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CN-122017063-A - Content determination method for simultaneously detecting two polypeptides in cantharis body

CN122017063ACN 122017063 ACN122017063 ACN 122017063ACN-122017063-A

Abstract

The invention discloses a content determination method for simultaneously detecting two polypeptides in cantharis, which takes characteristic polypeptides BM-1 (AGDDAPRAVF) and BM-2 (YSF) as indexes and adopts high performance liquid chromatography-tandem mass spectrometry technology to perform quantitative analysis. The method comprises the steps of sample enzymolysis, chromatographic separation and mass spectrum detection, and has the characteristics of strong specificity, high sensitivity and good accuracy. Meanwhile, a matched detection kit is provided, and the kit comprises key components such as a reference substance, an enzymolysis reagent and chromatographic flow. The method can be used for quality evaluation of Mylabris, quality control of decoction pieces and authenticity identification, and provides a new technical means for perfecting Mylabris quality evaluation system.

Inventors

  • ZHANG JIANYONG
  • ZHANG XIAOHONG
  • YUAN XIAOYAN
  • ZHANG HUAN
  • LU DINGCHANG
  • WANG PEILI
  • LI XIAOFEI

Assignees

  • 遵义医科大学

Dates

Publication Date
20260512
Application Date
20260129

Claims (9)

  1. 1. A method for measuring the content of two polypeptides in cantharis includes such steps as precisely weighing cantharis powder, adding water, mixing, regulating pH value, adding pepsin, enzymolysis in water bath at 37 deg.C, deactivating by boiling water bath, cooling, centrifugal separation to obtain supernatant, adding methanol to deposit macroprotein, centrifugal separation to obtain supernatant, passing through 0.22 microns organic filter membrane, preparing reference solution, precisely weighing polypeptides BM-1 and BM-2, dissolving in ultrapure water, ultrasonic vibration, preparing single reference stock solution, diluting to obtain mixed reference solution, HPLC-MS analysis, and determining the characteristic peak of polypeptide by Skyline software.
  2. 2. The method according to claim 1, wherein the sample weight of the defatted cantharis powder in the step (1) is 0.15 g, water is added to 9 mL, pH is adjusted to 2, pepsin is added to make the enzyme activity 1250U/mL, the enzymolysis is performed in a 37 ℃ water bath for 30min, the boiling water bath is inactivated for 10min, centrifugation is performed at 8000 rpm for 5min to obtain supernatant, equal volume of methanol is added, centrifugation is performed at 4 ℃ and 12000 rpm for 15 min, and the supernatant is subjected to an organic filter membrane of 0.22 μm.
  3. 3. The method of claim 1, wherein the mass concentration gradient of BM-1 in the mixed control solution in step (2) is 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 ng/mL, the mass concentration gradient of BM-2 is 5, 10, 20, 40, 80, 100, 200, 400 ng/mL, and the sample is introduced through a 0.22 μm microporous filter.
  4. 4. The method according to claim 1, wherein the HPLC conditions in the step (3) are that the chromatographic column is Thermo Kinetex C18 columns, 2.1 mm ×150 mm,2.6 μm, the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is 0.1% formic acid acetonitrile, the flow rate is 0.2 mL/min, the column temperature is 25 ℃, the sample injection amount is 1 μl, the gradient elution procedure is that 0-0.5 min keeps 8% B,0.5-8 min is increased from 8% B to 70% B,8-9.5 min is decreased from 70% B to 8% B, and 9.5-10 min keeps 8% B.
  5. 5. The method of claim 1, wherein the mass spectrometry conditions in step (3) are positive ion multi-reaction monitoring mode, and the ion source parameters include gas curtain gas 40 psi, ionization voltage 5500V, ion source temperature 500 ℃, spray gas 50 psi, auxiliary heating gas 50 psi, BM-1 parent ion m/z 1018.6, sub ion m/z 660.1, collision energy 62V, declustering voltage 143V, BM-2 parent ion m/z 416.3, sub ion m/z 136.1, collision energy 42V, declustering voltage 65V.
  6. 6. The method of claim 1, wherein step (3) further comprises a methodological verification step comprising: 1) Taking 6 parts of sample solution for continuous sample injection, and calculating RSD of BM-1 and BM-2 peak areas; 2) Taking sample solutions of the test samples at different time points of 0, 4, 12, 24 and 48 h, and calculating RSD of the peak area in 48 h; 3) Taking mixed reference substance solution to continuously sample for 6 times, and calculating RSD of peak area; 4) Gradually diluting the reference substance solution, wherein the signal-to-noise ratio S/N is more than or equal to 3 and is more than or equal to 10 as the detection limit; 5) Standard polypeptide solution is added into cantharis sample at 50%, 100% and 150% level, and recovery rate and RSD are calculated.
  7. 7. The method of claim 1, wherein the polypeptides BM-1 and BM-2 are purified chemically and are greater than 98% pure.
  8. 8. A kit for cantharis polypeptide content determination, comprising: 1) The polypeptides BM-1 and BM-2 control of claim 7; 2) Pepsin; 3) Mobile phase reagent for chromatography, 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile; 4) Methanol, hydrochloric acid and ultrapure water for sample pretreatment.
  9. 9. Use of the method or kit according to any one of claims 1-8 in quality assessment or quality control of cantharis medicinal materials.

Description

Content determination method for simultaneously detecting two polypeptides in cantharis body Technical Field The invention belongs to the field of analysis and test, relates to a method for detecting the content of traditional Chinese medicine polypeptides, and particularly relates to a method for simultaneously detecting the content of two polypeptides in cantharis. Background The Mylabris is dried body of Mylabris (Mylabris PHALERATA PALLAS) or Mylabris (Mylabris cichorii Linnaeus) belonging to Mylabris of Genkwaceae, has effects of removing blood stasis, removing toxic substances, and removing sore, and contains cantharidin, protein, amino acid, fatty acid, nucleoside and volatile components. The cantharis polypeptide obtained by adopting an enzymolysis method has no immunosuppression effect on spleen and thymus growth while resisting tumor. However, the structural characteristics of cantharis polypeptide are not further analyzed and studied by separation and purification. The polypeptide is a protein hydrolysate, is a molecular polymer composed of 2-20 amino acids, widely exists in nature, and has the characteristics of high safety, strong biological activity, high targeting specificity and the like. The method for establishing the quality analysis based on polypeptide combined with HPLC-MS/MS is widely applied to the field of traditional Chinese medicine quality control. Based on the method, a scientific basis is provided for the quality research of the cantharis by establishing a HPLC-MS detection method based on the synthetic polypeptide. Disclosure of Invention The invention aims to provide a method for measuring the content of cantharis polypeptide based on HPLC-MS. The invention selects the cantharis polypeptide with activity as an analysis object, and establishes simultaneous quantification of the characteristic polypeptide content in cantharis through HPLC-MS. The method has the advantages of stability, reliability, good reproducibility and high instrument precision, can be used for quality evaluation research of cantharis, and provides a theoretical basis for quality evaluation. The two polypeptide information of the invention are as follows: TABLE 2 multiple reaction parameters for cantharis polypeptides In order to achieve the above purpose, the technical scheme adopted is as follows: A content determination method for simultaneously detecting two polypeptides in cantharis body comprises the following steps: (1) Preparing test solution by precisely weighing defatted Mylabris powder, adding water, mixing, adjusting pH, adding pepsin, performing enzymolysis in 37deg.C water bath, inactivating in boiling water bath, cooling, centrifuging to obtain supernatant, adding methanol to precipitate large protein, centrifuging again, and collecting supernatant, and filtering with 0.22 μm organic filter membrane to obtain test solution; (2) Preparing reference substance solution, namely precisely weighing polypeptides BM-1 and BM-2, respectively dissolving with ultrapure water, ultrasonically oscillating to prepare single reference substance stock solution, and diluting to obtain mixed reference substance solution with serial concentration; (3) HPLC-MS analysis, namely respectively carrying out HPLC-MS analysis on the reference substance solution and the sample solution, adopting Skyline software to establish an analysis method, determining a polypeptide characteristic peak, and calculating the polypeptide content in the sample according to the peak area and the concentration of the reference substance; wherein the sequence of the polypeptide BM-1 is AGDDAPRAVF and the sequence of the polypeptide BM-2 is YSF. Further, the sample weighing amount of the defatted cantharis powder in the step (1) is 0.15 g, water is added to 9 mL, the pH is adjusted to 2, pepsin is added to enable the enzyme activity of the defatted cantharis powder to be 1250U/mL, the obtained product is subjected to enzymolysis in a 37 ℃ water bath for 30 min, the obtained product is inactivated in a boiling water bath for 10min, centrifugation is carried out at 8000 rpm for 5min, supernatant is taken, equal volume of methanol is added, centrifugation is carried out at 4 ℃ and 12000 rpm for 15 min, and the obtained product is subjected to organic filter membrane of 0.22 mu m. Further, the mass concentration gradient of BM-1 in the mixed reference substance solution in the step (2) is 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 ng/mL, and the mass concentration gradient of BM-2 is 5, 10, 20, 40, 80, 100, 200, 400 ng/mL, and the sample is introduced through a microporous filter membrane of 0.22 μm. Further, the HPLC condition in the step (3) is that the chromatographic column is Thermo Kinetex C columns, 2.1 mm ×150 mm and 2.6 μm, the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is 0.1% formic acid acetonitrile, the flow rate is 0.2 mL/min, the column temperature is 25 ℃, the sample injection amount is 1 μl, the