A method of enriching a sample for amyloid ß peptides

Abstract

A method of enriching a sample for amyloid β peptides, said method comprising: (i) contacting the sample with a solid support having immobilised thereon an anti-amyloid β peptide antibody; (ii) washing said solid support with a washing solution which has a pH between 10.5 and 10.7; (iii) applying an elution solution which has a pH between 10.5 and 10.7 to said solid support; and (iv) collecting the eluted sample.

Description

A method of enriching a sample for amyloid β peptides This application claims priority from and the benefit of United Kingdom patentapplication No.2410666.8, which was filed on 22 July 2024. The entire contents ofthis application are incorporated herein by reference. The present invention relates to methods of analysing samples for the presence ofamyloid β (Aβ) peptides and of enriching samples for Aβ peptides, in particular ofpreparing a sample containing Aβ peptides for analysis by Liquid Chromatography- Mass Spectrometry (LC-MS). Alzheimer’s disease (AD) is the most common type of dementia worldwide with significant socioeconomic implications. The pathogenesis of AD is marked by formation and deposition of insoluble protein aggregates in the brain. Proteinaceous plaque formation in AD is a combination of amyloid beta (Aβ) peptides, derived from the action of β and γ secretases on the amyloid precursor protein, and formation of intracellular neurofibrillary tangles (NFTs) whose major constituent is microtubule- binding protein tau. Aβ peptides are found in both plasma and cerebrospinal fluid(CSF), are 43 amino acids or less in length, and can polymerize to form longinsoluble fibrils consisting of parallel-aligned hydrogen-bonded β-sheets. Theamyloid peptide Aβ 1-40 forms the insoluble β-sheet but is seeded by Aβ 1-42. Thedeposition of these insoluble aggregates from extracellular Aβ deposits as well asintracellular NFTs in the brain is considered the critical event in the developmentand pathology of AD, making Aβ peptides potential biomarkers of disease severity.Traditionally, Aβ peptides as AD biomarkers have been quantified using immunoassay techniques, but these are time consuming, subject to cross-reactivity, and with high batch variation. Methods of quantitation of Aβ peptides in human cerebrospinal fluid (hCSF) havebeen described which use ultra-performance liquid chromatography-tandem massspectrometry (UPLC-MS / MS) (Lame et al. Analytical Biochemistry 419

[2011] 133-139; and Waters application note ‘Amyloid Beta Peptides Quantification by SPE- LC-MS / MS with Automated Sample Preparation for Preclinical Research andBiomarker Discovery). These methods use solid phase extraction (SPE) to preparethe sample which does not provide the selectivity or sensitivity required fordetection of Aβ peptides in plasma.Iino et al. (Journal of Applied Laboratory Medicine, July 2021, 834-845) describe amethod for quantifying Aβ peptides in plasma (in which the concentration of Aβpeptides is low and the level of interfering factors is high) which uses immunoaffinityenrichment followed by LC-MS / MS. In this protocol, Aβ peptides are eluted from theantibodies used for immunoprecipitation (IP) at a pH of 11.64. This Iino methodprovides an approach for analysis of plasma samples, but can lead to backpressure build up during LC-MS due to build-up of interfering non-targetcomponents, limiting application of the method for routine analysis.Thus, there remains a need for a method of preparing samples of Aβ peptides, inparticular preparing such samples for analysis by LC-MS. The present inventor hasfound that an immunoprecipitation method in which the washing and elutingsolutions have controlled pHs provides a robust method of sample preparation.In one embodiment, the present invention provides a method of enriching a samplefor amyloid β (Aβ) peptides, said method comprising(i) contacting the sample with a solid support having immobilised thereonan anti-amyloid β peptide antibody; (ii) washing said solid support with a washing solution which has a pHbetween 10.5 and 10.7; (iii) applying an elution solution which has a pH between 10.5 and 10.7 tosaid solid support; and (iv) collecting the eluted sample.The sample to be enriched may be any material containing or suspected ofcontaining Aβ peptides, preferably it will be a body fluid or tissue sample, such asblood, plasma or any other blood derived fraction, urine, CSF or brain tissue. Preferably the body is a human body but may be a livestock, companion or other animal.The solid support may be any suitable carrier from those widely used in methods of immobilisation, separation and purification etc., provided it can have antibodies immobilised thereon. The solid support may take the form of particles, sheets, membranes, filters, plates or wells etc. Preferably the support is stirable and preferably it is particulate. Conveniently the particulate solid support comprises beads or pellets, e.g. sphericalbeads. Monodisperse particles, i.e. those which are substantially uniform in size arepreferred and are well known in the art. The particles may be non-magnetic polymeric beads, e.g. agarose beads, but to aid manipulation and separation, magnetisable particles are preferred. “Magnetisable” particles are capable of havinga magnetic moment imparted to them when placed in a magnetic field. A solidsupport comprising magnetisable particles can be readily manipulated by magnets, e.g. to remove or aggregate the particles from or within the sample. Superparamagnetic beads are especially preferred, e.g. the widely used Dynabeads such as Dynabeads MyOneTMStreptavidin T1 beads. In order to assist immobilisation of the antibodies, the solid support may have a binding ligand conjugated thereto, such as streptavidin. Immobilised on the solid support are antibodies which bind with high selectivity forAβ peptides, known as anti-amyloid β peptide antibodies. Antibodies may bindselectively to any part of the Aβ peptides derived from Aβ precursor protein (APP).Aβ peptides of interest include those consisting of the first 40 (from the N terminus)amino acids or the first 42 amino acids, known respectively as:Aβ 1-40 – DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVAβ 1-42 – DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAAs used herein, the term “anti-amyloid β peptide antibody” includes antibodyfragments and derivatives capable of selective binding to Aβ peptide(s). Theseantibodies (including antibody fragments such as Fab and fragment constructs suchas scFv) can selectively bind to any part of Aβ peptides and preferably bind Aβ 1-40and Aβ 1-42 peptides. Preferably the anti-Aβ peptide antibody binds selectively tothe sequence of amino acids 1 to 16 within human Aβ. When the solid support iscoated with streptavidin, the antibody is preferably biotinylated. Preferred antibodiesfor use in the methods of the invention include Biotin anti-Aβ, 1-16 antibody from Biolegend, clone 6E10. Alternatively viewed, the present invention provides a method of preparing an Aβpeptide sample for (subsequent) analysis, e.g. by LC-MS, said method comprising(i) contacting a sample with a solid support having immobilised thereon an anti-amyloid β peptide antibody;(ii) washing said solid support with a washing solution which has a pH between10.5 and 10.7;(iii) applying an elution solution which has a pH between 10.5 and 10.7 to saidsolid support; and(iv) collecting the eluted sample.A method of enriching a sample for Aβ peptides (and preparing an Aβ peptidesample for analysis) is described in detail in the examples herein.Starting samples, e.g. plasma samples, may be centrifuged prior to use and aninternal standard solution containing (optionally labelled) Aβ peptides may be addedthereto prior to contacting the sample with the solid support described herein. After contacting the sample with the solid support, the sample and the solid support arepreferably mixed. Mixing may conveniently comprise one or more cycles of rapidmixing (1000-1200 rpm for 10-30 seconds) followed by gentler mixing (400-600 rpm for 5-15 minutes).The contacting step (i), which may include mixing, allows the antibodies to bind toAβ peptides present in the sample in accordance with well-known principles ofimmunoprecipitation (IP).As is standard in IP methods, the solid support with Aβ peptides bound thereto arewashed to remove unbound proteins and other components from the sample. If a particulate magnetic (magnetisable) solid support is used, it may be placed in amagnetic array to allow pull down of the particles and convenient removal of thesupernatant, e.g. by pipetting.The inventor has found that an improved method is provided when the final washingstep prior to elution of Aβ peptides from the solid support is performed with awashing solution which has a pH between 10.5 and 10.7 preferably at or around pH10.6. This washing solution preferably comprises 0.05% aqueous ammonia.pH may be determined in any convenient way, preferably using a pH meter rather than indicator, for accuracy, e.g. a pH meter incorporating a glass electrode and a reference electrode (which may be separate or combined). The pH meter is ideally calibrated with reference to solutions where the pH value is known. The pH valuesrecited here assume an ambient temperature.This and other washing steps are preferably performed with mixing. For this finalwashing step, the solid support and the washing solution may be mixed for 5 to 15minutes, preferably 8 to 12 minutes, more preferably for 10 (or about) 10 minutes. The mixing speed can be chosen to complement the mixing duration and is conveniently done at 600-1500 rpm, preferably 800-1200 rpm e.g. around 1000 rpm. The wash solution is removed prior to elution.Prior to the above final washing step (ii), the solid support may be washed usingdifferent wash solutions. Preferably the penultimate washing step is performed using a washing solution which has a pH between 9 and 10, for example using100mM HCl adjusted to a pH of around 9.4 (e.g.9.2 to 9.6) with ammonia. Thiswashing step typically involves mixing with the solid support, e.g. for around 5 minutes at about 1000 rpm; the skilled reader will be aware that mixing conditions can be varied, e.g. longer mixing times may allow slower mixing speeds. Prior to the final and penultimate washing steps, the support may be washed with a washing solution of approximately neutral pH, e.g. a solution composed of PBS + 0.03% CHAPS. This washing solution may be mixed with the solid support, forexample at around 1000 rpm for about 1 minute. This is conveniently the firstwashing step and washing solutions are removed before each new addition.The next step in the method of enrichment / sample preparation is elution, step (iii),in which an elution solution is applied to the solid support. The elution solution has a pH between 10.5 and 10.7, preferably at or around pH 10.6. The inventor hassurprisingly found that having a final washing solution and an elution solution withabout the same pH gives the best recovery of Aβ peptides and produced samplesbest suited to subsequent analysis, e.g. by LC-MS. These conditions optimisedretention of Aβ peptides on the solid support while removing as much as possible ofthe non-specifically bound matrix or contaminating components which can cause pressure increases over time in LC-MS runs. This elution solution preferably comprises 0.05% aqueous ammonia and preferably, and conveniently, has the same composition as the final washing solution. The elution solution is typically applied to the solid support in a lower volume than the washing solutions, e.g. at less than half the volume of the final washing solution, preferably at less than one third of the volume of the final washing solution. Theelution solution is preferably mixed with the solid support, preferably for 30 minutesto 3 hours, more preferably 30 to 90 minutes, most preferably 45 to 75 minutes, e.g.about an hour. The period of mixing with elution solution is preferably at least threetimes, more preferably at least five times as long as the final mixing step with the washing solution, step (iii). Mixing may conveniently be at 800 to 1400 rpm, preferably 1000 to 1200 rpm, more preferably around 1100 rpm.After mixing, the elution solution is collected to provide an eluted sample. Theeluted sample is enriched for Aβ peptides, preferably it is enriched for Aβ 1-40and / or Aβ 1-42 peptides as compared to the original sample. Preferably the profileof Aβ peptides in the eluted sample is representative of the Aβ peptides present inthe original sample. If magnetic (magnetisable) beads are used as the solid support, as is convenient, a magnetic array may be used to pull down the beadsand the eluted sample may be collected in well plates.As discussed herein, the eluted sample is an enriched Aβ peptide sample suitablefor subsequent analysis. Thus, in a further aspect, the present invention provides amethod of analysing Aβ peptides in a sample comprising performing the method ofenrichment of the invention as described herein and then applying the elutedsample to a liquid chromatography (LC) column. Preferably the eluted sample canbe applied (e.g. injected into) directly to the LC column without addition or fractionation / purification (thus the sample applied to the LC column has the samepH as the elution solution as defined herein), this offers a very convenient integration between the IP and LC phases of the total method. It has further been found by the present inventor that the optimum pH of the sample and the mobile phase used to perform LC are the same, at pH 10.5-10.7, preferably about pH 10.6. This pH provides maximum sensitivity and extends the lifetime of the LC column. The optimum pH is the same for the final washing step and theeluting step of the IP, and therefore for the sample and also the mobile phase of theLC, providing a robust and simple method. Thus, the pH of the washing solution,the elution solution (so also the eluted sample used as the sample for LC) and themobile phase is 10.6. The LC column may be any convenient set up used in the art for reverse phase LC,e.g. as described in Lame et al. supra and the Waters application note mentionedabove and described in the Examples herein. An interior surface of the LC column preferably has an alkylsilyl coating (e.g. as described in US11,709,155). Conveniently an acetonitrile (ACN) gradient is generated by varying the composition of the mobile phase. The ACN concentration in the mobile phase mayconveniently increase and then decrease over time and eluted fractions arecollected for subsequent analysis, e.g. using a mass spectrometer (MS). The LC column is preferably operably linked to the MS and suitable integrated systems areavailable from Water Corporation and described in Lame et al. supra and theabove-mentioned Waters application note.The person skilled in the art is aware of suitable MS machines and these includethe Xevo TQ Absolute Mass Spectrometer (Waters Corporation). Commerciallyavailable LC-MS systems typically contain an interface that transfers the separated components from the LC column into the MS ion source. The present invention will now be described with reference to the following non- limiting Examples and the Figures, in which:Figure 1 shows LC-MS chromatograms for beta amyloid peptide 1-40 (first fivechromatograms) and beta amyloid peptide 1-42 (second five chromatograms) for various pH levels at 0% acetonitrile content in the sample container.Figure 2 provides response curves for beta amyloid peptide 1-40 (top) and betaamyloid peptide 1-42 (bottom) in solutions at various pH and in various levels of acetonitrile.Figure 3 shows Chromatographic separation of beta amyloid 1-40 and 1-42peptides at mobile phase pH 11 after 1 (left) and 15 (middle) injections. Chromatographic performance, as indicated by peak shape, resolution and retention time, was restored by replacing the analytical separation LC column (right).Figure 4 are backpressure traces from an LC-MS system run without additional pH10.6 injection system solvent washes or final pH 10.6 bead wash, demonstrating instant pressure build up (A) vs. with additional pH 10.6 injection system solvent washes and final pH 10.6 bead wash (B), which maintains pressure stability.Examples Materials and Methods SolutionsInternal standard: uniformly labelled humanAβ peptide(1-42) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA, and Aβ peptide(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV were prepared in WASH1 solution.WASH1: PBS + 0.03% CHAPS detergent (pH ≈ neutral)WASH2: 100 mM HCl adjusted to pH 9.4 with ammoniaWASH3 / Elution solution: 0.05% Ammonia in H2O (pH 10.5 - 10.7)antibody: Biotin anti – Aβ, 1-16 antibody from Biolegend, clone 6E10Instrumentation• ACQUITY I-Class FL UPLC System• Xevo Absolute Mass Spectrometer• Andrew Alliance Andrew+ Pipetting RobotProtocol for enrichment of Aβ peptides – preparation of sample for LC-MSSamples of human plasma were centrifuged at 4000g for 5 minutes prior to use.400 µL plasma was transferred to an Axygen 1.1mL 96-well collection plate.50 µL of 2ng / mL of 15N Internal standard solution was added to each sample andmixed for 1 minute.100 µL of washed Dynabead MyOne Streptavidin T1 beads with immobilisedbiotinylated Aβ 1-16 antibody were added to each sample and mixed briefly with aplate mixer at 1100 rpm for 20 seconds, followed by 500 rpm mix for 10 minutes atroom temperature. This was repeated 5 times. The collection plate was added to a magnetic array, allowing the beads to pull downfor 2 minutes. The entire liquid volume was removed and 650 µL of WASH 1 wasadded to the sample and mixed for 1 minute at 1000 rpm. The plate was transferredback to the magnetic array and beads were left to pull down for 2 minutes prior towash removal. The wash step was repeated another two times.650 µL of WASH 2 was added to the sample and mixed for 5 minutes at 1000 rpm.The plate was transferred back to the magnetic array and beads were left to pulldown for 2 minutes prior to wash removal.250 µL of WASH 3 was added to the sample and mixed for 10 minutes at 1000rpm.The plate was transferred back to the magnetic array and beads were left to pulldown for 2 minutes prior to wash removal.75 µL of ELUTION Solution was added to each sample. The collection plate wasmixed at 1100rpm for 1 hour. The plate was transferred back to the magnetic array and beads were left to pull down for 1 minute prior to elution transfer to a washed Waters QuanRecovery 96-well collection plate. The collection plate was sealed and an autosampler magnetic plate (SISCAPA, Canada) was secured to the collectionplate prior to injection into the liquid chromatography column.Liquid Chromatography / Mass Spectrometry (LC-MS) Conditions / Parameters Mobile Phases: Mobile Phase A / Water + 0.05% ammonia solution.200µL of 28% Ammonia Elution Solventadded to a bottle containing 400 mL of MilliQ waterpH ~10.5 - 10.7Mobile Phase B90:10 ACN:H2O contained 0.05% ammonia solution. 200pH ~10.5 - 10.7µL of 28% ammonia is added to a bottle containing 360mL of acetonitrile and 40mL of MilliQ waterColumn and Guard column used:ACQUITY Premier Peptide BEH C18 Column, 300 Å, 1.7 µm, 2.1 xWaters 18601076350 mm ACQUITY Premier Peptide BEH C18 VanGuard FIT Cartridge, 300Waters 186010775Å, 1.7 µm, 2.1 x 5 mmWeak wash solvent: 60% acetonitrile, 0.05% ammonia, 1500 µLStrong wash solvent: 60% acetonitrile, 0.05% ammonia, 1500 µLSeal Wash: 20% methanol (aqueous) Column temperature: 35°CInjection Volume: 50 µLInjection mode: Full Loop Separation Inlet Gradient: Flow Rate Mobile Phase Mobile Phase Time (min) Curve (mL / min) A (%) B (%) 0.00 0.250 87.5 12.5 Initial2 0.250 87.5 12.5 63.5 0.250 80 20 65.25 0.250 20 80 65.5 0.500 20 80 66.3 0.500 20 80 66.4 0.500 87.5 12.5 66.9 0.250 87.5 12.5 6Run time: 7 mins (8 minutes injection to injection) Blank Inlet Gradient: Time FlowMobile Phase Mobile Phase curve (mL / min) A (%) B (%) 00.300 90 10 Initial0.3 0.300 20 80 110.6 0.300 90 10 110.9 0.300 20 80 111.2 0.300 90 10 111.5 0.300 20 80 111.8 0.300 90 10 112.1 0.300 20 80 112.4 0.300 90 12.5 11Run time: 2.8 mins (4 mins injection to injection)Xevo Absolute MS - ParametersThe instrument was tuned for unit resolution for MS1 (0.7 Da FWHM) and theresolution for MS2 (1.2 Da FWHM).MS ConditionsPolarity ESI+Capillary (kV) 2.0Cone (V) see MRM tableSource Temperature (°C) 150Desolvation Temperature (°C) 650Cone Gas Flow (L / h) 150Desolvation Gas Flow (L / h) 1000MS / MS Mode Collision Energy see MRM tableResolution: MS1 (0.7 Da FWHM) and MS2 (1.2 DaFWHM)FWHM = Full Width Half Maximum (resolution definition); ESI = ElectrosprayIonization MRM Transitions MRM Identifier Cone (V) CollisonCompound (V)Aβ40 1083.3 > 1053.9 Quantifier 60 22Aβ40 1096.4 > 1066.5 SIL 60 22Aβ42 1129.3 > 1078.6 Quantifier 60 22Aβ42 1142.9 > 1091.6 SIL 60 22Interscan Scan Delay (s):0.02 Interscan Channel Delay (s):0.01SIL: stable isotope-labelled peptideThe LC-MS set up was adapted fromhttps: / / www.waters.com / nextgen / us / en / library / application-notes / 2019 / amyloid-beta- peptides-spe-lc-ms-ms-with-automated-sample-preparation-for-preclinical-research-and- discovery.html and Lame et al. in Analytical Biochemistry 419 (2011)133- Investigations into the effect of pH on LC-MS analysis and on Immunoprecipitation based sample preparation prior to LC-MS. Methods Combined 50 ng / mL solutions of beta amyloid peptides 1-40 and 1-42 wereprepared in aqueous ammonia solution at various pH levels (9.2 - 11.2) andacetonitrile concentrations (0 - 30%). All solutions were prepared in Quan Recoveryplates and injected onto an LC-MS system as described above.pH was also studied as a variable during development of a bead-based immunoprecipitation (IP) sample preparation method. Results A pH optimization example at 0% acetonitrile is illustrated in Figure 1, showing representative LC-MS chromatograms at different pH levels. An overall summary is provided in Figure 2, showing the combined effect of changing pH and acetonitrile concentration. The point at which the pH was optimal to counteract the effect of reducing concentration of acetonitrile was found to be in between pH 10.5 and 10.7. The surface adsorption reduction induced by altering solution pH, typically interpreted as non-specific binding in the sample container, was found to be less prevalent at higher acetonitrile concentrations. Above a sample or mobile phase pH of 11, LC column integrity can be negatively affected. Maintaining pH between 10.5 and 10.7 provided the optimal balance between reducing a-specific binding and prevention of LC column stationary phase degradation. This pH of about 10.6 is represented by an ammonia concentration of 0.05%. The effect of LC column life deterioration on chromatographic resolution isshown in Figure 3, demonstrating the separation of beta amyloid peptides 1-40 and 1-42 after 1 and 15 injections using a mobile phase pH of 11. Chromatographic performance was restored by replacing the analytical separation column, shown in Figure 3 (right).Net, the optimum final wash solution for IP was the same as the elution solution at apH value of 10.6. Washing the IP beads for 10 minutes at pH 10.6 proved to beoptimum for washing off as little analyte as possible, while removing as much of the non-specifically bound matrix or contaminating components. The presence of non- specifically bound interfering matrix components manifests itself by gradual system pressure increase over time, as shown in Figure 4, which illustrates the difference in backpressure between a system run without vs. with an additional injection system wash using a pH 10.6 solution. An extra blank solvent sample was injected with a specific gradient between each sample during the batch. Conclusions The sample and mobile phase pH optimum for the separation of beta amyloid peptides using reversed phase liquid chromatography was found to be about pH 10.6. Although this optimum maximizes in a narrow pH range from 10.5 to 10.7, itstill reduces peptide interaction with surfaces of sample containers and thechromatographic system, which subsequently affords maximizing method sensitivity. It also means that critical sample and mobile phase pH values are not exceeded, which in turn provides extended chromatographic column lifetimes.A pH of 10.5 - 10.7 also proved to be the optimum for washing matrix componentsand eluting beta amyloid from the IP beads during sample preparation. This meant that both the sample for injection onto LC and the mobile phase had the same pH, adding to method robustness and simplicity.

Claims

CLAIMS 1. A method of enriching a sample for amyloid β peptides, said methodcomprising (i) contacting the sample with a solid support having immobilisedthereon an anti-amyloid β peptide antibody; (ii) washing said solid support with a washing solution which has a pHbetween 10.5 and 10.7; (iii) applying an elution solution which has a pH between 10.5 and 10.7to said solid support; and (iv) collecting the eluted sample.

2. The method of claim 1, wherein the solid support is stirable and preferablycomprises magnetisable particles, more preferably superparamagneticbeads.

3. The method of any preceding claim, wherein step (i) further comprisesmixing the solid support and the sample.

4. The method of any preceding claim, wherein step (iii) further comprisesmixing the elution solution and the sample.

5. The method of claim 4, wherein the elution solution and the solid supportare mixed for 30 to 90 minutes, preferably for about 1 hour.

6. The method of any preceding claim, wherein in step (ii) the solid supportand the washing solution are mixed for 5 to 15 minutes, preferably for about 10 minutes.

7. The method of claim 4 or claim 5, wherein mixing of the solid support andthe elution solution is performed in a centrifuge at 800 to 1400 rpm,preferably 1000 to 1200 rpm, more preferably around 1100 rpm.

8. The method of any preceding claim, wherein prior to washing step (ii), thesolid support is washed with a washing solution which has a pH between 9 and 10, optionally this further washing step is preceded by an additionalwashing step with a washing solution which has an approximately neutral pH.

9. The method of any preceding claim, wherein the washing solution in step (ii)and the elution solution in step (iii) have the same composition.

10. The method of any preceding claim, wherein the antibody is reactive toamino acid residues 1 to 16 of amyloid β.

11. The method of any preceding claim wherein the solid support is streptavidincoated and the antibody is biotinylated.

12. The method of any preceding claim. wherein the eluted sample is enrichedfor amyloid β 1-40 and / or 1-42 peptides.

13. The method of any preceding claim, wherein the sample is a plasmasample, preferably a human plasma sample.

14. A method of analysing amyloid β peptides in a sample comprisingperforming the enrichment method of claim 1 and then applying said eluted sample to a liquid chromatography column.

15. The method of claim 14, wherein the eluted sample is applied to the liquidchromatography column without changing the solution thereof, such that the sample applied to the liquid chromatography column has a pH between 10.5 and 10.7.

16. The method of claim 14 or claim 15, wherein the pH of the mobile phaseused to perform the liquid chromatography is between 10.5 and 10.7.

17. The method of any preceding claim, wherein the pH of the washing solution,the elution solution and the mobile phase is 10.6.

18. The method of any one of claims 14 to 17, wherein one or more elutedfractions form the liquid chromatography column are analysed using a mass spectrometer.

19. The method of claim 18, wherein the liquid chromatography column isoperably linked to said mass spectrometer.

20. The method of any of claims 14 to 19, wherein an interior surface of theliquid chromatography column has an alkylsilyl coating.

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