Chelating agent based eluents for solid phase extraction of phosphorylated compounds

Abstract

The present technology relates to a method for improving the recovery rate of phosphorylated compounds (e.g., phosphorylated glycans). In particular, the present technology utilizes a solution containing certain chelating agent additives to wash and elute the phosphorylated compounds from a solid phase extraction column.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 944,153, filed December 5, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the use of chelating agent additives for improving the solid phase extraction recovery of compounds, and in particular phosphorylated glycans. More specifically, the present disclosure relates to the use of chelating agent additives that are soluble in both aqueous and acetonitrile solutions and used in the elution step to obtain high yields and unbiased recoveries of phosphorylated compounds, including various glycan species, during solid phase extraction (SPE). Background Art

[0004] Extraction of oligosaccharides from a sample matrix is ​​an important step in many different types of analysis. In one example, oligosaccharides attached to proteins are released chemically or enzymatically, and the resulting so-called "glycans" are profiled using liquid chromatography. However, it is often necessary to derivatize the released glycans prior to analysis so that they can be detected by UV, fluorescence, or mass spectrometry.

[0005] For this type of analysis, it is often necessary to extract the oligosaccharides by solid phase extraction (SPE) to minimize interference with the derivatization reaction or chromatographic separation. Common derivatization methods using the GlycoWorks RapiFluor-MS kit (commercially available from Waters Corporation, Milford, MA) produce derivatized N-glycan samples in a matrix of reaction byproducts that can complicate many forms of chromatography, including hydrophilic interaction chromatography (HILIC) and reversed phase (RPLC) separations. Therefore, SPE is employed to enrich the oligosaccharides and improve the quality of the assay.

[0006] Historically, the recovery and analysis of phosphorylated glycan species has been a significant challenge. This is problematic because some specific types of proteins, such as many enzymes responsible for lysosomal function, are post-translationally modified with mannose-6-phosphate. Indeed, mannose-6-phosphate is essential for the uptake of lysosomal enzymes when used as enzyme replacement therapy.

[0007] It would be advantageous to develop SPE kits, elution solutions, and methods that can produce high yields and unbiased recoveries of various glycan species. In addition, it would be advantageous to develop an SPE procedure for extracting oligosaccharides that minimizes interference with derivatization reactions or subsequent chromatographic separations. It would also be advantageous to develop SPE kits, elution solutions, and methods for extracting other phosphorylated compounds, including but not limited to oligonucleotides, phosphorylated peptides, phosphorylated proteins, glyphosate / glyphosate-related substances, bisphosphonate drugs, and phospholipids. Summary of the invention

[0008] The present technology provides an optimized SPE elution solution with a selected chelating agent additive to minimize the loss of phosphorylated polysaccharides during solid phase extraction. Without wishing to be bound by theory, it is believed that the chelating agent additive acts to disrupt the adsorption of phosphorylated polysaccharides to metal contaminants in the SPE device. Phosphate groups exhibit a natural tendency to chelate metals. Although not limited to this theory, chelating agents may also play a role in disrupting the interaction of Lewis acids / bases with adsorbent heteroatoms. GlycoWorks (available from Waters Corporation) HILIC SPE is based on aminopropyl-bonded silica adsorbents. The nitrogen atoms of the adsorbent may create sites for Lewis acid-base interactions that can only be displaced by introducing a strong chelating agent. Regardless of the mechanism of action, the present technology using a chelating agent-based eluent is an effective means to improve the SPE recovery of phosphorylated polysaccharides.

[0009] In some embodiments, the chelating agent additive is selected because of its solubility properties. For example, in embodiments where it is desired to perform HILIC after SPE separation, it is advantageous to use a chelating agent additive that is soluble in acetonitrile. Certain chelating agents such as etidronate are precipitated from high concentration acetonitrile solutions. These precipitates can cause blockage or interfere with subsequent HILIC separation / equipment. In other embodiments where RPLC is to be performed after SPE, a chelating agent that is soluble in aqueous solution is desired. Divalent or more valent carboxylic acids with a concentration between about 1mM and about 200mM are both soluble in aqueous solution and soluble in high percentage acetonitrile. An example of a trivalent carboxylic acid that can be used in the present technology is citric acid (solid form is citrate, such as sodium citrate). Additional exemplary trivalent acids that are useful in the present technology include isocitric acid, 2,3 hydroxycitric acid, tricarballylic acid, ethanetricarboxylic acid (HETA) and aconitic acid. Other divalent or more valent carboxylic acids can also be utilized. It is reasonable to think that succinic acid, maleic acid, fumaric acid, oxaloacetic acid and ketoglutaric acid have some effectiveness in the present technology.Tetravalent carboxylic acid, such as EDTA (ethylenediaminetetraacetic acid), butane tetracarboxylic acid, NTA, DTPA, DOTA, IDHA, EDDHA may also have some effectiveness.In alternative embodiments, dimethylglyoxime and diethylenetriamine may also be used in the present technology.Generally speaking, the concentration of divalent or more valent carboxylic acid in the elution solution should be about 1M or less than 1M, and preferably less than about 200mM (for example, between about 0.5mM to 200mM).In some embodiments, concentration is about 100mM or less than 100mM, and in the range of about 1mM to 100mM.In certain embodiments, concentration is in the range between 10mM and 100mM.In other embodiments, concentration is in the range between 40mM and 60mM, such as for example about 50mM.

[0010] In one aspect, the present technology relates to a method for performing solid phase extraction. The method includes injecting a sample containing polysaccharides into a solid phase extraction column. The SPE column includes a stationary phase, wherein the polysaccharides are retained by the stationary phase. The method also includes washing the SPE column with a divalent or higher valent carboxylic acid at a concentration between about 1 mM and about 100 mM to elute the polysaccharides from the SPE column.

[0011] On the other hand, the present technology relates to a method for performing solid phase extraction. The method includes injecting a sample containing polysaccharides into a solid phase extraction column. The SPE column includes a stationary phase, wherein the polysaccharides are retained by the stationary phase. The method also includes washing the SPE column with citric acid with a concentration between about 1mM and about 100mM to elute the polysaccharides from the SPE column. In one embodiment, the concentration is between about 40mM and 60mM (e.g., 45mM, 50mM, 55mM) to elute the polysaccharides from the SPE column.

[0012] According to another aspect, the present technology relates to a method for performing solid phase extraction. The method includes injecting a sample containing glycans into a solid phase extraction column containing a stationary phase, wherein the glycans are retained by the stationary phase. The method also includes washing the solid phase extraction column with an elution solution to elute the glycans from the solid phase extraction column, wherein the elution solution includes (i) a salt for adjusting the ionic strength of the elution solution and (ii) a divalent or higher valent carboxylic acid at a concentration between about 1 mM and about 100 mM.

[0013] Some embodiments of this aspect of the present technology include one or more of the following features. In some embodiments, the salt is selected from the group consisting of ammonium formate, ammonium acetate, sodium formate, sodium acetate, ammonium chloride, sodium chloride, potassium chloride, triethylammonium acetate, and tetramethylammonium acetate. In certain embodiments, the elution solution is prepared with a co-solvent selected from the group consisting of acetonitrile, methanol, ethanol, propanol, butanol, tetrahydrofuran, isopropanol.

[0014] According to another aspect, the present technology relates to a method for performing solid phase extraction. The method includes injecting a sample containing polysaccharides into a solid phase extraction column containing a stationary phase, wherein the polysaccharides are retained by the stationary phase. The method also includes washing the solid phase extraction column with an elution solution to elute the polysaccharides from the solid phase extraction column, wherein the elution solution includes (i) a salt for adjusting the ionic strength of the elution solution and (ii) citric acid at a concentration between about 1 mM and about 100 mM. In certain embodiments, the concentration of citric acid is between about 40 mM and about 60 mM.

[0015] Some embodiments of this aspect of the present technology include one or more of the following features. In some embodiments, the salt is selected from the group consisting of ammonium formate, ammonium acetate, sodium formate, sodium acetate, ammonium chloride, sodium chloride, potassium chloride, triethylammonium acetate, and tetramethylammonium acetate. In certain embodiments, the elution solution is prepared with a co-solvent selected from the group consisting of acetonitrile, methanol, ethanol, propanol, butanol, tetrahydrofuran, isopropanol.

[0016] On the other hand, the present technology relates to a method for performing solid phase extraction. In particular, solid phase extraction is performed to elute oligonucleotides. Similar to the phosphate polysaccharides and polysaccharides of the previous aspects of the present technology, oligonucleotides are polyanionic in nature and tend to bind to plasma proteins, sample plates and vials non-specifically. In addition to the sample matrix, this tendency to bind to the contact surface has led to poor recovery when performing common analytical extraction techniques such as SPE. The use of a polycarboxylate chelating agent added during the elution step of a weak anion exchange SPE is another embodiment of the present invention, and the polycarboxylate chelating agent produces oligonucleotides with improved recovery by reducing non-specific binding to the surface and sample matrix. The method according to this aspect of the present technology includes injecting a sample comprising one or more oligonucleotides into a solid phase extraction column comprising a stationary phase, wherein the one or more oligonucleotides are retained by the stationary phase. The method further includes washing a solid phase extraction column with a divalent or higher valence carboxylic acid (e.g., citric acid, polycarboxylic acid) with a concentration between about 10mM and about 1M to elute one or more oligonucleotides from the solid phase extraction column.

[0017] In another aspect, the present technology relates to a method for performing solid phase extraction, the method comprising injecting a sample comprising one or more oligonucleotides into a solid phase extraction column comprising a stationary phase, wherein the one or more oligonucleotides are retained by the stationary phase. The method also includes washing the solid phase extraction column with an elution solution to elute the one or more oligonucleotides from the solid phase extraction column, wherein the elution solution comprises (i) a salt for adjusting the ionic strength of the elution solution and (ii) a polycarboxylic acid (or other divalent or higher carboxylic acid) at a concentration between about 10 mM and about 1 M. In certain embodiments, the concentration of the polycarboxylic acid is between about 40 mM and about 60 mM.

[0018] While the last two aspects of the present technology relate to the extraction of oligonucleotides and the previous aspects relate to the extraction of phosphorylated glycans, other biological samples may benefit from the improved SPE protocol and elution solutions of the present technology. For example, the present technology is also applicable to SPE for the extraction of any phosphorylated compound, including the following applicable items: glyphosate (and related organophosphorus compounds), phospholipids, bisphosphonate drugs, peptides and proteins.

[0019] In some embodiments, the concentration of the divalent or higher carboxylic acid is between about 0.5 mM and about 1 M. The concentration of the divalent or higher carboxylic acid may be between about 1 mM and about 100 mM. The concentration of the divalent or higher carboxylic acid may be between about 40 mM and about 60 mM (e.g., 45 mM, 50 mM, 55 mM).

[0020] In some embodiments, the wall of the solid phase extraction column is formed of plastic. In some embodiments, the solid phase extraction column is a filling syringe. The solid phase extraction column can be an orifice plate. In some other embodiments, a solution containing a divalent or higher valent carboxylic acid is used to mitigate the loss of sample into the container. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present technology will be more fully understood through the following detailed description made in conjunction with the accompanying drawings, in which:

[0022] Figure 1A is a fluorescence chromatogram of selected phosphoglycan markers eluted from an SPE column using an elution solution containing 50 mM sodium citrate (pH 7) (enlarged view).Glycan-2P and glycan-1P are glycan markers containing two and one phosphorylated residues, respectively.

[0023] Figure 1B The elution solution was eluted from the SPE column using 50 mM etidronate sodium (pH 7). Figure 1A Another fluorescence chromatogram of the same selected phosphoglycan marker is shown.

[0024] Figure 1C The elution solution was eluted from the SPE column using 50 mM sodium succinate (pH 7). Figure 1A Another fluorescence chromatogram of the same selected phosphoglycan marker is shown.

[0025] Figure 1D The elution solution was eluted from the SPE column using 50 mM sodium phosphate (pH 7). Figure 1A Another fluorescence chromatogram of the same selected phosphoglycan marker is shown.

[0026] Figure 1E is eluted from the SPE column using a control elution solution (i.e., without chelating agent additive). Figure 1A Another fluorescence chromatogram of the same selected phosphoglycan marker is shown.

[0027] Figure 1F is a graph showing the relative abundance of selected phosphoglycan markers (Glycan-1P, Glycan-2P) for various chelator additives used in the elution solution. Figure 1A-1E The calculated relative abundance percentage of the selected marker is equal to (peak area of ​​the marker) / (peak area of ​​the internal standard)×100.

[0028] Figure 2A-2D are fluorescence chromatograms of selected phosphoglycan markers spiked with etidronate at different sample preparation steps. Figure 2A shows the chromatogram of a sample matrix spiked with 50 mM etidronate sodium (pH 7) before SPE cleanup; Figure 2B shows the chromatogram with 50 mM etidronate sodium (pH 7) spiked into the SPE elution solution; Figure 2C shows a chromatogram with 50 mM etidronate sodium (pH 7) spiked into the SPE eluate (i.e., spiked after elution); and Figure 2D The chromatogram of the control (no additive administered) is shown.

[0029] Figure 3 is a graph showing the percent recovery of glycan-2P and glycan-1P plotted against the sodium citrate concentration in the SPE elution solution. DETAILED DESCRIPTION

[0030] The present technology solves the problems of the prior art by solving the loss of phosphorylated polysaccharides during solid phase extraction. By adding a chelating agent to the elution solution, and in particular a chelating agent that is soluble in both aqueous solution and acetonitrile, an effective means of improving the recovery of polysaccharides has been surprisingly found. Using this technology, the inventors have found a high recovery SPE scheme that utilizes a chelating agent additive to achieve unprecedented recovery of not only monophosphorylated polysaccharide species, but also diphosphorylated polysaccharide species. One theory for improving the recovery is that the chelating agent additive acts to destroy the adsorption of phosphorylated polysaccharides to metal contaminants in the SPE device. The phosphate group exhibits a natural tendency to chelate metals. Another theory that may be a supplement or alternative to the first theory is that the chelating agent may also play a role in destroying the interaction of Lewis acid / base with adsorbent heteroatoms. GlycoWorks (available from Waters Corporation) HILIC SPE is based on an aminopropyl-bonded silica adsorbent. The nitrogen atoms of the adsorbent may produce sites for Lewis acid-base interactions that can only be replaced by introducing a strong chelating agent. Regardless of the mechanism of action, the present technique using a chelating agent-based eluent is an effective means of improving SPE recoveries of phosphorylated glycans.

[0031] In one embodiment, the SPE scheme of the present technology is performed with HILIC separation mode and GlycoWorks HILIC SPE micro-elution plate (available from Waters Corporation, Milford, MA). In other embodiments, alternative HILIC adsorbents can be used, such as, for example, aminopropyl-bonded silica or amides containing hydrophilic-lipophilic balance polymers. Examples of other effective adsorbents in the present invention include, but are not limited to, Sep-Pak aminopropyl (available from Waters Corporation), Oasis HLB (available from Waters Corporation), Glycoclean H (available from Agilent Technologies, Santa Clara, CA), Strata X (available from Phenomenex Inc., Torrance, CA) and BioZen N-Glycan HILIC (available from Phenomenex Inc., Torrance, CA). In some embodiments, these SPE separations can involve mixed mode retention, and more specifically, an added ion exchange retention mechanism. In other embodiments, the separation mechanism can be based on reverse phase.

[0032] The present technology relates to an improved SPE protocol that minimizes the loss of phosphorylated glycans during extraction. In general, the methods of the present technology can be used in a wide variety of applications for studying / dissecting glycan analysis. Typically, the released glycans require chemical derivatization or enrichment prior to chromatographic separation and detection. Examples of other possible applications include, but are not limited to, SPE of oligonucleotides, glyphosate, phospholipids, bisphosphonate drugs, peptides, and proteins.

[0033] In general, the present technology relates to a SPE scheme that utilizes a chelating agent additive to minimize the loss of polysaccharides during extraction. The chelating agent additive can be selected to be both soluble in aqueous solution and soluble in a high acetonitrile solution. For example, the chelating agent additive can be a divalent or higher valent carboxylic acid (e.g., citric acid). The chelating agent additive is present in the eluent. In other words, the chelating agent additive is a part of the eluent solution, which is used to wash the stationary phase in the SPE column to form an eluent. Typically, the chelating agent additive is present in a concentration less than about 200 mM. For example, the eluent solution can include citric acid in a concentration range of 1 mM to 100 mM.

[0034] In addition to the chelating agent additive, salt can be added to the elution solution to adjust the ionic strength of the solution. The example of the salt used in this technology includes but is not limited to: ammonium formate, ammonium acetate, sodium formate, sodium acetate, ammonium chloride, sodium chloride, potassium chloride, triethylammonium acetate and tetramethylammonium acetate. In certain embodiments, the elution solution is prepared with a cosolvent. Cosolvents can be used for subsequent separation steps (e.g., HILIC or RPLC). The example of cosolvents includes but is not limited to: acetonitrile, methanol, ethanol, propanol, butanol, tetrahydrofuran, isopropanol.

[0035] In some embodiments, an elution solution is used for HILIC SPE of polysaccharides, the elution solution consisting of a predominantly aqueous solution (less than 30% co-solvent) and a chelating agent additive present at a concentration in the range of 1 mM to 100 mM. In the eluent, other salts may be added to adjust the ionic strength, including but not limited to ammonium formate, ammonium acetate, sodium formate, sodium acetate, ammonium chloride, sodium chloride, potassium chloride, triethylammonium acetate, and tetramethylammonium acetate. Similarly, the co-solvent may be selected from organic solvents miscible with water, including but not limited to acetonitrile, methanol, ethanol, propanol, isopropanol, butanol, or amyl alcohol. In the eluent, the chelating agent may be selected from etidronate, citrate, succinate, EDTA, methylene diphosphonate, polyphosphonate in ammoniated, sodiated, potassiated, or protonated form.

[0036] In certain embodiments, citrate is preferred due to its solubility in a variety of solvent systems, which may facilitate the injection of the SPE eluent into different forms of chromatography, such as reverse phase chromatography, hydrophilic interaction chromatography, anion exchange chromatography, or mixed mode chromatography. In some embodiments, the eluent is prepared at a neutral pH (pH 7), while in other embodiments, it can be prepared at a pH as low as pH 3 or as high as pH 10. In an exemplary embodiment, the SPE eluent consists of a 95 / 5 solution of water:acetonitrile containing 200 mM ammonium acetate, 50 mM ammonium citrate (pH 7).

[0037] The inventors have also determined that certain chelating agent additives, such as, for example, etidronate additives, have poor solubility in high acetonitrile solutions, which limits their usefulness, especially when it is necessary to perform HILIC after SPE extraction. With the precipitation of etidronate, there may also be sample loss due to co-precipitation of analytes. In addition, the precipitate may cause LC failure, as may occur with a blocked injection needle. Therefore, it is advantageous to use additives that are soluble in both predominantly aqueous solutions and in high acetonitrile solutions. Using such additives, it becomes possible to convert the SPE eluate into a diluent suitable for RPLC or HILIC, respectively. By visual inspection, we have observed that 50mM etidronate will precipitate in a 89.5:7.5:3 (v / v) ACN / water / DMF solution, which is a recommended diluent composition for performing HILIC on N-glycan samples labeled with RapiFluor-MS. In contrast, we did not observe such problems with 50mM citrate. Based on this observation, citrate is preferred over etidronate and other chelators, including EDTA.In some embodiments, the chelator additive is selected for its solubility properties, and in particular for its ability to be soluble in the HILIC sample diluent.

[0038] Citrates (eg, citric acid) and other divalent or higher valent carboxylic acids appear to be soluble in both aqueous and high acetonitrile solutions, making these chelating agent additives optimal for the SPE protocol of the present technique.

[0039] Example 1: Phosphorylation of N-glycans using GlycoWorks RapiFluor-MS and Chelating Agent-Based SPE Eluents Optimization of high-recovery preparation of polysaccharides

[0040] The protocol for recovering glycans includes: preparing reagents (i.e., optimized elution solution / buffer), preparing a biological sample containing glycans, and performing a HILIC SPE cleanup of the biological sample to elute the glycans. The reagents used were prepared using the following procedure:

[0041] a) Reagents used:

[0042] 1.5% (w / v) RapiGest solution:

[0043] Dissolve 10 mg of RapiGest in 200 μL of 5× GlycoWorks Fast Buffer. Vortex to mix.

[0044] 2. Rapid labeling reagent (prepare before use):

[0045] Dissolve 9 mg of RapiFluor-MS reagent powder in 131 μL of the provided GlycoWorks reagent solvent, anhydrous DMF. Aspirate and dispense the solution 5-10 times to ensure all reagent is dissolved.

[0046] 3. Optimized SPE elution buffer: containing 50 mM of the selected chelating agent additive

[0047] Transfer 10 mL of GlycoWorks SPE Elution Buffer to a 15 mL conical tube. Add 0.5 mL of 1 M sodium citrate solution (pH 7) to the tube. Vortex to mix. The final concentration of sodium citrate is 50 mM.

[0048] Biological samples were prepared by using the following procedure:

[0049] b) Deglycosylation and rapid labeling of glycosylamine

[0050] 1. Dilute the glycoprotein to 2 mg / mL with MilliQ water. Vortex to mix.

[0051] 2. Add 15.3 μL of MilliQ water to the 1 mL tube.

[0052] 3. Dispense 7.5 μL of 2 mg / mL glycoprotein into the same 1 mL tube.

[0053] 4. Add 6 μL of 5% RapiGest solution, aspirate and dispense to mix.

[0054] 5. Heat denature the mixture using a heat block at least 90°C for 3 min.

[0055] 6. Remove the 1 mL tube from the heating block and allow it to cool at room temperature for at least 3 min.

[0056] 7. Add 1.2 μL PNGase F to the mixture, aspirate and dispense to mix.

[0057] 8. Incubate at 50°C for 5 min using another heating block.

[0058] 9. Remove the 1 mL tube from the heating block and allow it to cool at room temperature for at least 3 min. Prepare the rapid labeling reagent during the incubation.

[0059] 10. Add 12 μL of Rapid Labeling Solution, aspirate and dispense to mix.

[0060] 11. Incubate at room temperature for 5 minutes

[0061] 12. Dilute with 358 μL acetonitrile

[0062] Perform SPE cleanup of samples with prepared reagents (e.g., elution solution) using the following procedure:

[0063] c) HILIC SPE cleanup using optimized SPE elution buffer

[0064] 1. Combine the HILIC micro-elution plate with the waste tray. Drive the flow using vacuum (2.5-4 in Hg) or positive pressure (approximately 3 psi) manifold.

[0065] 2. Condition the HILIC micro-elution plate with 200 μL MilliQ water

[0066] 3. Equilibrate the plate with 200 μL 15:85 (v / v) water / acetonitrile

[0067] 4. Load the acetonitrile diluted samples onto the plate

[0068] 5. Wash the wells with two 600 μL volumes of 1:9:90 (v / v / v) formic acid / water / acetonitrile.

[0069] 6. Replace the waste tray with a 96-well collection plate

[0070] 7. Elute the glycans using three 30 μL volumes of optimized SPE elution buffer

[0071] Released glycans (from lysosomal enzymes obtained from Genzyme Corp, Cambridge, MA) were prepared and labeled using the optimized protocol described above. After SPE cleanup, all eluted fractions were transferred to QuanRecovery sample vials (Waters Corporation, Milford, MA) and analyzed by LC-FLR-MS with a 1 μL injection. Glycans with two phosphorylated residues were observed to have a retention time of approximately 25 min and were labeled as so-called "glycan-2P" species [1015.85 m / z, 2+; (Hex)5(HexNAc)2(Man-6-P)2]. Monophosphorylated glycans were also observed to have a retention time of 25.8 min and were labeled as so-called "glycan-1P" species [1222.96 m / z, 2+; (Hex)6(HexNAc)3(NeuNAc)1(Man-6-P)1]. These two species were selected as markers for monitoring yield and recovery. A non-phosphorylated glycan with an observed retention time of about 28.8 min was also monitored and used as an internal standard [Int. Std.; 894.36 m / z, 3+(Hex)5(Fuc)1(HexNAc)4(NeuNAc)2] to calculate the relative abundance of phosphorylated glycan markers. The relative abundance of phosphorylated glycan markers was calculated based on the peak area of ​​the phosphorylated glycan markers observed by fluorescence chromatography detection.

[0072] Figure 1A-1E The fluorescence chromatograms shown for SPE extracted RapiFluor-MS labeled N-glycans were generated using the following parameters and protocol:

[0073]

[0074]

[0075] Example 2: Relative abundance of phosphorylated glycans in SPE elution solutions using different chelating agent additives

[0076] In this example, the recovery of phosphorylated glycans from solid phase extraction based on the presence or absence of various chelating agent additives was analyzed. To investigate the effect of different chelating agent additives on the recovery of phosphorylated glycans, 1 mL of 0.5 M sodium phosphate, sodium succinate, sodium citrate, and sodium etidronate were each spiked into 10 mL of GlycoWork SPE elution buffer to obtain a final concentration of 50 mM. The pH of the concentrated chelating agent solution was adjusted to a neutral pH with sodium hydroxide to avoid acid-promoted hydrolysis of sialic acid residues. Samples prepared without chelating agent additives were used as controls. Figure 1A-1F As shown, the presence of 50 mM chelating agent additives, especially citrate and etidronate, in the SPE elution buffer resulted in higher relative abundance and, therefore, higher recovery of the doubly phosphorylated glycan (glycan-2P). In fact, the glycan-2P species increased from 16% to 89%, and the relative abundance of glycan-1P also increased from 82% to 104%. In addition, it was observed that the recovery of the two phosphorylated glycans did not increase with the addition of 50 mM ammonium acetate to the standard SPE elution buffer, which contained a 5% acetonitrile solution (neutral pH) containing 200 mM ammonium acetate. This indicates that the chelating properties of the additives, rather than the increased ionic strength, contribute to the improved recovery of the phosphoglycans.

[0077] Example 3: Timing of chelating agent additive application during sample preparation

[0078] Furthermore, the point at which chelators are implemented during the SPE process was investigated. To determine the optimal procedure and timing for applying chelators, chelator additives were incorporated at different stages of sample preparation and the corresponding effects on phosphoglycan recovery were summarized, e.g. Figure 2A-2D In this study, 0.5 M etidronate sodium was spiked into the sample mixture to achieve a final concentration of 50 mM at the following times: a) before SPE cleanup (step b.12), and the resulting chromatogram is shown in Figure 2A b) in SPE elution buffer (as described above), and the resulting chromatogram is shown in Figure 2B or c) in the SPE clean-up eluate after elution, and the resulting chromatogram is shown in Figure 2C middle. Figure 2Dis a chromatogram of a sample prepared without a chelating agent additive and applied as a control. The results of these experiments indicate that etidronate spiked into the SPE eluate did not improve the recovery of phosphoglycans and that spiking etidronate into the sample mixture prior to SPE cleanup had an adverse effect on the overall intensity of the released glycans due to the poor solubility of sodium etidronate under acetonitrile SPE loading conditions. Therefore, it was determined that adding a chelating agent additive to the SPE elution buffer (eluate) was the most effective method to improve the recovery of phosphoglycans without adversely affecting the overall mass balance / recovery.

[0079] Example 4: Effect of Chelating Agent Concentration on the Recovery Rate of Phosphopolysaccharide

[0080] It was also found that the concentration of the chelating agent additive in the SPE elution buffer was a key factor in maximizing recovery. To determine the optimal concentration of the chelating agent additive, 0.5 M sodium citrate solution (pH 7) was spiked into the SPE elution buffer at a final concentration of 0 mM (control), 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, or 100 mM. The released glycans were prepared and analyzed as described in Example 1. The percent recovery of phosphoglycans was calculated using the fluorescence peak data and plotted as a function of citrate concentration (see for details). Figure 3 ). The percent recovery of the phosphoglycan marker prepared with 100 mM citrate was assigned as 100% because those data correspond to the stationary phase data and are ostensibly the maximum recovery of this type of glycan. Figure 3 As shown, by increasing the concentration of citrate from 0 mM to 50 mM, the percent recovery of glycan-2P and glycan-1P increased from 14% to 100% and 74% to 100%, respectively. No further improvement was observed beyond this concentration. This result suggests that the SPE elution buffer spiked with 50 mM citrate additive is optimized to provide maximum recovery of phosphorylated glycans.

[0081] In summary, an optimized SPE elution buffer with a selected chelating agent additive is presented as an invention to minimize the loss of phosphorylated glycans during solid phase extraction. It is reasonable to believe that this chelating agent acts to disrupt the adsorption of phosphorylated glycans to metal contaminants in the SPE device. Phosphate groups exhibit a natural tendency to chelate metals. Although not limited to theory, chelating agents may also play a role in disrupting Lewis acid / base interactions with adsorbent heteroatoms. GlycoWorks HILIC SPE is an aminopropyl-bonded silica adsorbent. Nitrogen atoms of the adsorbent may create sites for Lewis acid-base interactions that can only be displaced by the introduction of a strong chelating agent. Regardless of the mechanism of action, this work has shown that the use of a chelating agent-based eluent is an effective means to improve SPE recoveries of phosphorylated glycans.

Claims

1. A method for performing solid phase extraction, the method comprising: injecting a sample comprising phosphoglycans into a solid phase extraction column comprising a stationary phase, wherein the phosphoglycans are retained by the stationary phase; as well as The solid phase extraction column is washed with citric acid at a concentration between 1 mM and 100 mM to elute the phosphoglycan from the solid phase extraction column.

2. The method of claim 1, wherein the concentration is between 40 mM and 60 mM to elute the phosphoglycan from the solid phase extraction column.

3. A method for performing solid phase extraction, the method comprising: injecting a sample comprising phosphoglycans into a solid phase extraction column comprising a stationary phase, wherein the phosphoglycans are retained by the stationary phase; as well as The solid phase extraction column is washed with an elution solution to elute the phosphoglycan from the solid phase extraction column, wherein the elution solution comprises (i) a salt for adjusting the ionic strength of the elution solution and (ii) citric acid or succinic acid at a concentration between 1 mM and 100 mM.

4. The method of claim 3, wherein the salt is selected from the group consisting of ammonium formate, ammonium acetate, sodium formate, sodium acetate, ammonium chloride, sodium chloride, potassium chloride, triethylammonium acetate, and tetramethylammonium acetate.

5. The method according to claim 4, wherein the solution is prepared with a co-solvent selected from the group consisting of acetonitrile, methanol, ethanol, propanol, butanol, tetrahydrofuran, isopropanol.

6. A method for performing solid phase extraction, the method comprising: injecting a sample comprising phosphoglycans into a solid phase extraction column comprising a stationary phase, wherein the phosphoglycans are retained by the stationary phase; as well as The solid phase extraction column is washed with an elution solution to elute the phosphoglycan from the solid phase extraction column, wherein the elution solution comprises (i) a salt for adjusting the ionic strength of the elution solution and (ii) citric acid at a concentration between 1 mM and 100 mM.

7. The method of claim 6, wherein the salt is selected from the group consisting of ammonium formate, ammonium acetate, sodium formate, sodium acetate, ammonium chloride, sodium chloride, potassium chloride, triethylammonium acetate, and tetramethylammonium acetate.

8. The method according to claim 7, wherein the solution is prepared with a co-solvent selected from the group consisting of acetonitrile, methanol, ethanol, propanol, butanol, tetrahydrofuran, isopropanol.

9. The method of claim 6, wherein the concentration is between 40 mM and 60 mM.

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