Compounds for system suitability testing of inert LC systems and columns

Abstract

The present disclosure relates to a kit for evaluating system inertness. The kit includes a positive control comprising a metal interaction portion and a negative control without a metal interaction portion. In some embodiments, the kit also includes a container for containing a system suitability solution (e.g., an equimolar mixture of a positive control and a negative control).

Description

[0001] Sequence Listing

[0002] This application contains a sequence list, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on September 8, 2021, is named W-4289-US02_SL.txt and is 907 bytes in size.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority and the benefit of U.S. Provisional Patent Application No. 63 / 079,151, filed September 16, 2020, entitled "Compounds for Use in System Suitability Testing of Inert LC Systems and Columns". This application also claims priority and the benefit of U.S. Provisional Patent Application No. 63 / 079,160, filed September 16, 2020, entitled "Evaluation of System Inertness". The contents of both applications are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure relates to compounds for system suitability testing in inert LC systems and columns. More specifically, the technique relates to probe compounds for detecting exposed metals in liquid chromatography, chromatographic columns, and coupled detectors. Background Art

[0006] Liquid chromatography (LC) is an analytical separation technique that separates mixtures of chemicals based on the differential interactions (defined as the principal interactions) between the compounds in the mixture and the stationary phase. These principal interactions are the intended interactions between the mixture, the designed stationary phase, and the modulations from the specifically chosen mobile phase / environmental conditions. These interactions depend on many controlled variables, such as mobile phase composition, temperature, and flow rate. Because the analytical quality factor of a given separation depends on a large number of variables that directly affect the range and extent of the principal chemical interactions, careful preparation of the mobile phase, appropriate temperature control equipment, and the use of reproducible columns, along with operation at a consistent set point, are crucial. Summary of the Invention

[0007] The degree to which probe compounds interact with the metals exposed in the chromatographic system and column can be used to design and execute suitability experiments that produce useful information. One technique for assessing the suitability of inert LC systems and columns is to use probe compounds as positive and negative controls. Compounds that can be selected and adapted as a pair to serve as positive and negative controls can be chosen. An example of a positive and negative control is adenosine-5'-triphosphate (ATP) and adenosine. Chemically modified analogs of adenosine and ATP, as well as other nucleotides, can also be used to accommodate alternative detection modalities, such as fluorescence detection. A set of ATP analogs and associated non-interacting negative controls are 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (Bz-ATP) and benzophenone. Similarly, non-hydrolyzable nucleotide analogs are of interest because the natural pyrophosphate bonds are known to be readily hydrolyzed. Other chemical components can be used for system suitability criteria.

[0008] Confirming instrument performance is a critical aspect of any analytical measurement. When analyzing compounds susceptible to metal adsorption, it is not just the mechanical instrument performance that needs to be confirmed. Verifying the inertness of the selected LC is also important. This disclosure presents a new method for evaluating the inertness of a chromatography system.

[0009] Concerns about standard stability led to the use of a nucleotide analog called AMPcP, which, unlike ATP and ADP, does not contain a pyrophosphate bond, making it hydrolytically stable. Using flow injection analysis of AMPcP, higher and more consistent recoveries were observed from inert LC systems relative to metal alloy-based instruments.

[0010] It was also confirmed that AMPcP could be paired with caffeine as a negative control, in which the use of AMPcP successfully identified the presence of the metal, while caffeine helped to verify the overall mechanical properties.

[0011] In one aspect, the present disclosure includes a kit for evaluating system inertness. The kit includes a positive control and a negative control, the positive control comprising a nucleotide diphosphate with a non-hydrolyzable α-β bond or a chemically modified analog thereof, wherein the positive control contains a metal interaction portion and the negative control does not contain a metal interaction portion. In some embodiments, the kit also includes a container for a solution (e.g., a system adaptability solution) for forming a mixture of a negative control and a positive control. In some embodiments, the nucleotide diphosphate with a non-hydrolyzable α-β bond is adenosine 5'-(α, β-methylene) diphosphate (AMPcP). In some embodiments, the solution of the mixture of the negative control and the positive control is an equimolar mixture. In some embodiments, the negative control and the positive control have the same chromophore, have the same maximum UV absorption wavelength, and exhibit the same extinction coefficient at this wavelength. In some embodiments, the negative control consists of or is essentially composed of adenosine (e.g., a diluted solution of adenosine in water or a water / polar solvent mixture). In some embodiments, the negative control consists of or consists essentially of caffeine (eg, a dilute solution of caffeine in water or a water / polar solvent mixture).

[0012] In another aspect, the present disclosure includes a kit for evaluating system inertness. The kit includes a solution having a positive control and a negative control, the positive control comprising adenosine-5'-triphosphate (ATP) or a chemically modified analog thereof, the negative control not containing a metal interacting portion; and a container for holding the solution. In some embodiments, the negative control and the positive control have the same chromophore, have the same maximum UV absorption wavelength, and exhibit the same extinction coefficient at that wavelength. In some embodiments, the solutions of the negative control and the positive control are an equimolar mixture. In some embodiments, the chemically modified analog of adenosine-5'-triphosphate (ATP) is a 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) compound. In some embodiments, the negative control consists of or consists essentially of adenosine. In some embodiments, the negative control consists of or consists essentially of caffeine.

[0013] In another aspect, this disclosure includes a kit for assessing system inertness. The kit includes: a positive control containing a metal-interacting moiety; a negative control lacking a metal-interacting moiety; and a container holding a system-adaptive solution. In some embodiments, the negative and positive controls have the same chromophore, the same maximum UV absorption wavelength, and exhibit substantially the same extinction coefficient at that wavelength. In some embodiments, the negative and positive controls are compounds, and the difference between the negative and positive controls is a series of three phosphate groups. In some embodiments, the positive control is adenosine-5'-triphosphate (ATP) or a chemical analog thereof, and the negative control is adenosine or caffeine. In some embodiments, the positive control is adenosine 5'-(α,β-methylene)diphosphate (AMPcP) or a chemical analog thereof, and the negative control is adenosine or caffeine. In some embodiments, the positive control is a nucleotide diphosphate having a non-hydrolyzable α-β bond. In some embodiments, the negative control consists of or is substantially composed of adenosine. In some embodiments, the negative control consists of or is substantially composed of caffeine. In some embodiments, the negative control and the positive control have different spectral characteristics. In some embodiments, the solutions of the negative control and the positive control are equimolar mixtures. In some embodiments, the positive control is vinylidene adenosine 5'-(α,β-methylene)bisphosphate having the following structure:

[0014]

[0015] In one aspect, this disclosure includes a kit for assessing system inertness. The kit includes a container with a solution containing a positive control comprising a metal-interacting moiety, wherein the positive control is vinylidene adenosine 5'-(α,β-methylene)bisphosphate having the following structure:

[0016]

[0017] Embodiments of this aspect of the invention may also include one or more of the following features. The kit may also include a syringe for injecting a positive control into at least a portion of the liquid analysis system (e.g., into the fluid handling system of an LC system with the column removed; into an LC column; into an LC system with the column in place, etc.). Some embodiments of the kit also include a container for a negative control that does not contain metal-interacting components (e.g., adenosine, caffeine, etc.).

[0018] The foregoing aspects and features of this disclosure can be combined to provide a number of advantages over the prior art. In some embodiments, the use of the compounds of this disclosure in system suitability testing of inert LC systems and columns has many beneficial effects. For example, this disclosure uses probe compounds to detect metals exposed in liquid chromatography, chromatographic columns, and coupled detectors. These compounds (e.g., suitability samples) can help analysts determine the suitability of a system for a particular application before (and without needing to) expend resources (e.g., time and money) on separations that will not provide meaningful results. That is, by determining the inert suitability of the system before the desired separation or application, the analyst will know not to waste resources on separations / applications destined to fail. This information can prevent the waste of time and money, as knowledge of the system's suitability for a particular application will eliminate or significantly reduce experiments destined to yield poor-quality results. Attached Figure Description

[0019] The present technology will be more fully understood through the following detailed description taken in conjunction with the accompanying drawings, in which:

[0020] Figure 1 is a schematic diagram of a chromatography system including a chromatography column and various other components according to an exemplary embodiment of the present disclosure.

[0021] Figure 2 are representative examples showing the sensitivity and repeatability of AMPcP applied to the detection of metals in a chromatographic flow path according to exemplary embodiments of the present disclosure.

[0022] Figure 3 is a representative example of a test demonstrating the metal sensitivity evaluation of a probe compound without reliance on chromatography, according to an exemplary embodiment of the present disclosure.

[0023] Figure 4 This is a flowchart illustrating a method for evaluating the suitability of a system according to an exemplary embodiment of the present disclosure.

[0024] Figure 5 This is a representative example of chromatographic testing of inert surfaces using metal-sensitive compounds and inert analogues, as demonstrated by exemplary embodiments of the present disclosure.

[0025] Figure 6 This demonstrates how UHPLC repeated injection of AMPcP and caffeine using a partially configured (as opposed to a fully configured) hybrid surface technique (HST) readily identifies exposed metal surfaces by increasing peak area relative to standard deviation and decreasing absolute recovery.

[0026] Figure 7A It is a chromatogram showing the caffeine peak and AMCP peak on a chromatographic system equipped with PEEK (metal-free).

[0027] Figure 7B Display for comparison Figure 7A The tool provided in the software compares the peak shapes of AMPcP and caffeine.

[0028] Figure 8A It is a chromatogram showing the caffeine peak and AMPcP peak on a chromatographic system equipped with a chromatographic surface coated with an ethylene-bridged hybrid (an inert material covering a metal surface).

[0029] Figure 8B Display used for comparison Figure 8A Peak comparison tool for AMPcP and caffeine peak shapes provided in .

[0030] Figure 9A is a chromatogram showing the caffeine and AMPcP peaks on a chromatography system equipped with an intentionally degraded ethylene bridged hybrid coated chromatography surface (due to expected metal exposure due to coating degradation).

[0031] Figure 9B Display used for comparison Figure 9A Peak comparison tool for AMPcP and caffeine peak shapes provided in . Detailed Implementation

[0032] Generally, this disclosure relates to characterization methods for inert LC systems used to evaluate the appropriate operation of inert surfaces, particularly when analyzing compounds known to exhibit high metal-to-metal interactions. Compounds may be selected and suitably used as a pair to serve as positive and negative controls. The degree of interaction between the probe compound and the metals exposed in the chromatographic system and column can be used to design and execute suitable experiments that produce useful information.

[0033] One approach to system suitability testing involves preparing a mixture containing the analyte of interest or an alternative reagent molecule exhibiting the same chemical properties. If the method is published in various global pharmacopoeias, standards can be commercially available; otherwise, scientists will have to manually identify and prepare standards suitable for the test. A known test mixture is injected prior to sample analysis to confirm system suitability. Referring to these measurements against expected or historical results confirms appropriate system performance and can be used to monitor method performance over time.

[0034] Assessing the system readiness for analyzing compounds adsorbed onto metal surfaces can be relatively complex. In some cases, using suitability standards that do not contain metal-sensitive compounds offers little help in detecting exposed metal surfaces. One option for addressing these issues is to use metal-sensitive probe compounds to examine the flow path of the instrument along with that of any detector. In this paper, the use of the hydrolyzed stable nucleotide analog AMPcP for system readiness measurements is proposed. This molecule has been shown to be as effective as adenosine triphosphate in binding metals, but is not readily degraded during storage and long-term use. One technique involves methods utilizing such metal-adsorbing compounds, which are simply used together with non-adsorbed caffeine standards to perform flow injection tests. These methods have shown effectiveness in detecting metals and rapidly investigating the inertness of LC equipment.

[0035] One technique for assessing the suitability of an inert LC system via positive and negative controls uses adenosine-5'-triphosphate (ATP) and adenosine. Chemically modified analogs of adenosine and ATP, along with other nucleotides, can also be used to accommodate alternative detection modalities, such as fluorescence detection. A set of ATP analogs and associated non-interacting negative controls are 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (Bz-ATP) and benzophenone.

[0036] Another group of molecules that can be used to achieve similar purposes as Bz-ATP include, but are not limited to, vinylidene adenosine, 6-aminohexyl adenosine and aminopurines and nucleotides, nucleotides or nucleobases labeled with MANT (methyl-o-aminobenzoyl), TNP (trinitrophenyl), luciferin, pyrene, 7-propargylamino-7-deaza, DY-647P1, and fluorescent nucleobases and nucleotides.

[0037] Non-hydrolyzable nucleotide analogs are also of interest because the native pyrophosphate bond may be readily hydrolyzed. Some exemplary nucleotides with non-hydrolyzable bonds include ATPαS, ApCpp, AppCp, AppNHp, and ATPγS. Other equivalents include, but are not limited to, those based on guanine, cytosine, uridine, and thymine. Nucleotide diphosphates with non-hydrolyzable α-β bonds can be used for certain applications due to their improved stability.

[0038] Some other chemical moieties considered for use in system suitability standards include probes based on or similar to the following: 2-hydroxypyridine-1-oxide, tetracycline, fructose-1,6-diphosphate, pyranine, deferiprone, deferasirox, bisphosphonate-containing molecules, tiludronic acid, risedronic acid, diphenylglyoxime, α-benzoin oxime, or salicylaldoxime. Molecules based on tricarboxylic acid moieties can also be used.

[0039] Compounds can be used to perform tests to assess the health of the system, including as a maintenance procedure. Specific times for testing the LC system can be scheduled, such as after a set amount of time, a set number of uses, or after using a specific compound. At a given time, it may be necessary to test only a portion of the LC system. For example, evaluating only a part of the system can be used for fault diagnosis.

[0040] The content being tested can also vary. Evaluation tests that simultaneously measure multiple performance characteristics are desirable. This is achieved using various chromatographic methods with mixtures of compounds. For example, a single experiment can use several compounds and combine chromatographic methods to simultaneously evaluate gradient delivery and exposure of metal surfaces.

[0041] The quality factor of LC separation depends on many uncontrollable variables, among which "secondary interactions" are a common source of performance degradation. One example is the poorly controlled metal content found in the stationary phase silica particles. The uncontrolled presence of metals in the particles leads to variable secondary interactions, which can degrade the quality and reproducibility of analytical separations. Additional sources of secondary interactions include various metallic components within the column and LC flow path components (e.g., column tubing, frit, syringe needle, and tubing). To mitigate unwanted interactions involving system surfaces (e.g., the surfaces of LC flow path components), analysts have relied heavily on passivating or modulating hardware surfaces and have even altered analytical methods by using ion-pairing reagents, chemical derivatizations, and chelating agents as mobile phase additives, among other things.

[0042] Another proposed approach to addressing the detrimental effects of secondary interactions involves the use of LC components that prevent the analyte from contacting the metallic surface. These techniques include PEEK or other non-metallic components, PEEK-lined steel components, and metallic components that have already been chemically modified or coated on the surface. An example of a coated surface is one using a surface technology based on a vapor-deposition-based combination of organosilicon and carboxysilane. This emerging technique is described in more detail in patent application (US20190086371A) published by Lauber et al. LC systems based on such techniques are generally described as inert LCs. The entire contents of US20190086371 are incorporated herein by reference.

[0043] Another approach is to passivate the system with a liquid solvent, such as nitric acid. The solvent method provides temporary passivation, that is, it provides the system with temporary inertness (or an inert coating).

[0044] There are many methods that can be used alone or in combination to address the presence of secondary interactions and their impact on the quality of a given chromatographic separation. There is a need for compounds that can help evaluate whether the coating / passivation method provides an inert system for the samples being processed. Therefore, there is a need for compounds that can be used alone or in combination with other compounds to evaluate the system suitability testing of inert LC systems and columns. First, we will discuss the Figure 1 Discuss the system / device and the coating that inerts the system / device.

[0045] Figure 1 This is a representative schematic diagram of a chromatography system 100 that can be used to separate analytes such as peptide compounds from a sample. System 100 includes several components, including: a fluid manager system 105 (e.g., controlling the flow of the mobile phase through the system); tubing 110 (which may also be replaced by or used in conjunction with microfabricated fluid conduits); a fluid connector 115; a frit 120; a column 125; a sample injector 135 including a needle (not shown) for inserting or injecting a sample into the mobile phase; a vial or sample container 130 for holding the sample prior to injection; and a detector 150, such as a mass spectrometer. The column 125 may be a reversed-phase column. The inner surfaces of the components of the chromatography system 100 form fluid flow paths with wetting surfaces. The components of the fluid flow paths may have a length-to-diameter ratio of at least 20, 25, 30, 35, or 40. The fluid flow paths may include the wetting surface (not shown) of an electrospray needle.

[0046] At least a portion of the wetting surface may have a coating, such as an alkylsilyl coating. The coating can adjust the hydrophobicity of the wetting surface. The coating can be applied by vapor deposition. Therefore, the methods and apparatus of this disclosure may include a pressure-resistant material (e.g., stainless steel) for the flow system and a wetting surface of the fluid flow path that provides suitable hydrophobicity so that harmful interactions or undesirable chemical effects on the sample can be minimized.

[0047] In some examples, the coating of the flow path is non-binding with respect to analytes such as metal-sensitive compounds (eg, peptides). Thus, analytes such as peptide compounds do not bind to the coating of the flow path.

[0048] The coating can be provided throughout the system by tubing or fluid conduit 110 extending from the fluid manager system 105 to the detector 150. The coating can also be applied to portions of components of the fluid path. That is, one or more components or portions of components can be coated instead of the entire fluid path. For example, the interior portion of column 125, its molten core 120, and fluid connector 115 can be coated while the rest of the flow path remains unmodified. Furthermore, removable / replaceable components can be coated. For example, the sample container 130 containing the sample and the molten core 120 can be coated.

[0049] In some examples, System 100 will need to be cleaned / removed before the evaluation begins in order to establish a baseline for determining suitability before testing begins. Ensuring System 100 is at the baseline helps demonstrate the absence of contaminants. It can also be used to validate the preparation process of System 100 after its manufacturing is complete. For example, after manufacturing System 100, it can be used... Figure 4 Method 400.

[0050] The flow path of the fluid system may be defined at least partially by the inner surface of the tubing. The flow path of the fluid system may also be described at least partially by the inner surface of the micro-machined fluid conduit. Furthermore, the flow path of the fluid system may be described at least partially by the inner surface of the column or at least partially by a channel through the molten metal. The flow path of the fluid system is also described at least partially by the inner surface of the sample injection needle or by an inner surface extending from the inner surface of the sample injection needle throughout the column. Additionally, the flow path may be described as extending from a sample container (e.g., a vial) disposed upstream of and in fluid communication with the inner surface of the sample injection needle located throughout the fluid system to the port of the connector / detector.

[0051] In some implementations, only the wetting surface of the column and components located upstream of the column are coated with, for example, an alkylsilane coating, while the wetting surface downstream of the column is not coated. The coating can be applied to the wetting surface via vapor deposition. Similarly, the “wetting surface” of laboratory equipment or other fluid handling devices can benefit from an alkylsilane coating. The “wetting surface” of these devices includes not only the fluid flow path but also the elements located within it. For example, the molten material and / or membrane within a solid-phase extraction device comes into contact with the fluid sample. Therefore, not only the inner walls within a solid-phase extraction device but also any molten material / membrane is included within the scope of the “wetting surface.” The term “wetting surface” refers to all surfaces within an apparatus (e.g., a chromatographic column, a chromatographic injection system, a chromatographic fluid handling system, laboratory glassware, a solid-phase extraction device, the tip of a pipette, a centrifuge tube, a beaker, a dialysis chamber, etc.) that come into contact with fluid, especially a fluid containing the analyte of interest.

[0052] In embodiments featuring an inert coating along or on the wetted surface, at least a portion of the wetted surface is coated with an alkylsilyl coating. The alkylsilyl coating is inert to at least one of the analytes in the sample.

[0053] In some embodiments, the alkylsilyl coating is an organosilica coating.In certain embodiments, the alkylsilyl coating is a hybrid inorganic / organic material that forms a wetting surface or coats a wetting surface.

[0054] The coating (e.g., an alkylsilyl coating) can have a contact angle with water of at least about 15°. In some embodiments, the coating can have a contact angle of less than or equal to 30°. The contact angle can be less than or equal to about 115°. In some embodiments, the contact angle of the coating is between about 15° and about 90°, in some embodiments between about 15° and about 105°, and in some embodiments between about 15° and about 115°. For example, the contact angle of the coating can be about 0°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°, 95°, 100°, 105°, 110°, or 115°.

[0055] The thickness of the coating (e.g., an alkylsilyl coating) can be at least approximately For example, the thickness may be between about to approximately The thickness of the coating can be about or The thickness of a coating (e.g., a vapor deposited alkylsilyl coating) can be detected optically by the naked eye. For example, higher opacity and coloration indicate a thicker coating. When the coated part is viewed under full spectrum light (such as sunlight), the color changes from yellow, to purple, to blue, to slightly green, and then back to yellow, from thin to thick. For example, when the thickness of the alkylsilyl coating is When the coating is yellow, it reflects light with peak wavelengths between 560 nm and 590 nm. When the thickness of the alkylsilane coating is... When the coating is applied, it can exhibit a purple hue and reflect light with peak wavelengths between 400 nm and 450 nm. When the thickness of the alkylsilane coating is... When the coating is blue, it can reflect light with a peak wavelength between 450 nm and 490 nm. See, for example, Faucheu et al., Relating Gloss Loss to Topographical Features of a PVDF Coating, published October 6, 2004; Bohlin, Erik, Surface and Porous Structure of Pigment Coatings, Interactions with flexographic ink and effects of print quality, Dissertation, Karlstad University Studies, 2013: 49.

[0056] The coating may be a product of vapor-deposited bis(trichlorosilyl)ethane, bis(trimethoxysilyl)ethane, bis(trichlorosilyl)octane, bis(trimethoxysilyl)octane, bis(trimethoxysilyl)hexane, or bis(trichlorosilyl)hexane. The coating may include a siloxane moiety imparted by a vapor-deposited reagent.

[0057] In some respects, at least a portion of the wetted surface is coated with multiple layers of the same or different alkylsilanes, wherein the thickness of the alkylsilane coating is related to the number of layering steps performed (e.g., the number of alkylsilane coating deposits on the wetted surface).

[0058] The chromatographic apparatus may have multiple alkylsilyl coatings. For example, a second alkylsilyl coating may be in direct contact with a first alkylsilyl coating.

[0059] In one aspect, the coating is n-decyltrichlorosilane, (3-glycidoxypropyl)trimethoxysilane (GPTMS), hydrolyzed (3-glycidoxypropyl)trimethoxysilane (GPTMS), 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, trimethylchlorosilane, trimethyldimethylaminosilane, methoxy-polyvinyloxy(3)silane, propyltrichlorosilane, propyltrimethoxysilane, (heptadecanofluoro-1,1,2,2-tetrafluoroethylene) (Hydrodecyl)tri(dimethylamino)silane, (heptadecano-1,1,2,2-tetrahydrodecyl)trichlorosilane, (heptadecano-1,1,2,2-tetrahydrodecyl)trimethoxysilane, vinyltrichlorosilane, vinyltrimethoxysilane, allyltrichlorosilane, 2-[methoxy(polyvinyloxy)3propyl]trichlorosilane, 2-[methoxy(polyvinyloxy)3propyl]trimethoxysilane or 2-[methoxy(polyvinyloxy)3propyl]tri(dimethylamino)silane.

[0060] Flow path components may be made of stainless steel and include, but are not limited to, tubing, micromachining fluid conduits, column ingots, column inlet tubing, and sample injection needles, coated by vapor deposition with one or more of the disclosed alkylsilyl groups. In one aspect, these coated components are annealed to alter their chemical or physical properties.

[0061] Flow path components made of materials other than stainless steel or other metals (e.g., polymers, glass, etc.) are coated via vapor deposition using one or more of the disclosed coatings. In particular, sample vials that can be connected to an injection needle may be coated.

[0062] The wetted surface of laboratory glassware, or at least portions thereof, may be coated with one or more disclosed alkylsilyl coatings via vapor deposition. In some examples, the vapor-deposited coating minimizes adsorption loss of the sample. The vapor-deposited coating may be neutral (low ionic properties) and hydrophilic (exhibiting a contact angle with water of less than 60°). This coating can be used to mitigate problems with many different types of materials, including glass and polymer compositions such as polypropylene or polyethylene.

[0063] Alternatively, commercially available vapor-deposited coatings may be used in the disclosed systems, apparatus, and methods, including but not limited to... and (Both are commercially available from SilcoTek Corporation, Bellefonte, PA.) The manufacturing method is described in U.S. application serial number 14 / 680,669, entitled “Thermal Chemical Vapor Deposition Coated Article and Process,” filed on April 7, 2015, which claims priority to and the benefit of U.S. provisional application serial number 61 / 976,789, filed on April 8, 2014. The contents of each patent application are incorporated herein by reference in their entirety.

[0064] In one aspect, alkylsilyl coatings enhance the corrosion performance of metals, such as in metal chromatography columns. Depending on density and thickness, the coating acts as a barrier, preventing water and corrosive molecules from reacting with the base metal. Increasing hydrophobicity and density improves corrosion performance.

[0065] In some embodiments, the alkylsilyl coating is modified with a silanizing agent to obtain the desired surface properties. The silanizing agent can be a non-volatile zwitterion. The non-volatile zwitterion can be a sulfobetaine or a carboxybetaine. In some embodiments, the silanizing agent is an acidic or basic silane. The silanizing agent can introduce a polyethylene oxide moiety.

[0066] Other components of the LC system may also include the coatings described herein, such as tubing, frits, and / or connectors. LC systems can be used to separate metal-sensitive analytes, such as biomolecules, proteins, glycans, peptides, oligonucleotides, pesticides, bisphosphonates, anionic metabolites, and zwitterions (e.g., amino acids and neurotransmitters), and the LC system includes multiple coated components to minimize or eliminate the presence of metals along wetted surfaces within the fluid flow path.

[0067] Coating provides a method of how to make a system (such as LC system and column) suitable for use. Another possible alternative to coating can be to passivate the system using a liquid solvent (such as, for example, nitric acid). The system can also be made inert by using a non-metallic surface (including polyetheretherketone or diamond-like carbon). The coating can also be a surface or composition deposited by charge vapor deposition or atomic layer deposition. After making the system inert, it is then necessary to test the system to determine whether the system is suitable. Compounds according to the present technology are used to test whether the system is suitable. This article describes a technique for determining the suitability of a system using the compound, such as an inert LC system and column.

[0068] One technique for evaluating the suitability of inert LC systems coupled with optical or MS detectors involves the chromatographic separation and detection of equimolar solutions of adenosine-5'-triphosphate (ATP) and adenosine. These two compounds have the same chromatin and therefore the same maximum UV absorption wavelength of 259 nm, and exhibit the same extinction coefficient at said wavelength, E = 15400 at pH 7.0. The difference between the two compounds is a series of three phosphate groups, which are known to interact with metals. Because ATP contains a metal-interacting moiety while adenosine does not, adenosine serves as a structurally similar negative control compound. Furthermore, the significant difference in hydrophobicity between the positive and negative control compounds (such as ATP and adenosine) (Log P -5.5 vs -1.05) ensures their resolution in reversed-phase chromatographic separation. Two compounds with a log P value difference greater than one are desirable. Adenosine 5'-(α,β-methylene)diphosphate (AMPcP) is another pairing of adenosine (Log P -4.8 vs -1.05, respectively).

[0069] Chemically modified analogs of adenosine and ATP, as well as other nucleotides, can also be used to accommodate alternative detection modalities, such as fluorescence detection. A set of ATP analogs and associated non-interacting negative controls are 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) and benzophenone. As a result of the benzoylbenzoyl O-linking to the ATP compound, an increased degree of reversed-phase retention can be expected, which favorably promotes the suitability of separation-based methods. In addition, Bz-ATP is expected to produce increased UV absorption due to the benzoylbenzoyl functional group. Similarly, Bz-ATP can be readily measured in both positive and negative modes of LC-MS, while ATP is readily observed only in negative mode.

[0070] In addition to the aforementioned benefits, chromatographic analysis of Bz-ATP and benzophenone in an equimolar mixture, when optionally employing fluorescence detection, enables the evaluation of inert flow paths / columns. An additional benefit of this fluorescent analog method for evaluating LC inertness is its ability to operate at significantly reduced molar loadings, where secondary interactions are more readily detected, addressing the challenges associated with LC-MS signal intensity variations.

[0071] Another group of molecules that can be used to achieve the same purpose as Bz-ATP includes, but is not limited to, vinylidene adenosine, 6-aminohexyl adenosine and aminopurine, as well as nucleotides, nucleotides or nucleobases labeled with MANT (methyl-o-aminobenzoyl), TNP (trinitrophenyl), luciferin, pyrene, 7-propynylamino-7-deaza, and DY-647P1, and fluorescent nucleobases and nucleotides (such as those described in Nature Chemistry, Vol. 9, pp. 1043-1055 (2017)).

[0072] Non-hydrolyzable nucleotide analogs are also of interest because the native pyrophosphate bonds are readily hydrolyzed. Therefore, nucleotide-based standards present challenges for manufacturing, stable storage, and global transport without degradation. Some exemplary nucleotides with non-hydrolyzable bonds include ATPαS, ApCpp, AppCp, AppNHp, and ATPγS. Other equivalents include, but are not limited to, those based on guanine, cytosine, uridine, and thymine. Nucleotide diphosphates with non-hydrolyzable α-β bonds may be advantageous for certain applications due to their improved stability, such as the example described in Structure 1, adenosine 5'-(α,β-methylene) diphosphate (AMPcP). These can also be labeled with additional substituents or constructed from alternative nucleobase-like structures to better facilitate detection, whether by UV, MS, or fluorescence.

[0073]

[0074] Structure 2 is an example of such variants shown with vinylene-AMPcP, where the addition of a vinylene substituent coupled to the nucleobase moiety of the molecule via a ring-closed configuration imparts increased hydrophobicity, enabling positive-mode MS detection as well as fluorescence detection and providing more sensitive UV processing for UV spectrophotometric measurements. An additional beneficial effect of chemical modification of the test probe for suitability is the ability to tune the molecule's metal-chelating capacity. Tuning the strength of these interactions allows for the development and application of test probes (negative and positive controls). The test probe interacts strongly enough to detect secondary interactions in the chromatographic instrument and column, but not so strongly that the signal is unobservable (e.g., complete adsorption of the test probe). This provides an additional degree of flexibility in the design of system suitability, which is helpful because detectors typically used with liquid chromatography span many orders of magnitude in terms of detection dynamic range.

[0075]

[0076] Some other chemical moieties considered for use in system suitability standards include probes based on or similar to the following: 2-hydroxypyridine-1-oxide, tetracycline, fructose-1,6-diphosphate, pyranine, deferiprone, deferasirox, bisphosphonate-containing molecules, tiludronic acid, risedronic acid, diphenylglyoxime, α-benzoin oxime, or salicylaldoxime. Molecules based on tricarboxylic acid moieties can also be used.

[0077] Biomolecular scaffolds can also be used to prepare standards for system suitability testing. Synthetic polypeptides are one such scaffold. Peptides can be chemically synthesized to contain one or more phosphorylated residues, such as phosphoserine, phosphothreonine, or phosphotyrosine. In some examples, O-phosphorylated residues are more stable and more resistant to hydrolysis than N-phosphorylated residues (e.g., phosphohistidine). Optionally, peptides can be synthesized to contain one or more fluorescent amino acid residues, including but not limited to tryptophan. In one example, a metal-sensitive probe based on the sequence H2N-GEGWG(pT)G(pT)GK-OH (SEQ ID NO: 1) was used, and the chromatographic behavior was compared to that of H2N-GEGWGTGTGK-OH (SEQ ID NO: 2). Standards based on chemically synthesized oligonucleotides can also demonstrate practicality. On these biomolecular scaffolds, additional substituents can be added to advantageously alter the chromatographic effect and detectability.

[0078] The present disclosure includes methods for testing LC systems for metal interactions using system suitability standards, such as adenosine 5'-(α,β-methylene) diphosphate (AMPcP) or adenosine triphosphate (ATP). These standards interact with metals (i.e., metal-interacting moieties), which adsorb to exposed metal components. Consequently, a decrease in the amount of standard measured by the injected amount indicates the presence of exposed metal and the likelihood of system unsuitability.

[0079] Nucleotide analogs of AMPcP have been shown to adsorb onto titanium frit as readily as adenosine triphosphate (ATP), remain resistant to hydrolysis, and are stable for long-term storage and repeated use (applicable to any reagent proposed for system suitability testing).

[0080] AMPcP can be used in flow injection assays (column-free direct injection) to monitor losses and peak shape disturbances that can potentially exist in any chromatograph made with one or more metal-based components. In this method, AMPcP is injected sequentially without a column, and various peak properties are monitored and ultimately correlated with the amount of metal in the flow path. The ability of this method to distinguish inert chromatographic surfaces from metals was validated by comparing the peak area, peak shape, and injection repeatability of AMPcP using ultra-high performance liquid chromatography (UHPLC) equipped with conventional MP35N metal alloy components with equivalent UHPLC equipped with a hybrid organic-inorganic surface with ethylene bridging.

[0081] This disclosure illustrates how AMPcP can be used to identify varying degrees of exposed metal within the flow path of an LC instrument itself. When metal-sensitive compounds, such as AMPcP, are injected into a metal-based flow path, poor reproducibility is commonly observed during multiple injections, particularly variations in peak area and peak height, as well as an overall decrease in analyte signal. Systems with exposed metal surfaces may also exhibit increased peak area during repeated injections, a result of analyte adsorption onto the exposed metal and thus passivation of it. This adsorption process can lead to greater recoveries in subsequent injections, but this is not always a predictable phenomenon, and the passivation state is reversible. Not wishing to be bound by theory, it is believed that peak area and peak area changes detected after a flow injection of AMPcP are diagnostic of the presence of a metal surface. Peak area and peak area changes can be used alone or in combination with analyte recoveries or absolute analyte responses to assess system suitability. The extent of the change can depend on several factors, such as the specific compound being analyzed, system history, and the mobile phase. To ensure removal of any previously injected sample, a rinsing step using multiple injections of 0.3% ammonia solution can be employed. Rinsing can be an effective method for removing components and past analyte samples from any metal in the flow path.

[0082] In some examples, because metal interactions can be significantly affected by pH, the testing procedures of this disclosure use a neutral pH and do not use a buffer. Using a neutral pH and not using a buffer can ensure a large effect because at a neutral pH, AMPcP will have a negative charge, while the metal surface of the flow path will have a positive charge.

[0083] Furthermore, AMPcP's sensitivity to exposed metal surfaces may be more pronounced with increased organic composition, which could be a result of affecting the dielectric constant of the mobile phase. The system setup of this disclosure may include the use of a zero-dead-volume titanium connector instead of a column, and flow conditions of 80:20 acetonitrile:water at 0.5 mL / min.

[0084] Figure 2Repeated injection of AMPcP was demonstrated on LC with a hybrid surface bridged by ethylene and on chromatography with a biocompatible MP35N flow path (i.e., a flow path with an exposed metal surface). Figure 2 This is a representative example demonstrating the sensitivity and repeatability of AMPcP applied to the detection of metals in the chromatographic flow path. Figure 2 Overlay of six repeated injections of 1 mL of AMPcP on an LC equipped with an ethylene-bridged hybrid chromatographic surface (top, 202) and an equivalent LC configuration equipped with a metal MP35N component (bottom, 204), as collected using 80:20 acetonitrile:water at a flow rate of 0.5 mL / min.

[0085] for Figure 2 When AMPcP was injected onto the metal MP35N flow path, there was a significantly lower average peak area (48%) and average peak height (47%). In addition, the peak area RSD for the metal-based biocompatible system was 6.4%, while the peak area RSD for the LC equipped with ethylene-bridged hybrid (HST) surface technology was 0.4%. This improved peak area reproducibility and overall increased analyte recovery stems from the elimination of metal interactions.

[0086] Figure 3 The present invention is a representative example of an evaluation test for metal sensitivity of a probe compound in accordance with an exemplary embodiment of the present invention that demonstrates that the metal sensitivity of the probe compound is not dependent on chromatography. The extent to which a probe compound (e.g., a chemically synthesized compound) interacts with the metal exposed in the chromatographic system and column can be used to design and perform applicability experiments that produce information useful to the analyst. Excessively interacting compounds may produce zero detectable signal, whereas minimally interacting compounds may not be able to detect metal exposed in the system and / or column. To this end, as described in Example 3, an injection test was developed using a chromatographic system with an inert flow channel (except for a single component, such as a column) and exposed metal. This evaluation enables the degree to which a group of compounds adsorb to metal components (e.g., active preheaters or inline metal frits) to be characterized and compared. Figure 3 Show different Positive Comparison of control (ATP-AMPcP) interactions and adsorption on metal components.

[0087] like Figure 3 As shown, the area recovery (%) of the probe compounds AMPcP and ATP was measured and compared. This method enables the assessment of the tendency of the probe compounds to bind to exposed metal relative to each other under the test conditions. In addition, the test can be adjusted by using components with more or less exposed metal in the flow path.

[0088] Figure 3The ability to measure the area recovery (%) of probe compounds (AMPcP and ATP in this case) by injection testing with a straight-line titanium (Ti) fused mass was confirmed. Figure 3 In this assay, test conditions included a 10 ng injection volume and 10 mM ammonium acetate solvent. The flow rate was set to 0.2 mL / min, and the Ti frit was an uncoated 4.6 mm Ti frit. The concentration of the two probe compounds, AMPCP and ATP, in water was 0.01 mg / mL. The recoveries of the test probe compounds were compared at equimolar loadings to assess their tendency to bind exposed metals relative to each other under the test conditions.

[0089] In addition to the methods described for designing and synthesizing probe compounds to detect exposed metals in liquid chromatography, chromatographic columns, and coupled detectors, manufacturing methods and considerations are also described. Because the magnitude of the effect of secondary interactions between the various metal-sensitive compounds and metal surfaces on chromatographic performance is molecularly loaded, methodologies designed to measure the degree of metal exposure control the molecular loading of the test probes. Some of the previously described probe compounds are purified or derived from compositions that are not entirely composed of the probe compound. Specifically, the desired compound is typically crystallized with counterions and / or water. The amount of counterions and / or water varies depending on the batch of the source or purified material.

[0090] Furthermore, hygroscopic reagents absorb water from the atmosphere, thus altering the water content from the initial measurement (e.g., the percentage of water by mass in the reagent solid material). Preparing test samples without considering this change (e.g., including only a positive control or a suitability sample including both a positive and a negative control) will result in variable test concentrations of metal-sensitive compounds and complicate system suitability test results and analysis.

[0091] These challenges can be addressed by preparing standards, in which the chemical components are prepared in the stock solution, indicating the presence of counterions and / or water in the batch material, aliquoting it in controlled volumes, and lyophilizing it into individual vials. Additionally, stabilizing compounds, such as trehalose, can be added for chemical stability and to prepare visible solid particles in the standard vials. Importantly, the stabilizing compound should not interfere with the intended system suitability test, meaning that the compound should exhibit little to no UV, fluorescence, or MS signal and minimal to no MS inhibition. Preparing system suitability standards in this manner improves the consistency of test sample concentrations, thereby enhancing the reliability and robustness of system suitability techniques employing these standards.

[0092] A kit for evaluating system inertness can be used in conjunction with these stock solutions. For example, the kit can include a container containing a solution comprising a positive control comprising a metal-interactive moiety and a negative control not comprising a metal-interactive moiety.

[0093] Figure 4 This is a flowchart illustrating a method 400 for evaluating the suitability of a system according to an exemplary embodiment of this disclosure. The method has certain optional steps, as indicated by the dashed outlines around specific steps. Method 400 may begin with components of an isolated system 402. It may be desirable to test only a portion of the LC system at a given time. It may be necessary to test only a portion of the LC system at a given time. When a component of the LC system (e.g., a sample syringe) is replaced, that component can be evaluated. Evaluating only a portion of the system (e.g., evaluating only components of the system) can be used for fault diagnosis of the system.

[0094] As noted, the components of isolation system 402 are optional. In some examples, it will be necessary to evaluate the suitability of the entire system / apparatus. A suitability sample can flow through system 404, such as system 100. The suitability sample may contain one or more compounds. After the sample flows through the system, a chromatographic detector can be used to evaluate the suitability of the sample and thus assess whether the components of the flow path are inert.

[0095] During or after the application sample flow 404, method 400 includes an evaluation system 406. Numerous methods exist, individually or in combination, to address the presence of secondary interactions and their impact on the quality of a given chromatographic separation. Inert system characterization methods are essential for evaluating the proper operation of inert surfaces.

[0096] In some implementations, system suitability analysis is performed simultaneously with the injection of sensitive probes that interact with the surface (positive control) and non-interacting probes (negative control). This allows for comparison of peak area ratios to confirm system suitability. If the ratio of positive to negative controls is sufficiently high, the system is considered to have good system health and is suitable for analysis.

[0097] Figure 5 This is a representative chromatographic example demonstrating the chromatographic testing of inert surfaces using metal-sensitive compounds and inert analogues, according to exemplary embodiments of this disclosure. Specifically, Figure 5 Representative chromatographic examples demonstrating the ability to easily separate ATP (positive control probe) and adenosine (negative control probe) by reversed-phase chromatography are shown. Comparisons of peak area ratio, peak area reproducibility, peak shape, and peak shape reproducibility allow characterization of the surface inertia and suitability for use in liquid chromatography and columns. This comparison will use one or more compounds known to bind metals to measure surface inertia and suitability. As demonstrated in Example 1 (described below) and Figure 5As shown in Figure 2, the relative area between ATP and adenosine can be used to assess the extent of metal interactions in the system while accounting for non-secondary interaction variations (e.g., injection volume). Additionally, evaluation of the peak shape of the ATP compound can be used to characterize system suitability.

[0098] In some examples, the method of characterizing the system includes introducing a sample having a positive control and a negative control (e.g., a suitability sample) into the system. The positive control is a sensitive probe that interacts with the system, while the negative control does not substantially interact with the system. In some examples, a compound that does not substantially interact with the system can be defined as a compound that does not contain a metal-interacting portion or a molecule that does not contain one or more strongly electronegative portions (such as acidic residues with a pKa of less than 5). The method may also include detecting the positive control and the negative control, and determining the system suitability by comparing the amount of the positive control detected with the amount of the negative control.

[0099] Determining system suitability by comparing the amount of the positive control detected with the negative control can include determining the ratio of the positive control detected to the negative control. If the ratio of the positive control to the negative control is high enough, it is considered that the system has good system health and is suitable for analysis. In some examples, high enough is greater than 0.5, 0.6, 0.7, 0.8, 0.9 or less than 1.1, wherein a ratio of 1.0 indicates that the analyte interacts with the system flow path without loss. The ratio considered to be high enough can vary based on the selected negative control and positive control. For example, a positive control (such as ATP or AMPcP) that interacts strongly with exposed metals can have a lower limit of a ratio range of 0.75 or 0.8.

[0100] In some examples, the analyst can tune the system to target a ratio of 1.0, and the analyst can determine a lower range of acceptable values ​​for the ratio based on the needs of the analyst / system. Determining system suitability by comparing the amount of positive control detected to the amount of negative control includes determining the inertness of the system to the positive control and / or indicating the integrity of an inert coating deposited along the flow path of the system.

[0101] Determining system suitability may involve testing a component of the system, such as a sample injector. The sample may adversely affect other components. At any given time, it may only be necessary to test a portion of the LC system. For example, evaluating only a part of the system can be used for fault diagnosis of the system.

[0102] Positive and negative controls can be detected using a liquid chromatography-optical, liquid chromatography-mass spectrometry, or liquid chromatography-optical-mass spectrometry detector configuration, where optical means ultraviolet / visible (UV / Vis) absorbance or fluorescence detector. The flow of the suitability sample can also be split.

[0103] More than one chromatographic method can be used to evaluate the system. The evaluation of the system using more than one chromatographic method can be performed simultaneously. For example, employing more than one chromatographic method involves introducing a sample containing multiple compounds (i.e., a suitability sample). These multiple compounds include a mixture of negative controls, positive controls, and resolved neutral substances. For example, in addition to positive and negative controls as surface probes, the compounds may include parabens or benzophenone ladders. Examples of paraben ladders may include at least one of methylparaben, ethylparaben, propylparaben, or butylparaben. Examples of benzophenone ladders may include at least one or more of acetophenone, phenylacetone, butylparaben, benzophenone, or phenylpentanone. Intentional changes to hydrophobicity help ensure successful resolution by reversed-phase chromatography. Furthermore, well-characterized neutral compounds, such as 2-acetylfuran, caffeine, or acetanilide, can also be used in a similar manner. Selection is based at least in part on differences in hydrophobicity, which can result in groups of compounds that will resolve well and enable evaluation of various performance aspects, including gradient delivery, flow rate accuracy, and column efficiency and health. The method can include evaluating gradient delivery and exposed metal surfaces in a single injection.

[0104] Characterization of the system can be a pre-determined maintenance procedure. For example, maintenance procedures can be scheduled after a pre-determined number of uses or a pre-determined amount of time. In other embodiments, characterization can occur before long or extremely long separations or runs (e.g., one or more days of operation) to ensure suitability before initiating long procedures. In some embodiments, characterization can occur before separating expensive or valuable samples. In some embodiments, characterization can occur before separating highly metal-sensitive samples or samples / applications that tend to have known system environmental suitability issues.

[0105] In some examples, no chromatographic column is used, and repeatedly injected suitability samples (e.g., samples including a positive control probe) are allowed to flow through the system and reach the detector. If the detector indicates that the compound in the sample is increasing, this indicates that the system is not inert. For example, there may be exposed metal, and the sample slowly passivates the exposed metal, so that an increased amount of suitability sample is detected after each injection.

[0106] When testing repeated injections, observing consistency across injections is diagnostic. For the sample being tested, a well-functioning system will show consistent results across injections, and these results should be reproducible. Systems with exposed metals will show variations across injections. The number of injections used to evaluate the system can vary based on the system components and the compound in the sample. In some examples, there are at least 3 injections, 3–50 injections, and 4–20 injections.

[0107] While using AMPcP to assess system inertness is a novel approach, it can also be used as part of a methodology to test the performance of a system's mechanical components. In situations where AMPcP alone is injected and the response is lower than expected, one might speculate whether the result is due to metal interactions or to mechanical aspects of the system. To distinguish the effects of metal interactions from other system performance issues, another reference check is performed.

[0108] Figure 6 This image shows an overlay of representative chromatograms (top chromatogram, 602; AMPcP, 604; caffeine, 606) on an ethylene-bridged HSTLC system with representative chromatograms (bottom chromatogram, 608; AMPcP, 614; caffeine, 612) on the same system, where a portion of these chromatograms were exchanged with the MP35N equivalent. That is, Figure 6 The diagram shows an overlay of the injection of caffeine (606, 612) and AMPcP (604, 614) on an ethylene-bridged HSTLC system and a partially inert system, in which a component (representing approximately 40% of the flow path surface area) is replaced by an MP35N component (i.e., a component with exposed metal).

[0109] Figure 6 This study demonstrates how repeated injections of AMPcP and caffeine using a partially configured (vs. fully configured) UHPLC with Hybrid Surface Technology (HST) readily identify exposed metal surfaces by increased peak area relative standard deviation and decreased absolute recovery. Caffeine injections were investigated as part of system suitability measurements. Caffeine served as a negative control for metal binding. Caffeine is not expected to interact with metal surfaces, thus providing an instrument-specific representation of peak shape and dispersion, as well as overall mechanical system performance.

[0110] for Figure 6 The results described herein, experimental protocol included an initial injection of 0.3% ammonium hydroxide to clean any metal surface, followed by multiple injections of AMPcP, and finally multiple injections of caffeine. Conditions included an 80:20 acetonitrile:water flow rate of 0.5 mL / min. AMPcP and caffeine were analyzed repeatedly after six replicate injections of 0.3% ammonium hydroxide (aqueous solution) and equilibration for two minutes with the described mobile phase and flow rate.

[0111] Comparison of representative caffeine injections running on this fully and partially inert system ( Figure 6, top chromatogram 602 compared to bottom chromatogram 608) show almost identical peak areas, peak heights, and peak shapes. For the completely inert system, the average values ​​of peak area, peak height, and area RSD were 16100 μAU*sec, 32000 μAU, and 1.0%, respectively, and for the partially inert system, the average values ​​of peak area, peak height, and area RSD were 16300 μAU*sec, 32800 μAU, and 1.0%, respectively. There was good consistency in the values ​​obtained on the two systems, which confirmed that caffeine did not experience any significant interaction with the metal. In addition, this information confirms that the two LC configurations were assembled equivalently and operated appropriately. It is important to be able to distinguish between system inertness and mechanical properties. Adding caffeine to the test protocol allowed these observations to be decoupled.

[0112] In contrast to the caffeine results (606 and 612), the AMPcP injections (604 and 614) proved to be significantly different when observed on the fully and partially inert systems. For the fully inert system, the average values ​​for the AMPcP peak area and peak height were 18,000 μAU*sec and 34,400 μAU, respectively, while for the partially inert system, the same values ​​were 12,900 μAU*sec and 18,100 μAU. This corresponds to a 28% decrease in average peak area and a 47% decrease in average peak height. The relative standard deviation of the AMPcP peak area on the fully inert system was 0.4%, while the same measured values ​​on the partially inert system increased to 4.3%.

[0113] The above data demonstrates an effective method for determining system inertness and system performance. The data collected above used an inert system where a single part was intentionally made metallic. In some examples, this test can be used to determine the presence of unknown amounts of metal in a flow path. The observation of repeatability (or lack thereof) may be most diagnostic of metal problems. Additionally, the peak area ratio between AMPcP and caffeine may prove effective. Figure 6 In the HPLC-MS / MS analysis, the completely inert system showed a peak area ratio of 1.1 (as predicted), while the partially inert system showed a peak area ratio of only 0.79, indicating that some metal-sensitive AMPcP has been lost to the flow path.

[0114] Figure 7A , Figure 8A and Figure 9A Provides chromatograms of caffeine to AMPcP peaks in three different systems. Figure 7A The chromatographic system for the peaks was equipped with PEEK (an inert, metal-free system). Figure 8A The chromatographic system for obtaining the peaks of γ-γ was equipped with an ethylene-bridged hybrid-coated chromatographic surface (a metal system made inert via an organosilica coating deposited on the chromatographic surface). Figure 9AThe chromatographic system for the peak is equipped with a hybrid chromatographic surface that is intentionally degraded ethylene-bridged (a system with the intended metal exposure due to the degradation of the coating). Figure 7A , Figure 8A and Figure 9A The rapid visual comparison illustrates how to use the system suitability solution (an equimolar solution containing AMPcP and caffeine) to show that suitability (peak shapes of equimolar positive and negative control probes) is substantially similar to inapplicability (the peak shape of caffeine differs from that of AMPcP).

[0115] While mathematical calculations of repeatability and peak area ratio are useful for determining system inertia, a final type of data processing was explored: tools that compare the obtained caffeine and AMPcP chromatograms, output difference plots, and also quantify the percentage difference between the traces. The peak observer tool was designed to acquire chromatograms of caffeine and AMPcP (i.e., Figure 7A , Figure 8A and Figure 9A (those shown) and normalize their peak heights before plotting the intensity differences across the collected data points. Figure 7B , Figure 8B and Figure 9B ). The peak heights are normalized to account for any potential differences in sample preparation. The Peak Viewer tool thus identifies meaningful differences in peak shape and tailing between the two chromatograms. For a completely inert system, comparison of the caffeine and AMPCP peaks should give an overall low level of difference, as the peak shape will be based solely on system dispersion effects. However, if the system contains some amount of exposed metal in the flow path, the differences in the caffeine peak shape (702, 702', and 702') and the AMPCP peak shape (704, 704', and 704') would be predicted to be more significant. The prototype Peak Viewer tool was used to process the full PEEK flow path ( Figure 7B ), coated surface bridged with inert ethylene ( Figure 8B Compared to the previous year, the differences were determined to be 19.88% and 20.84%. Both were low-level differences and were within the same range of differences (i.e., within 1% of each other). Meanwhile, for flow injection results from chromatographic systems with coated surfaces including ethylene bridging, a difference of 55.69% was determined, where individual components had been intentionally and actively degraded by exposure to 96 L of aqueous solution at pH 10 and 90°C for 57 days. Figure 9B The significant increase in the percentage difference on LC with forced degradation demonstrates the usefulness of peak visualization tools for determining the presence of metals within the chromatographic flow path. The ease with which this tool can be implemented makes considering this type of system-ready measurement attractive.

[0116] Example

[0117] For the reagents and standards used in the examples, all reagents were obtained from Millipore-Sigma (Burlington, MA). LC-MS grade acetonitrile was obtained from Honeywell (Muskegon, MI). Deionized water was prepared using a Millipore Milli-Q system. The liquid chromatography system, including the column, was treated with the aforementioned organosilica vapor deposition to reduce metal interactions.

[0118] Example 1 uses UV detection.

[0119] Example 1

[0120] Reverse-Phase LC-UV Detection of Adenosine-5'-Triphosphate (ATP) and Adenosine

[0121] Example 1 is a technique for assessing surface metal exposure, which involves separating and measuring ATP / adenosine using a chromatographic column. Adenosine serves as a negative control, where no metal interaction-related effects on separation and recovery are observed. For example, insufficient syringe delivery volume will result in a reduction in the area of ​​metal-sensitive ATP and adenosine control compounds.

[0122] As demonstrated in Example 1 and Figure 5 As shown in Figure 2, the relative area between ATP and adenosine can be used to assess the extent of metal interactions in the system while accounting for non-secondary interaction variations (e.g., injection volume). Additionally, evaluation of the peak shape of the ATP compound can be used to characterize system suitability.

[0123] A test sample (e.g., suitability sample) consisting of 20 ng / μL ATP and 9.5 ng / μL adenosine in water is prepared from a 1 mg / mL stock solution of ATP and adenosine in water. These solutions are then diluted into individual sample vials to produce the test sample. Freshly prepared test samples are recommended because ATP is expected to be degraded to ADP via hydrolysis. Similarly, the ammonium acetate mobile phase used is also freshly prepared daily, as ammonium acetate buffer is known to be volatile.

[0124] Separation details are shown in Table 1. After analyzing at least five injections of the test sample, the results of peak area, reproducibility, and peak shape for ATP and adenosine can be evaluated. Effective metal interaction attenuation is indicated by comparable peak areas, comparable and low peak area variation, and comparable and low peak tailing for the two compounds.

[0125] Table 1: Separation details of Example 1

[0126]

[0127]

[0128] Example 2

[0129] Reverse-Phase LC-UV Detection of Adenosine 5'-(α,β-methylene) Diphosphate (AMPcP) and Adenosine

[0130] Example 2 is a technique for assessing surface metal exposure, involving the separation and measurement of AMPcP and adenosine using a chromatographic column. Adenosine serves as a negative control, where no non-metal interaction-related effects on separation and recovery are observed. For example, insufficient syringe delivery volume will result in a reduction in the area of ​​metal-sensitive AMPcP and the adenosine control compound.

[0131] Test samples consisting of 17 ng / μL AMPcP and 10.7 ng / μL adenosine in water were prepared from 1 mg / mL stock solutions of AMPcP in water and adenosine in water. These solutions were then diluted into individual sample vials to produce the test samples. The ammonium acetate mobile phase was prepared fresh daily, as ammonium acetate buffer salts are known to be volatile.

[0132] The separation details are shown in Table 2. After analyzing at least five injections of the test sample, the results of the peak areas, reproducibility, and peak shapes of AMPcP and adenosine can be evaluated. Effective metal interaction attenuation is indicated by comparable peak areas, comparable and low peak area variations, and comparable and low peak tailing for both compounds.

[0133] Table 2: Separation details of Example 2

[0134]

[0135]

[0136] Example 3

[0137] Controlled Metal Exposure Testing for Evaluation of Positive Probe Compounds

[0138] Example 3 is an example of a technique used to evaluate a compound's ability to detect controlled levels of exposed metals in a chromatography system. Method details are provided in Table 3. After analyzing at least five injections of the test sample, the peak area and reproducibility results for the probe compound can be evaluated. Comparison of the results with other probe compounds analyzed under the same conditions provides an understanding of the compound's ability to detect exposed metals.

[0139] Table 3: Separation details of Example 3

[0140]

[0141] The foregoing aspects and features of this disclosure offer numerous advantages over the prior art. In some embodiments, the use of the compounds of this disclosure in system suitability testing of inert LC systems and columns has many beneficial effects. For example, this disclosure uses probe compounds to detect exposed metals in liquid chromatography, chromatographic columns, and coupled detectors. These compounds (e.g., suitability samples) can help analysts determine the suitability of a system for a particular application before (and without needing to) expend resources (e.g., time and money) on separations that will not provide meaningful results. This information can prevent the waste of time and resources, as knowledge of the system's suitability for a particular application will eliminate or significantly reduce experiments destined to yield poor-quality results. <110> WATERS TECHNOLOGIES CORPORATION <120> Compounds used for system suitability testing of inert liquid chromatography systems and columns. <130> W-4289-US02 <140> <141> <150> 63 / 079,151 <151> 2020-09-16 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MOD_RES <222> (6) <223> Phosphothreonine <220> <221> MOD_RES <222> (8) <223> Phosphothreonine <400> 1 Gly Glu Gly Trp Gly Thr Gly Thr Gly Lys 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 2 Gly Glu Gly Trp Gly Thr Gly Thr Gly Lys 1 5 10

Claims

1. A kit for evaluating system inertness, the kit comprising: a first vial comprising a positive control comprising a nucleotide diphosphate having a non-hydrolyzable α-β bond or a chemically modified analog thereof, wherein the positive control contains a metal interacting moiety; as well as A second vial, said second vial comprising a negative control, said negative control not containing a metal interacting moiety. 2 . The kit according to claim 1 , wherein the nucleotide diphosphate having a non-hydrolyzable α-β bond is adenosine 5′-(α,β-methylene) diphosphate (AMPcP).

3. The kit according to claim 1, further comprising a container for forming a solution of the negative control and the positive control.

4. The kit according to claim 1, wherein the negative control and the positive control have the same chromophore, have the same maximum UV absorption wavelength, and exhibit the same extinction coefficient at the wavelength.

5. The kit of claim 1, wherein the negative control consists of or consists essentially of adenosine.

6. The kit of claim 1, wherein the negative control consists of or consists essentially of caffeine.

7. A kit for evaluating system inertness, the kit comprising: a first vial comprising a positive control comprising adenosine-5'-triphosphate (ATP) or a chemically modified analog thereof; as well as A second vial, said second vial comprising a negative control, said negative control not containing a metal interacting moiety.

8. The kit according to claim 7, wherein the negative control and the positive control have the same chromophore, have the same maximum UV absorption wavelength, and exhibit the same extinction coefficient at the wavelength.

9. The kit according to claim 7, further comprising a container for forming a solution of the negative control and the positive control.

10. The kit according to claim 7, wherein the chemically modified analog of adenosine-5'-triphosphate (ATP) is a 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) compound.

11. The kit of claim 7, wherein the negative control consists of or consists essentially of adenosine.

12. The kit of claim 7, wherein the negative control consists of or consists essentially of caffeine.

13. A kit for evaluating system inertness, the kit comprising: a positive control comprising a metal interacting moiety; a negative control, the negative control not containing a metal interacting moiety, wherein the negative control and the positive control are compounds, and wherein the difference between the negative control and the positive control is a series of three phosphate groups; and Container for system suitability solution.

14. The kit of claim 13, wherein the negative control and the positive control have the same chromophore, have the same maximum UV absorption wavelength, and exhibit substantially the same extinction coefficient at said wavelength.

15. The kit of claim 13, wherein the positive control is adenosine-5'-triphosphate (ATP) or a chemical analog thereof, and the negative control is adenosine or caffeine.

16. The kit of claim 13, wherein the positive control is adenosine 5'-(α,β-methylene) diphosphate (AMPcP) or a chemical analog thereof, and the negative control is adenosine or caffeine.

17. The kit according to claim 13, wherein the positive control is a nucleotide diphosphate having a non-hydrolyzable α-β bond.

18. The kit of claim 13, wherein the negative control consists of or consists essentially of adenosine.

19. The kit of claim 13, wherein the negative control consists of or consists essentially of caffeine.

20. The kit of claim 13, wherein the negative control and the positive control have different spectral characteristics.

21. The kit of claim 13, wherein the system suitability solution comprises an equimolar mixture of the negative control and the positive control.

22. The kit according to claim 13, wherein the positive control is vinylidene adenosine 5'-(α,β-methylene) diphosphate having the following structure:

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