CN101266231A - A method for identifying paederin - Google Patents

Abstract

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

A method for identifying paederin

technical field

The invention relates to a method for identifying paederin, in particular to a method for identifying paederin by means of liquid chromatography-mass spectrometry.

Background technique

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.

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.

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.

Contents of the invention

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.

In order to solve the problems of the technologies described above, the technical solution of the present invention is as follows:

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.

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.

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/z 340.

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 of drawings

Fig. 1 is the 0～8min chromatogram of the poisonous paederus extract diluent scanned in negative ion mode;

Fig. 2 is the 0～8min chromatogram of the poisonous paederus extract diluent scanned in positive ion mode;

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

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

Fig. 5 is the product ion mass spectrum of m/z=526.4 ion;

Fig. 6 is the product ion mass spectrum of m/z=548.4 ion;

Fig. 7 is the product ion mass spectrum of m/z=502.2 ion;

Fig. 8 is the chemical molecular structural formula of the compound of paederin.

Detailed ways

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.

Example

1. Sample processing

The Paederus venom extract was diluted 100-fold with 50% HPLC grade methanol.

2. Data acquisition

a) Acquisition of parent ion peak data

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.

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) Acquisition of product ion peak data

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.

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.

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.

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.

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.

Claims (3)

1. a discriminating method of paederin, comprising sample processing and data acquisition process, characterized in that the sample processing is to dilute the paederin extract 100 times with 50% HPLC grade methanol; the data Acquisition is firstly using liquid chromatography-mass spectrometry to scan the diluent of Paederus venomosa extract in negative ion mode and positive ion mode to obtain parent ion peak data; Dissociate and scan for product ion peak data. 2. The method for identifying paederin according to claim 1, wherein the liquid chromatography conditions are as follows: the chromatographic column is Shiseido C18/100×2.1mm/5u, and the mobile phase has a volume ratio of 70 : 30 methanol-water and a solution system containing 1 mM ammonium formate, the flow rate is 0.2ml·min ^-1 , the column temperature is 30°C, and the injection volume is 10 μl; the mass spectrometry conditions are: Turbo Ionspray ion source, triple quadrupole Rod mass detector, spray gas 13L/min, air curtain gas 12L/min, spray voltage 5KV, focus voltage 400V, inlet voltage 10V, outlet voltage 10V, auxiliary heater temperature 450°C and scanning mass range 400-800amu. 3. the differential method of paederin according to claim 1, is characterized in that, described parent ion peak data is: chromatographic peak retention time t _R =4.0min, quasi-molecular ion peak [MH] ^- is m /z=502.4, the adducted ion peak [M+HCOO] with the formate group ^is m/z=548.4, and the adducted ion peak [M+Na] ⁺ with the sodium ion is m/z=526.4; 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/z 340.

Images