Techniques for system suitability testing of inert liquid chromatography systems and columns

Abstract

The present disclosure relates to methods of characterizing a system comprising a chromatography column. The methods can comprise introducing a sample comprising a positive control and a negative control into a system comprising a chromatography column, wherein the positive control is a sensitive probe that interacts with the system and the negative control does not substantially interact with the system; detecting the positive control and the negative control after passing the sample through the chromatography column; and determining system suitability by comparing the amount of positive control detected to the negative control. In some embodiments, determining system suitability (e.g., sample inertness to the system) is accomplished by determining the ratio of positive control to negative control detected.

Description

[0001] Cross-references to related applications

[0002] This patent application claims priority and the benefit of U.S. Provisional Application 63 / 079,091, filed September 16, 2020, entitled "Techniques for System Suitability Testing of Inert LC Systems and Columns". This patent application also claims priority and the benefit of U.S. Provisional Application 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

[0003] This disclosure relates to techniques for system suitability testing. More specifically, this technique relates to techniques for evaluating whether a system (such as an inert liquid chromatography system) is operating properly. Background Technology

[0004] 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

[0005] Generally, the present invention relates to methods for characterizing systems (such as pressurized flow systems, e.g., liquid chromatography systems). In embodiments, the method involves determining the suitability of the system. That is, in some embodiments, the method involves confirming the suitability of the system for a specific application, such as the chromatographic separation of metal-sensitive analytes. Many applications of liquid chromatography target the measurement of compounds known to interact with exposed metal surfaces. As a result of such interactions, many compounds are known to exhibit poor chromatographic properties (retention, peak shape), recovery (peak area), or interactions so strong that these compounds cannot be fully measured. Furthermore, existing methods for mitigating these interactions may have varying effectiveness or performance over time. The present invention allows analysts to confirm the suitability of a system for such applications, thereby avoiding the allocation of resources (i.e., time, cost) to experiments destined to produce poor quality results.

[0006] In one aspect, the present invention relates to a method for characterizing a system comprising a chromatographic column. The method according to this aspect includes introducing a sample comprising a positive control and a negative control into the system comprising the chromatographic column, wherein the positive control is a sensitive probe that interacts with the system, while the negative control substantially does not interact with the system. The method further includes (after passing the sample through the chromatographic column) detecting the amounts of the positive and negative controls; and determining system suitability by comparing the detected amounts of the positive control with the detected amounts of the negative control.

[0007] Implementations of the above aspects of this technology may include one or more of the following features. In some embodiments, the step of determining system suitability by comparing the amount of detected positive control with that of negative control includes determining the ratio of detected positive control to negative control. In some embodiments, the inertness of the system is determined by the amount of detected positive control. In some embodiments, the step of determining system suitability by comparing the amount of detected positive control with that of negative control indicates the integrity of the inert coating deposited along the flow path of the system. In some embodiments, the positive control and negative control are structurally similar. In some embodiments, the positive control comprises a metal-sensitive or metal-chelating molecule. In some embodiments, the step of determining system suitability by comparing the amount of detected positive control with that of negative control includes detection using a configuration of liquid chromatography-optics, liquid chromatography-mass spectrometry, or liquid chromatography-optics-mass spectrometry, wherein the optics is a UV / Vis absorption or fluorescence detector. In some embodiments, detecting the positive control and negative control includes splitting the sample stream. In some embodiments, the method further includes employing more than one chromatographic method to evaluate the system by introducing a sample containing a plurality of compounds, wherein the plurality of compounds includes a mixture of negative control, positive control, and resolved neutral substances. The system can be evaluated simultaneously using more than one chromatographic method. In some embodiments, the peak area and peak height of the positive and negative controls have a standard deviation of less than 3.0%. In some embodiments, the peak shape of the negative and positive controls has an asymmetry of less than 3.0% (10%). In some embodiments, the method is characterized by determining system suitability at previously determined maintenance steps of the system (e.g., previously determined replacement steps of the column or syringe in use). In some embodiments, maintenance steps are scheduled after a previously determined number of uses or a previously determined amount of time. In some embodiments, the system includes a liquid chromatography column, a syringe, and a detector. In some embodiments, detecting the positive and negative controls includes detecting a value associated with the positive control (i.e., a first value) and a value associated with the negative control (i.e., a second value). The value associated with the positive control (the first value) can be a value of at least one of peak area, peak height, peak width, or peak symmetry. In some embodiments, the value associated with the positive control is a peak area value and the relative standard deviation of peak area reproducibility is less than 3%. In some embodiments, the value associated with the positive control is the peak height, and the relative standard deviation of peak height reproducibility is less than 2.5%. In some embodiments, the value associated with the positive control includes the peak shape, and the symmetry of 10% of the peak shapes is less than 3.0.

[0008] In another aspect, the present invention relates to a method for characterizing a system comprising a chromatographic column. This method includes: introducing a sample into a fluid system comprising a flow path disposed within the fluid system, the flow path including a surface, wherein the surface is substantially inert to at least one analyte in the sample, wherein the sample comprises a negative control and a positive control; and determining the inertness of the system by detecting the ratio of the positive control to the negative control.

[0009] Implementations of the above aspects of this technology may include one or more of the following features. In some embodiments, in addition to positive and negative controls, the sample also contains multiple compounds. At least one of the multiple compounds is a metal-insensitive compound, and the performance properties of the chromatographic column are measured. In some embodiments, the positive and negative controls are structurally similar. In some embodiments, the flow path comprises a chromatographic column in a straight line between the sample syringe and the detector.

[0010] The foregoing aspects and features of this disclosure offer numerous advantages over the prior art. For example, this disclosure characterizes inert LC systems by evaluating the appropriate operation of inert surfaces, particularly when analyzing compounds known to exhibit high metal-metal interactions. This information prevents a waste of time and resources, as knowledge of the system's suitability for a particular application eliminates or significantly reduces experiments destined to yield poor results. Attached Figure Description

[0011] The present technology will be more fully understood through the following detailed description taken in conjunction with the accompanying drawings, in which:

[0012] Figure 1 This is a schematic diagram of a chromatographic system including a chromatographic column and various other components according to an exemplary embodiment of this disclosure.

[0013] Figure 2 This is a flowchart illustrating a method for evaluating the suitability of a system according to an exemplary embodiment of the present disclosure.

[0014] Figure 3 This is a representative chromatographic example of an exemplary embodiment of the present disclosure, which demonstrates the chromatographic testing of inert surfaces using metal-sensitive compounds and inert analogues.

[0015] Figure 4A The chromatographic assays using ATP (positive control) and adenosine (negative control) are shown, employing a system with components having coated flow paths.

[0016] Figure 4B The chromatographic assays utilizing ATP and adenosine are shown, employing a system with components having an uncoated flow path. Detailed Implementation

[0017] Generally speaking, this disclosure relates to a characterization method for inert LC systems used to evaluate the proper operation of inert surfaces, particularly when analyzing compounds known to exhibit high metal-metal interactions.

[0018] Tests can be performed to assess the health of the system, including as part of maintenance procedures. 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, such as the column. Evaluating only a part of the system can be used for system troubleshooting.

[0019] 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 combine chromatographic methods to simultaneously evaluate gradient delivery and exposure of metal surfaces.

[0020] 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.

[0021] 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., which is incorporated herein by reference in its entirety. LC systems based on such techniques are generally described as inert LCs.

[0022] With advancements in improving system inertia and expanding instrument options, new techniques are needed to help confirm system readiness for intended applications and to help determine overall system inertia. Few tests are designed for this purpose, requiring the detection and measurement of interactions that could adversely affect the analysis of metal-sensitive analytes. System suitability testing can be performed in pharmaceutical / biopharmaceutical industries and government laboratories to provide confidence in the accuracy of analytical measurements for a particular method. System readiness is one aspect of system suitability testing, confirming the proper functioning of the components that make up the system, including solvent delivery modules, sample delivery modules, any required column heating / cooling modules, and the module ultimately used for detection. Besides ensuring proper system operation, system suitability is also a validation of method performance. Such validation is an important aspect of generating reliable data.

[0023] Many methods, individually or in combination, can be used to address the presence of secondary interactions and their impact on the quality of a given chromatographic separation. It is necessary to evaluate whether the coating provides an inert system for the treated sample. Therefore, characterization methods for inert LC systems are essential for assessing the proper operation of inert surfaces, especially when analyzing compounds known to exhibit high metal-metal interactions. This article describes techniques for system suitability testing of inert LC systems (e.g., chromatographic systems / devices). First, regarding… Figure 1 Discuss the system / device and the coating that inerts the system / device.

[0024] Figure 1 This is a representative schematic diagram of a chromatography system / apparatus 100 for separating analytes such as peptide compounds from a sample. System 100 includes several components, including: a fluid manager system 105 (e.g., controlling flow through the mobile phase of 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. Column 125 may be a reversed-phase column. The inner surfaces of the components of chromatography system / apparatus 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.

[0025] 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.

[0026] In some examples, the coating of the flow path is non-binding to analytes such as metal-sensitive compounds (e.g., peptides). Therefore, analytes such as peptide compounds do not bind to the coating of the flow path.

[0027] 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 fused block 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 vial 130 containing the sample and the fused block 120 can be coated.

[0028] In some examples, System 100 will need to be cleaned / trimmed before the evaluation begins in order to establish a baseline for suitability determination 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 2 Method 200.

[0029] 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 as being defined at least partially by the inner surface of the microfabrication 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 extending from the inner surface of the sample injection needle throughout the inner surface of 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 throughout the fluid system to the port of the connector / detector.

[0030] 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, molten material and / or membranes within a solid-phase extraction device come into contact with a fluid sample. Therefore, not only the inner walls of 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 fluids, especially fluids containing the analyte of interest.

[0031] In an embodiment characterized by an inert coating along or on the wetted surface, at least a portion of the wetted surface is coated with an alkylsilane coating. The alkylsilane coating is inert to at least one of the analytes in the sample.

[0032] In some embodiments, the alkylsilyl coating is an organic silica coating. In some embodiments, the alkylsilyl coating is a hybrid inorganic / organic material that forms or coats a wetting surface.

[0033] The coating (e.g., an alkylsilyl coating) may have a contact angle with water of at least about 15°. In some embodiments, the coating may have a contact angle of less than or equal to 30°. The contact angle may 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 it is between about 15° and about 105°, and in some embodiments it is between about 15° and about 115°. For example, the contact angle of the coating may 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°.

[0034] The thickness of the coating (e.g., an alkylsilyl coating) can be at least approximately For example, the thickness can be between approximately to approximately Between. The coating thickness can be approximately

[0035] or The thickness of a coating (e.g., a vapor-deposited alkylsilane coating) can be optically inspected by the naked eye. For example, higher opacity and tinting indicate a thicker coating. When a coated part is observed under full-spectrum light (such as sunlight), the color changes from yellow to purple, then to blue, then to a slightly greenish hue, and finally back to yellow as the coating thickens. For example, when the thickness of an alkylsilane 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... At this time, the coating can exhibit a blue color and reflect light with peak wavelengths 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 and Erik, Surface and Porous Structure of Pigment Coatings, Interactions with flexographic ink and effects of print quality, Dissertation, Karlstad University Studies, 2013:49.

[0036] The coating can 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 comprises a siloxane moiety imparted by the vapor-deposited reagent.

[0037] 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).

[0038] The chromatographic apparatus may have multiple alkylsilyl coatings. For example, a second alkylsilyl coating may be in direct contact with a first alkylsilyl coating.

[0039] 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.

[0040] Flow path components can be made of titanium, such as Made of alloys, stainless steel, or other metals (purchased from Best Stainless & Alloys, Houston, TX). Flow path components include, but are not limited to, tubing, microfabrication fluid conduits, column slabs, column inlet tubing, and sample injection needles. Flow path components may be coated by vapor deposition using one or more of the disclosed alkylsilanes. In some examples, the coated components are annealed to alter their chemical or physical properties.

[0041] For flow path components made of materials other than stainless steel or other metals, the flow path components can be coated by vapor deposition using one or more of the disclosed coatings. In particular, sample vials that can be connected to injection needles can be coated.

[0042] 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.

[0043] 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 No. 14 / 680,669, filed April 7, 2015, entitled “Thermal Chemical Vapor Deposition Coated Article and Process,” which claims priority and benefit to U.S. Provisional Application No. 61 / 976,789, filed April 8, 2014. The full contents of each patent application are incorporated herein by reference.

[0044] In one aspect, alkylsilyl coatings enhance the corrosion resistance of metals, for example, in metal chromatography columns. Depending on their density and thickness, the coating acts as a barrier, preventing water and corrosive molecules from reacting with the base metal. Increased hydrophobicity and density improve corrosion resistance.

[0045] In some embodiments, alkylsilane coatings are modified with a silanizing agent to obtain desired surface properties. The silanizing agent can be a non-volatile zwitterion. The non-volatile zwitterion can be sulfobetaine or carboxybetaine. In some embodiments, the silanizing agent is an acidic or basic silane. The silanizing agent can incorporate a polyethylene oxide moiety.

[0046] Other components of the LC system may also include the coatings described herein, such as tubing, frits, and / or connectors. The LC system can be used to separate metal-sensitive analytes, such as biomolecules, proteins, glycans, peptides, oligonucleotides, pesticides, bisphosphonates, anionic metabolites, and zwitterions (such as amino acids and neurotransmitters). This LC system includes multiple coated components to minimize or eliminate the presence of metals on wetted surfaces along the fluid flow path.

[0047] Coating provides a method for making a system (such as an LC system and a column) suitable for use. Another possible alternative to coating is to passivate the system using a liquid solvent (such as, for example, nitric acid). The system can also be inertized 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 inertizing the system, it is then necessary to test the system to determine its suitability. This article describes techniques for system suitability testing, such as inert LC systems and columns.

[0048] Figure 2This is a flowchart illustrating a method 200 for evaluating system suitability according to an exemplary embodiment of this disclosure. The method has certain optional steps, as indicated by the dashed outlines around specific steps. Method 200 may begin with components of isolated system 202. 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 column) 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.

[0049] As noted, components of isolation system 202 are optional. In some examples, it will be necessary to evaluate the suitability of the entire system / device. A suitability sample can flow 204 through the system, such as system / device 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.

[0050] During or after the application sample flow 204, method 200 includes an evaluation system 206. 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.

[0051] 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.

[0052] One technique used to evaluate 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 share a chromophore and therefore a 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 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)bisphosphate (AMPcP) is another pairing of adenosine (Log P -4.8 vs -1.05, respectively). In some examples, AMPcP (positive probe) is paired with caffeine (negative probe, which does not interact with metals).

[0053] In some examples, when using metal-sensitive compounds to detect exposed metals in the flow path, a decreased analyte response can be expected because some amount of analyte binds to the metal surface. With subsequent injections, the active binding sites may become saturated, and the total analyte response may begin to increase. This process is reversible and exists at some equilibrium. To better control the detection of exposed metals, it may be important to employ a rinsing step to remove any previously bound sample material that may mask the metal surface and introduce undesirable variability. In some examples, the rinsing step may apply a solution of 0.3% ammonia prepared from a 1:100 dilution of 30% ammonium hydroxide with water for six repeated injections.

[0054] Metal adsorption can be dependent on mobile phase conditions. High pH lowers the surface potential of metals, which may be one reason for using ammonia solutions to prepare the flow path for testing. Analyte adsorption to metals can be primarily caused by electrostatic interactions, which also means that mobile phases with different compositions can be used to desorb samples and / or mitigate secondary interactions in a particular method. Therefore, an unbuffered water / acetonitrile mobile phase used for flow injection testing can be used to exacerbate metal binding and the magnitude of observed test results.

[0055] Figure 3 This is a representative chromatographic example according to an exemplary embodiment of the present disclosure, demonstrating the chromatographic testing of inert surfaces using metal-sensitive compounds and inert analogues. Specifically, Figure 3Representative chromatographic examples are shown, demonstrating the ability to easily separate ATP and adenosine by reversed-phase chromatography. Comparisons of peak area ratio, peak area reproducibility, peak shape, and peak shape reproducibility allow for 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 shown in Example 1 and... Figure 3 As shown, the relative areas between ATP and adenosine can be used to assess the degree of metal interaction in the system, while taking into account changes in non-secondary interactions (e.g., injection volume). Furthermore, the assessment of the peak shape of the ATP compound can be used to characterize the system's suitability.

[0056] In some examples, methods for characterizing a system include introducing samples with positive and negative controls (e.g., suitability samples) into the system. The positive control is a sensitive probe that interacts with the system, while the negative control substantially does not interact with the system. In some examples, a compound that substantially does not interact with the system may be defined as a compound lacking a metal-interacting moiety or a molecule lacking one or more strongly electronegative moieties (such as acidic residues with a pKa below 5). The method may also include detecting the positive and negative controls and determining system suitability by comparing the amount of the detected positive control with that of the negative control.

[0057] Determining system suitability by comparing the amount of detected positive control with that of negative control may include determining the ratio of detected positive control to negative control. If the ratio of positive control to negative control is sufficiently high, the system is considered to have good system health and is suitable for analysis. In some examples, sufficiently high is greater than 0.5, 0.6, 0.7, 0.8, 0.9, or less than 1.1, where a ratio of 1.0 indicates no loss of interaction between the analyte and the system flow path. A ratio considered sufficiently high can vary based on the selected negative and positive controls. For example, a positive control that strongly interacts with the exposed metal (e.g., ATP or AMPcP) may have a lower limit in the ratio range of 0.75 or 0.8.

[0058] In some examples, the analyst may adjust the system to target a ratio of 1.0, and the analyst may determine a lower range of acceptable ratio values ​​based on the analyst / system's needs. Determining system suitability by comparing the amount of detected positive control with that of negative control includes determining the system's inertness to positive control and / or indicating the integrity of the inert coating deposited along the system's flow path.

[0059] Determining system suitability may involve testing a component of the system, such as a chromatographic column. 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.

[0060] Positive and negative controls can be detected using liquid chromatography-optics, liquid chromatography-mass spectrometry, or liquid chromatography-optics-mass spectrometry configurations, where optical refers to UV / Vis absorbance or fluorescence detectors. The flow of suitability samples can also be split.

[0061] 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. The selection is based at least in part on differences in hydrophobicity, which can result in a set of compounds that will be well-disaggregated and allow for evaluation of various performance aspects, including gradient delivery, flow rate accuracy, and column efficiency and health. The method may include evaluating gradient delivery and exposed metal surfaces in a single injection.

[0062] 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.

[0063] The techniques described above for evaluating the inertness of LC systems require the use of an inert chromatographic column. The suitability tests described above analyzed the results of a single injection of a suitability sample.

[0064] Comparison of peak area, peak shape, and any changes in these metrics during repeated injections also allows analysts to characterize the flow path surface of the liquid chromatography to mitigate metal interactions. In some examples, only a positive control probe is used, and a series of sequential injections are performed using a column aligned in a straight line between the autosampler and detector. If minimal changes in recovery, peak area, peak height, peak width, and / or peak symmetry are observed across repeated injections, the system is deemed suitable for analysis.

[0065] 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.

[0066] Table 1 shows the minimum specifications for the chromatographic suitability test using ATP (positive control) and adenosine (negative control). The minimum specifications depend on the instrument platform. For example, different variants of ultra-high performance liquid chromatography (UHPLC) with different flow path sections (composed of tubing of different materials and lengths) and platforms with different coupled detectors or configurations (LC-optical, LC-MS, LC-optical-MS with or without split flow, where optical refers to UV / Vis absorbance or fluorescence detectors) can provide different results. The underlying reason involves the amount of exposed metal introduced into the flow path from sample injection to detection. As the exposed metal increases, there is a corresponding change in the resulting measurements using the test probe.

[0067] Table 1: Minimum Specifications for System Inertia

[0068] Inert system specifications (*RSD = Relative Standard Deviation) measure Chromatographic testing Peak area reproducibility <2.4%RSD* Peak height reproducibility <1.8%RSD* Peak shape 10% asymmetry <1.9 Peak area ratio ATP / adenosine > 0.8

[0069] As described herein, the minimum specification depends on the instrument platform and includes sample components such as positive controls (e.g., ATP vs. AMPcP) and negative controls (e.g., adenosine or caffeine). In some examples, the peak area reproducibility and peak height reproducibility of the inert system specification may have RSDs of less than about 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or any intermediate number. In some examples, the peak shape may have an asymmetry of less than about 5, 4, 3, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or any intermediate number in the range of 10%. Depending on the sample components of the positive and negative controls (e.g., ATP and adenosine), the peak area ratio can be greater than approximately 5, 4, 3, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or any intermediate number.

[0070] Given that adenosine and ATP are readily measurable by UV absorbance and mass spectrometry, the aforementioned evaluation methods are well-suited for LC-optical and LC-MS. Typically, a form of detection, either separable or selective, is required to compare the recoveries of positive and negative controls in a single run. One form of selective detection using optical methods can be achieved by using positive and negative control molecules with unique spectral properties. For example, a positive control molecule might have a unique molar absorptivity and a maximum wavelength at 280 nm, while a negative control molecule might have a UV absorbance profile with a 40 nm redshift.

[0071] Figure 4A and Figure 4B The chromatographic assays using adenosine-5'-triphosphate (ATP) and adenosine are shown. Figure 4A This demonstrates a chromatographic assay utilizing ATP and adenosine, employing a system with a component having a coated flow path. (Compared to...) Figure 4A on the contrary, Figure 4B The chromatographic assays utilizing ATP and adenosine are shown, employing a system with components having uncoated flow paths. Figure 4A and Figure 4BThe measurement results include the relative standard deviation (RSD) of area and height, as well as the 10% reduction in asymmetry and peak area ratio. Table 2 lists the measurement results that can be used to assess system inertia. Figure 4A The peak area ratio (0.86) is higher than that of the peak area ratio ... Figure 4B The peak area ratio (0.75) is closer to 1. Therefore, by comparison... Figure 4A The coating flow path relative to Figure 4B The peak area ratio of biological flow pathways Figure 4A The system was evaluated as being better than Figure 4B The system is more inert.

[0072] Table 2: Chromatographic Test Measurement Results

[0073]

[0074] Example

[0075] 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. Example 1 used UV detection.

[0076] Example 1

[0077] Reverse-phase LC-UV for the detection of adenosine-5'-triphosphate (ATP) and adenosine

[0078] Example 1 is a technique for evaluating surface metal exposure, involving the separation and measurement of ATP / adenosine via a chromatographic column. Adenosine is used as a negative control, where the effects of nonmetallic interactions on separation and recovery will be observed. For example, insufficient syringe delivery volume will result in a reduction in the area of ​​both metal-sensitive ATP and the adenosine control compound.

[0079] As shown in Example 1 and Figure 3 As shown, the relative areas between ATP and adenosine can be used to assess the degree of metal interaction in the system, while taking into account changes in non-secondary interactions (e.g., injection volume). Furthermore, the assessment of the peak shape of the ATP compound can be used to characterize the system's suitability.

[0080] Test samples (e.g., suitability samples) consisting of 20 ng / μL ATP and 9.5 ng / μL adenosine in water are 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 test samples. 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.

[0081] Separation details are shown in Table 3. Peak area, reproducibility, and peak shape of ATP and adenosine can be assessed after at least five injections of the test sample. Effective metal-metal interaction attenuation is indicated by comparable peak areas of both compounds, comparable and low peak area variability, and comparable and low peak tailing.

[0082] Table 3: Separation details of Example 1

[0083]

[0084]

[0085] Example 2

[0086] Reversed-phase LC-UV for the detection of adenosine 5'-(α,β-methylene)bisphosphate (AMPcP) and adenosine

[0087] Example 2 is a technique for evaluating surface metal exposure, which involves using a chromatographic column to separate and measure AMPcP and adenosine. Adenosine is used as a negative control, where the effects of nonmetallic interactions on separation and recovery will be observed. For example, insufficient syringe delivery volume will result in a reduction in the area of ​​both metal-sensitive AMPcP and the adenosine control compound.

[0088] Test samples consisting of 17 ng / μL AMPcP and 10.7 ng / μL adenosine in water were prepared from a 1 mg / mL stock solution of AMPcP and adenosine in water. These solutions were then diluted into individual sample vials to produce test samples. The ammonium acetate mobile phase was prepared fresh daily because ammonium acetate buffer salts are known to be volatile.

[0089] Separation details are shown in Table 4. Peak area, reproducibility, and peak shape of AMPcP and adenosine were evaluated after at least five injections of the test sample. Effective metal-metal interaction attenuation was indicated by comparable peak areas of both compounds, comparable and low peak area variability, and comparable and low peak tailing.

[0090] Table 4: Separation details of Example 2

[0091]

[0092]

[0093] The foregoing aspects and features of this disclosure offer numerous advantages over the prior art. In some embodiments, the methods for evaluating system suitability have many beneficial effects. For example, this disclosure characterizes inert LC systems by evaluating the appropriate operation of inert surfaces, particularly when analyzing compounds known to exhibit high metal-metal interactions. These embodiments and features are combinable.

Claims

1. A method for characterizing a liquid chromatography system comprising a chromatographic column, the method comprising: A sample containing a positive control and a negative control is introduced into a fluid system of a liquid chromatography system containing a chromatographic column, the fluid system including a flow path disposed inside the fluid system, the flow path including a surface, wherein the positive control is a sensitive probe having a secondary interaction with the surface of the flow path, and the negative control is a molecule that does not contain a metal-interacting part or does not contain one or more strongly electronegative parts. After the sample is passed through the chromatographic column, the positive control and the negative control are detected. as well as System suitability is determined by comparing the amount of detected positive controls with that of negative controls.

2. The method of claim 1, wherein determining system suitability by comparing the amount of detected positive control with that of negative control includes determining the ratio of detected positive control to negative control.

3. The method of claim 1, wherein determining system suitability by comparing the amount of detected positive control with that of negative control includes determining the inertia of the system to the positive control.

4. The method of claim 1, wherein the system suitability is determined by comparing the amount of detected positive control with that of negative control to indicate the integrity of the inert coating deposited along the flow path of the system.

5. The method according to claim 1, wherein the positive control and the negative control have the same parent structure.

6. The method of claim 1, wherein the positive control comprises a metal-sensitive or metal-chelating molecule.

7. The method of claim 1, wherein detecting the positive control and the negative control comprises using a configuration of liquid chromatography-optics, liquid chromatography-mass spectrometry, or liquid chromatography-optics-mass spectrometry, wherein the optics is a UV / Vis absorbance or fluorescence detector.

8. The method of claim 7, wherein detecting the positive control and the negative control comprises shunting the sample stream.

9. The method of claim 1, further comprising employing more than one chromatographic method to evaluate the system by introducing a sample having a plurality of compounds, wherein the plurality of compounds includes the negative control, the positive control, and a mixture of resolved neutral substances.

10. The method of claim 9, wherein the evaluation of the system using more than one chromatographic method is performed simultaneously.

11. The method according to claim 1, wherein the peak area and peak height of the positive control and the negative control have a relative standard deviation of less than 3.0%.

12. The method of claim 1, wherein the peak shapes of the negative control and the positive control have an asymmetry of less than 10% of 3.

0.

13. The method of claim 1, wherein determining system suitability occurs at a previously determined system maintenance step.

14. The method of claim 13, wherein the previously determined maintenance steps are scheduled after a previously determined number of uses or a previously determined amount of time.

15. The method of claim 1, wherein the system comprises a liquid chromatography column, a syringe, and a detector.

16. The method of claim 1, wherein detecting the positive control and the negative control comprises detecting values ​​associated with the positive control and values ​​associated with the negative control.

17. The method of claim 16, wherein the value associated with the positive control includes at least one of peak area, peak height, peak width, or peak symmetry.

18. The method of claim 16, wherein the values ​​associated with the positive control include peak area values, and the relative standard deviation of peak area reproducibility is less than 3%.

19. The method of claim 16, wherein the values ​​associated with the positive control include peak height values, and the relative standard deviation of peak height reproducibility is less than 2.5%.

20. The method of claim 16, wherein the values ​​associated with the positive control include peak shape values, and 10% of the peak shape values ​​have a symmetry of <3.

0.

21. The method of claim 1, wherein, in addition to the positive control and the negative control, the sample further comprises a plurality of compounds, wherein at least one of the plurality of compounds is a metal-insensitive compound, and the performance properties of the chromatographic column are measured.

22. The method of claim 15, wherein the flow path comprises a chromatographic column in a straight line between the syringe and the detector.

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