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Liquid-mass spectrometry detection method for identifying species of pangolin

A detection method, liquid chromatography-mass spectrometry technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of weak specificity, damage, and the influence of subjective factors, and achieve strong specificity, overcome trait identification, The effect of high accuracy

Active Publication Date: 2018-02-13
广东省药品检验所
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] In pangolin quality standards and related research materials, traditional trait identification can distinguish different species of pangolins, but trait identification is an empirical identification, which may be affected by human subjective factors, and pangolins are deformed, brittle, and easily broken after processing. Even the crushed pangolin finished products (pangolin formula granules), pangolin-containing prescription preparations, etc., mostly no longer exist in appearance, size, and size, so it is impossible to judge the source of pangolins; use PCR-DNA and other general detection methods for protein components, The specificity is not strong. During the processing of pangolins, factors such as temperature and time of sand scalding cause different degrees of collagen damage, which directly affects the stability and reproducibility of the results of the above method, and this method is only applicable to authentic and Identification of pure and counterfeit products, but helpless to the problem of some counterfeit products mixed with genuine products

Method used

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  • Liquid-mass spectrometry detection method for identifying species of pangolin
  • Liquid-mass spectrometry detection method for identifying species of pangolin
  • Liquid-mass spectrometry detection method for identifying species of pangolin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Establishment of Enzymatic Peptide Map

[0021] 1.1 Instruments and materials: Agilent 6540UHDAccurate-Mass Q-TOF LC / MS; Chromatographic column: Agilent Zorbax 300SB-C18 (3.5μm, 2.1×100mm). Trypsin was purchased from Sigma, Promega and China National Institutes for Food and Drug Control. Dithiothreitol, iodoacetamide, and formic acid were purchased from Sigma. Ammonium bicarbonate was analytically pure. Chinese pangolin control medicinal materials were obtained from China National Institutes for Food and Drug Control. Tree pangolin and Indian pangolin working reference medicinal materials were identified by Guangdong Provincial Institute for Drug Control for use in this study.

[0022] 1.2 Sample treatment: take a pangolin sample, smash it into powder, pass it through a No. 6 sieve, weigh 10 mg of the powder, add 1 ml of 8M urea, add 10 μl of 1M dithiothreitol, heat in a water bath at 60°C for 30 minutes, let it cool, and add 1M dithiothreitol. Iodoacetami...

Embodiment 2

[0027] The investigation of embodiment 2 different enzymolysis conditions

[0028] In order to ensure the complete enzymatic hydrolysis of trypsin, different enzymatic hydrolysis conditions were investigated. Four copies of 10 mg and 20 mg of the Chinese pangolin control medicinal material were taken respectively, and trypsin (1 μg / μl) 10, 20, 50, and 100 μl were added to each of the four copies, and pressed For the "sample processing" operation described in Example 1, inject samples according to the method under the "chromatographic and mass spectrometry conditions" described in Example 1, and record the peptide map. Select 10 chromatographic peaks (m / z 731.4053, 511.2301, 832.2751, 605.3276, 543.3145, 617.0039, 934.9308, 910.4491, 1032.5203, 891.4426) at different time points of 0-70min to extract the chromatographic peaks, and calculate the peak areas of the 10 chromatographic peaks. The result shows (see table 1), when substrate is 20mg, each extraction ion chromatogram pe...

Embodiment 3

[0031]Examination of Example 3 Precision of Liquid-mass Spectrometry Method

[0032] Take 10 mg of Chinese pangolin control medicinal material, operate according to the "sample treatment" described in Example 1, repeat the sample injection 6 times according to the chromatographic and mass spectrometry conditions described in Example 1, and record the peptide map. Select 10 chromatographic peaks (m / z731.4053, 511.2301, 832.2751, 605.3276, 543.3145, 617.0039, 934.9308, 910.4491, 1032.5203, 891.4426) at different time points of 0-70min to extract the chromatographic peaks, and calculate the retention time of the 10 chromatographic peaks, Accurate mass and peak area. Result shows (seeing table 2), the retention time of each chromatographic peak, accurate mass number and peak area are consistent substantially, and the RSD of retention time and accurate mass number is all less than 0.3%, and the RSD of peak area is less than 10%, meets the requirement.

[0033] Table 2. Retention t...

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Abstract

The invention discloses a liquid-mass spectrometry detection method for identifying species of pangolin. The liquid-mass spectrometry detection method comprises the following steps of (1) preparing atest sample solution; (2) analyzing a sample by a liquid-mass spectrometry detection method; (3) utilizing a feature ion qualitative identification method to identify the sample. By adopting the technical scheme, the liquid-mass spectrometry detection method has the advantages that the base source of Chinese pangolin, manis tricuspis and Indian pangolin can be identified; the accuracy is high, thespecificity is strong, and the defects of character identification and DNA (deoxyribonucleic acid) detection are overcome.

Description

technical field [0001] The invention relates to a detection method for identifying the origin of traditional Chinese medicines, in particular to a detection method and application for identifying the species of pangolins, and belongs to the fields of drug analysis technology and germplasm resource identification. Background technique [0002] The 2015 edition of the "Chinese Pharmacopoeia" stipulates that pangolins are derived from the scales of the Chinese pangolin Manispentadactyla Linnaeus, an animal of the carp family. , milk obstruction, carbuncle sore, rheumatic arthralgia, stroke paralysis, numbness and spasm embolism. Its processed products are generally used clinically: Pao Shan Jia and Vinegar Shan Jia. Paogolin is made by scalding raw pangolin; vinegar golin is made by scalding and quenching with vinegar. [0003] The pangolin is a national second-class wildlife protection animal. Its curative effect is definite, but it is relatively difficult to raise, medicina...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 刘潇潇罗卓雅李华谢健鸣乔莉
Owner 广东省药品检验所
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