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CN-122017061-A - Method for detecting polysorbate 80 based on combined technology of MALDI-MS and UHPLC-Q-TOF/MS

CN122017061ACN 122017061 ACN122017061 ACN 122017061ACN-122017061-A

Abstract

The invention discloses a method for detecting polysorbate 80 based on a combined technology of MALDI-MS and UHPLC-Q-TOF/MS, which comprises the steps of taking a polysorbate 80 sample, preparing a polysorbate 80 solution by using acetonitrile, respectively carrying out MALDI-MS detection and UHPLC-Q-TOF/MS detection on the polysorbate 80 solution, obtaining PS80 main component distribution according to a MALDI-MS map, namely polyoxyethylene chain delta m/z 44.0+/-0.2, esterified derivative sodium adduct ion gradient peak clusters, carrying out qualitative analysis on components of the polysorbate 80 by combining with characteristic intervals of m/z 44.0+/-0.2 in the MALDI map, and identifying monoester, diester and trace unesterified sorbitol anhydride in the polysorbate 80 according to retention time in a UHPLC-Q-TOF/MS detection result. The invention realizes the molecular weight distribution analysis and component separation and identification of PS 80.

Inventors

  • ZHAO SHUN
  • HU ZHAOLIANG
  • Shao Fangxian
  • SHI HAIWEI
  • YUAN YAOZUO

Assignees

  • 江苏省药品监督检验研究院

Dates

Publication Date
20260512
Application Date
20260120

Claims (10)

  1. 1. A method for detecting polysorbate 80 based on a combined technology of MALDI-MS and UHPLC-Q-TOF/MS is characterized by comprising the steps of taking a polysorbate 80 sample, preparing a polysorbate 80 solution by using acetonitrile, respectively carrying out MALDI-MS detection and UHPLC-Q-TOF/MS detection on the polysorbate 80 solution, obtaining PS80 main component distribution according to a MALDI-MS map, namely polyoxyethylene chain delta m/z 44.0+/-0.2, esterified derivative sodium adduct ion gradient peak clusters, carrying out qualitative analysis on components of the polysorbate 80 by combining characteristic intervals of m/z 44.0+/-0.2 in the MALDI map, and carrying out matching with a polysorbate 80 theoretical component database according to the retention time of UHPLC-Q-TOF/MS detection results, and identifying monoester, diester and trace unesterified sorbitol anhydride in the polysorbate 80.
  2. 2. The method for detecting polysorbate 80 according to claim 1, wherein the preparation of the polysorbate 80 solution is characterized in that a polysorbate 80 sample is dissolved in acetonitrile to prepare a solution with the concentration of 0.2-1 mg/mL, and the solution is filtered through a 0.22 μm organic filter membrane to obtain the polysorbate 80 solution.
  3. 3. The method for detecting polysorbate 80 according to claim 1, wherein the conditions for MALDI-MS detection are as follows: a substrate, namely 2, 5-dihydroxybenzoic acid; Cationic reagent, na+ solution; The sample application method comprises mixing matrix solution, cationic reagent and polysorbate 80 solution, precisely applying the mixture to the center of 48-hole stainless steel target plate hole, and drying at room temperature in dark until solvent volatilizes completely to form homogeneous co-crystal; the mass spectrum condition is that the positive ion mode is reflected, the scanning range m/z is 500-2500, the laser energy is 90-100, and the frequency is 50 Hz.
  4. 4. The method for detecting polysorbate 80 according to claim 3, wherein the matrix solution is a solution with DHB concentration of 5-15 mg/mL prepared by using 50% v/v acetonitrile aqueous solution as a solvent, and the cationic reagent is a sodium chloride aqueous solution with sodium chloride concentration of 3-6 mg/mL.
  5. 5. The method for detecting polysorbate 80 according to claim 4, wherein the matrix solution is a solution prepared by using 50% v/v acetonitrile aqueous solution as a solvent and having a DHB concentration of 10 mg/mL, and the cationic reagent is a sodium chloride aqueous solution having a sodium chloride concentration of 5 mg/mL.
  6. 6. The method of claim 3, wherein the volume ratio of the matrix solution, the cationic reagent and the polysorbate 80 solution is 10:5:10.
  7. 7. The method for detecting polysorbate 80 according to any one of claims 3 to 6, wherein the sample application method is a mixed sample application method, wherein 10 μl of matrix solution, 5.0 μl of cationic reagent and 10 μl of polysorbate 80 solution are added into a 0.6mL centrifuge tube, vortex mixing is carried out, 1 μl of the mixture is sucked by a micropipette, and the mixture is precisely applied to the center of the 48-hole stainless steel target plate hole, and dried at room temperature in a dark place for about 10min-40min until the solvent is completely volatilized.
  8. 8. The method for detecting polysorbate 80 according to claim 1, wherein the UHPLC-Q-TOF/MS detection conditions are as follows: Agilent ZORBAX RRHD SB-C8 (100 mm ×2.1 mm, 1.8 μm) or equivalent chromatography column; mobile phase A is 0.05-0.2% v/v formic acid aqueous solution, preferably 0.1% v/v formic acid aqueous solution, mobile phase B is 0.1% (v/v) formic acid acetonitrile; Gradient elution procedure is 0-30 min,5% B-100% B, 30-33 min,100% B, 33-36 min,100% B-5% B, 36-46 min,5% B; the flow rate is 0.4-0.6 mL/min; column temperature is 45-55 ℃; a sample injection volume of 20 mu L; the mass spectrum condition is positive ion mode, the scanning range m/z is 100-3000, in the secondary mass spectrum analysis, collision induction dissociation mode is adopted, and the collision energy is respectively set to 80 eV and 120 eV for double-charge ions and single-charge ions.
  9. 9. The method for detecting polysorbate 80 according to claim 8, wherein the chromatographic column is Agilent ZORBAX RRHD SB-C8 (100 mm X2.1 mm, 1.8 μm).
  10. 10. The method for detecting polysorbate 80 according to claim 8, wherein the mobile phase A is 0.1% v/v formic acid aqueous solution.

Description

Method for detecting polysorbate 80 based on combined technology of MALDI-MS and UHPLC-Q-TOF/MS Technical Field The invention belongs to the field of pharmaceutical auxiliary material analysis, and relates to a method for detecting Polysorbate 80 (Polysorbate 80, PS80) based on a combined technology of matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS), in particular to a method for measuring molecular weight distribution and analyzing component separation efficiency of Polysorbate 80 based on a combined technology of MALDI-MS and UHPLC-Q-TOF/MS. Background Polysorbate 80 is a nonionic surfactant widely used in biopharmaceutical preparations and is commonly used for the stability protection of protein drugs. However, polysorbate 80 has a complex structure and is mainly formed by connecting sorbitol, polyoxyethylene chains and fatty acids (such as oleic acid) through ester bonds, and various isomers with polymerization degrees and esterification forms exist, and the composition difference among different batches is large, so that the quality control is challenged. At present, common analysis methods of polysorbate 80 comprise HPLC-UV, ESI-MS and the like, and the methods have the problems of insufficient resolution, serious component overlapping, incapability of comprehensively characterizing complex components and the like. Thus, there is a need for a novel assay that is highly sensitive, high resolution, easy to operate, and suitable for complex sample matrices to achieve comprehensive quality assessment of polysorbate 80. Disclosure of Invention The invention aims to solve the problems of low resolution, difficult component identification, long analysis period and the like in the prior art, and provides a method for detecting polysorbate 80 based on a combined technology of MALDI-MS and UHPLC-Q-TOF/MS. In order to achieve the above purpose, the present invention provides the following technical solutions: A method for detecting polysorbate 80 based on a combined technology of MALDI-MS and UHPLC-Q-TOF/MS comprises the steps of taking a polysorbate 80 sample, preparing a polysorbate 80 solution by using acetonitrile, respectively carrying out MALDI-MS detection and UHPLC-Q-TOF/MS detection on the polysorbate 80 solution, obtaining PS80 main component distribution according to a MALDI-MS map, carrying out qualitative analysis on components of polysorbate 80 by combining a characteristic interval of m/z 44.0+/-0.2 in the MALDI map, and carrying out matching with a polysorbate 80 theoretical component database according to the retention time of a UHPLC-Q-TOF/MS detection result, and identifying monoester, diester and trace unesterified sorbitol anhydride in polysorbate 80. The preparation of the polysorbate 80 solution comprises the steps of dissolving a polysorbate 80 sample in acetonitrile to prepare a solution with the concentration of 0.2-1 mg/mL, and filtering the solution through a 0.22 mu m organic filter membrane to obtain the polysorbate 80 solution. Preferably, the concentration of polysorbate 80 solution is 1 mg/mL when MALDI-MS detection is performed, and the concentration of polysorbate 80 solution is 0.2 mg/mL when UHPLC-Q-TOF/MS detection is performed. The MALDI-MS detection conditions are as follows: a matrix of 2, 5-dihydroxybenzoic acid (DHB); A cationic reagent, na+ solution, for promoting [ M+Na ] + ion formation; The sample application method comprises mixing matrix solution, cationic reagent and polysorbate 80 solution, precisely applying the mixture to the center of 48-hole stainless steel target plate hole, and drying at room temperature in dark until solvent volatilizes completely to form homogeneous co-crystal; the mass spectrum condition is that the positive ion mode is reflected, the scanning range m/z is 500-2500, the laser energy is 90-100, and the frequency is 50 Hz. The matrix solution is prepared by taking a 50% v/v acetonitrile aqueous solution as a solvent, wherein the concentration of DHB is 5-15 mg/mL. Preferably, the concentration of DHB in the matrix solution is 10 mg/mL. Preferably, the cationic reagent is a sodium chloride aqueous solution with the concentration of 3-6 mg/mL. Specifically, the cationic reagent is sodium chloride aqueous solution with the concentration of 5 mg/mL. The volume ratio of the matrix solution, the cationic reagent and the polysorbate 80 solution is 10:5:10. Specifically, the sample application mode is to add 10 mu L of matrix solution, 5.0 mu L of cationic reagent and 10 mu L of polysorbate 80 solution into a 0.6mL centrifuge tube by adopting a mixed sample application method, vortex and mix uniformly, suck 1 mu L of mixed solution by adopting a micropipette, precisely sample the mixed solution at the center of the hole of a 48-hole stainless steel target plate, and dry at room temperature in a dark place for abou