Improved analysis of oligosaccharides using LC systems and columns in which internal metal surfaces of liquid chromatography system and chromatography column have been modified with layer of inert material

Abstract

The present disclosure relates to a method for separating one or more unmodified oligosaccharides having a degree of polymerization of 6 or greater from a sample using a hydrophilic interaction liquid chromatography (HILIC) column connected to a liquid chromatography system comprising a coated flow path. The methods disclosed herein entail, among other things, a liquid chromatography system wherein at least one component of the LC system includes a fluid contacting alkylsilyl coating. When implementing a standard HILIC method, the methods of the present technology may be used to reduce the loss and / or leave-away of the isolated analyte, thereby improving the analysis.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 489,505, filed March 10, 2023, the entire disclosure of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure relates to the use of hydrophilic interaction liquid chromatography columns and related liquid chromatography systems, including coated flow paths for the analysis of unmodified oligosaccharides. More specifically, the present technology relates to methods for isolating one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher from a sample using a hydrophilic interaction liquid chromatography column comprising a fluid-contacting alkylsilyl coating on at least some portion of its flow path. The methods of the present technology can be used to reduce loss and / or carry-over of the isolated analytes, thereby improving the analysis of unmodified oligosaccharides (e.g., galacto-oligosaccharides). BACKGROUND

[0004] Since high pressure amenable chromatography systems with minimal dispersion are desirable, material selection for chromatography flow paths is generally a choice of metals by nature. However, metal-analyte interactions in liquid chromatography (LC) are a big problem for certain analyte groups such as phosphorylated peptides, oligonucleotides, proteins, phosphorylated glycans, and organic acids. These analytes contain strong metal complexing or chelating functional groups such as phosphates, carboxylates (conjugate bases of carboxylic acids), amines, and amides that interact with metals (metal ions or metal oxides) in LC systems, including columns.

[0005] While analytes containing strong metal complexing groups cause problems in LC such as loss of analyte recovery, peak tailing, and carry-over, to our knowledge, there has been no report on any detrimental effects caused by the interaction of unmodified carbohydrates with metal surfaces.

[0006] Carbohydrates play an important role in living organisms. They can act as energy sources, structural components, and functional molecules that keep the human body healthy. Among carbohydrates, oligosaccharides, such as beta-galactooligosaccharides (GOS), have recently received increasing attention for their potential health benefits, such as prebiotics. Analysis of oligosaccharides typically involves hydrophilic interaction chromatography (HILIC), for example, using standard published protocols, such as AOAC Official Method 2021.01 for the analysis of beta-galactooligosaccharides (GOS) in infant formula and adult nutritional formulas (available from AOAC International, Rockville, MD). See, e.g., Cuany et al., Journal of AOAC INTERNATIONAL, 105(1), 2022, 142-158. This reference is incorporated by reference herein in its entirety.

[0007] In this study, when using traditional stainless steel hardware in the LC system, problems such as analyte loss and carryover were observed when separating unmodified oligosaccharides having a degree of polymerization of 6 or higher under certain conditions. Specifically, for the analysis of dextran molecules having a degree of polymerization (DP) of six or higher under recommended conditions with the International Standard method (i.e., AOAC Official Method 2021.01 for the separation of galactooligosaccharides by HILIC-FLR (available from AOAC International, Rockville, MD)), analyte loss and carryover were observed.

[0008] Accordingly, there is a continuing need to reduce analyte loss and carryover in traditional stainless steel LC systems, in an effort to promote improved chromatographic separations. SUMMARY

[0009] It has been demonstrated that a substantially inert alkylsilyl coating, which shields the metal surfaces of the LC system from interacting with metal-sensitive analytes, provides a reliable solution to mitigate metal-analyte interaction problems in LC systems for the analysis of strongly metal-chelating or complexing analytes (e.g., oligonucleotides). Accordingly, to date, the analytes that have benefited from the effects of alkyl coatings on LC systems are primarily compounds containing strong metal complexing or chelating functional groups, such as phosphates, carboxylates (the conjugate base of carboxylic acids), amines, and amides. For unmodified oligosaccharides, which do not have additional functional groups on the common oligosaccharides (e.g., neutral sugars), to date, no effects of alkyl coatings on LC have been reported to show an impact on the analysis of unmodified oligosaccharides, as oligosaccharides are not expected to be strong metal interaction analytes in the art.

[0010] However, in the present disclosure, it is demonstrated that an alkylsilyl coating used in LC systems, which is designed to minimize negative analyte / surface interactions for metal sensitive analytes, is also a viable solution to the analyte loss and carryover problem for the analysis of oligosaccharides having a degree of polymerization (DP) of six or higher.

[0011] In some examples, oligosaccharides having a degree of polymerization (DP) of six or higher are tested with and without a coating on the column hardware. It is also found that oligosaccharides having a DP of less than six do not encounter any analyte loss or carryover problems in traditional LC systems.

[0012] The significance of the findings of the present disclosure is that it is the first time that the size of the analyte can play an important role in the analysis of weak metal interacting analytes (e.g., oligosaccharides, neutral sugars).

[0013] To address the problems experienced in separating oligosaccharides having a degree of polymerization (DP) of six or higher in metal fluid systems, an LC system coated with an alkylsilyl coating designed to minimize negative analyte / surface interactions is used in the methods of the present disclosure.

[0014] The methods disclosed herein improve the liquid chromatography separation for oligosaccharides having a degree of polymerization (DP) of six or higher when implementing a standard hydrophilic interaction liquid chromatography (HILIC) method (i.e., AOAC Official Method 2021.01 (available from AOAC International, Rockville, MD). For example, the methods of the present technology can be used to reduce the loss and / or carryover of separated analytes, resulting in better quality analysis.

[0015] In one aspect, provided herein is a method of separating and analyzing a sample comprising one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher. The method comprises: providing a hydrophilic interaction liquid chromatography (HILIC) column, wherein at least one component of the HILIC column comprises a fluid-contacting alkylsilyl coating on a metal surface; injecting the sample onto the HILIC column; flowing the sample through the HILIC column; and separating and analyzing the sample, wherein the carryover of the one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher is reduced to at most 1 / 10 as compared to an uncoated HILIC column.

[0016] In some examples of the above aspects, the analyzing step comprises analyzing the separated sample and a blank sample injected after the separated sample with a detector to determine the amount of carryover.

[0017] In some examples of the foregoing aspect, carryover of the one or more unmodified oligosaccharides having a degree of polymerization of 6 or more is reduced to at most 1 / 15 compared to an uncoated HILIC column. In some examples of the foregoing aspect, carryover of the one or more unmodified oligosaccharides having a degree of polymerization of 6 or more is reduced to 1 / 20 compared to an uncoated HILIC column.

[0018] In some examples of the foregoing aspect, the fluid-contacting alkylsilyl coating comprises a vapor deposition product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

[0019] In some examples of the foregoing aspect, the fluid-contacting alkylsilyl coating has a contact angle of less than or equal to 30° or less than or equal to 90°.

[0020] In some examples of the foregoing aspect, the fluid-contacting alkylsilyl coating has a thickness of at least

[0021] In some examples of the foregoing aspect, the method further comprises connecting the HILIC column to a liquid chromatography system, wherein an interior surface of the liquid chromatography system is coated with the fluid-contacting alkylsilyl coating.

[0022] In some examples of the foregoing aspect, the one or more unmodified oligosaccharides having a degree of polymerization of 6 or more are selected from the group consisting of galactooligosaccharides (GOS), fructooligosaccharides, and combinations thereof.

[0023] In some examples of the foregoing aspect, the sample comprises dextran.

[0024] In some examples of the foregoing aspect, the total weight concentration of the one or more unmodified oligosaccharides having a degree of polymerization of 6 or more in the sample is from about 0.0005% to about 5% of the sample. In some examples of the foregoing aspect, wherein the total weight concentration of the one or more unmodified oligosaccharides having a degree of polymerization of 6 or more in the sample is from about 0.003% to about 1% of the sample.

[0025] In some examples of the foregoing aspect, the analyzing comprises analyzing the separated sample with a UV detector, a fluorescence detector, a mass spectrometer, or a mass spectrometer coupled with a UV detector and / or a fluorescence detector.

[0026] ​In another aspect, the present disclosure provides a method of separating and analyzing one or more unmodified oligosaccharides having a degree of polymerization of 6 or greater from a sample. The disclosed method comprises: adding a fluorescent labeling reagent to the sample to label at least one of the one or more unmodified oligosaccharides having a degree of polymerization of 6 or greater, thereby forming a fluorescently labeled sample; injecting the fluorescently labeled sample onto a hydrophilic interaction liquid chromatography (HILIC) column, wherein at least one component of the HILIC column comprises a fluidic contact alkylsilyl coating on a metal surface; flowing the injected sample through the HILIC column; separating the injected sample into components, wherein at least one of the components comprises a fluorescently labeled unmodified oligosaccharide having a degree of polymerization of 6 or greater; and analyzing the at least one of the components with a UV detector and / or a fluorescence detector.

[0027] In some examples of the above aspect, prior to injecting, the method further comprises:

[0028] adding a known amount of an internal standard to the sample; diluting the fluorescently labeled sample with an acetonitrile / water solvent mixture to form a diluted sample for injection; and

[0029] wherein analyzing the at least one of the components comprises using a detected peak area of the internal standard to quantify the at least one of the components.

[0030] In some examples of the above aspect, the fluidic contact alkylsilyl coating comprises a vapor deposition product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

[0031] In some examples of the above aspect, the fluidic contact alkylsilyl coating has a contact angle of less than or equal to 30° or less than or equal to 90°.

[0032] In some examples of the above aspect, the fluidic contact alkylsilyl coating has a thickness of at least .

[0033] In some examples of the above aspect, the one or more unmodified oligosaccharides having a degree of polymerization of 6 or greater are selected from the group consisting of galactooligosaccharides (GOS), fructooligosaccharides, and combinations thereof. In some embodiments, the galactooligosaccharides are selected from the group consisting of beta-galactooligosaccharides, alpha-galactooligosaccharides, and galacturonans. In some examples of the above aspect, the unmodified oligosaccharide having a degree of polymerization of 6 or greater is a beta-galactooligosaccharide.

[0034] In some examples of the above aspect, the UV detector or the fluorescence detector is coupled to a mass spectrometer.

[0035] In some examples of the above aspect, the internal standard comprises laminar trisaccharide.

[0036] In some examples of the above aspect, the method further comprises adding an enzyme selected from the group consisting of amyloglucosidase, beta-galactosidase, and combinations thereof to the sample prior to adding the internal standard.

[0037] In some examples of the above aspect, the sample comprising the added enzyme is held at a temperature between 60 °C and 80 °C for at least 2 hours.

[0038] In some examples of the above aspect, wherein the fluorescent labeling reagent is 2- aminobenzamide or 6-amino-N-[2-(diethylamino)ethyl]-2-quinolinecarboxamide.

[0039] In some examples of the above aspect, wherein the sample is a nutritional bar, a nutritional liquid, or a nutritional powder.

[0040] In some examples of any of the above aspects, the liquid chromatography system has a flow path defined by one or more of a sample storage vessel, a sample injection needle, and tubing. When a HILIC column is connected to the liquid chromatography system, the HILIC column becomes part of the flow path. One or more portions of the flow path can include a fluid-contacting alkylsilyl coating. For example, only the HILIC column can include a fluid-contacting alkylsilyl coating. Alternatively, at least some portions of the flow path (e.g., the sample injection needle and tubing) together with the HILIC column can include a fluid-contacting alkylsilyl coating.

[0041] Generally, the present methods enhance analyte recovery, reproducibility, and carryover by (without wishing to be bound by theory) minimizing negative analyte / surface interactions that can cause sample loss. The present disclosure addresses issues with HILIC separation of unmodified oligosaccharides having a degree of polymerization of 6 or higher from samples, such as carryover, analyte loss. BRIEF DESCRIPTION OF DRAWINGS

[0042] The present technology will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0043] Figure 1 A method 100 of isolating one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher from a sample according to a number of embodiments of the present disclosure is shown.

[0044] Figure 2A and Figure 2B A conventional standard stainless steel column (i.e., uncoated column and uncoated system tubing, etc.) ( Figure 2A) and coated Premier columns (i.e., columns and systems coated according to the present technology) Figure 2B ) and coated Premier columns (i.e., columns and systems coated according to the present technology)

[0045] Figure 3 A plot showing the relationship of relative peak areas of glycans (relative to internal standard) obtained on coated (Premier) and traditional standard BEH Glycan Amide columns (uncoated).

[0046] Figure 4 A plot showing the glycan response on standard uncoated columns after the 1st, 18th, and 21st injections, providing a loss of analytes for DP 6 or higher. As DP increases, the loss of analytes increases.

[0047] Figure 5A and Figure 5B Chromatograms showing the glycan ladder (containing DPs 2 to 11 and internal standard) after injection onto traditional columns and solvent blank after running the glycan ladder in the system, respectively.

[0048] Figure 6A and Figure 6B Chromatograms showing the glycan ladder (containing DPs 2 to 11 and internal standard) after injection onto coated (i.e., alkylsilyl coated) Premier columns and solvent blank after running the glycan ladder in the system, respectively.

[0049] Figure 7 A plot showing the calibration curve showing the linear relationship between peak area ratio (maltotriose to internal standard) and maltotriose molarity. The fit equation and R 2 (0.99998) are shown in the plot.

[0050] Figure 8A and Figure 8B Chromatograms from Assay 1 and Assay 2 for a typical infant formula sample, respectively. The retention time window for oligosaccharides with hexose units 2 to 6 is indicated in the chromatograms. DETAILED DESCRIPTION

[0051] In the present disclosure, certain conditions are applied in traditional LC, and issues such as analyte loss and carryover are observed during the analysis of oligosaccharides having a degree of polymerization (DP) of six or higher compared to oligosaccharides having a DP less than six. Specifically, in AOAC Official Method 2021.01 for the separation of galactooligosaccharides (GOS) by hydrophilic interaction liquid chromatography (HILIC) (available from AOAC International, Rockville, MD), analyte loss is observed for glucan molecules having a degree of polymerization (DP) of six or higher. The analyte loss and carryover observed for the analysis of oligosaccharides having a DP of six or higher is observed to be resolved when at least a portion of the wetted surfaces (metallic surfaces) of the LC system are coated with an alkylsilyl coating that minimizes possible metal-analyte interactions.

[0052] The significance of this finding is that it demonstrates for the first time that the size of a molecule can play an important role and should not be ignored in LC when mitigating metal-analyte interaction issues. This finding broadens the potential impact of alkylsilyl coatings on LC analysis and indicates that not only metal-sensitive analytes containing strong metal-interaction functional groups (such as oligonucleotides), but also analytes with long enough chain length (i.e., oligosaccharides having a DP of six or higher) and weak metal-interaction groups (e.g., hydroxyl groups and acetal groups) can benefit from alkylsilyl coatings in LC analysis. The finding presented herein makes the need for alkylsilyl coatings that prevent or mitigate any possible interaction of the analyte with the metallic surfaces of the LC system under AOAC Method 2021.01 conditions mandatory. Without wishing to be bound by theory, oligosaccharides with larger structures (i.e., having a DP of six or higher) can present a structural zone (microenvironment) with chemical properties that amplify the adverse interaction with the metallic surface. Thus, the alkylsilyl coatings of the present technology are used to modify the flow path of the LC system (or at least some portion of the flow path, such as the HILIC column) to resolve the interaction of the flow path with the analyte. That is, the alkylsilyl coatings used in the methods of the present disclosure minimize the surface reactions with oligosaccharides having a DP of six or higher and allow the analyte to pass along the flow path without blocking, attaching to the surface, or changing the analyte properties.

[0053] The reduction / elimination of these interactions is advantageous because it allows for accurate quantification and analysis of samples containing oligosaccharides having a DP of six or higher.

[0054] In one aspect, provided herein is a method 100 of isolating one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher from a sample. (SeeFigure 1 ).

[0055] As used herein, "oligosaccharide" refers to a compound containing two or more monosaccharide units linked by glycosidic bonds.

[0056] As used herein, "degree of polymerization" (DP) refers to the number of monomers or monomer units covalently linked together to form a polymer, for example, the number of monosaccharide units in a glucan.

[0057] The method 100 presented herein optionally includes the step of adding 110 an enzyme to the sample to form a partially hydrolyzed analyte comprising at least one oligosaccharide having a degree of polymerization higher than 6. The enzyme can be selected from the group consisting of amyloglucosidase, beta-galactosidase, and combinations thereof.

[0058] In some embodiments, the sample is selected from the group consisting of infant formula, adult nutritional formula, nutritional bar, nutritional liquid, and nutritional powder.

[0059] As used herein, the term "infant formula" refers to a liquid, solid, semi-solid, and semi-liquid human milk replacement or substitute suitable for consumption by an infant.

[0060] As used herein, the term "adult nutritional formulation" refers to synthetic formulas including nutritional liquids, nutritional powders, nutritional solids, nutritional semi-solids, nutritional semi-liquids, nutritional supplements, and any other nutritional food product as known in the art. The nutritional powders can be reconstituted to form nutritional liquids, all of which are suitable for oral consumption by a human.

[0061] As used herein, the term "nutritional liquid" refers to nutritional products in the form of ready-to-drink liquids, concentrated forms, and nutritional liquids made by reconstituting the nutritional powders described herein prior to use.

[0062] As used herein, the term "nutritional powder" refers to nutritional products in flowable or scoopable form that can be reconstituted with water or another aqueous liquid prior to consumption and includes spray-dried and dry-mixed / dry-blended powders.

[0063] The method 100 includes forming a sample having unmodified oligosaccharides with a degree of polymerization of 6 or higher. The method can include adding 120 an internal standard (e.g., laminaribiose) to the sample. The internal standard serves two purposes in the provided method: to normalize variations that can be introduced in the derivatization and dilution steps in sample preparation, and to act as a retention time marker for system suitability checks. For example, peak area ratios (compared to the internal standard) can be used in calibration and quantitation, and the relative retention time (RRT) of the oligosaccharides compared to the internal standard can be used to verify system suitability requirements. After adding a known amount of the appropriate internal standard to the sample, a fluorescent labeling reagent is added 130 to the sample to label at least one unmodified oligosaccharide having a degree of polymerization higher than 6. In some embodiments, the fluorescent labeling reagent can be selected from a fluorophore having an emission wavelength of about 400 nm to about 800 nm and having an amine and / or N-hydroxysuccinimide (NHS). In some embodiments, the fluorescent labeling reagent is 2-aminobenzamide (2-AB). In some embodiments, the fluorescent labeling reagent is 6-amino-N-[2-(diethylamino)ethyl]-2-quinolinecarboxamide (6-ADEQ). In some embodiments, the concentration of the fluorescent labeling reagent can be higher than the concentration of the analytes in the sample. After mixing the sample with the fluorescent labeling reagent at a temperature (e.g., between 40 °C and 80 °C) for a period of time (e.g., 1 hour), the sample is diluted 140 with an acetonitrile / water solvent mixture. In some examples, the sample is diluted at least 2-fold, at least 2.5-fold, at least 3-fold, at least 5-fold of its volume. After diluting the sample 140, the sample is injected 150 onto a hydrophilic interaction liquid chromatography column that can be connected to a liquid chromatography system. In some embodiments, at least one component of the hydrophilic interaction liquid chromatography column and the liquid chromatography system (e.g., Waters Acquity® Premier System available from Waters Corporation of Milford, MA 01757) includes a fluid-contacting alkylsilyl coating on a metal surface (e.g., a flow path). TM Arc TM At least one component of the hydrophilic interaction liquid chromatography column and the liquid chromatography system (e.g., Waters Acquity® Premier System available from Waters Corporation of Milford, MA 01757) includes a fluid-contacting alkylsilyl coating on a metal surface (e.g., a flow path).

[0064] At least one component of a liquid chromatography system can be selected from a number of components including: a fluid manager system (e.g., to control mobile phase flow through the system); tubing (which can also be replaced or used with microfabricated fluidic conduits); fluid connectors (e.g., fluidic caps); frits; chromatography columns (e.g., XBridge Premier columns available from Waters Corporation, Milford, MA 01757); sample injectors (including needles for inserting or injecting samples into mobile phase); vials, sedimenters, or sample reservoirs for holding samples prior to injection; detectors (such as mass spectrometers); and pressure regulators for controlling flow pressure. The interior surfaces of the components of the chromatography system form fluid flow paths having wetted surfaces.

[0065] At least a portion of the wetted surfaces can be coated with an alkylsilyl coating. The coating can be applied by vapor deposition. Thus, the methods and devices of the present technology provide the advantage of being able to use high pressure resistant materials (e.g., stainless steel) to form the flow system, but also being able to tailor the wetted surfaces of the fluid flow paths to provide appropriate hydrophobicity, so that adverse interactions or undesirable chemical effects on the sample can be minimized. In some examples, the coating of the flow paths is non-binding or low-binding or non-interacting with respect to analytes such as oligosaccharides having a DP of 6 or higher.

[0066] The alkylsilyl coating can be provided throughout the system from the tubing or fluidic conduits all the way to the detector (e.g., UV detector). The coating can also be applied to portions of the fluidic paths (e.g., at least a portion of the fluidic paths). That is, it can be selected to coat one or more components or portions of components rather than the entire fluidic path. For example, the interior portions of the column and its frits and end caps can be coated, while the rest of the flow path can remain unmodified. Further, removable / replaceable components can be coated. For example, the vials or sedimenters housed in the sample reservoirs can be coated; other replaceable components such as frits can also be coated.

[0067] Generally, the alkylsilyl coating is applied by vapor deposition techniques. A precursor is loaded into a reactor in which the component to be coated is located. The vaporized precursor reacts on the surface of the component to be coated to form a first layer of deposited material. Vapor deposition can be applied in a step function to apply multiple layers of deposited material to the surface to increase the thickness of the coating and / or to apply layers of different materials (e.g., alternating between a first material and a second material) to form the coating.

[0068] Further information on coating and fluid contact coating deposition in accordance with the present technology is available in US 2019 / 0086371, which reference is incorporated herein by reference. US 2019 / 0086371 also describes tailoring the coating to meet the needs of a particular sample / analyte.

[0069] In some embodiments, the fluid contact alkylsilyl coating is made using a vapor of bis(trichlorosilyl)ethane, bis(trimethoxysilyl)ethane, bis(trichlorosilyl)octane, bis(trimethoxysilyl)octane, bis(trimethoxysilyl)hexane, and bis(trichlorosilyl)hexane.

[0070] In some embodiments, at least a portion of the wetted surface of the flow path is coated with multiple layers of the same or different alkylsilyl groups, where the thickness of the alkylsilyl coating is related to the number of delamination steps performed (e.g., the number of deposited layers of the alkylsilyl coating on the wetted surface of the flow path of the chromatography system / device). In this way, the thickness of the fluid contact coating can be controlled and tailored to be increasingly thick to achieve the desired separation.

[0071] In some embodiments, the alkylsilyl coating is formed from one or more of the following precursor materials: bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. Other embodiments of alkylsilyl coatings suitable for use with the present technology are described in U.S. Patent Publication No. 2019 / 0086371 (see specifically Table 1) and U.S. Application Publication No. 2022 / 0118443, both of which are hereby incorporated by reference. For example, the alkylsilyl coating can comprise or consist of a C2 coating, which is the product of vapor-deposited bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

[0072] In some embodiments, prior to coating, all metal parts are passivated according to nitric acid passivation. The passivated parts and silicon wafer are then introduced into a vapor deposition chamber and a vacuum is established. In some embodiments, the first step is a 15 minute 200 watt 200 cc / minute oxygen plasma cleaning step, and the next step is a first vapor deposition cycle. In some embodiments, each vapor deposition cycle contains a silane vapor deposition, followed by the introduction of water vapor to perform silane hydrolysis. In one example, silane vapor is delivered for 5 seconds at a pressure of 2.0 Torr, and water vapor is subsequently delivered for 5 seconds at a pressure of 50 Torr. After delivery, the silane and water are allowed to react with the substrate for 15 minutes. The cycle is repeated to produce a desired number of layers and coating thickness.

[0073] After injecting the sample onto the hydrophilic interaction liquid chromatography column 150, the injected sample is flowed through the column 160 and separated 170 using hydrophilic interaction liquid chromatography. The separated sample is then passed through a detector for analysis. In some embodiments, the detector is a UV detector and / or a fluorescence detector. In certain embodiments, multiple detectors can be used. For example, a UV detector and / or a fluorescence detector can be coupled to a mass spectrometer.

[0074] Methods and Examples

[0075] Example 1 : Comparative Example against 2-AB labeled dextran ladder with DP values 2 to 11

[0076] Using the above method, a glucan ladder having DP values ranging from 2 to 11 was separated in a system using uncoated hardware and coated hardware according to the present technology. In each of the examples, the chromatograms compared indicated that a consistent and high response was obtained for the glucan ladder having DP 6 or higher using the PremierGlycan BEH Amide column (with alkylsilyl coating). When using the conventional Glycan BEH Amide column (without coating), an inconsistent and low response was observed (for DP > 6).

[0077] Samples containing glucan molecules were labeled with 2-AB molecules and HILIC separation of the 2-AB labeled glucan ladder was performed under conditions recommended in AOAC Official Method 2021.01 (available from AOAC International, Rockville, MD).

[0078] The standard sample was prepared in 1 mL in an aqueous solution for analysis. First, 250 pL of water was added to 500 pL of glucan solution. Then, an internal standard (tunicamycin) was added to the sample. After 20 pL of solution was taken from this glucan internal standard solution, 100 pL of water and 100 pL of 2-AB labeling reagent were added. The solution was mixed in a water bath at 65 °C for 1 hour. After 1 hour, 1 mL of acetonitrile / water solution (75 / 25 v / v) was added to the mixture before analysis. The analysis conditions are shown below.

[0079] Methods :

[0080] LC system: ACQUITY H-Class Plus (QSM) with ACQUITY FLR detector. Arc TM Premier System (BSM)

[0081] Column: XBridge TMGlycan BEH Amide Column, XBridge TM Premier Glycan BEH Amide Column, 2.5 pm, 2.1 x 150 mm.

[0082] Column temperature: 25 °C

[0083] Injection volume: 2 pL

[0084] Run time: 60 minutes.

[0085] Mobile phase A: Acetonitrile

[0086] Mobile phase B: 100 mM ammonium formate pH 4.4

[0087] FLR detector: Excitation l = 330 nm; Emission l = 420 nm

[0088] Elution program: (see Table 1 below)

[0089] Table 1 : Elution program

[0090]

[0091] Figures 2A to 2B A comparison of 2AB labeled glycan ladder chromatography is shown on a traditional standard column (i.e., uncoated column and system tubing, etc.) ( Figure 2A ) and a coated Premier column (i.e., column and system coated according to the present technology) ( Figure 2B ). The difference in relative peak height found using the Premier Glycan BEH Amide column for oligosaccharides with DP 6 and higher compared to the conventional Glycan BEH Amide column indicates that the response obtained in the coated column and system is higher compared to the conventional column and system. For example, the peak height for DP 8 is about 5 EU for the coated column, while the DP 9 peak is only about 4.8 EU for the traditional uncoated column.

[0092] Figure 3 The relationship of the relative peak area of glycans obtained on the coated (Premier) and traditional standard BEH Glycan Amide column (uncoated) (relative to the internal standard) is shown. Figure 3 The injections on the traditional column show inconsistent and low response starting from DP6 (glycan glucose units 6). In contrast, the first and eighteenth injections on the Premier column show identical results, demonstrating the consistency of the method. The response is normalized to the I.S. (tunicamycin). The same sample (containing IS and glycans with different DP) is used in the comparison.

[0093] Figure 4 The dextran response on a standard uncoated column is shown, providing loss of analyte after DP 6 or higher. For these uncoated columns, analyte loss increased as DP increased.

[0094] Figure 5A and Figure 6A Chromatograms of a dextran ladder (containing DPs 2 to 11 and an internal standard) are shown after injection into a traditional column (i.e., no coating) and traditional system Figure 5A ) and a coated column and coated system Figure 6A . Figure 5B and Figure 6B Chromatograms of a solvent blank are shown immediately after running a dextran ladder in a traditional column and system Figure 5B ) and in a coated column and system Figure 6B . While Figure 6B shows no carryover (i.e., no measurable carryover) for a dextran ladder with DP 6 or higher, it is Figure 5B shows carryover from a dextran ladder with DP 6 or higher (measured at 4%) in the same region of the chromatogram, demonstrating the positive impact of the coating on eliminating carryover for dextran ladders with DP 6 or higher.

[0095] Carryover results in different systems were investigated. Carryover was considered to be the presence of a small analyte peak that appeared when a blank was injected after a sample that produced a large peak was injected. When it occurred, a peak attributable to the previously analyzed sample that could co-elute with or interfere with the desired analyte could be observed in subsequent chromatograms. The percentage of carryover was calculated by the following formula:

[0096] Carryover % = (Carryover peak area / Internal standard peak area) x 100%.

[0097] While approximately 4% carryover was observed after running a dextran ladder with DP 6 or higher on a non-Premier column (uncoated) connected to a non-Premier system (uncoated, ACQUITY H Class PLUS) Figure 5B , only 0.2% carryover was observed when the same sample was run on a non-Premier column (uncoated) connected to a Premier system (coated, Arc Premier System). No carryover was observed when the sample was run on a Premier column (coated column) connected to a Premier system (coated, Arc Premier System), see Figure 6B . These results demonstrate the impact of the coating on eliminating carryover for analysis of oligosaccharides with DP 6 or higher.

[0098] Example 2: Determination of GOS in infant formula using conventional column and LC system (AOAC Official Method 2001.08) Calibration linearity Figure 7 2021.01)

[0099] Determination of GOS in infant formula (AOAC Official Method 2021.01) was performed on an ACQUITY UPLC H-Class System coupled with an ACQUITY UPLC FLR Detector and an ACQUITY UPLC Glycan BEH Amide Column (standard column or regular settings without HPS). The analytical performance for linearity, sensitivity, repeatability, and accuracy is described below.

[0100] Sensitivity

[0101] The relationship between peak area ratio (maltotriose / I.S.) and maltotriose molar concentration (nmol / mL) is shown in Repeatability and accuracy The linear model fits the data very well with a coefficient of determination (R 2 ) of 0.99998 by least squares regression. This calibration curve is applicable for all oligosaccharides as they all have the same response factor (one 2-AB label per molecule and thus the same amount of fluorescent signal).

[0102] Table 2. Repeatability and accuracy of determination of GOS in infant formula

[0103] The limit of quantitation (LOQ) was estimated using the lowest concentration standard solution. The standard deviation (SD) of the response of the lowest concentration standard solution (n = 5) was used to estimate the LOQ (at 10 times the SD) (assuming MW of 342 g / mol and sample size of 5 g). The estimated LOQ value was 0.003 g / 100 g, which is within the expected range of method performance.

[0104] Sample analysis

[0105] Repeatability was evaluated by the results of 2 infant formula samples measured twice a day over 2 different days (see Table 2). For both samples, a relative standard deviation (RSD) of less than 1.0% was obtained, indicating good repeatability performance. Difference values of 3.4% and -6.4% were obtained, which are within the range of performance requirements (within ±10%).

[0106] Figures 8A to 8B

[0107] Note: 1) Average from multiple labs.

[0108] Table 3. Sample analysis results

[0109] Example 3: Comparative study against the influence of alkylsilyl coating Typical chromatograms obtained in the determination of GOS in infant formula are shown. The retention time windows for different hexose units (or DP) are labeled in these chromatograms. The GOS content is calculated from the difference between the Assay 1 result and the Assay 2 result. Table 3 shows the comparison of the GOS content determined in our lab with the average and reproducibility (RSDR) in the multi-lab test (MLT) study. The relative difference between the observed result and the average MLT result (relative difference in Table 3) is well within the RSDR except for one relative difference from sample G which is slightly larger than its RSDR. These results demonstrate the excellent accuracy of using the ACQUITY UPLC H-Class PLUS System to analyze the GOS content in these MLT samples.

[0110] Figures 5A to 5B

[0111]

[0112] Figures 6A to 6B

[0113] MaxPeak High Performance Surfaces (i.e., MaxPeak HPS) are alkylsilyl coatings that have been found to be very useful in mitigating issues related to metal-analyte interactions in LCs such as analyte loss, carryover, and peak tailing (Waters White Paper, 720006930EN 2020; Waters Application Notes, 720007003EN 2020; Waters Application Notes, 720007014EN 2020; Waters Application Notes, 720006721EN 2020; Waters Application Notes, 720007019EN 2020). The effect of MaxPeak HPS on the analysis of GOS in food was investigated by side-by-side comparison methods. Two LC systems and column settings were configured. One consisted of an ACQUITY H-Class Plus System and an ACQUITY UPLC Glycan BEH Amide Column (2.5 pm, 2.1 x 150 mm) and is referred to herein as the “regular setting,” see Example 2. The other consisted of an Arc Premier System with an XBridge Premier Glycan BEH Amide Column (2.5 pm, 2.1 x 150 mm) (HPS setting). The two settings were essentially identical except that only the HPS setting was combined with an alkylsilyl coating deposited on the fluidic contact metal surface.

[0114] Carryover peaks or residual peaks from previous injections were only found in the regular setting in the glucosan oligosaccharides with DP 6 and higher (see ​ ). No carryover was found in any of the glucosan oligosaccharides when the HPS setting was used (see ​ ). The degree of carryover also increased with increasing oligosaccharide DP. No carryover was observed in the small glucosan oligosaccharides (DP < 6) using the regular setting. The MW-dependent behavior in carryover was the same as observed in the analyte loss study, which supports the hypothesis that the MW (or size) of the glucosan oligosaccharides can induce surface adsorption under certain LC conditions. And most importantly, the fact that no carryover or analyte loss was found for all of the glucosan oligosaccharides on the HPS setting demonstrates that the alkylsilyl coating technology can effectively mitigate the analyte loss and carryover problems for glucosan oligosaccharides on regular systems and columns.

[0115] While the present disclosure has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the technology encompassed by the appended claims.

Claims

1. A method for separating and analyzing a sample containing one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher, the method comprising: i. Providing a hydrophilic interaction liquid chromatography (HILIC) column, wherein at least one component of the HILIC column includes a fluid-contact alkylsilyl coating located on a metal surface; ii. Inject the sample onto the HILIC column; iii. Pass the sample through the HILIC column; as well as iv. Separate and analyze the sample, wherein the residue of one or more unmodified oligosaccharides with a degree of polymerization of 6 or higher is reduced to at most 1 / 10 compared to an uncoated HILIC column.

2. The method of claim 1, wherein the analysis comprises: The separated sample and the blank sample injected after the separated sample were analyzed using a detector to determine the amount of residue.

3. The method of claim 1, wherein the residue of the one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher is reduced to at most 1 / 15 compared to an uncoated HILIC column.

4. The method of claim 1, wherein the residue of the one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher is reduced to 1 / 20 compared to an uncoated HILIC column.

5. The method of claim 1, wherein the fluid-contact alkylsilyl coating comprises a vapor-deposited product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

6. The method according to claim 1, further comprising: The HILIC column is connected to a liquid chromatography system, wherein the internal surface of the liquid chromatography system is coated with the fluid-contact alkylsilyl coating.

7. The method of claim 1, wherein the one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher are selected from the group consisting of galactooligosaccharides (GOS), fructooligosaccharides, and combinations thereof.

8. The method of claim 1, wherein the sample comprises dextran.

9. The method of claim 1, wherein the total weight concentration of the one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher in the sample is from about 0.0005% to about 5% of the sample.

10. The method of claim 1, wherein the total weight concentration of the one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher in the sample is from about 0.003% to about 1% of the sample.

11. The method of claim 1, wherein the analysis comprises: The separated sample is analyzed using a UV detector, a fluorescence detector, a mass spectrometer, or a mass spectrometer coupled to a UV detector and / or a fluorescence detector.

12. A method for separating and analyzing one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher from a sample, the method comprising: i. Adding a fluorescent labeling reagent to the sample to label at least one unmodified oligosaccharide among the one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher, thereby forming a fluorescently labeled sample; ii. Inject the fluorescently labeled sample onto a hydrophilic interaction liquid chromatography (HILIC) column, wherein at least one component of the HILIC column includes a fluid-contact alkylsilyl coating on a metal surface; iii. Allow the injected sample to flow through the HILIC column; iv. Separate the injected sample into components, wherein at least one of the components comprises a fluorescently labeled unmodified oligosaccharide having a degree of polymerization of 6 or higher; as well as v. To analyze at least one of the components using a UV detector and / or a fluorescence detector.

13. The method of claim 12, further comprising, prior to injection: a. Add a known amount of internal standard to the sample; b. Dilute the fluorescently labeled sample with an acetonitrile / water solvent mixture to form a diluted sample for injection; and The analysis of at least one component in the composition includes: quantifying at least one component in the composition using the detected peak area of ​​the internal standard.

14. The method of claim 12, wherein the fluid-contact alkylsilyl coating comprises a vapor-deposited product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

15. The method of claim 12, wherein the one or more unmodified oligosaccharides having a degree of polymerization of 6 or higher are selected from the group consisting of galactooligosaccharides (GOS), fructooligosaccharides, and combinations thereof.

16. The method of claim 12, wherein the UV detector or the fluorescence detector is coupled to the mass spectrometer.

17. The method of claim 13, wherein the internal standard comprises laminarin.

18. The method of claim 13, wherein the method comprises: Before adding the internal standard, an enzyme selected from the group consisting of: amylase, β-galactosidase, and combinations thereof is added to the sample.

19. The method according to claim 12, wherein the fluorescent labeling reagent is 2-aminobenzamide or 6-amino-N-[2-(diethylamino)ethyl]-2-quinolinecarboxamide.

20. The method of claim 12, wherein the sample is a nutrient bar, a nutrient liquid, or a nutrient powder.

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