Evaporation-based sample preparation workflow for mass spectrometry

Abstract

The present invention relates to a method for detecting and / or quantifying an analyte in a sample using mass spectrometry. The method of the invention comprises extracting an analyte from a sample using solid phase extraction (SPE) to obtain an SPE extract containing the analyte, concentrating the analyte, where the concentration comprises evaporating a solvent from the SPE extract, and detecting and / or quantifying the analyte in the sample using mass spectrometry.

Description

Technical Field

[0001] The present invention relates to a method for detecting and / or quantifying an analyte in a sample using mass spectrometry. The method of the present invention comprises the steps of extracting an analyte from a sample using solid-phase extraction (SPE) to obtain an SPE extract containing the analyte; concentrating the analyte, the concentration step including partially evaporating a solvent from the SPE extract; and detecting and / or quantifying the analyte in the sample using mass spectrometry.

Background Art

[0002] In automated sample preparation systems and HPLC systems for LC-MS applications, there are sample volume and analyte losses associated with dead volume, which cause a decrease in sensitivity. For example, significant sample loss can occur when eluting samples obtained from the concentration of analytes by solid-phase extraction (SPE) are transferred from a primary reaction vessel to a secondary reaction vessel. Further sample loss can occur during the LC injection process from the dead volume of the sample or HPLC vial and from full-loop injection using overfilling to ensure high accuracy and reproducibility.

[0003] Elution of analytes in SPE often requires an organic solvent (e.g., 80% MeOH). However, the content of organic solvent in the sample needs to be minimized before LC-MS injection to maintain acceptable chromatographic performance, especially for μLC systems. Therefore, such eluents are often diluted 1:1 with water, for example, to lower the organic solvent concentration (e.g., to 30 - 40% MeOH or less). This dilution causes a further decrease in analyte detection sensitivity.

[0004] Therefore, there is a high need to improve the sample preparation workflow for mass spectrometry so that analyte loss can be minimized and higher analyte detection sensitivity can be achieved, particularly in the context of sample preparation processes using SPE techniques (e.g., magnetic particle-based SPE techniques). [Overview of the project]

[0005] This specification describes a method for detecting and / or quantifying an analyte of interest in a sample using mass spectrometry, a) A step of extracting an analyte from a sample using solid-phase extraction (SPE) to obtain an SPE extract containing the analyte; b) A step of concentrating the analyte by partially evaporating the solvent from the SPE extract obtained in a); and c) A step of detecting and / or quantifying the analyte in the sample using mass spectrometry. A method is provided that includes this.

[0006] In particular, the present invention also relates to the following items.

[0007] 1. A method for detecting and / or quantifying an analyte in a sample using mass spectrometry, a) A step of extracting an analyte from a sample using solid-phase extraction (SPE) to obtain an SPE extract containing the analyte; b) A step of concentrating the analyte, comprising evaporating the solvent from the SPE extract obtained in a); and c) A step of detecting and / or quantifying the analyte in the sample using mass spectrometry. Methods including Preferably, a method for detecting and / or quantifying an analyte in a sample using mass spectrometry, a) A step of extracting an analyte from a sample using solid-phase extraction (SPE) to obtain an SPE extract containing the analyte, wherein the SPE extract contains 50% to 100% by volume of an organic solvent, and the analyte is a steroid, preferably selected from the group consisting of testosterone and estradiol; b) A step of concentrating the analyte, wherein the concentration includes partially evaporating the solvent from the SPE extract obtained in a); the volume of the SPE extract subjected to partial evaporation is reduced by 50% to 95%, preferably 60% to 90%, and more preferably 70% to 80%; b1) A step of diluting the concentrated analyte obtained from step b) with a diluent solvent to obtain a diluted analyte, wherein the diluted analyte contains less than 50 volume% of an organic solvent or further organic solvent prior to step c); and c) A step of detecting and / or quantifying an analyte in a sample using mass spectrometry, wherein the mass spectrometry is mass spectrometry coupled to liquid chromatography (LC-MS). Methods that include...

[0008] 2. The method according to item 1, wherein the evaporation of the solvent from the SPE extract is partial evaporation of the solvent from the SPE extract.

[0009] 3. The method according to item 1 or 2, wherein the volume of the SPE extract subjected to concentration is reduced by 50% to 95%, 60% to 90% in one embodiment, 70% to 80% in one embodiment, and 73% to 87% in one embodiment.

[0010] 4. The method according to item 3, further comprising adjusting the volume after evaporation using a diluent solution to a final volume corresponding to 5% to 40%, in one embodiment 10% to 30%, and in one embodiment 13% to 27%, of the volume of the sample to be subjected to the SPE in a).

[0011] 5. The method according to item 4, wherein the diluent solution is an aqueous solution having an organic solvent concentration of 10% by volume, particularly 5% by volume, and particularly less than 0% by volume.

[0012] 6. The method described in item 4, wherein the diluent solution is water.

[0013] The method according to any one of items 1 to 6, wherein the volume of the sample subjected to SPE in 7.a) is 250 μl or less, in one embodiment 200 μl or less, in one embodiment 150 μl or less, and in one embodiment 150 μl.

[0014] 8. The method according to any one of items 1 to 7, wherein the volume of the sample subjected to SPE is 150 μl, and the volume of the SPE extract is reduced to a final volume of 10 μl to 60 μl, in one embodiment 20 μl to 50 μl, and in one embodiment 40 μl by concentration.

[0015] 9. The method according to any one of items 1 to 8, wherein the SPE extract contains 50% to 100% by volume of an organic solvent.

[0016] 10. The method according to item 9, wherein the organic solvent is selected from the group consisting of acetonitrile and methanol.

[0017] 11. The method according to any one of items 1 to 10, wherein the analyte is a steroid.

[0018] 12. The method according to any one of items 1 to 11, wherein the analyte is a steroid hormone, and in one embodiment, a steroid hormone selected from the group consisting of androgen, estrogen, glucocorticoid, mineralocorticoid, and gestagen.

[0019] 13. The method according to any one of items 1 to 12, wherein the analyte is androgen or estrogen.

[0020] 14. The method according to item 13, wherein the androgen is testosterone.

[0021] 15. The method according to item 13, wherein the estrogen is estradiol.

[0022] 16. The method according to any one of items 1 to 15, wherein the sample is a fluid, particularly a biological fluid.

[0023] 17. The method according to any one of items 1 to 16, wherein the sample is a obtained body fluid, in one embodiment, a human body fluid.

[0024] 18. The method according to any one of items 1 to 17, wherein the sample is serum or plasma.

[0025] 19. The method according to any one of items 1 to 18, further comprising a pretreatment step for releasing the analyte from the analyte-binding protein.

[0026] 20. The method according to any one of items 1 to 19, wherein the SPE is a batch-type SPE.

[0027] 21. The method according to any one of items 1 to 20, wherein the solid phase used in the SPE is formed by magnetic particles, particularly magnetic microbeads.

[0028] 22. The method according to any one of items 1 to 21, wherein the solid phase of the SPE is formed by particles (e.g., magnetic particles) configured to capture the analyte from the sample and release the analyte when treated with an elution solvent.

[0029] 23. The method according to any one of items 1 to 22, wherein the solid phase of the SPE is formed by particles (e.g., magnetic particles) coated with an antibody that specifically binds to the analyte.

[0030] 24. The method according to any one of items 1 to 23, wherein the solid phase of the SPE is formed by a porous polymer matrix that can bind or adsorb the analyte of the sample and release the analyte when treated with an elution solution.

[0031] 25. Solid phase extraction (SPE) is: a) a step of capturing the analyte on the solid phase; b) optionally, one or more washing steps of the solid phase; and c) a step of eluting the analyte from the solid phase to obtain an SPE extract containing the analyte The method according to any one of items 1 to 24, comprising.

[0032] 26. The method according to item 25, wherein the step of eluting the analyte includes adding an elution solvent to a solid phase and incubating the solid phase in the presence of the added elution solvent.

[0033] 27. The method according to item 25 or 26, wherein the volume of the elution solvent added corresponds to 50% to 150% of the volume of the sample subjected to the SPE, in one embodiment to 90% to 120% of the volume of the sample subjected to the SPE, and in one embodiment to 100% of the volume of the sample subjected to the SPE.

[0034] 28. The method according to any one of items 25 to 27, wherein the eluting solvent contains ACN in particular at concentrations of 40 to 100% by volume, particularly 45 to 90% by volume, and particularly 60 to 80% by volume.

[0035] 29. The method according to any one of items 25 to 27, wherein the eluting solvent contains MeOH in a concentration of particularly 70 to 100 by volume, particularly 80 to 90 by volume, or particularly 80 by volume.

[0036] 30. The method according to any one of items 1 to 29, further comprising the step of adding an internal standard (ISTD) for quantification to the sample before step a).

[0037] 31. The method according to item 30, wherein ISTD is an analyte artificially labeled with one or more heavy isotopes.

[0038] 32. The method according to any one of items 1 to 31, wherein the mass spectrometry is coupled to liquid chromatography (for example, the analysis is LC-MS analysis or more specifically LC-MS / MS analysis).

[0039] 33. The method according to item 32, wherein liquid chromatography (LC) is HPLC or rapid LC.

[0040] 34. The method described in item 32 or 33, wherein LC is microLC.

[0041] 35. The method according to any one of items 32-34, wherein LC is ultra-high performance liquid chromatography (UHPLC).

[0042] 36. The method described in any one of items 1 to 35, wherein mass spectrometry is performed using a mass spectrometer employing electrospray ionization (ESI).

[0043] 37. The method according to any one of items 1 to 36, wherein the mass spectrometry is performed using a mass spectrometer that is a tandem mass spectrometer, in particular a triple quadrupole mass spectrometer.

[0044] 38. The method described in any one of items 1 through 37, wherein the method is automated. [Brief explanation of the drawing]

[0045] [Figure 1] Comparison of analyte signal (A) and recovery (B) for analyte testosterone using no evaporation (simple dilution), complete evaporation, and partial evaporation workflows. Samples containing 60 pg / mL of testosterone in 60% MeOH were distributed into three 40 μL aliquot groups. In the first group, the sample was diluted with 40 μL of H2O to a total final volume of 80 μL, serving as the control group (100% recovery). In the second and third groups, the samples were evaporated to complete dryness (complete evaporation) and then reconstituted with 40 μL of 30% MeOH, or evaporated to a volume of 10 μL (partial evaporation) and then diluted with 30 μL of 30% MeOH to a total final volume of 40 μL. All samples contained the same amount of analyte, and the final organic matter content was 30% MeOH. [Figure 2]Schematic diagrams of the standard dilution workflow (top) and the full and partial evaporation workflows (bottom), optimized to maximize detection sensitivity and chromatographic performance. The full evaporation workflow used in Figure 1 (center) is also shown. All workflows proceed by mixing 150 μL of sample and internal standards, pre-treating the analyte to deconjugate it from bound proteins, concentrating the analyte with antibody-coated magnetic beads, and minimizing unbound matrix components by washing twice with water. The analyte is released using 60 μL or 150 μL of 80% MeOH elution buffer, followed by the transfer of 40 μL or 130 μL of eluate to a new reaction vessel for the standard and evaporation workflows, respectively. In the normal workflow, 40 μL of eluate is diluted with 67 μL of water, while in the evaporation workflow, the sample is either evaporated to completely dry (full evaporation) or evaporated to 20–40 μL (partial evaporation), and the sample is optionally diluted with water to obtain a final volume of 40 μL. For all workflows, 20 μL is injected for LC-MS analysis. [Figure 3] Extraction ion chromatograms of 2 pg / mL estradiol spiked with UniDil, comparing a standard workflow (Figure 3) and an optimized partial evaporation workflow (Figure 4). Analyte chromatographic peaks were clearly detectable using the evaporation assay but not with the standard assay. [Figure 4] Extraction ion chromatograms of 2 pg / mL estradiol spiked with UniDil, comparing a standard workflow (Figure 3) and an optimized partial evaporation workflow (Figure 4). Analyte chromatographic peaks were clearly detectable using the evaporation assay but not with the standard assay. [Figure 5] The analyte / ISTD peak area ratio was the same for both the standard and optimized evaporation workflows (A), and the linear fit of analyte peak area and analyte concentration demonstrated that the evaporation workflow had a steeper slope for estradiol added to UniDil (Figure 5) and testosterone added to GoldenWestSerum (Figure 6) (B). [Figure 6] The analyte / ISTD peak area ratio was the same for both the standard and optimized evaporation workflows (A), and the linear fit of analyte peak area and analyte concentration demonstrated that the evaporation workflow had a steeper slope for estradiol added to UniDil (Figure 5) and testosterone added to GoldenWestSerum (Figure 6) (B). [Modes for carrying out the invention]

[0046] The present invention relates to a method for detecting and / or quantifying a target analyte in a sample using mass spectrometry, a) A step of extracting an analyte from a sample using solid-phase extraction (SPE) to obtain an SPE extract containing the analyte; b) A step of concentrating the analyte, comprising evaporating the solvent from the SPE extract obtained in a); and c) A step of detecting and / or quantifying the analyte in the sample using mass spectrometry. This includes methods.

[0047] In a preferred embodiment, the present invention relates to a method for detecting and / or quantifying an analyte in a sample using mass spectrometry, a) A step of extracting an analyte from a sample using solid-phase extraction (SPE) to obtain an SPE extract containing the analyte, wherein the SPE extract contains 50% to 100% by volume of an organic solvent, and the analyte is a steroid, preferably selected from the group consisting of testosterone and estradiol; b) A step of concentrating the analyte, wherein the concentration includes partially evaporating the solvent from the SPE extract obtained in a); the volume of the SPE extract subjected to partial evaporation is reduced by 50% to 95%, preferably 60% to 90%, and more preferably 70% to 80%; b1) A step of diluting the concentrated analyte obtained from step b) with a diluent solvent to obtain a diluted analyte, wherein the diluted analyte contains less than 50 volume% of an organic solvent or further organic solvent prior to step c); and c) A step of detecting and / or quantifying an analyte in a sample using mass spectrometry, wherein the mass spectrometry is mass spectrometry coupled to liquid chromatography (LC-MS). This includes methods.

[0048] In a preferred embodiment, the present invention relates to a method for detecting and / or quantifying an analyte of interest in a sample using mass spectrometry, a) A step of extracting the analyte from a sample using a solid-phase extract (SPE) to obtain an SPE extract containing the analyte; b) A step of concentrating the analyte by partially evaporating the solvent from the SPE extract obtained in a); and c) A step of detecting and / or quantifying the analyte in the sample using mass spectrometry. This includes methods.

[0049] As demonstrated by the attached examples and the method of the present invention, the inventors have unexpectedly found that applying partial evaporation to an SPE extract containing the analyte of interest (e.g., steroids, particularly androgens or estrogens) can improve analyte recovery in an MS sample preparation workflow compared to a previously used dilution workflow. Furthermore, the inventors have demonstrated that partial evaporation of the SPE extract results in remarkably superior sample recovery and, consequently, analyte detection sensitivity (e.g., steroids, particularly androgens or estrogens) compared to the use of complete evaporation to dryness and subsequent reconstruction at a defined volume. In addition to the advantages of higher analyte recovery and detection sensitivity, partial evaporation has the advantage of being much faster than complete evaporation. Therefore, reducing the solvent evaporation time reduces the overall sample preparation time. Shorter sample preparation times allow for higher sample turnover, which is particularly important when discussing fully automated MS sample preparation and measurement systems, which typically need to have the highest possible sample throughput.

[0050] "Concentrating the analyte by partially evaporating the solvent from the SPE extract" means evaporating the SPE extract in such a way that the volume of the analyte in the SPE extract decreases relative to the concentration of the analyte in the extract, and the concentration of the analyte in the resulting solution increases.

[0051] As used herein, "partial evaporation" means that the solvent of a liquid sample (SPE extract / eluate in this specification) does not evaporate completely, but rather some of the solvent remains. In other words, the solvent of a liquid sample subjected to evaporation (SPE extract / eluate in this specification) does not evaporate completely.

[0052] "Evaporating the solvent" means evaporating the liquid so that the analyte does not evaporate. Preferably, the analyte also does not precipitate during evaporation.

[0053] The partial evaporation according to the present invention is configured to increase the analyte concentration. Furthermore, the partial evaporation is preferably configured to reduce the content of organic solvents (e.g., volatile organic solvents such as acetonitrile or methanol). By reducing the content of organic solvents in the SPE extract, the LC performance in LC-MS is improved.

[0054] In some embodiments, the method is carried out in the following order: a), then b), then c).

[0055] In some embodiments, the method is carried out in the following order: a), then b), then b1), then c).

[0056] In several embodiments, partial evaporation settings (e.g., temperature and / or vacuum settings) may be configured to allow organic solvents (e.g., acetonitrile or methanol) to evaporate. In several embodiments, the partial evaporation settings may be selected so that organic solvents evaporate preferentially over other solvents such as water. Solvents with higher vapor pressures or lower boiling points in aqueous mixtures (e.g., acetonitrile and methanol) evaporate faster than water content that is close to or above its boiling point (combination of temperature and pressure). This effectively reduces the relative organic content through evaporation.

[0057] The concentration step has the advantage of increasing the concentration of the analyte used for mass spectrometry, which can improve the limit of quantification for the analyte concentration in the sample. In particular, by combining concentrations involving SPE and partial evaporation, a surprising increase in signal was achieved. The increase in analyte concentration due to the concentration step also allows for the use of lower sample volumes for a given limit of quantification, increasing the volume of analyte used for mass spectrometry at a given volume. Furthermore, analyte concentration using partial evaporation of the solvent has the advantage of removing volatile solvents, such as organic solvents, contained in SPE extracts (e.g., ACN or methanol, which may be used to extract the analyte from the solid phase). Such organic solvents can interfere with liquid chromatography in LC, including in mass spectrometry workflows.

[0058] Evaporation or partial evaporation can be achieved using various evaporation systems or chambers known in the art. The evaporation system or chamber may be part of a mass spectrometry system. Evaporation may be fully automated, i.e., without manual handling steps. In several embodiments, evaporation systems that do not use centrifugation may be used. In preferred embodiments, the evaporation system may use vacuum and heating for evaporation (e.g., SpeedVac vacuum concentrator, ThermoFisher).

[0059] In several embodiments, the volume of the SPE extract subjected to concentration by evaporation is reduced by 50% to 95%, particularly 60% to 90%, and particularly 70% to 80% (relative to the volume of the SPE extract subjected to concentration / evaporation). In particular, the SPE extract can be concentrated by reducing the solvent volume by 73% to 87% using evaporation. In Appendix Example 1, it is demonstrated that such a degree of solvent concentration / evaporation from the SPE extract can be used to achieve increased sample recovery and detection sensitivity for both dilution and complete evaporation workflows.

[0060] For example, if a magnetic particle-based SPE is performed and 150 μl of total SPE extract is obtained, the magnetic particles may be pelletized, and 130 μl of the SPE extract may be transferred to another tube and subjected to evaporation. For example, reducing the processing volume of 130 μl of SPE extract to a volume of 20 μl is equivalent to a volume reduction of 84.6%.

[0061] In several embodiments, the volume of the SPE extract subjected to evaporation may be reduced by evaporation by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0062] In several embodiments, the volume of the SPE extract subjected to evaporation may be reduced by evaporation by up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 99%.

[0063] In several embodiments, the volume of the SPE extract subjected to evaporation is reduced by evaporation to a final volume of 5% to 50%, particularly 10% to 40%, and especially 20% to 30%. In particular, the SPE extract can be concentrated by evaporation that reduces the solvent volume to a final volume of 13% to 27%.

[0064] In several embodiments, this method may include adjusting the volume after evaporation to the final volume subject to LC-MS using a diluent solution. The final volume after adjustment with the diluent solution may correspond to 5% to 40%, 10% to 30% in one embodiment, or 13% to 27% of the volume of the sample subjected to SPE. Exemplary but non-limiting, a volume of 130 μl of SPE extract may be subjected to a step of concentrating the analyte using evaporation, the volume may be reduced to 10 μl by concentration, and finally, the volume may be adjusted to 40 μl using a diluent solution.

[0065] In several embodiments, the volume obtained after evaporation of the solvent from the SPE extract can be adjusted by adding a diluent solution so that the final volume obtained corresponds to the injection volume into the LC and, optionally, the dead volume of the reaction vessel containing the solution. For example, the volume obtained after evaporation of the solvent from the SPE extract can be adjusted by adding a diluent solution so that 15–30 μl, particularly 20 μl, can be removed from the reaction vessel used for injection into the LC.

[0066] The diluent solution used in the context of this disclosure may be, for example, an aqueous solution or water. Preferably, the aqueous solution used as the diluent solution contains an organic solvent concentration of 20% by volume or less, preferably 10% by volume or less, more preferably 5% by volume or less, and most preferably 0% by volume. In some embodiments, the diluent solution may be water. Using a low concentration of organic solvent, or even having no organic solvent content at all, has the advantage of being able to maintain a low organic solvent content, which is advantageous when the concentrated solution is subjected to LC-MS. In particular, when using a hydrophobic LC stationary phase such as a C18 matrix, high concentrations of organic solvent typically cause broadening of the LC peak width.

[0067] The method of the present invention is applicable to various analytes and samples. The method may include detecting and / or quantifying only the target analyte, and the target analyte and additional analytes may be detected.

[0068] In several embodiments, the analyte of interest to be detected and / or quantified by the method of the present invention may be a steroid, particularly a steroid hormone. In particular, the analyte may be a steroid selected from the group consisting of androgens, estrogens, glucocorticoids, mineralocorticoids, and gestagens.

[0069] Exemplary but non-exclusive examples of androgens include testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), androstenedione (A4), androstenediol (A5), dihydrotestosterone (DHT), and androsterone.

[0070] In certain embodiments, the androgen is testosterone.

[0071] Exemplary but non-exclusive examples of estrogens are estrone (E1), estradiol (E2), estriol (E3), and estetrol (E4).

[0072] In certain embodiments, estrogen is estradiol.

[0073] A representative but non-restrictive example of a glucocorticoid is cortisol.

[0074] Aldosterone is an exemplary but non-exclusive example of a mineralocorticoid. Exemplary but non-exclusive examples of gestagens are progesterone (P4), 16α-hydroxyprogesterone, 17α-hydroxyprogesterone, 20α-dihydroprogesterone, 20β-dihydroprogesterone, 5α-dihydroprogesterone, 5β-dihydroprogesterone, 3β-dihydroprogesterone, 11-deoxycorticosterone, and dihydrodeoxycorticosterone.

[0075] In several embodiments, the analyte is a steroid selected from the group consisting of 11-deoxycortisol, 17-alpha-hydroxyprogesterone (17OHP), 21-deoxycortisol, aldosterone, androstenedione (A4), cortisol, cortisone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), estradiol (E2), progesterone, and testosterone (T). In preferred embodiments, the analyte is selected from aldosterone, androstenedione (A4), dehydroepiandrosterone, dihydrotestosterone (DHT), estradiol (E2), progesterone, or testosterone (T). In particularly preferred embodiments, the analyte is testosterone (T) or estradiol (E2).

[0076] The method of the present invention uses previously obtained samples. Therefore, the method of the present invention is an in vitro method. The sample may, in particular, be a sample obtained from a human individual.

[0077] In principle, any sample containing or suspected to contain the analyte of interest can be subjected to the method of the present invention. While the sample subjected to solid-phase extraction must be liquid, in principle, solid samples (e.g., dried blood spots) can also be subjected to the method of the present invention. In the case of solid samples, an additional sample preparation step is included before SPE, which includes reconstituting the dried blood spot in a liquid. Each method and means for reconstituting a solid sample into a liquid so that the analyte (e.g., steroids) is recovered in the liquid is known in the art (Rossi et al., Clin Chem Lab Med 2011;49(4):677-684; Kim et al., Ann Lab Med 2015;35:578-585).

[0078] In several embodiments, the sample may be a liquid sample, such as a biological fluid. In certain embodiments, the sample may be a body fluid. Exemplary but non-limiting examples of body fluids include whole blood, serum, plasma, urine, semen, (female) follicular fluid, and saliva. In particular, the sample may be a blood sample selected from whole blood, serum, and plasma. In certain embodiments, the sample may be serum or plasma. Depending on the type of sample used, a sample preparation step may be required before the sample is subjected to SPE, and the volume of the sample may need to be adjusted. Those skilled in the art know how to perform such sample preparation.

[0079] The volume of the sample subjected to SPE may be varied depending on the type of sample and the type and / or concentration of the analyte to be detected in the sample. A smaller sample volume has the advantage of reducing the volume of reagents required for analysis and the total analysis time (e.g., by shortening the time for liquid chromatography), and providing an opportunity to subject additional sample material to various analyses. The combination of SPE extraction and concentration steps in the method of the present invention contributes to improved sensitivity and therefore allows for the maintenance of a low sample volume.

[0080] For example, the sample volume may be 250 μl or less, 200 μl or less in one embodiment, 150 μl or less in one embodiment, and 150 μl in another embodiment. In several embodiments, sample volumes of 150 μl to 250 μl, or more specifically 150 μl to 200 μl, may be used. In a particular embodiment, the sample volume may be 150 μl.

[0081] Depending on the type of sample and the analyte, a portion of the analyte may form complexes with one or more proteins (e.g., steroids may be bound to steroid-binding proteins and / or sex hormone-binding globin) or other sample components (e.g., serum components or albumin). The method of the present invention may include a step of debinding the analyte to be detected and / or quantified from its binding partner, such as a protein (e.g., sex hormone-binding globin). The debinding step may be performed in particular before the SPE. Performing a debinding / pretreatment step may increase the availability of one or more steroids for detection and / or quantification. The pretreatment step may be a “deproteinization” step, i.e., a step of debinding some or all of one or more steroids from one or more proteins to which they are bound.

[0082] The pretreatment / release step may include adding a release agent (e.g., a deproteinizer) to the sample. The release agent (e.g., a deproteinizer) is an agent that, when added to the sample, releases one or more steroids from their binding partners (proteins such as sex hormone-binding globin and / or other components of the sample) in the sample. In some embodiments, a release composition (e.g., a deproteinizer composition) may be used. The release composition (e.g., a deproteinizer composition) is a mixture of two or more substances containing at least one agent that, when added to the sample, causes the release of one or more steroids from their binding partners (proteins such as sex hormone-binding globin and / or other components of the sample). Deproteinizers and compositions, as well as techniques for using them, are known in the art. In the context of this disclosure, the release agent (e.g., a deproteinizer) may be an organic solvent, such as an organic solvent selected from acetonitrile (ACN), methanol (MeOH), and dimethyl sulfoxide (DMSO). Examples of liberation compositions (e.g., deproteinizing compositions) include, but are not limited to, mixtures comprising at least two from the group consisting of acetonitrile (ACN), methanol (MeOH), and dimethyl sulfoxide (DMSO). The volume of the liberation agent (e.g., deproteinizing agent) and / or liberation composition (e.g., deproteinizing composition) added to the sample may be adjusted depending on the agent and / or agent composition and the type of bond it is to interfere with. For example, ACN may be added to the sample to a final concentration of 1 to 10 vol%, particularly 2 to 5 vol%, particularly about 2 vol%, or exactly 2 vol%. MeOH may be added to the sample to a final concentration of, for example, 2.5 to 30 vol%, particularly 5 to 15 vol%, particularly 7.5 vol%. DMSO may be added to the sample to a final concentration of 2 to 20 vol%, particularly 3 to 10 vol%, particularly 5 vol%. The pretreatment step in the context of the present invention may further or alternatively include lowering or raising the pH of the sample. It is known in the art that changes in pH can interfere with the binding of steroids to sample components. For example, the pH can be acidified to 5 or less, particularly 4 or less, particularly 3 or less, and particularly 2 or less.

[0083] In preferred embodiments, the method of the present invention may include a pretreatment step prior to step a), which includes adding an organic solvent as a decontaminating agent (e.g., at a neutral pH). For example, the organic solvent acetonitrile (ACN) may be added to the sample at the final concentration as described above.

[0084] In several embodiments, pretreatment conditions known in the Art may also be used. Non-limiting examples of pretreatment conditions are described by reference in their entirety in Gervasoni, J. et al. (Clin Biochem, 2016, 49(13-14): pp. 998-1003, which are incorporated herein by reference.

[0085] The method of the present invention includes the step of “extracting an analyte from a sample using solid-phase extraction (SPE) to obtain an SPE extract containing the analyte.” “Extracting an analyte from a sample” means that the complexity of the sample is reduced; that is, the analyte is partially or completely separated or purified from other samples. The reduction of sample complexity by extraction facilitates mass spectrometry and reduces background signal. In some embodiments, the step of “extracting an analyte from a sample” may also be called “concentrating an analyte from a sample.” “Concentration” in this context means that an SPE extract is produced in which the amount of analyte is increased compared to other sample components. In some embodiments, the abundance of analyte relative to at least one other sample component may be increased.

[0086] As used in the context of the present invention, “solid-phase extraction” (SPE) refers to a method for partially or completely separating an analyte or group of analytes from other components contained in a liquid mixture and / or sample. The SPE method depends on the solid-phase and liquid-phase distribution of the analyte and one or more other compounds contained in the mixture and / or sample. In a first embodiment of solid-phase extraction according to this disclosure, the analyte may have a higher binding affinity to the solid phase than one or more other compounds in the mixture or sample. Due to the high binding affinity of the analyte to the solid phase, the analyte can be partially or completely separated, i.e., extracted, from the other compounds in the mixture and / or sample. In a second alternative embodiment, the analyte may have a lower binding affinity to the solid phase than the binding affinity of one or more other compounds contained in the mixture and / or sample subjected to solid SPE. In this embodiment, one or more steroids remain in the liquid phase, and one or more other components of the sample are removed by binding to the solid phase. Therefore, as used herein, the term solid-phase extraction includes various embodiments such as: (i) retention of an analyte in a solid phase (sometimes including one or more washing steps) that allows for partial or complete removal of other compounds in the liquid phase; and (ii) retention of other compounds in a solid phase and extraction of the analyte in the liquid phase. In several embodiments (i), the SPE typically involves elution using a suitable elution solution to release the reversibly bound analyte from the solid phase. The elution solution may be selected depending on the binding principle of the solid phase.

[0087] As used in the context of the present invention, “solid-phase extraction” includes, but is not limited to, techniques such as classical solid-phase extraction methods using solid-phase extraction cartridges / columns or solid-phase tips. In particular, the term “solid-phase extraction” in the context of the present invention includes particle-based, and especially bead-based, workflows. The term “solid-phase extraction” in the context of this disclosure includes different separation principles. In several embodiments, the analyte (i.e., one or more steroids) may be delayed by a solid phase (e.g., beads), while one or more other sample components remain in the liquid phase. In other embodiments, the analyte (i.e., one or more steroids) may remain in the liquid phase, while one or more other sample components may be bound to the solid phase.

[0088] In the context of the present invention, the “solid phase” used in solid-phase extraction may include, but is not limited to, a surface or particles (e.g., microparticles such as microbeads). In certain embodiments, the solid phase may be beads, particularly microbeads. The beads (e.g., microbeads) may be nonmagnetic, magnetic, or paramagnetic. In a particularly preferred embodiment, the solid phase may be magnetic microbeads. The beads (e.g., microbeads, particularly magnetic microbeads) may be manufactured from a variety of different materials. The beads (e.g., magnetic beads) may have a variety of sizes (e.g., within the μm range) and may include surfaces with or without pores.

[0089] In certain embodiments of the present invention, the solid phase may be an optional solid phase; that is, a solid phase material that is held in a suspension and is dispenseable. Non-limiting examples of such dispenseable solid phases include particles, particularly beads, more specifically microbeads, and even more specifically magnetic microbeads. Dispensable solid phases have the advantage of being able to be used efficiently in random access mode in automated sample preparation and mass spectrometers, which may require the use of different solid phases for different analytes. Furthermore, the amount of dispenseable solid phase material can be more easily adjusted.

[0090] The solid phase (e.g., particles, especially magnetic particles, especially magnetic microbeads) may be coated to enable binding / capture of the analyte. Suitable coatings for capturing / binding one or more steroids are known in the art. In several embodiments, the solid phase (e.g., particles, particularly magnetic particles, particularly magnetic microbeads) may be coated with antibodies or fragments thereof that specifically bind to the analyte. In certain embodiments, immunobeads that bind to / capture the analyte (i.e., magnetic particles such as microbeads having antibodies or antigen-binding fragments bound to their surface) may be used as the solid phase for SPE. In other embodiments, the solid phase may be coated with a porous polymer matrix that allows for the delay of the analyte.

[0091] In several embodiments, particles such as those described in International Publication No. 2018189286 or International Publication No. 2019141779 may be used in SPEs for capturing analytes (e.g., steroids) from a sample. These documents, in particular the particles or beads described therein, are incorporated herein by reference in their entirety.

[0092] Examples of solid-phase extraction in the context of the present invention include flow-through solid-phase extraction and batch solid-phase extraction.

[0093] Flow-through solid-phase extraction means holding the solid phase in a container (e.g., a cartridge) and applying the sample (or pre-treated sample) to the solid phase in a flow-through process. Depending on the case, the flow-through may include an incubation time defined as the time during which the sample is in contact with the solid phase while the flow-through is blocked. For example, flow-through solid-phase extraction may be performed using a solid-phase extraction cartridge / column or solid-phase tip. Flow-through solid-phase extraction may include one or more washing steps in which the residual liquid phase is removed.

[0094] As used herein, “batch-based solid-phase extraction” refers to a solid-phase-based separation method that does not include a flow-through step. “Batch-based solid-phase extraction” includes the steps of bringing a sample (or pre-treated sample) into contact with a solid phase, optionally incubating the sample in the presence of the solid phase for a defined time (as required for analytic binding or binding of one or more other sample components, depending on the separation principle), and separating the solid phase from the liquid phase by means other than flow-through (e.g., pelletizing). In particular, batch-based solid-phase extraction includes embodiments in which the solid phase is formed by particles (especially magnetic particles such as beads), and the separation of the solid phase from the liquid phase in the SPE is achieved by pelletizing the beads. Pelleting the beads may be achieved by centrifugation or other means. In certain embodiments, pelletizing the beads does not necessarily involve centrifugation. In certain preferred embodiments, the beads may be magnetic, and the beads may be pelletized by magnetic force.

[0095] In certain embodiments, the solid phase used in the SPE may be a batch SPE, preferably a batch SPE using (micro)beads, and more preferably a batch SPE using magnetic (micro)beads. The batch SPE may be based on the binding / capture of the analyte to the solid phase used in the SPE. In other embodiments, other sample components may be bound to the solid phase, and the analyte may remain in the liquid phase.

[0096] A solution containing the analyte obtained by SPE, or a large portion thereof, is referred to herein as the “SPE extract.” Depending on the separation principle used in SPE (i.e., delay of the analyte on the solid phase or in the liquid phase), the “SPE extract” may correspond to the liquid phase of the sample obtained after incubation with the solid phase (in these embodiments, the analyte does not bind to the solid phase), or to the eluate obtained by elution from the solid phase using an elution solvent / solution (in these embodiments, the analyte binds to the solid phase and is subsequently eluted). In certain embodiments, the analyte may be delayed in the solid phase, and the SPE extract may correspond to the eluate obtained by eluting from the solid phase using an elution solvent.

[0097] In several embodiments, the SPE extract may contain 50% to 100% by volume of an organic solvent. The organic solvent may be methanol or acetonitrile.

[0098] In certain embodiments, the SPE is a) Attachment / capture of analytes to the solid phase; b) One or more washing steps, if applicable; and c) A step of eluting an analyte from a solid phase, wherein the obtained eluate is called an SPE extract and contains the analyte. It may include.

[0099] The binding of the analyte to the solid phase may involve incubation of the solid phase and the sample for a predetermined time under conditions that allow for the detection of analyte capture.

[0100] In a particularly preferred embodiment, the SPE may be a magnetic particle-based workflow. A magnetic particle-based workflow is a) The process of binding / capturing analytes to magnetic particles; b) One or more washing steps, if applicable; and c) A step of eluting an analyte from a solid phase, wherein the obtained eluate is called an SPE extract and contains the analyte. It may include.

[0101] Magnetic bead-based workflows are d) Further comprising separating the SPE extract from the beads.

[0102] This separation can be achieved by pelletizing the magnetic beads (e.g., by magnetic force) in a first reaction vessel and transferring the eluate to another reaction vessel.

[0103] Therefore, in certain embodiments, the present invention relates to a method for detecting or quantifying an analyte in a sample using mass spectrometry, a) A step of extracting the analyte from the sample using a magnetic particle-based workflow (for example, as described elsewhere herein) to obtain an eluate containing the analyte; b) A step of concentrating the analyte in an eluent, wherein the concentration includes partially evaporating the solvent from the eluent obtained in a); and c) A step of detecting and / or quantifying the analyte in the sample using mass spectrometry. This provides a method that includes [something].

[0104] In other words, "extracting an analyte from a sample using solid-phase extraction (SPE) to obtain an SPE extract containing the analyte" may, in embodiments of the present invention, mean "extracting an analyte from a sample using a magnetic particle-based workflow to obtain an eluate containing the analyte."

[0105] A "magnetic particle-based workflow" refers to a method of extracting analytes from a sample using magnetic particles (e.g., magnetic microbeads). The magnetic particles can bind to the analyte while other sample components are partially or completely removed in the liquid phase.

[0106] In a particularly preferred embodiment, the present invention uses magnetic particles coated with an antibody or antigen-binding fragment that specifically binds to an analyte (e.g., multiple copies of one antibody or antigen fragment). These magnetic particles are also referred to herein as immunobeads. Thus, in one aspect, the present invention relates to a method for detecting or quantifying an analyte in a sample using mass spectrometry, a) A step of purifying the sample from the sample using magnetic particles coated with sample-specific antibodies (i.e., immunobeads) to obtain an eluate containing the sample; b) A step of concentrating the analyte in an eluent, wherein the concentration includes partially evaporating the solvent from the eluent obtained in a); and c) A step of detecting and / or quantifying the analyte of the sample using mass spectrometry (e.g., LC-MS). Regarding methods including

[0107] The SPE of the present invention may include one or more washing steps using a washing solution. This method may include, for example, one or two washing steps using a washing solution. The washing step may be performed after the analyte has bound / captured to the solid phase but before the analyte has eluted from the solid phase.

[0108] In embodiments where the analyte needs to be bound to a solid phase and eluted to form an SPE extract, this can be achieved using an elution solvent. The elution solvent may be added to the solid phase, and the solid phase may be incubated in the presence of the elution solvent. The incubation time may be adjusted depending on the solid phase used and how harsh the elution solvent is.

[0109] The composition of the elution solvent for SPE (including magnetic particle-based workflows) can be selected depending on the solid phase, the analyte, and the principle of interaction between the analyte and the solid phase.

[0110] In several embodiments, the elution solvent may contain acetonitrile (ACN) in concentrations particularly 40–100% by volume, particularly 45–90% by volume, particularly 50–70% by volume, and particularly 60% by volume. The aforementioned ACN-containing elution solvents may be particularly used in embodiments using particles such as magnetic particles disclosed elsewhere in this specification. These elution solvents may also be used, in particular, when immunobeads are used.

[0111] In several embodiments, the elution solvent may contain methanol in concentrations particularly of 60–100% by volume, particularly of 70–90% by volume, and particularly of 80% by volume. The elution solvent may also be a mixture of methanol and water. The methanol-containing elution solvents described above may be particularly used in embodiments using particles such as magnetic particles disclosed elsewhere in this specification. These elution solvents may also be used, in particular, when immunobeads are used.

[0112] The method of the present invention may include the use of one or more internal standards (ISTDs), particularly isotope-labeled internal standards. ISTDs can be used for the quantification of analytes. Preferably, one or more internal standards are added to the sample in predetermined known amounts before the SPE and any pretreatment steps.

[0113] An "internal standard (ISTD)" is typically a compound that exhibits similar physicochemical properties to the analyte of interest when subjected to a mass spectrometry detection workflow (i.e., including any sample preparation, concentration, and actual detection steps). Furthermore, the ISTD is typically selected so as not to occur spontaneously in a sample being measured in a substantial amount (e.g., less than 1% of the analyte volume). The ISTD exhibits similar or identical chemical properties to the analyte of interest, but is still clearly distinguishable from the analyte of interest by mass spectrometry. For example, during chromatographic separation such as gas or liquid chromatography, the ISTD has approximately the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. The ISTD, however, exhibits a different molecular weight than the analyte of interest from the sample. This allows for the distinction of ions from the ISTD and ions from the analyte by mass spectrometry using different mass / charge (m / z) ratios. Both are subjected to fragmentation to obtain daughter ions. These daughter ions can be distinguished by their respective m / z ratios and their respective parent ions. As a result, separate determination and quantification of signals from ISTD and analyte are possible. Since ISTD is added in known amounts, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of analyte. Thus, by adding ISTD, it becomes possible to relatively compare the amounts of detected analytes, enabling clear identification and quantification of the target analyte present in the sample when the analyte reaches the mass spectrometer. Although not always the case, typically, ISTD is an isotopically labeled variant of the target analyte (e.g., 2 H, 13 C, and / or 15 (This includes at least three of the labels such as N.)

[0114] An exemplary but non-limiting ISTD for quantifying testosterone is 13C3-testosterone (e.g., available from Cerilliant). An exemplary but non-limiting ISTD for quantifying estradiol is 13C3-estradiol (e.g., available from Cerilliant).

[0115] Mass spectrometry may include liquid chromatography (LC). In a particularly preferred embodiment, mass spectrometry may be LC-MS or LC-MS-MS.

[0116] In some embodiments, liquid chromatography may be high-pressure liquid chromatography (HPLC). HPLC may be based on different column materials known in the art. The flow rate of HPLC can be adapted to the analyte and, if necessary. In certain embodiments, the flow rate of HPLC may be 0.20 to 1.0 ml / min, particularly 0.44 ml / min.

[0117] In some embodiments, the liquid chromatography of mass spectrometry may be rapid LC.

[0118] In some embodiments, liquid chromatography of mass spectrometry may be microLC.

[0119] In some embodiments, the liquid chromatography of mass spectrometry may be UHPLC, for example, UHPLC using microLC.

[0120] In several embodiments, the HPLC separation principle may be reverse-phase HPLC (RP-HPLC). RP-HPLC may be, but is not limited to, C18-HPLC.

[0121] Ionization in mass spectrometry may be based on various techniques as described elsewhere in this specification. In one embodiment, electrospray ionization (ESI) is used.

[0122] The MS instrument used for mass spectrometry may be a tandem mass spectrometer, particularly a triple quadrupole instrument.

[0123] In certain embodiments, the method of the present invention may be automated. "Automated" means that one or fewer, preferably manual, handling steps are not required, except for the step of applying the sample and reagents to the system. Manual handling steps include, in particular, the manual addition of reagents to the sample and the transfer of the sample from one device to another during processing.

[0124] In several embodiments, the method of the present invention may not include a centrifugation step. In particular, SPE and / or concentration can be performed without centrifugation. By preventing the centrifugation step (e.g., by using a magnetic bead-based SPE workflow), automation can be made easier, and the sample preparation system has the advantage of not requiring a centrifuge. Furthermore, the time required to separate the solid phase during SPE can be reduced.

[0125] In several embodiments, the method of the present invention may not include liquid-liquid extraction, particularly during sample preparation. In other words, the SPE and concentration steps may be the only sample preparation steps (e.g., LC-MS, especially LC-MS / MS) prior to mass spectrometry. Liquid-liquid extraction steps are typically cumbersome and consume organic solvents.

[0126] The methods of the present invention may be performed, in particular, using a random-access compatible system in random-access compatible mode. "Random access" preferably means that the reagents and system settings of the present invention described herein are compatible with other assays addressing different analytes without requiring system adaptation or equilibration, in particular manual system adaptation or equilibration (including changes to mass spectrometry and / or LC settings).

[0127] The mass spectrometry of the present invention can be performed using multiple reaction monitoring (MRM) mode.

[0128] In one embodiment, the present invention also relates to a method for preparing a sample for mass spectrometry (e.g., LC-MS / MS) to detect and / or quantify an analyte, a) A step of extracting the analyte from a sample using a solid-phase extract (SPE) to obtain an SPE extract containing the analyte; b) A step of concentrating the analyte, wherein the concentration step includes evaporating the solvent from the SPE extract obtained in a). This provides a method that includes [something].

[0129] The above-mentioned methods for detecting and / or quantifying the analyte are applicable mutatis mutandis to methods for preparing samples for mass spectrometry (e.g., LC-MS / MS) to detect or quantify the analyte.

[0130] The term “mass spectrometry” (“Mass Spec” or “MS”) relates to an analytical technique used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or “m / z”. MS techniques generally involve (1) ionizing a compound to form a charged compound, and (2) detecting the mass-to-charge ratio. Compounds can be ionized by any suitable means and then detected. A “mass spectrometer” generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized, and these ions are then introduced into a mass spectrometer, in which, due to a combination of magnetic and electric fields, the ions follow a path in space depending on their mass (“m”) and charge (“z”). The term “ionization” refers to the process of generating ions of an analyte that have a net charge equal to one or more electron units. Anions have a net negative charge of one or more electron units, while cations have a net positive charge of one or more electron units. The MS method can be performed in either an "anion mode," where anions are generated and detected, or a "cation mode," where cations are generated and detected.

[0131] Tandem mass spectrometry, or MS / MS, involves multiple steps of selection and detection in mass spectrometry, with analyte fragmentation occurring between steps. In a tandem mass spectrometer, ions are generated in an ion source and separated by mass-to-charge ratio in the first stage of mass spectrometry (MS1). Ions with a specific mass-to-charge ratio (precursor or parent ions) are selected, and fragment ions (also called daughter ions) are generated by collision-induced dissociation, ion-molecular reactions, and / or photodissociation. The resulting ions are then separated and detected in the second stage of mass spectrometry (MS2).

[0132] Typically, mass spectrometry involves the following three steps: (1) The sample containing the analyte of interest is ionized, usually by adduct formation with cations, often by protonation to cations. Ionization sources include, but are not limited to, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). (2.) Classify and separate the ions according to their mass and charge. High electric field asymmetric waveform ion mobility spectroscopy (FAIMS) can be used as an ion filter. (3.) The separated ions are detected, for example, in multiple reaction mode (MRM), and the results are displayed on a chart.

[0133] The term "electrospray ionization" or "ESI" refers to a method in which a solution is passed through a short capillary tube, and a high positive or negative potential is applied to the end of the capillary tube. The solution that reaches the end of the tube is vaporized (atomized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets passes through an evaporation chamber, which is slightly heated to evaporate the solvent and prevent condensation. The smaller the droplets, the higher the electrical surface charge density becomes until ions and neutral molecules are released due to the natural repulsion between like charges.

[0134] The term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry method similar to ESI. However, APCI generates ions through ionic molecular reactions occurring within an atmospheric pressure plasma. The plasma is maintained by an electrical discharge between a spray capillary and a counter electrode. The ions are typically extracted into the mass spectrometer using a series of differentially pumped skimmer stages. Solvent removal can be improved using counterflow drying and preheating of N2 gas. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar entities.

[0135] "Multiple reaction mode" or "MRM" is a detection mode of an MS instrument in which a precursor ion (also called the parent ion) and one or more fragment ions are selectively detected and / or quantified.

[0136] Mass spectrometers separate and detect ions of slightly different masses, thus easily distinguishing different isotopes of a given element. Therefore, mass spectrometry is an important method for the accurate mass determination and characterization of analytes, including, but not limited to, low molecular weight analytes, peptides, polypeptides, or proteins. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits, and functional interactions, and the whole-body analysis of proteins in proteomics. De novo sequencing of peptides or proteins by mass spectrometry can usually be performed without prior knowledge of the amino acid sequence.

[0137] Mass spectrometry determination may be combined with additional analytical methods, including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and / or separation techniques based on ion mobility.

[0138] In the context of this disclosure, a sample may be a sample derived from an “individual” or “subject.” Typically, the subject is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In a preferred embodiment, the sample is obtained from a human.

[0139] The term "chromatography" refers to the process by which a chemical mixture, carried by a liquid or gas, is separated into its components as chemical entities flow around or over a fixed liquid or solid phase, interacting with the surrounding environment.

[0140] The term "liquid chromatography" or "LC" refers to the process of selectively delaying one or more components of a fluid solution as a fluid uniformly permeates through a column or capillary pathway of finely divided substances. The delay is due to the distribution of the mixture components between one or more stationary phases and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). When the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase), it is called normal-phase liquid chromatography (NPLC), and when the stationary phase is less polar than the mobile phase (e.g., a water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase), it is called reverse-phase liquid chromatography (RPLC).

[0141] High-performance liquid chromatography (HPLC) is a method of liquid chromatography in which the degree of separation is increased by passing a mobile phase, typically a densely packed column, through a stationary phase under pressure. Typically, the column is packed with a stationary phase consisting of irregular or spherical particles, a porous monolithic layer, or a porous membrane. Historically, HPLC has been divided into two distinct subclasses based on the polarity of the mobile and stationary phases: NP-HPLC and RP-HPLC.

[0142] MicroLC refers to an HPLC method that typically uses columns with a narrow (norrow) inner diameter of less than 1 mm, for example, about 0.5 mm. "Ultra-high-performance liquid chromatography" or "UHPLC" refers to a method using 120 MPa (17,405 lbf / in). 2 ) or refers to the HPLC method using a pressure of approximately 1200 atmospheres.

[0143] Rapid LC refers to an LC method (microLC, UHPLC) that uses a short column with the above-mentioned inner diameter and length of less than 2 cm, for example, 1 cm, and applies the above-mentioned flow rate and pressure. Short rapid LC protocols include a trapping / washing / elution process using a single analytical column and achieve LC in a very short time of less than 1 minute.

[0144] Furthermore, well-known LC methods include hydrophilic interaction chromatography (HIC), size exclusion LC, ion exchange LC, and affinity LC.

[0145] LC separation may be single-channel LC or multi-channel LC, including multiple LC channels arranged in parallel. In LC, analytes can be separated according to their polarity or log-P value, size, or affinity, as is generally known to those skilled in the art.

[0146] As used herein, “detecting” or “detecting” an analyte in a sample means at least determining whether or not the analyte is present in the sample. Detecting an analyte may or may not include quantifying the analyte, i.e., determining an absolute or relative amount of the analyte.

[0147] As used herein, “quantifying” or “determining” an analyte in a sample means determining the presence and amount of the analyte in the sample. The amount may be an absolute or relative amount of the analyte in the sample. An absolute amount may be any quantitative measure, such as concentration or mass. A relative amount may be any relative quantitative measure. For example, the amount of an analyte may be detected by comparing it to the amount of a component in another sample, an internal standard added to the sample, or a reference sample containing the same one or more analytes.

[0148] As used herein in the context of adding a drug and / or composition to a sample, “final concentration” refers to the concentration of the drug and / or composition in the mixture obtained by adding the drug and / or composition to the sample.

[0149] The term "solvent" includes any solvent or mixture of solvents that hold the analyte of interest (e.g., one or more steroids) in solution. Exemplary but non-limiting examples of components of a solvent or solvent mixture are water, alcohol (e.g., methanol or ethanol), and acetonitrile.

[0150] "Steroids" are a group of molecules known in the art. Steroids are typically compounds having a core structure of four rings, also called steroid rings A, B, C, and D. The core ring structure of a steroid usually consists of 17 carbon atoms, which are linked by four fused rings: three C6 cyclohexane rings (rings A, B, and C) and one C5 cyclopentane ring (ring D). Steroids vary depending on the functional groups attached to their four rings and the oxidation state of the rings.

[0151] Some steroids also involve changes in the ring structure, in which one of the four rings is open.

[0152] It should be understood that the word "comprise," as well as its variations such as "comprises" and "comprising," means to include a specified integer or process, or a group of integers or processes, but does not mean to exclude any other integer or process, or a group of integers or processes.

[0153] As used herein and in the appended claims, the singular forms "a," "an," and "the" also include individual plural terms unless the context specifically indicates otherwise.

[0154] Furthermore, the terms “particularly,” “more specifically,” “specifically,” “more specifically,” or similar terms (e.g., preferably or more preferably) as used below will be used in conjunction with features of specific or alternative embodiments without limiting the possibility of alternatives. The disclosed method / system may be performed by using alternative features, as will be recognized by those skilled in the art. Similarly, features introduced by “in one embodiment of the disclosed method / system,” “in an embodiment,” or similar expressions are intended to be additional and / or alternative features, without any limitation on alternative embodiments, without any limitation on the scope of the disclosed method / system, and without any limitation on the possibility of combining the thus introduced features with any other features of the disclosed method / system, or any other features of the disclosed method / system.

[0155] Ratios, concentrations, quantities, and other numerical data may be expressed or presented herein in the form of “ranges.” In the context of this disclosure, such range forms are used merely for convenience and conciseness and should therefore be understood to be interpreted flexibly to include not only the numbers explicitly listed as limits of the range, but also all individual numbers or subranges that are contained within that range as if each number and subrange were explicitly listed. For example, the numerical range “4% to 20%” should be interpreted to include not only the explicitly listed value of 4% to 20%, but also the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, and subranges such as 4 to 10%, 5 to 15%, 10 to 20%, etc. This same principle applies to ranges that list minimum or maximum values. Furthermore, such interpretation should apply regardless of the width or characteristics described of the range.

[0156] When used in relation to a number, the term "approximately" means to encompass a range of numbers that have a lower limit 5% less than the given number and an upper limit 5% greater than the given number. In this disclosure, references to solvents and solutions may be given in %, (v / v)%, or vol% of a compound. Unless otherwise specified, a solution or solvent is an aqueous solution. For example, 80% MeOH, 80(v / v)%, or 80 vol% MeOH refers to an aqueous mixture containing 80 vol percent of MeOH.

[0157] The following examples and figures are provided to aid in understanding the present invention, and the true scope of the invention is set forth in the appended claims. It is understood that modifications may be made to the procedures described without departing from the spirit of the invention. [Examples]

[0158] Examples material and method Preparation of analyte-specific magnetic immunobeads To prepare magnetic beads coated with analyte-specific antibodies (immunobeads), Elecsys® streptavidin bead suspension was used, and the beads were coated with analyte-specific antibodies. For coating, magnetic separation was used to separate the magnetic beads (1 mg / ml), and the beads were washed with PBS buffer and vortexed. The washing was repeated twice. After the final washing step, the supernatant was removed, and a solution containing biotin-labeled antibody against the analyte was added to the beads. Each anti-analyte antibody solution (50 μg / ml) was added in a volume equal to the volume of the beads, and the mixture was incubated overnight at 4°C. Finally, three washing steps using PBS were performed to remove unbound antibodies. The washed beads were resuspended in a volume equivalent to the original volume of beads to a final concentration of 1 mg / mL.

[0159] To produce testosterone-specific magnetic immunobeads, biotin-labeled monoclonal anti-testosterone antibodies were conjugated to the beads. To produce estradiol-specific magnetic immunobeads, biotin-labeled monoclonal anti-estradiol antibodies were conjugated to the beads.

[0160] sample In the context of the present invention, various samples were used. The samples particularly included solutions to which a specified amount of each analyte was added. The matrix to which each analyte was spiked is specified in each example. The matrices used throughout the examples included 60 vol% MeOH solution, UniDil, and Golden West Serum (Golden West Diagnostic LLC; catalog number MSG4000).

[0161] Internal standards used in quantitative mass spectrometry As internal standards, heavy isotope-labeled isotopes of each analyte were used. 13C3-testosterone (Cerilliant) was used for the detection of testosterone. 13C3-estradiol (Cerilliant) was used for the quantification of estradiol. Unless otherwise specified, 10 μl of 10 ng / ml internal standard solution was spiked to a 150 μl sample volume before sample preparation and immunobead-based solid-phase extraction.

[0162] Sample pretreatment The analytes used in the following examples are steroids. Steroids can be bound by binding to proteins in relation to the sample, such as serum or plasma. Therefore, pretreatment was performed to debound the steroids from the bound proteins before concentrating the analytes by immunobead-based solid-phase extraction. For pretreatment, 50 μl of 30 vol% MeOH aqueous solution was added to 150 μl of the sample, and the sample was mixed by vortexing.

[0163] Solid-phase extraction of immunobead-based analytes Immunobadze-based solid-phase extraction was performed to extract and / or concentrate analytes in the sample. For this purpose, analyte-specific magnetic immunobeads (see above) were added to a pre-treated sample spiked with an internal standard. Specifically, 40 μl of 1 mg / mL solution was added. The mixture was vortexed and incubated at 37°C for 7.5 minutes to allow the analyte to bind to the beads. The beads were then washed two or three times with 200 μl of water. Finally, the analyte was eluted from the beads using aqueous 80% methanol elution. A volume of 150 μl of elution solution was used in experiments involving a subsequent evaporation step (full or partial). A volume of 60 μl of elution solution was used in workflows without evaporation. For elution, the magnetic beads were separated magnetically after the final washing step, the supernatant was removed, the elution solution was added to the beads, the mixture of beads and elution solution was vortexed, and incubated at 37°C for 2 minutes. Finally, the beads were separated by magnetic force, and 130 μl (workflow including evaporation) or 40 μl (workflow without evaporation) of the eluent supernatant (also called solid-phase extract) containing the analyte was removed and pipetted into a new container.

[0164] Subsequently, the recovered eluate was either evaporated completely or partially (see below), or, in a workflow without evaporation, 40 μl of the recovered eluate was diluted to a final volume of 107 μl using LC-DIL.

[0165] evaporation Evaporation of the immunobead-based SPE or spike solution eluate was performed by applying a vacuum and heating at 50°C to 100°C using a custom-made apparatus. The starting volume was 130 μl unless otherwise specified.

[0166] The sample was evaporated to dryness to ensure complete evaporation. The remaining pellet was then divided into 40 μl LC-DILs using a vortex.

[0167] To partially evaporate the solvent, evaporation was carried out until a final solvent volume of 20–40 μl remained. The final volume of the sample before being subjected to LC-MS was adjusted to 40 μl (if necessary) using LC-DIL.

[0168] High-performance liquid chromatography (HPLC) connected to a mass spectrometry (MS) system. High-performance liquid chromatography (HPLC) was performed using an Agilent 1200 Infinity II LC System (Waldbron, Germany) and a PAL LC injection and autosampler system (Zwingen, Switzerland). The instruments were controlled via an AB Sciex Analyst device driver. Chromatographic separation was performed using a C18 HPLC column (1.0 or 2.1 mm inner diameter × 50 mm) packed with SunShell 2.6 μm fused core particles from ChromaNik (Osaka, Japan). The LC solvents used were (A) water and (B) 0.2 mM NH4F in methanol, with a flow rate of 440 μl / min. The LC gradient was established by grading from 39%–60% solution B to 90%–98% solution B within 0.7–1.2 minutes. The volume injected into the LC-MS system was 20 μl, regardless of the sample preparation workflow (with or without evaporation).

[0169] Mass spectrometry (MS) Mass spectrometry detection was performed using an AB Sciex (Darmstadt, Germany) Triple Quad 6500+LC-MS / MS system or an equivalent MS instrument.

[0170] For testosterone measurement, the MS setting was selected to positive mode and optimized for sensitivity. For estradiol measurement, the MS setting was selected to negative mode and also optimized for sensitivity.

[0171] Data Analysis The MS system used settings optimized for the analyte and associated software for machine control and data analysis. Chromatographic peaks of analyte-selective MRM transitions were integrated by Gaussian fitting to generate peak areas and signal-to-noise ratios (S / N) for the analyte and internal standards, which were directly compared from different workflows (i.e., dilution, partial evaporation, and complete evaporation workflows) to compare improvements or recovery of sensitivity. The accuracy and sensitivity of the LC-MS method were further evaluated using the analyte / ISTD ratio to compare various workflows.

[0172] Example 1: Concentration of testosterone in a pure (neat) sample using complete and partial evaporation This example compares three different workflows for performing LC-MS on spiked samples: (1) sample dilution; (2) complete evaporation; and (3) partial evaporation.

[0173] Specifically, samples containing 60 pg / ml of testosterone in 60 volume% MeOH were prepared. These samples mimic elutes / extracts from bead-based SPEs, particularly the elutions from immunobead-based SPEs described in the methods described above, and are also used below herein.

[0174] For samples used in the dilution workflow, the sample was diluted with 40 μL of H2O to a total final volume of 80 μL. For samples that were completely evaporated, the sample was evaporated and completely dried, then reconstituted with 40 μL of 30% MeOH. For samples used for partial evaporation, the sample was evaporated to 10 μL (partial evaporation), then diluted with 30 μL of 30% MeOH to a total final volume of 40 μL. All samples were ensured to ultimately contain 30% MeOH of organic matter.

[0175] The signal intensity of the final sample subjected to LC-MS and testosterone was measured (see Figure 1A). By setting the dilution workflow intensity to 100%, the testosterone recovery rate of the two evaporation workflows was calculated as a percentage (see Figure 1B).

[0176] Since all samples contained the same amount of analyte, both fully evaporated and partially evaporated samples were expected to have a 200% analyte recovery rate. While increased recovery rates were achieved with both full and partial evaporation, the recovery rate was remarkably better when using partial evaporation. Another advantage of partial evaporation is the shorter duration of the evaporation process. Reducing the time required for evaporation is particularly important in the context of automated sample preparation and LC-MS analysis systems.

[0177] Considering these results, partial evaporation was chosen for the experiments shown in further examples. Whenever these examples and the figures referring thereto refer to "evaporation," it refers to "partial evaporation" as described above.

[0178] Example 2: Comparison of partial evaporation and dilution of immunobead eluate in an MS sample preparation workflow for estradiol detection. Diagnostic MS measurements typically do not involve pure samples, but rather complex samples containing the analyte in a complex matrix with other components, such as blood-based samples (e.g., serum or plasma). To measure these samples, a sample preparation workflow is typically used that purifies the analyte as much as possible from the remaining components. For this purpose, methods such as SPE may be used. In this example, immunobead-based SPE is used, involving eluting the analyte from the beads with an elution solution that has a high content of organic solvents (e.g., MeOH or acetonitrile). High concentrations of these solvents can impair the LC resolution of LC-MS systems and methods, for example, by peak broadening. Therefore, SPE elutions are typically diluted before LC-MS to reduce the concentration of organic solvents before LC. However, such dilution reduces the analyte concentration, making it more difficult to detect low concentrations of the analyte in the initial sample, especially in samples containing complex matrices that typically exhibit higher background signals than pure samples.

[0179] To compare the performance of partial evaporation and sample dilution in the context of the entire MS workflow, experiments were conducted as described in the Materials and Methods section above, performing the entire MS workflow including (1) addition of an internal standard, (2) SPE based on immunobeads of the analyte, (3) partial evaporation or dilution of the SPE eluate, and (4) LC-MS.

[0180] The samples used in the dilution MS workflow and the evaporation MS workflow were 150 μl of UniDil (Elecsys® Diluent Universal) spiked with estradiol at concentrations of 0.5 pg / ml, 2 pg / ml, 5 pg / ml, 10 pg / ml, and 15 pg / ml, respectively. The dilution workflow and partial evaporation used are schematically shown in Figure 2.

[0181] The MS spectra obtained from the dilution workflow and partial evaporation workflow of a 2 pg / ml sample are shown in Figures 3 and 4, respectively.

[0182] As is evident from Figure 3, using the dilution workflow, the estradiol signal could not be detected in this particular experiment.

[0183] In contrast, the estradiol signal was clearly detectable using a partial evaporation workflow (see Figure 4), demonstrating that improved sensitivity was achieved with this workflow.

[0184] Therefore, this experiment demonstrates that partial evaporation can improve the signal-to-noise ratio and analyte detection sensitivity (limit of quantification) even when measuring analytes in more complex matrices and providing highly purified analyte samples in a multi-step sample preparation workflow, followed by high-resolution LC separation.

[0185] Figure 5B shows the signal intensity for both the partial evaporation workflow and the dilution workflow plotted against analyte concentration. This figure shows that for each sample analyzed, the partial evaporation workflow showed increased signal (peak area) compared to the dilution workflow. Furthermore, a linear measurement range was achieved for both workflows in the analyzed concentration range above LoQ. However, the partial evaporation workflow exhibits a steeper slope of linearity. A steeper slope is a clear advantage, as it increases the dynamism of the signal with respect to a given concentration difference, and therefore can improve accuracy.

[0186] Figure 5A shows the ratio of the analyte signal to the internal standard (ISTD) at the measured analyte concentrations. Both the dilution and partial evaporation workflows exhibit similar ratios and increase linearly with the same slope across the measured concentration range.

[0187] Example 3: Comparison of partial evaporation and dilution of immunobead eluate in an MS sample preparation workflow for testosterone detection. We repeated comparisons of partial evaporation and dilution workflows using GoldenWestSerum samples supplemented with analyte testosterone at concentrations of 25 pg / ml, 150 pg / ml, and 250 pg / ml.

[0188] As in Example 2 above, the entire MS workflow, including (1) the addition of an internal standard, (2) SPE based on immunobeads of the analyte, (3) partial evaporation or dilution of the SPE eluate, and (4) LC-MS, was carried out as specified in the Materials and Methods section above.

[0189] The samples used in the dilution MS workflow and the evaporation MS workflow were 150 μl of Golden West Serum spiked with testosterone at 25 pg / ml, 150 pg / ml, and 250 pg / mL, respectively. The dilution workflow and partial evaporation used are schematically shown in Figure 2.

[0190] Figure 6B shows the signal intensity for both the partial evaporation workflow and the dilution workflow plotted against analyte concentration. This figure shows that for each sample analyzed, the partial evaporation workflow showed increased signal (peak area) compared to the dilution workflow. Furthermore, a linear measurement range was achieved for both workflows in the analyte concentration range above the limit of quantification (LOQ). However, the partial evaporation workflow exhibits a steeper slope of linearity. A steeper slope is a clear advantage, as it increases the dynamism of the signal with respect to a given concentration difference, and therefore can improve accuracy.

[0191] Figure 6A shows the ratio of analyte signal to internal standard (ISTD) at the measured analyte concentrations. For both dilution and partial evaporation workflows, the ratios are similar and increase linearly with the same slope across the measured concentration range.

[0192] This patent application claims priority to European Patent Application No. 20215190.8, the contents of which are incorporated herein by reference.

Claims

1. A method for detecting and / or quantifying an analyte in a sample using mass spectrometry, a) A step of extracting the analyte from the sample using solid-phase extraction (SPE) to obtain an SPE extract containing the analyte, wherein the SPE extract contains 50% to 100% by volume of an organic solvent, and the analyte is a steroid selected from the group consisting of testosterone and estradiol; b) A step of concentrating the analyte, wherein the concentration includes partially evaporating the solvent from the SPE extract obtained in a); the volume of the SPE extract subjected to partial evaporation is reduced by 50% to 95%; b1) A step of diluting the concentrated analyte obtained from step b) with a diluent solvent to obtain a diluted analyte, wherein the diluted analyte contains less than 50% by volume of the organic solvent or further organic solvent prior to step c); and c) A step of detecting and / or quantifying the analyte in the sample using mass spectrometry, wherein the mass spectrometry is mass spectrometry coupled to liquid chromatography (LC-MS). Methods that include...

2. The method according to claim 1, wherein, in (b), the volume of the SPE extract is reduced by 60% to 90%.

3. The method according to claim 1 or 2, wherein, in b), the volume of the SPE extract is reduced by 70% to 80%.

4. The method according to any one of claims 1 to 3, further comprising using a diluent solution to adjust the volume after partial evaporation to a final volume corresponding to 5% to 40% of the volume of the sample to be supplied to the SPE in a).

5. The method according to any one of claims 1 to 4, further comprising using a diluent solution to adjust the volume after partial evaporation to a final volume corresponding to 10% to 30% of the volume of the sample supplied to the SPE in a).

6. The method according to any one of claims 1 to 5, further comprising using a diluent solution to adjust the volume after partial evaporation to a final volume corresponding to 13% to 27% of the volume of the sample provided to the SPE in a).

7. a) The method according to any one of claims 1 to 6, wherein the volume of the sample provided to the SPE in a) is 250 μl or less.

8. a) The method according to any one of claims 1 to 7, wherein the volume of the sample provided to the SPE in a) is 200 μl or less.

9. a) The method according to any one of claims 1 to 8, wherein the volume of the sample provided to the SPE in a) is 150 μl or less.

10. a) The method according to any one of claims 1 to 9, wherein the volume of the sample provided to the SPE in a) is 150 μl.

11. The method according to any one of claims 1 to 10, wherein the organic solvent is selected from the group consisting of acetonitrile and methanol.

12. The method according to any one of claims 1 to 11, wherein the sample is a fluid.

13. The method according to any one of claims 1 to 12, wherein the sample is a biological fluid.

14. The method according to any one of claims 1 to 13, wherein the sample is serum or plasma.

15. The method according to any one of claims 1 to 14, further comprising a pretreatment step for releasing the analyte from the analyte-bound protein.

16. The method according to any one of claims 1 to 15, wherein the solid phase used in the SPE is formed by magnetic particles.

17. The method according to any one of claims 1 to 16, wherein the solid phase used in the SPE is formed by magnetic microbeads.

18. The method according to any one of claims 1 to 17, wherein the solid phase of the SPE is formed of particles coated with an antibody that specifically binds to the analyte.

19. The aforementioned solid-phase extraction (SPE) is a) A step of capturing the analyte in a solid phase; and c) A step of eluting the analyte from the solid phase to obtain the SPE extract containing the analyte. The method according to any one of claims 1 to 18, including the method described in any one of claims 1 to 18.

20. Between a) and c), b) one or more washing steps of the solid phase; The method according to claims 1 to 19, including the method described in claims 1 to 19.

21. The method according to claim 19 or 20, wherein the volume of the added elution solvent corresponds to 50% to 150% of the volume of the sample subjected to SPE.

22. The method according to any one of claims 19 to 21, wherein the volume of the added elution solvent corresponds to 90% to 120% of the volume of the sample subjected to SPE.

23. The method according to any one of claims 19 to 22, wherein the volume of the added elution solvent corresponds to 100% of the volume of the sample subjected to the SPE.

24. The method according to any one of claims 1 to 23, further comprising the step of adding an internal standard for quantification (ISTD) to the sample before step a).

25. The method according to any one of claims 1 to 24, wherein the liquid chromatography (LC) is HPLC or rapid LC.

26. The method according to claim 25, wherein the HPLC is microLC (μLC) and / or ultrahigh-performance liquid chromatography (UHPLC).

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