discrimination method of malicious pederin provided by the invention, owing to adopted the liquid chromatograph mass spectrography system, make this discrimination method have good sensitivity, identification result is accurate and effective and reliable, and easy to operate, for the research clinically of malicious rove beetle extract provides convenience.

Description of drawings

0～8min chromatogram that Fig. 1 scans under negative ion mode for malicious rove beetle extract dilution;

0～8min chromatogram that Fig. 2 scans under positive ion mode for malicious rove beetle extract dilution;

Fig. 3 is parent ion peak (t _R=4.0min) the mass spectrogram under negative ion mode;

Fig. 4 is parent ion peak (t _R=4.0min) the mass spectrogram under positive ion mode;

Fig. 5 is the daughter ion mass spectrogram of m/z=526.4 ion;

Fig. 6 is the daughter ion mass spectrogram of m/z=548.4 ion;

Fig. 7 is the daughter ion mass spectrogram of m/z=502.2 ion;

Fig. 8 is the chemical molecular structural formula of malicious pederin compound.

Embodiment

The invention will be further described below by embodiment, and its purpose only is better to understand content of the present invention and unrestricted protection scope of the present invention.

Embodiment

1, sample preparation

Malicious rove beetle extract is diluted 100 times with 50%HPLC level methyl alcohol.

2, data are obtained

A) the parent ion peak data obtains

Liquid phase chromatogram condition is: chromatographic column is that Shiseido C18/100 * 2.1mm/5u, moving phase are that volume ratio is that 70: 30 methyl alcohol one water and solution system, the flow velocity that contains the 1mM ammonium formate are 0.2mlmin ^-1, column temperature is that 30 ℃, sample size are 10 μ l; The mass spectrum condition is: 450 ℃ of Turbo Ionspray ion gun, triple quadrupole bar mass detector, spraying gas 13L/min, gas curtain gas 12L/min, spray voltage 5KV, focus voltage 400V, inlet voltage 10V, outlet voltage 10V and auxiliary heating actuator temperatures; The mass spectrum working method is set at Q1 scanning, the quality of scanning scope is 400～800amu, the malicious rove beetle extract dilution of scanning under negative ion mode and positive ion mode obtains 0～8min chromatogram (seeing illustrated in figures 1 and 2) of scanning under different ionization patterns respectively, and visible two figure are at t _RThere is a total peak at=4.0min place.

By this total peak (t _R=4.0min) mass spectrogram under negative ion mode and positive ion mode (seeing Fig. 3 and shown in Figure 4) is as seen: the ion of m/z=502.4 is the quasi-molecular ion of malicious pederin compound molecule, can be expressed as [M-H] ^-The ion of m/z=548.4 is the adduct ion of malicious pederin compound molecule and formate, can be expressed as [M+HCOO] ^-The m/z=526.4 ion is the adduct ion of malicious pederin compound molecule and sodion, can be expressed as [M+Na] ⁺

B) the daughter ion peak data obtains

Injection system behind the employing micro-sampling pump plunger shaft carries out daughter ion scanning to m/z=526.4,548.4,502.2, and malicious rove beetle extract dilution sample introduction speed is 10ul/min, and collision gas is 7L/min.

As seen from Figure 5, the daughter ion mass spectrum of m/z=526.4 ion has m/z=494.2, m/z=462.2 and m/z=340, and wherein m/z=494.2, m/z=462.2 are [M+Na] ⁺Lose CH ₃The daughter ion of OH, m/z=340 are the daughter ion of the formation of carbonyl α side chain fracture.

As seen from Figure 6, the daughter ion mass spectrum of m/z=548.4 ion has m/z=502.2, m/z=470 and m/z=438, and wherein m/z=502.2 has proved m/z=548.4 ([M+HCOO] ^-) and [M-H] ^-Relation, m/z=470, m/z=438 are [M-H] ^-+Lose CH ₃The daughter ion of OH.

As seen from Figure 7, the daughter ion mass spectrum of m/z=502.2 ion has m/z=470 and m/z=438, and they are [M-H] ^-+Lose CH ₃The daughter ion of OH.

Comprehensive above-mentioned data analysis as seen, t _RIn the molecular structure of compounds of=4.0min chromatographic peak correspondence a plurality of CH are arranged ₃The O group, the ESI mass spectrum of this compound easily forms adduct ion, molecular weight is 503, fits like a glove with the chemical molecular architectural feature of malicious pederin compound, and the discrimination method that it can be said that bright malicious pederin of the present invention can accurately identify the malicious pederin in the malicious rove beetle extract.

Claims

1. the discrimination method of a malicious pederin comprises sample preparation and data acquisition, it is characterized in that, described sample preparation is with 100 times of 50%HPLC level methyl alcohol dilutions with malicious rove beetle extract; It is at first to utilize liquid chromatograph-mass spectrometer to scan malicious rove beetle extract dilution respectively under negative ion mode and positive ion mode that described data are obtained, to obtain the parent ion peak data; Injection system carries out collision induced dissociation and scanning after adopting the micro-sampling pump plunger shaft then, to obtain the daughter ion peak data.

2. the discrimination method of malicious pederin according to claim 1, it is characterized in that described liquid phase chromatogram condition is: chromatographic column is that Shiseido C18/100 * 2.1mm/5u, moving phase are that volume ratio is that 70: 30 methyl alcohol one water and solution system, the flow velocity that contains the 1mM ammonium formate are 0.2mlmin ^-1, column temperature is that 30 ℃, sample size are 10 μ l; Described mass spectrum condition is: Turbo Ionspray ion gun, triple quadrupole bar mass detector, spraying gas 13L/min, gas curtain gas 12L/min, spray voltage 5KV, focus voltage 400V, inlet voltage 10V, outlet voltage 10V, 450 ℃ of auxiliary heating actuator temperatures and quality of scanning scope are 400～800amu.

3. the discrimination method of malicious pederin according to claim 1 is characterized in that, described parent ion peak data are: chromatographic peak retention time t _R=4.0min, quasi-molecular ion peak [M-H] ^-Be m/z=502.4, with the adduct ion peak [M+HCOO] of formate ^-Be m/z=548.4, with the adduct ion peak [M+Na] of sodion ⁺Be m/z=526.4; Described daughter ion peak data is: m/z 470, m/z 438, m/z 502.2, m/z 470, m/z 438, m/z494.2, m/z462.2, m/z340.