RU-2022126759-A - Methods for quantitative detection of beta-amyloid peptides in plasma using mass spectrometry

Inventors

• АЛЬЮЭ БЛАСКО Хосе Антонио
• САРАСА КОРОНАС Мария Летиция

Assignees

• АРАКЛОН БАЙОТЕК, С.Л.

Dates

Publication Date

20260508

Application Date

20210429

Priority Date

20200429

Claims (20)

1. 1. A method for preparing a plasma sample containing beta-amyloid peptides for analysis by mass spectrometry, wherein the method is characterized in that it comprises the following steps:
2. a) Contact of the said plasma sample with a denaturing agent;
3. b) Carrying out a first stage of solid phase extraction of the solution obtained in stage a) to obtain a first eluate;
4. c) Performing a second step of solid phase extraction on said first eluate obtained in step b) to obtain a second eluate; and
5. d) Drying said second eluate obtained in step c) and processing it for analysis by mass spectrometry,
6. wherein the sample obtained in step d) comprises intact beta-amyloid peptides Aβ40 and Aβ42.
7. 2. The method according to claim 1, wherein the second stage of solid-phase extraction is a cation-exchange solid-phase extraction selected from a cation-exchange solid-phase extraction performed on a strong cation exchanger, a weak cation exchanger, or a mixed-type cation exchanger in reversed-phase mode, or is an anion-exchange solid-phase extraction selected from anion-exchange solid-phase extraction performed on a strong anion exchanger, a weak anion exchanger, or a mixed-type anion exchanger in reversed-phase mode.
8. 3. The method according to any one of the preceding claims, wherein in step a) the plasma sample is contacted with an acid denaturing agent to obtain a solution with a pH that is less than or equal to about 4.5, wherein the acid denaturing agent is a solution of formic acid in water with a concentration of from 40% to 70% (v/v).
9. 4. The method according to claim 3, wherein each of the first and second stages of solid-phase extraction includes at least two washing stages, characterized in that the first washing stages of the first and second stages of solid-phase extraction are performed with a solution comprising an acid, and the second washing stages of the first and second stages of solid-phase extraction are performed with a solution comprising a water-miscible polar organic solvent.
10. 5. A method according to any one of the preceding claims, wherein each of the first and second solid-phase extraction stages comprises at least two washing stages, characterized in that the first washing stage of the first solid-phase extraction is performed with a solution comprising an acid, and the first washing stage of the second solid-phase extraction is performed with a solution comprising a base, or vice versa, and the second washing stages of the first and second solid-phase extraction stages are performed with a solution comprising a water-miscible polar organic solvent.
11. 6. The method according to any one of the preceding paragraphs 1-5, wherein the solution of step d) for treating the dried eluate is an aqueous solution comprising a surfactant, a reducing agent, a water-miscible polar organic solvent and an acid.
12. 7. The method according to claim 6, wherein the solution of step d) for treating the dried eluate is an aqueous solution comprising Triton X-100 in a concentration of 0.01% to 0.8% (v/v) and tris-carboxyethylphosphine in a concentration of 0.1% to 0.2% (w/v).
13. 8. The method according to claim 7, in which the solution of step d) for treating the dried eluate is an aqueous solution comprising Triton X-100 in a concentration of 0.01% to 0.8% (v/v), tris-carboxyethylphosphine in a concentration of 0.1% to 0.2% (w/v), acetonitrile in a concentration of 3% to 7% (v/v), dimethylformamide in a concentration of 0.1% to 3% (v/v) and trifluoroacetic acid (TFA) in a concentration of 0.1% to 3% (v/v).
14. 9. A method for the quantitative detection of intact beta-amyloid peptides Aβ40 and Aβ42 in a plasma sample using mass spectrometry, characterized in that the method comprises steps a)-d) of the method for preparing a plasma sample according to any of the preceding claims and further comprises the following steps:
15. i) Carrying out a liquid chromatography step on the solution obtained in step d) to separate the analytes of interest;
16. ii) Performing ionization of the analytes isolated in step i) to generate one or more types of charged particles;
17. iii) Separation of said one or more types of charged particles according to the mobility of their ions;
18. iv) Detecting said one or more types of charged particles isolated in step iii) and measuring their relative abundance using mass spectrometry; and
19. v) Determining the amount or concentration of intact beta-amyloid peptides Aβ40 and/or Aβ42 in a plasma sample by comparing the relative abundances of one or more charged species determined in step iv) to a standard curve.
20. 10. An aqueous solution for treating dried eluate for analysis by mass spectrometry, comprising Triton X-100 at a concentration of 0.01% to 0.8% (v/v), tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w/v), acetonitrile at a concentration of 3% to 7% (v/v), dimethylformamide at a concentration of 0.1% to 3% (v/v) and trifluoroacetic acid (TFA) at a concentration of 0.1% to 3% (v/v).