Analysis systems and methods

Abstract

Disclosed herein is an analytical system (100, 100') including a mass spectrometer (60) and an ionization source (61) coupled to the mass spectrometer (60). The analytical system (100, 100') further comprises an analytical fluid system (10, 10') connectable to the ionization source (61) via a downstream valve (20) for injecting a sample into the mass spectrometer (60) via the ionization source (61), and a downstream pump (40) fluidly connectable to the ionization source (61) via the downstream valve (20), the downstream pump (40) being fluidly connected to a plurality of fluid containers containing respective fluids (41, 42, 43, 44), the fluids including at least one concentrated composition (44) for calibrating the mass spectrometer (60) and at least one diluent (42, 43) for diluting the at least one concentrated composition (44). The analytical system (100, 100') further comprises a controller (90) configured to control the downstream pump (40) to obtain at least one diluted composition (45) by automatically mixing the at least one concentrated composition (44) with at least one diluent (42, 43) at a predetermined dilution ratio, to inject the at least one diluted composition (45) into the ionization source (61), to acquire a mass spectrum (62) of the at least one diluted composition (45), and to perform a calibration (63) of the mass spectrometer (60) based on an evaluation (64) of the mass spectrum (62). Respective automated analytical methods including calibration of the mass spectrometer (60), such as a mass axis check and / or adjustment, with a selected chemical composition are also disclosed herein.

Description

[Technical field]

[0001] The present disclosure relates to automated analytical systems and methods that include calibration of mass spectrometers, such as mass axis checking and / or adjustment, with selected chemical compositions. [Background technology]

[0002] In mass spectrometry (MS), proper acquisition of a mass spectrum requires instrument adjustments to ensure good sensitivity and peak shape, and to ensure that the mass resolution is appropriate for the analytical requirements of the sample. Adjustments are followed by mass calibration, which involves adjusting the m / z scale of the instrument, typically by measuring a sample that produces a peak at a known m / z value, and adjusting the m / z axis, if necessary, to reflect the expected value. In addition, a background spectrum is typically measured before analyzing a sample to check for contaminants that may be present in the instrument.

[0003] Typically, especially in a high throughput analytical setting, mass axis checks are performed periodically to ensure accurate ion identification and mass axis adjustments are performed as needed or as part of routine work.

[0004] Calibration of mass spectrometers is described, for example, in US Patent Publication No. 2018 / 0047549. Mass axis check and / or adjustment compositions are also known in the art. For example, WO 2012 / 135682 proposes polyethylene compounds and mixtures thereof, and Zhou et al. (2012, Anal Chem 84:6016) used cesium iodide for calibration. In addition, commercially available calibration solutions are available.

[0005] Performing a Mass Axis Check (MAC) or Mass Axis Adjustment (MAA) typically requires intensive interaction and several manual steps by an experienced / qualified user, which is inconvenient and inappropriate for clinical use of mass spectrometers. Such manual steps may include ensuring the use of the correct calibration solution, manual preparation of the solutions including handling volatile and toxic solvents such as methanol, hardware modifications, e.g., to inject the calibration solution into the mass spectrometer, manual step-by-step initiation and execution of the procedure including evaluation and final adjustments that entail risks of error, injury, contamination and system damage.

[0006] When the mass spectrometer is coupled to an analytical fluidic system, such as a liquid chromatography (LC) system, the fluidic system also typically requires frequent maintenance and quality control procedures.

[0007] In particular, HPLC columns can undergo continuous degradation of their performance as the number of sample injections increases, resulting in, for example, increased background, shifts in retention times, reduced column lifetime, increased costs, and the need for frequent maintenance, primarily due to particle accumulation at the column head.

[0008] Backwashing an HPLC column can provide higher sample throughput, longer uptime, less maintenance, lower cost, higher data quality, and longer-lasting calibrations. This is typically done by manually disconnecting the column from the flow system and reconnecting the outlet side of the column to the flow inlet, reversing the flow direction through the column, while the flow exiting the column is typically directed to waste so as not to contaminate the detector. This manual procedure can take 15-20 minutes or more for each column before the column is reconnected in its original orientation with the correct flow direction, followed by re-equilibration and quality control.

[0009] This manual procedure is also laborious and time consuming and may require the intervention of outside service personnel or skilled laboratory personnel, during which time the analyzer or its components may be unavailable for use, in addition to incurring additional costs and introducing the risk of errors and malfunctions and even damage to the system.

[0010] Furthermore, for high-throughput applications requiring a large number of successive sample injection cycles, especially for random-access analysis of different samples, which may require different injection conditions and different LC separation conditions, high flexibility and speed in rapidly changing and adapting conditions for each sample in a scheduled sequence can be important.

[0011] In particular, the solvents / eluents used in LC separations may need to be frequently exchanged within the same fluid stream and between parallel fluid streams. Due to the nature of pumping, internal dead volumes, and fluid and surface effects, liquid exchange is a process of repeatedly diluting previously used liquid with newly used liquid, which may take a long time for equilibration and signal stabilization. This process may therefore present a bottleneck when the goal is to achieve high-throughput random-access sample processing, especially when the goal is to maintain a constant pace of sample injection and analysis, regardless of sample sequence and separation / analysis conditions. Summary of the Invention

[0012] Against the above background, embodiments of the present disclosure provide certain unobvious advantages and advancements over the prior art. In particular, the inventors have recognized a need for improved automated analytical systems that include mass spectrometers and methods that include calibration of mass spectrometers.

[0013] While embodiments of the present disclosure are not limited to any particular advantage or functionality, it should be noted that the present disclosure provides an analytical system including a mass spectrometer and an automated analytical method including the use of a mass spectrometer. This allows, among other functions, automatic calibration of the mass spectrometer, thereby ensuring continuous analytical performance of the system without manual intervention, thus increasing user convenience. Another advantage is that the risk of errors, malfunctions and system damage is eliminated while system downtime is minimized. Also, the risk of injury or health hazards is minimized. This is primarily achieved by using a downstream pump coupled to a mixing unit, which includes at least the functionality of diluting a concentrated calibration solution (composition) as needed and injecting the diluted composition into the mass spectrometer while bypassing any analytical fluidic system. Incorporating this functionality into the analytical system allows for the utilization of one or more standardized and optimized compositions in concentrated form, which the analytical system can automatically select and / or combine as needed, for example, based on the specific m / z range of a given mass spectrometer, based on the ionization mode, based on the sensitivity of the mass spectrometer, etc. The use of commercially available prepared concentrated compositions, in addition to eliminating the inconvenience of manual preparation and the above-mentioned risks, allows a wider selection of compositions to be carried (taking up less space) and also allows the use of less solvents in the compositions, which are typically volatile, flammable and of health and environmental concerns, thus reducing the risks of preparation, storage and transport as well as handling of such solutions. In particular, since such solvents are typically used anyway for analytical reasons in applications involving the use of mass spectrometry and / or liquid chromatography or other chromatographic techniques coupled with mass spectrometry, and therefore also present in the analytical system, it is convenient to use them also to dilute the concentrated calibration composition when and in the amount required. Moreover, according to one embodiment, it is possible to flexibly, dynamically and automatically adapt the calibration conditions in case of calibration failure or expected failure, for example by adapting the dilution factor.

[0014] In particular, the analytical system includes a mass spectrometer, an ionization source coupled to the mass spectrometer, an analytical fluid system connectable to the ionization source via a downstream valve for injecting a sample into the mass spectrometer through the ionization source, and a downstream pump fluidly connectable to the ionization source via the downstream valve, the downstream pump being fluidly connected to a plurality of fluid containers containing respective fluids, the fluids including at least one concentrated composition for calibrating the mass spectrometer and at least one diluent for diluting the at least one concentrated composition, and a controller configured to control the downstream pump to obtain at least one diluted composition by automatically mixing the at least one concentrated composition with the at least one diluent, inject the at least one diluted composition into the ionization source, obtain a mass spectrum of the at least one diluted composition, and perform calibration of the mass spectrometer based on evaluation of the mass spectrum.

[0015] An "analytical system" is an automated laboratory device dedicated to sample analysis, possibly for in vitro diagnostics. An analytical system may have different configurations as needed and / or according to a desired laboratory workflow. Additional configurations may be obtained by coupling several devices and / or modules together. A "module" is a work cell, typically smaller in size than the entire analytical system, with a dedicated function. This function may be analytical, but also pre-analytical or post-analytical, or it may be a supplementary function to either the pre-analytical, analytical or post-analytical functions. In particular, a module may be configured to cooperate with one or more other modules for performing a dedicated task of a sample processing workflow, for example by performing one or more pre-analytical and / or analytical and / or post-analytical steps. Thus, an analytical system may include either one analytical device, or any combination of such analytical devices with their respective workflows, and the pre-analytical and / or post-analytical modules may be coupled to individual analytical devices or shared by several analytical devices. Alternatively, the pre-analytical and / or post-analytical functions may be performed by units integrated into the analytical device. An analytical system may include functional units such as liquid handling units for pipetting and / or pumping and / or mixing of samples and / or reagents and / or system fluids, as well as functional units for sorting, storing, transporting, identifying, separating and detecting analytes in samples. In particular, an analytical system may include an analytical fluidic system or module, a mass spectrometer (MS) system or module, and an ionization source (IS) system or module as an interface between the analytical fluidic system and the mass spectrometer, which are distinguishable as individual replaceable units coupled to each other or at least partially integrated in a common system housing.

[0016] A "mass spectrometer (MS)" is an analytical module that includes a mass analyzer designed to further separate and / or detect analytes based on their mass-to-charge ratio. According to one embodiment, the mass spectrometer is a rapid scanning mass spectrometer. According to one embodiment, the mass spectrometer is a tandem mass spectrometer that can select parent molecular ions, generate fragments by collision-induced fragmentation, and separate the fragments or daughter ions according to their mass-to-charge (m / z) ratios. According to one embodiment, the mass spectrometer is a single or triple quadrupole mass spectrometer, as known in the art. In addition to quadrupoles, other types of mass analyzers may be used as well, including time-of-flight, ion trap, or combinations thereof.

[0017] An "ionization source (IS)" is an interface that couples an analytical fluidic system to an MS and is configured to generate charged analyte molecules (molecular ions) and convert the charged analyte molecules from the liquid to the gas phase. According to certain embodiments, the ionization source is an electrospray ionization (ESI) source, or a heated electrospray ionization (HESI) source, or an atmospheric pressure chemical ionization (APCI) source, or an atmospheric pressure photoionization (APPI) source, or an atmospheric pressure laser ionization (APLI) source. However, the interface may also include a dual ionization source, e.g. both an ESI source and an APCI source, or a modular, interchangeable ionization source.

[0018] Typical parts of an ionization source are a nebulizer and a sampling capillary, which are typically positioned orthogonal or coaxial to each other. The LC eluate leaving the LC channel is directed through a probe containing a nebulizer needle. In this way, the LC eluate is sprayed into a volume downstream of the nebulizer needle where ionization occurs, resulting in charged analyte molecules in the gas phase. A sampling device (e.g., a sampling capillary) is provided to collect ions in the gas phase and direct them to the mass spectrometer.

[0019] The ionization source may further include an assembly for providing a curtain gas (e.g., N2) that reduces ingress of background ions (e.g., solvent clusters) into the MS. The assembly may have a curtain plate and an orifice assembly for supplying the curtain gas. The ionization source may further include sources of auxiliary gas and nebulizer gas.

[0020] To optimize ionization conditions, it is also possible to adjust the solvent composition by adding a make-up stream just before the ionization source to adjust the pH, salt, buffer or organic content.

[0021] Such ionization sources are known in the art and will not be described further here.

[0022] As used herein, the term "controller" may refer to a processing unit such as a microprocessor, a microcontroller, a reduced instruction set circuit (RISC), an application specific integrated circuit (ASIC), a logic circuit, and any other circuit or processor capable of performing the functions / methods described herein and configured, in particular, to control a downstream pump to obtain at least one dilute composition by automatically mixing at least one concentrated composition with at least one diluent, inject the at least one dilute composition into the ionization source, obtain a mass spectrum of the at least one dilute composition, and perform calibration of the mass spectrometer based on evaluation of the mass spectrum.

[0023] The controller may be integrated into the analytical system or may be a separate logic entity that communicates with the analytical system via a wired or wireless direct connection or indirectly via a network interface device, a wired or wireless communication network such as a wide area network, e.g., the Internet or a healthcare provider's local area network or intranet. In some embodiments, the processor may be integral with a data management unit implemented on a computing device such as, for example, a desktop computer, a laptop, a smartphone, a tablet, a PDA, etc. The processor may include a server computer and / or may be distributed / shared across / among multiple analytical systems. Furthermore, the analytical system may include remote devices, servers and cloud-based elements that communicate with the controller or remote PC / server or cloud-based systems via wired or wireless (e.g., infrared, cellular, Bluetooth). The controller may also be configurable to control the analytical system such that workflows and workflow steps are executed by the analytical system.

[0024] As used herein, the term "analytical fluid system" refers to any fluid system that includes at least one fluid stream and is configured to prepare a sample for mass analysis and / or transport the prepared sample to a mass spectrometer, particularly to separate analytes of interest in the sample prior to detection by the mass spectrometer.

[0025] A "fluid stream" is a fluid pathway through which liquids can flow and in particular through which a sample from a sample injection point can be transported to a mass spectrometer and through which the sample can undergo a separation and / or purification process. The fluid connections through different parts of the fluid stream can be discontinuous because the fluid stream can include elements such as switching valves that can establish alternative connections and regulate the fluid flow between different parts of the fluid stream at different times.

[0026] According to one embodiment, the fluid stream may include at least one liquid chromatography (LC) column, and the analytical fluid system is or includes a liquid chromatography (LC) system.

[0027] A "liquid chromatography system or LC system" is an analytical device or a module or a unit within an analytical device for performing liquid chromatography. An LC system may be embodied as a single channel with one fluid stream or as a multi-channel system with multiple fluid streams including one or more LC columns arranged in parallel and / or series. An LC system may also comprise a sample injector, valves, liquid sources, fluid connections and components, e.g. for mixing liquids, degassing liquids, tempering liquids, etc., one or more sensors, such as pressure sensors, temperature sensors, and in particular at least one LC pump. This list is not exhaustive. At least one LC column may be interchangeable. In particular, an LC system may include more LC columns than fluid streams, and multiple LC columns may be selectable, e.g. interchangeably coupled to the same fluid stream. Capillaries may also be used to bypass an LC column. A fluid stream may include multiple substreams.

[0028] "Liquid chromatography or LC" is an analytical process in which a sample injected by a sample injector is subjected to chromatographic separation by an LC column, e.g., to separate analytes of interest from matrix components, e.g., remaining matrix components after sample preparation that may still interfere with subsequent detection, such as mass spectrometry detection, and / or to separate analytes of interest from each other to enable their individual detection. "High performance liquid chromatography" or HPLC, "ultra high performance liquid chromatography" or UHPLC, "micro liquid chromatography" or μLC, and "small bore liquid chromatography" or small bore LC are forms of liquid chromatography performed under pressure.

[0029] "LC column" may refer to any column, cartridge, capillary, etc. for performing separations of a chromatographic nature. A column is typically packed or loaded with a stationary phase through which a mobile phase is pumped to trap and / or separate, elute and / or transport analytes of interest under selected conditions, e.g. according to their polarity or logP value, size or affinity, as is commonly known. This stationary phase may be particulate or beaded or a porous monolith. However, the term "column" may also refer to capillaries or channels that are not packed or loaded with a stationary phase but rely on the surface area of ​​the inner capillary walls or geometric structures to perform separations. An example is provided by pillar array chromatography, where a separation bed is formed by etching away the interstitial volume from a solid silicon wafer, leaving an array of pillars. The resulting channels can be folded into a small footprint by coupling bed segments with optimized flow distributors that limit peak dispersion. This produces stationary phase support structures organized in reproducible, regular patterns.

[0030] The LC column may be interchangeable and / or may operate in parallel or in sequence with one or more other LC columns. The LC column may be, for example, a fast trap and elute LC column or "trap column" for short, an HPLC column or a UHPLC column, and may be of any size, including micro LC columns and small bore LC columns or pillar array LC columns. In the case of a trap column, a stationary phase can be selected that retains the analytes of interest, while any salts, buffers, detergents and other matrix components are not retained and are washed away. This process is typically followed by elution of the analytes, for example in a backwash mode, using a different mobile phase or solvent gradient. Depending on the analyte, separation of some analytes may be expected. On the other hand, for analytes with identical mass (isobars) and / or overlapping daughter ion spectra in multiple reaction monitoring (MRM), a more extensive chromatographic separation may be typical when it comes to mass spectrometry. In that case, separation on an HPLC or UHPLC column may be advantageous.

[0031] A "liquid chromatography pump or LC pump" is a high-pressure pump that can provide a consistent and reproducible volumetric flow rate through an LC channel, although its pressurizing capacity can vary. Pressures in HPLC can typically reach 60 MPa or about 600 atm, but UHPLC and μ-LC systems have been developed to operate at even higher pressures, e.g., up to 140 MPa or about 1400 atm, thus allowing the use of much smaller particle sizes (<2 μm) in LC columns. LC pumps may be configured as binary or quaternary pumps, for example, for conditions that require the use of an elution gradient, by gradually changing the ratio between up to four elution solvents.

[0032] According to one embodiment, the LC pump can generate a pressure of 60 MPa to 140 MPa, typically 75 MPa to 100 MPa, more typically 80 MPa.

[0033] According to one embodiment, the LC pump can be configured to operate at a flow rate of 1 μl / min to 500 μl / min or more, typically up to 1500 μl / min, more typically 100 μl / min to 300 μl / min, for example with an accuracy of about ±5% or less.

[0034] The term "liquid" with respect to a fluid stream refers to liquids commonly used in liquid chromatography, e.g., as a mobile phase or eluent (elution solvent), and as known in the art, e.g., liquids used as a solvent or mixture of solvents.

[0035] A "downstream pump" is an auxiliary pump that is at least functionally distinct from an LC pump, and possibly a multi-function pump, including at least the function of diluting a concentrated calibration solution (composition) if necessary and injecting the diluted composition into the ionization source while bypassing the analytical fluid system via the downstream valve. Another possible function is to connect to an ionization source between two successive fluid streams of an analytical fluid system to flush liquid from a conduit between the downstream valve and the ionization source with a cleaning liquid before liquid from the subsequent fluid stream enters the conduit while bypassing the analytical fluid system via the downstream valve. Another possible function is to connect to at least one fluid stream of an analytical fluid system that includes an HPLC column to backwash the HPLC column with a cleaning liquid via the downstream valve, thereby cleaning the HPLC column with a cleaning liquid. In general, the downstream pump is a low-pressure, high-volume (high-flow) pump compared to the LC pump, and is in fluid communication with the downstream valve. Typically, the downstream pump according to the present disclosure is also a lower-precision pump and therefore simpler and less expensive in construction compared to the LC pump. According to one embodiment of the present disclosure, the downstream pump is a positive pressure pump. Any pump suitable for generating positive pressure may be used to actively pump the wash or mobile phase from a liquid source through at least one HPLC column in a backwash and / or through a conduit from the valve to the detector via a flow selection valve, such as a membrane / diaphragm pump, a single plunger high speed pump, a syringe piston pump, a gear pump, etc. According to one embodiment, the positive pressure and active pumping may be achieved by applying air pressure, for example by a pressurized gas, for example by a nitrogen source, in a sealed liquid container connected to an upstream inlet valve.

[0036] According to one embodiment, the analytical fluid system includes a plurality of fluid streams, at least one fluid container includes a cleaning fluid, and the controller is further configured to control a downstream pump and a downstream valve to connect to the ionization source between two successive fluid streams to flush liquid from a previous fluid stream from the conduit between the downstream valve and the ionization source with the cleaning fluid before liquid from the subsequent fluid stream enters the conduit.

[0037] According to one embodiment, the analytical fluid system includes at least one fluid stream including an HPLC column, and the at least one fluid container includes a cleaning fluid, and the controller is further configured to control and connect a downstream pump and a downstream valve to the at least one fluid stream to backwash the at least one HPLC column with the cleaning fluid, thereby cleaning the at least one HPLC column with the cleaning fluid.

[0038] The controller may be configured to automatically backwash the at least one HPLC column at regular intervals and / or upon detection of a pressure increase above a predetermined threshold in the at least one fluid stream and / or upon detection of a performance degradation below a predetermined threshold, for example, by monitoring data such as an increase in signal background, a shift in analyte retention time, a change in peak shape during use of the at least one HPLC column.

[0039] According to one embodiment, the controller may be configured to manage a fluid stream-to-mass spectrometer connection time, which is the connection time between the at least one fluid stream and the ionization source, a downstream pump-to-mass spectrometer connection time, which is the connection time between the downstream pump and the ionization source, and a downstream pump-to-fluid stream connection time, which is the connection time between the at least one fluid stream and the downstream pump, by controlling switching of any one or more valves, including the downstream valve.

[0040] According to one embodiment, the connection time between the fluid stream and the mass spectrometer is fixed and is the same for each fluid stream. Furthermore, when the downstream pump is used to flush the liquid from the previous fluid stream from the conduit between the downstream valve and the ionization source, the downstream pump-mass spectrometer connection time may be fixed and is a fraction of the fluid stream-mass spectrometer connection time, thereby providing continuous switching at a constant pace, at least temporarily. If the downstream pump-mass spectrometer connection time is 20% or less, typically 10% or less, or more typically 5% or less of the fluid stream to mass spectrometer connection time, it may be particularly beneficial for the downstream pump conditions, e.g., with respect to flow rate and / or washing liquid, to be adapted so that the downstream pump-mass spectrometer connection time is minimized. In absolute terms, the downstream pump-mass spectrometer connection time may be on the order of a few seconds, typically 5 seconds or less, more typically 3 seconds or less.

[0041] According to one embodiment, the downstream pump is configured to pump the wash liquid through the conduit between the HPLC column and / or downstream valve and the ionization source at a flow rate higher than the flow rate of the fluid stream, e.g. several times higher, e.g. 5 times, 10 times, 20 times or more. For example, for a μ-LC with a typical flow rate of about 1 μL / min, a wash pump flow rate of 5 μL / min may already be beneficial. For a flow rate of the fluid stream of about 100 μL / min, the effective flow rate of the downstream pump may be about 500-1000 μL / min or more. According to one embodiment, the flow rate of the fluid stream is about 440 μL / min and the flow rate of the downstream pump is about 5-10 times higher. According to some embodiments, a flow rate of the downstream pump of up to 5000 μL / min is possible. The flow rate may be variable depending on whether the wash liquid is directed to the HPLC column or to the valve-detector conduit. According to one embodiment, the downstream pump is configured to inject the at least one dilute composition into the ionization source at a flow rate similar to the flow rate of the at least one fluid stream, e.g., about 500 μL / min or less, e.g., 440 μL / min, 100 μL / min or less, e.g., 50 μL / min or less, e.g., 30 μL / min.

[0042] A "washing liquid" may be a liquid suitable for washing away and eventually dissolving particulate matter, adsorbed matrix components, etc., that have accumulated on the HPLC column head during multiple sample injection cycles. A wash liquid may be a liquid suitable for washing away and eventually dissolving final sample traces from the valve-detector conduit while still being compatible with the detector used, for example, in a mass spectrometer. The wash liquid may be the same as or similar to the elution solvent used for chromatography, or may be different depending on the type of column, or the type of sample and analyte that is passed through the column. For example, for analytes for which reversed-phase chromatography is typically used, suitable solvents may be organic solvents such as methanol, acetonitrile, tetrahydrofuran, and / or isopropyl alcohol. These solvents may also be mixed with each other and / or with water. Acidic or basic additives may be added to adjust the pH. Typical additives may include formic acid, ammonium formate, ammonium acetate, etc. For example, for analytes for which normal-phase chromatography is typically used, suitable solvents may include solvents such as hexane, heptane, mixed with polar organic solvents such as ethyl acetate, chloroform, or 2-propanol. The wash solutions may be the same or different depending on the HPLC column, for backwashing the HPLC column and for washing the conduit between the downstream valve and the ionization source, respectively.

[0043] According to one embodiment, the at least one diluent for diluting the at least one concentrated composition and the washing liquid are the same liquid.

[0044] For purposes of this disclosure, and in relation to backwashing, the term "HPLC" also encompasses, for simplicity, UHPLC or μ-LC and other high performance columns such as small bore columns, and is distinguished from fast trap-and-elute LC columns, which do not typically undergo backwashing during an analytical run and for the sole purpose of cleaning the column, requiring manual intervention to perform a backwash by disconnecting the column, reversing the flow direction by reconnecting the column in the opposite direction, and directing the flow to waste rather than to the detector.

[0045] The term "valve" refers to a flow regulator for controlling, redirecting, restricting, or stopping flow, particularly an LC switching valve, i.e. a multiport valve that controls flow between elements connected to a port. This is typically accomplished by moving one or more valve conduits to switch communication between different elements. Elements may be fluidly connected to the ports via further conduits, such as pipes, tubing, capillaries, microfluidic channels, and by fittings such as screws / nuts and ferrules, or alternative liquid-tight seals held in place, for example, by clamping mechanisms. LC switching valves can typically tolerate liquid pressures at or above those used for HPLC.

[0046] In particular, a "downstream valve" is an LC switching valve located downstream with respect to the analytical fluid system in the normal flow direction through the fluid streams towards the ionization source. The downstream valve may include a port for each fluid stream, a port for a conduit leading to the ionization source, one or more downstream pump ports, and one or more waste ports.

[0047] According to one embodiment, the downstream valve has an inner valve conduit having an inner diameter of less than 0.6 mm, typically about 0.5 mm to 0.2 mm, more typically about 0.4 mm, and even more typically about 0.25 mm, however, the inner valve conduit can have any other diameter within the range typically used.

[0048] According to one embodiment, the downstream valve has a typical switching time of about 500 ms or less, however, the switching time can be longer than 500 ms.

[0049] According to one embodiment, the downstream pump is connected to the downstream valve via a wash selection valve, the wash selection valve being configured to alternately connect any one of the at least one fluid stream and a conduit leading to the ionization source via the downstream valve.

[0050] A "wash selection valve" is an LC switching valve disposed between the downstream pump and the downstream valve, with a downstream pump inlet port and one wash outlet port for each fluid stream to be backwashed, with fluid connections between the outlet ports and the respective fluid streams via the downstream valve, and ultimately one wash outlet port connectable to a valve-detector conduit via the downstream valve.

[0051] According to one embodiment, the wash selection valve is connectable to at least one fluid stream via a respective three-way valve fluidly connected to a downstream valve, the three-way valve comprising a wash selection valve inlet port, a downstream valve outlet port, and a waste outlet port.

[0052] The term "enriched composition" as used herein refers to a mixture of compounds used for calibration, selectively selected for their particular suitability in calibrating a mass spectrometer, and having a respective concentration higher than the final concentration obtained after dilution. In one embodiment, the composition comprises the indicated compound, in a further embodiment, the composition consists essentially of the indicated compound, and in a further embodiment, the composition consists of the indicated compound. In one embodiment, the composition is a liquid composition, and in a further embodiment, the composition is a liquid under standard conditions.

[0053] The term "diluted composition" refers to a composition obtained by automatically mixing at least one concentrated composition with at least one diluent to obtain the final concentration of each compound in the composition required for the calibration of a mass spectrometer.

[0054] As used herein, the term "standard conditions" refers to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e., in one embodiment, a temperature of 25° C. and an absolute pressure of 100 kPa, unless otherwise indicated, and in one embodiment, the standard conditions include a pH of 7. Furthermore, unless otherwise indicated, the term "about" refers to an indicated value with a technical precision generally accepted in the relevant field, in one embodiment, to an indicated value ±20%, in a further embodiment, to ±10%, and in a further embodiment, to ±5%. Furthermore, the term "essentially" indicates that there is no deviation affecting the indicated result or use, i.e., potential deviations do not cause the indicated result to deviate by more than ±20%, in a further embodiment, to ±10%, and in a further embodiment, to ±5%. Thus, "consisting essentially of" means including the specified components, but excluding materials present as impurities, unavoidable materials present as a result of the process used to provide the components, and other components other than the components added for purposes other than achieving the technical effect of the present invention. For example, a composition defined using the phrase "consisting essentially of" encompasses any known acceptable additives, excipients, diluents, carriers, and the like. In one embodiment, a composition consisting essentially of a set of components contains less than 5 wt.%, in a further embodiment less than 3 wt.%, in a further embodiment less than 1 wt.%, and in a further embodiment less than 0.1 wt.% of an unspecified component.

[0055] According to one embodiment, at least one concentrated composition comprises (i) cesium iodide, (ii) ethylamine and / or formic acid, and (iii) methanol and / or water, and optionally (iv) cyclosporin A and / or 5-(4-hydroxyphenyl)-5-phenylhydantoin and / or ammonium formate.

[0056] According to one embodiment, in at least one concentrated composition, (i) the cesium iodide has a concentration of 0.1 μg / mL to 100 mg / mL; (ii) ethylamine, when present, has a concentration of 0.01 μg / mL to 1 mg / mL; Formic acid, when present, has a concentration of 0.001% (v / v) to 10% (v / v); (iii) cyclosporine A, if present, has a concentration of 0.01 μg / mL to 1000 μg / mL; 5-(4-hydroxyphenyl)-5-phenylhydantoin, if present, has a concentration of 0.01 μg / mL to 1000 μg / mL; and ammonium formate, if present, has a concentration of 0.01 mM to 1 M; (iv) Adding a polar solvent such as methanol, water, or a mixture thereof up to 100%.

[0057] According to one embodiment, the at least one diluted composition is obtained by mixing the at least one concentrated composition with the at least one diluent in a ratio of 1:99 to 99:1, typically 1:99 to 20:80, for example 10:90.

[0058] The term "polar solvent" is understood by those skilled in the art. Preferably, the term relates to a solvent or mixture of solvents having a dipole moment of at least 1.5D, preferably at least 1.7D. Typically, the polar solvent is methanol, water, ethanol, acetonitrile, n-propanol, isopropanol, or a mixture of any combination of at least two of the aforementioned polar solvents. More typically, the polar solvent is methanol, water, or a mixture thereof.

[0059] According to one embodiment, in at least one concentrated composition, the concentration of cesium iodide is 500 μg / mL, the concentration of ethylamine, if present, is 10 μg / mL, the concentration of formic acid, if present, is 1% (v / v), the concentration of methanol is 30% (v / v), and water is added to 100%.

[0060] According to one embodiment, the at least one concentrated composition comprises cesium iodide, ethylamine, formic acid, methanol and water.

[0061] According to one embodiment, the at least one diluent is either methanol, acetonitrile, ethanol, propanol, isopropanol, or any combination thereof.

[0062] According to one embodiment, at least one diluted composition is obtained by mixing at least one concentrated composition with a combination of methanol and acetonitrile as diluents, the respective ratio being, for example, 10:45:45 to obtain a dilution ratio of 10:90.

[0063] In one embodiment, the composition includes the indicated components at the concentrations specified herein. Unless otherwise specified, a polar solvent, such as methanol, water or a mixture thereof, is added to 100%. In one embodiment, methanol is included in the composition at the indicated concentration, and water is added to 100%, and thus, in one embodiment, all unoccupied portions of the composition are water. As will be understood by those skilled in the art, the term "to a concentration of 100% of X" refers to the addition of an amount of compound X that provides the volume required for the other compounds of the composition to have the desired concentration. Thus, compound X that is added to 100% is typically a liquid, and in one embodiment is water or methanol, and in a further embodiment is water. In one embodiment, the purity of the components of the composition is independently selected from at least 90%, in one embodiment at least 95%, in a further embodiment at least 98%, in a further embodiment at least 99%, at least 99.5%, and in a further embodiment at least 99.9%.

[0064] The term "calibration" is used herein in a broad sense consistent with typical use by those skilled in the art. Thus, the term calibration includes the operation of establishing a relationship between the value of a quantity obtained with a measurement standard under certain conditions and the value of the corresponding quantity of a calibrated instrument, i.e., calibration in the strict sense. However, calibration may also be the verification of a measurement. The term calibration further includes the means of adjusting or re-adjusting a calibrated instrument or its output to match the value of said quantity obtained with a measurement standard, i.e., calibration in its usual broader sense. Thus, calibration may be, in particular, mass axis check (MAC) and / or mass axis adjustment (MAA) in mass spectrometry. In one embodiment, the composition is a universal calibration solution for positive ion mode and negative ion mode MS. In one embodiment, when the calibration composition includes at least ethylamine, formic acid, and cesium iodide, and at least one of methanol and water, the m / z values ​​of the main signals are 46, 133, 393, 653, 912, 1172, 1432, 1692, and / or 1952 in positive ion mode, and in one embodiment, 46.066, 132.905, 392.715, 652.524, 912.334, 1172.1 44, 1431.953, 1691.763, and / or 1951.572, and in negative ion mode, 45, 127, 387, 647, 906, 1166, 1426, 1686, and / or 1946, and in one embodiment 44.998, 126.905, 386.714, 646.524, 906.333, 1166.143, 1425.953, 1685.762, and / or 1945.572. In one embodiment, the composition further comprises at least one of cyclosporine A, 5-(4-hydroxyphenyl)-5-phenylhydantoin, and ammonium formate, and the m / z values ​​of the resulting additional ions are 601.921, 1202.841, 1219.841, 1224.841, 1334.751, and 269.0848 in positive ion mode, and 267.0848 and 1200.841 in negative ion mode.Thus, in one embodiment, the calibration is a calibration in the m / z range of 46-1952 in positive ion mode and / or a calibration in the m / z range of 45-1946 in positive ion mode.

[0065] Advantageously, in the research underlying the present disclosure, it has been found that the composition described herein is useful as a universal calibration solution in mass spectrometry suitable for use in positive and negative ion modes, providing the option to calibrate over a wide range of m / z values, and providing a suitable number of calibration points over the entire range.Furthermore, this solution is stable for at least one year at temperatures typical for the use of mass spectrometers.

[0066] In one embodiment, the calibration includes performing a mass axis check (MAC), i.e. verifying that the mass axis is correctly adjusted. The mass axis check includes, in one embodiment, evaluating the mass spectrum of the dilution composition specified herein and verifying that the signal is obtained within a predetermined reference range for any one or more or all of the m / z ratios indicated above. If the m / z ratios are obtained within the predetermined range, the method may indicate that the mass axis check is successful, and in one embodiment, the method is then terminated. If at least one of the m / z ratios is not obtained within the predetermined range, the method may indicate that the mass axis check is unsuccessful, and in such a case, the method may continue with the step of mass axis adjustment as specified herein below.

[0067] In one embodiment, the calibration includes performing a mass axis adjustment (MAA), i.e., adjusting or re-adjusting at least one parameter of the mass spectrometer such that the measured m / z values ​​are obtained within a predefined reference range for any one or more or all of the above m / z ratios. Adjustable parameters to achieve the aforementioned mass axis adjustment are known to those skilled in the art and may include hardware and software / setting adjustments / tuning, such as adjusting RF DACs (radio frequency digital-to-analog converters), DC (direct current) and voltage values, etc., and finally including adjustment of ion source parameters.

[0068] In particular, in the event of failure or anticipated failure of the mass axis check and / or mass axis adjustment based on evaluation of the mass spectrum of the at least one diluted composition, the controller may be further configured to perform any one or more actions selected from adapting the dilution factor of the at least one concentrated composition, adjusting one or more mass spec acquisition parameters, performing a maintenance procedure, before repeating the mass axis check and / or mass axis adjustment.

[0069] The reasons for possible failure of the mass axis check and / or mass axis adjustment may be due to the performance limitations of the downstream pump (usually lower than the LC pumps mentioned above), whose mixing ratio accuracy may be about + / - 5%, and whose mixing ratio accuracy may be about + / - 30%, thus resulting in signal intensity variations that may vary between about 30% and about 200% across different analytical systems. The signal strength and signal intensity variations may also be due to chemical degradation and / or aging of the components in the calibration solution, as well as contamination of the components of the mass spectrometer, aging or partial clogging of downstream pump components, including the mixing unit, capillaries, valves, etc. Contamination of the above mentioned components may introduce interferences that may eventually suppress or interfere with the signal, and the degree of suppression and interference may be different due to different ionization efficiencies of different physicochemical properties of the compounds in the calibration solution. Sufficient signal strength within the dynamic range of the mass spectrometer, i.e., a detectable signal that is distinct from background noise but not saturated (i.e., not too high and not too low), is required over a wide m / z range for successful mass axis check and mass axis adjustment.

[0070] The term "evaluating a mass spectrum or evaluating a mass spectrum" is understood by those skilled in the art. In one embodiment, the term relates to determining a correlation plot between a semi-quantitative or quantitative measure of one or more signals obtained from a sample or at least one dilution composition by a mass spectrometer and the m / z values ​​of the ions causing said signals. The graphical representation of the mass spectrum or m / z spectrum may be provided, for example, as a centroid graph and / or a continuum graph. The m / z spectrum may be a full scan spectrum obtained by scanning over the m / z measurable range of the mass spectrometer, or a partial scan spectrum obtained by scanning over a selected m / z range within the m / z measurable range. In particular, multiple complete or partial m / z scan data may be repeatedly acquired at intervals or consecutively in any given time frame by performing m / z scans one after the other. In particular, the mass spectral parameters may include any one or more of the shape or area of ​​one or more m / z peaks, the signal / noise ratio, the m / z peak height, the ratio of m / z peak heights, the background signal intensity, the m / z value of the peak maximum, the m / z mass position, the presence of one or more unexpected m / z peaks, and the height of one or more unexpected m / z peaks.

[0071] The conditions leading to failure may be cumulative, i.e., progressing over time, and such progression may be monitored each time a mass axis check and / or mass axis adjustment is performed, such that failure of the mass axis check and / or mass axis adjustment may be anticipated. Thus, even if the mass axis check and / or mass axis adjustment does not fail because the mass spectral parameters are still within the reference range, any of the actions mentioned herein may be scheduled before the next or subsequent mass axis check and / or mass axis adjustment to prevent an otherwise anticipated failure.

[0072] "Adapting the dilution factor of at least one concentrated composition" means changing the mixing ratio between at least one concentrated composition and at least one diluent, for example by increasing or decreasing the dilution factor, for example from 10:90 to 5:95 or 15:85 or 20:80, so that any one or more respective signals that resulted in a mass spectrum determination that was too low or too high for the reference range or dynamic range at the newly obtained concentration of the compound in the diluted composition will fall within that range at the next spectrum determination. Changing the mixing ratio may also include changing the concentrated composition, for example with a combination of different compounds and / or at least partially different concentrations of the same compounds, and / or changing at least one diluent or the relative mixing ratio, when two or more concentrated compositions are available.

[0073] The term "adjusting mass spectrometry acquisition parameters" can refer to adjusting the sensitivity of the mass spectrometer to increase or decrease the signal intensity, the overall signal intensity across the spectrum, or the overall signal intensity of an individual peak / signal, or adjusting the m / z range, adjusting the selection and relative collision-induced fragmentation of the parent molecular ion, adjusting ion source parameters that contribute to changes in the acquisition parameters such as curtain gas pressure, make-up flow conditions, voltages, etc.

[0074] The term "maintenance procedure" refers to a maintenance procedure of an ionization source (IS) and / or a mass spectrometer (MS) that is intended to solve the root cause of a technical problem of the ionization source and / or mass spectrometer, respectively, that caused the failure of the mass axis check and / or the mass axis adjustment. A possible root cause may be clogging or accumulation of contaminants, such as analyte and matrix components, in the ionization source and / or mass spectrometer, leading to a performance degradation of the analytical system. According to an embodiment, the IS and / or MS maintenance procedure is any one or more of an IS cleaning procedure, an IS and / or MS bakeout procedure, and an intervention procedure.

[0075] "IS cleaning procedure" refers to an automatically initiated and executed cleaning procedure of the ionization source, which includes liquid injection into the ionization source. The liquid injection may include purging through the nebulizer needle by increasing the flow rate, for example to 1 mL / min or even several mL / min. This may optionally further include increasing the temperature and / or gas pressure and / or changing the applied electrical potential.

[0076] "IS and / or MS bake-out procedure" refers to an automatically initiated and executed procedure that involves raising the temperature of the IS or IS parts and / or MS parts, especially metal parts, to a temperature of, for example, 200° C., in order to accelerate the desorption rate of the final adsorbate. Heating can be performed by evacuation followed by vacuum conditions, for example up to 10 -10 It may further be advantageous to more quickly re-establish the mbar pressure condition.

[0077] "Intervention Procedure" refers to a semi-automated procedure including manual cleaning and / or repair and / or replacement of the IS or IS parts and / or MS parts, automatic lowering of the IS and / or MS temperature before manual intervention and automatic raising of the IS and / or MS temperature after manual intervention, and / or automatic lowering or removal of vacuum conditions in the IS and / or MS before manual intervention and automatic re-establishment of vacuum conditions in the IS and / or MS after manual intervention. In this way, manual steps are reduced to a minimum and the process is greatly simplified. Furthermore, manual intervention time is reduced to a minimum because the system is automatically prepared for manual intervention and automatically returned to operational state after manual intervention.

[0078] According to certain embodiments, the IS and / or MS maintenance procedures further include any one or more of the following: automatic increase in gas flow, e.g., curtain gas, to reduce IS and / or MS temperature; automatic disconnection of the IS and / or MS from power, e.g., before manual intervention, and eventual automatic reconnection after manual intervention; automatic enabling / disabling of manual access to the IS and / or MS with automatic interlock start / stop; and automatic change of vacuum state of the IS and / or mass spectrometer.

[0079] Also disclosed herein is an automated analytical method involving the use of a mass spectrometer, the method comprising connecting a downstream pump to an ionization source coupled to a mass spectrometer via a downstream valve disposed downstream of an analytical fluid system, the downstream pump being fluidly connected to a plurality of fluid containers containing respective fluids, the fluids including at least one concentrated composition for calibrating the mass spectrometer and at least one diluent for diluting the at least one concentrated composition. The method further comprises controlling the downstream pump to obtain at least one diluted composition by automatically mixing the at least one concentrated composition with the at least one diluent at a predetermined dilution ratio, injecting the at least one diluted composition into the ionization source, acquiring a mass spectrum of the at least one diluted composition, and performing a calibration of the mass spectrometer based on an evaluation of the mass spectrum.

[0080] According to one embodiment, performing the calibration includes checking the mass axis and / or adjusting the mass axis.

[0081] According to one embodiment, in the event of failure or anticipated failure of the mass axis check and / or mass axis adjustment based on evaluation of the mass spectrum of at least one diluted composition, the method includes performing any one or more actions selected from adapting a dilution factor of the at least one composition, adjusting one or more mass spec acquisition parameters, performing a maintenance procedure, before repeating the mass axis check and / or mass axis adjustment.

[0082] According to one embodiment, the method includes controlling a downstream pump and a downstream valve to connect to an ionization source between two successive fluid streams of the analytical fluid system to flush liquid from a conduit between the downstream valve and the ionization source with a cleaning liquid before liquid from the subsequent fluid stream enters the conduit.

[0083] According to one embodiment, the method includes controlling a downstream pump and a downstream valve to connect to at least one fluid stream of an analytical fluid system including the HPLC column to backwash the HPLC column with a cleaning solution, thereby cleaning the HPLC column with the cleaning solution.

[0084] According to one embodiment, the method includes connecting a downstream pump to a downstream valve via a wash selection valve, and alternately connecting the wash selection valve to any one of the at least one fluid stream and an ionization source via the downstream valve.

[0085] According to one embodiment, the method includes connecting a wash selection valve to at least one fluid stream via a respective three-way valve fluidly connected to a downstream valve, the three-way valve including a wash selection valve inlet port, a downstream valve outlet port and a waste outlet port.

[0086] According to one embodiment, the method includes automatically backwashing at least one HPLC column during use of the at least one HPLC column at regular intervals and / or upon detection of a pressure increase in at least one fluid stream above a predetermined threshold and / or upon detection of a performance degradation below a predetermined threshold by monitoring data such as, for example, an increase in signal background, a shift in analyte retention time, a change in peak shape, etc.

[0087] According to one embodiment, the method includes automatically managing by a controller a fluid stream-to-mass spectrometer connection time, which is the connection time between at least one fluid stream and an ionization source, a downstream pump-to-mass spectrometer connection time, which is the connection time between the downstream pump and the ionization source, and a downstream pump-to-fluid stream connection time, which is the connection time between at least one fluid stream and the downstream pump, by controlling switching of any one or more valves, including a downstream valve, a wash selection valve, and a three-way valve.

[0088] According to one embodiment, the method includes fixing the fluid stream-mass spectrometer connection time, e.g., to the same duration for each fluid stream; when a downstream pump is used to flush liquid from a previous fluid stream from a conduit between the downstream valve and the ionization source, the method may also include fixing the downstream pump-mass spectrometer connection time, which can be a fraction of the fluid stream-mass spectrometer connection time, resulting in continuous switching at a constant pace, at least temporarily.

[0089] Other and further objects, features and advantages will become apparent from the following description of exemplary embodiments taken in conjunction with the drawings and the appended claims, which should be noted that the claims are defined by the description therein and not by the specific recitation of the features and advantages set forth herein. [Brief description of the drawings]

[0090] The following detailed description of the embodiments of the present disclosure can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:

[0091] [Figure 1] FIG. 1 shows a schematic diagram of an analytical system and method that includes using a downstream pump to calibrate a mass spectrometer. [Diagram 2] FIG. 2 illustrates, in schematic form, further details of the method of FIG. 1 in case of failure or expected failure of calibration according to an embodiment. [Diagram 3]FIG. 2 shows a schematic diagram of a further embodiment of the analytical system and method of FIG. 1, in which the downstream pump has an additional function. [Figure 4] FIG. 2 shows a schematic diagram of yet another embodiment of the analytical system and method of FIG. 1, including a downstream pump having yet another function;

[0092] Those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale, for example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help improve understanding of embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0093] FIG. 1 shows a schematic example of an analytical system 100 including a mass spectrometer 60 and an ionization source 61 coupled to the mass spectrometer 60, an analytical fluid system 10 connectable to the ionization source via a downstream valve 20 for injecting a sample into the mass spectrometer 60 via the ionization source 61, and a downstream pump 40 fluidly connectable to the ionization source 61 via the downstream valve 20, the downstream pump 40 being fluidly connected to a number of fluid containers containing respective fluids 41, 42, 43, 44, the fluids including at least one concentrated composition 44 for calibrating the mass spectrometer 60 and at least one diluent 42, 43 for diluting the at least one concentrated composition 44. The analytical system 100 further includes a controller 90 configured to control the downstream pump 40 to inject at least one diluted composition 45 into the ionization source 61 to obtain at least one diluted composition 45 by automatically mixing the at least one concentrated composition 44 with at least one diluent 42, 43 at a predetermined dilution ratio, to obtain a mass spectrum 62 of the at least one diluted composition 45, and to perform a calibration 63 of the mass spectrometer 60 based on an evaluation 64 of the mass spectrum 62.

[0094] In particular, the analytical fluid system 10 comprises a number of fluid streams 11, 12, 13, while the downstream valve 20 in this case comprises a respective fluid stream port 21, 22, 23 for each fluid stream 11, 12, 13 and a respective waste port 21', 22', 23' for each fluid stream 11, 12, 13 leading to waste 50. The downstream valve 20 further comprises a valve-ionization source port 25 connected to a conduit 30 leading to an ionization source 61 and alternatively connectable to each of the fluid streams 11, 12, 13 via the fluid stream ports 21, 22, 23, respectively. In particular, the downstream valve 20 further comprises a downstream pump inlet port 24 also connectable to the conduit 30 via the valve-ionization source port 25, and a downstream pump waste port 24' leading to waste 50 when connected to the downstream pump inlet port 24. It is clear that this is only an example and that the number of ports and connections can be adapted as required and depending on the number of fluid streams.

[0095] In this example, the downstream pump 40 is controlled by the controller 90 to inject the diluted composition 45 into the ionization source 61 at a flow rate similar to the flow rate 15 of the fluid streams 11, 12, 13, for example, about 500 μL / min or less, for example 440 μL / min, for example about 100 μL / min or less, for example 50 μL / min or less, for example 30 μL / min.

[0096] 1 also shows a schematic diagram of an automated analytical method including the use of a mass spectrometer 60, the method including connecting a downstream pump 40 to an ionization source 61 coupled to the mass spectrometer 60 via a downstream valve 20 disposed downstream of the analytical fluid system 10, the downstream pump 40 being fluidly connected to a plurality of fluid containers including respective fluids 41, 42, 43, 44, the fluids including at least one concentrated composition 44 for calibrating the mass spectrometer 60 and at least one diluent 42, 43 for diluting the at least one concentrated composition 44. The method further includes controlling the downstream pump 40 to obtain at least one diluted composition 45 by automatically mixing the at least one concentrated composition 44 with the at least one diluent 42, 43 at a predetermined dilution ratio, injecting the at least one diluted composition 45 into the ionization source 61, acquiring a mass spectrum 62 of the at least one diluted composition 45, and performing a calibration 63 of the mass spectrometer 60 based on an evaluation 64 of the mass spectrum 62.

[0097] In this example, the calibration includes a mass axis check and / or mass axis adjustment, where the concentrated composition 44 includes, for example, cesium iodide, ethylamine, formic acid, methanol, and water at 10 times the required concentration, and the diluted composition 45 is obtained by mixing the concentrated composition 44 with, for example, a combination of methanol and acetonitrile as diluents 42, 43, in a respective ratio of, for example, 10 (concentrated composition):45 (methanol):45 (acetonitrile), to obtain, for example, a dilution factor of 10:90, i.e., a 10-fold dilution.

[0098] 2 shows in schematic form further details of the analytical system 100 and the method of FIG. 1 in case of failure or expected failure of the mass axis check (MAC) and / or mass axis adjustment (MAA) based on evaluation 64 of the mass spectrum 62 of the diluted composition 45. In particular, referring to FIG. 2, the controller 90 is further configured to perform any one or more actions selected from adapting the dilution factor of the concentrated composition, adjusting one or more mass spectrometry (MS) acquisition parameters, performing maintenance procedures such as bake-out and / or IS cleaning procedures, before repeating the mass axis check (MAC) and / or mass axis adjustment (MAA). In particular, the method may include evaluating parameters of the mass spectrum such as signal intensity, peak shape, background and the presence of interferences. For example, in the case of signal intensities below the reference range, where at least one or more peaks, e.g. the highest m / z cluster, tend to have low signal intensities compared to more abundant m / z clusters, e.g. the cesium iodide cluster, the action may include decreasing the dilution factor, e.g. to obtain a less dilute calibration solution, or increasing the MS acquisition parameters, e.g. to improve detector sensitivity for individual m / z values ​​or ranges or the entire spectrum. On the other hand, in the case of signal intensities above the reference range, e.g. in the case of signal saturation, e.g. for the most abundant cluster, the action may include increasing the dilution factor, e.g. to obtain a more dilute calibration solution, or decreasing the MS acquisition parameters, e.g. to reduce detector sensitivity for individual m / z values ​​or ranges or the entire spectrum. Similarly, in the case of abnormal peak shapes, the action may include modifying, increasing or decreasing the MS acquisition parameters accordingly. In the case of background signals above the reference range, the action may include bake-out and / or IS cleaning procedures and / or decreasing the dilution factor, e.g. to obtain a higher signal-to-noise ratio. Similarly, if interferences are present, actions may include bake-out and / or IS cleaning procedures and / or reducing the dilution factor, such as to enhance the relative signal of the calibration solution component compared to the signal of the interference.

[0099] Figure 3 shows diagrammatically a further embodiment of the analytical system 100 and analytical method of Figure 1, in which the downstream pump 40 has a further function in addition to the function of providing at least one diluent composition described in relation to Figure 1 (represented in Figure 3 by dashed lines). In particular, the at least one fluid container contains at least one washing liquid 41, and the controller 90 is further configured to control the downstream pump 40 and the downstream valve 20 to connect to the ionization source 61 between two successive fluid streams 11, 12; 12, 13; 13, 11, in order to flush liquid from a previous fluid stream from the conduit 30 between the downstream valve 20 and the ionization source 61 with the washing liquid 41 before liquid from the subsequent fluid stream enters the conduit 30.

[0100] The method therefore comprises alternatingly connecting a plurality of fluid flows 11, 12, 13 to a conduit 30 via a downstream valve 20, and connecting a downstream pump 40 to the conduit 30 via the downstream valve 20 between two successive fluid flows 11, 12; 12, 13; 13, 11 to flush liquid from the previous fluid flow from the conduit 30 before liquid from the subsequent fluid flow enters the conduit 30.

[0101] The at least one washing liquid 41 can be, for example, water, acetonitrile, methanol, tetrahydrofuran or isopropyl alcohol, which can be pumped individually or mixed with each other in any combination and ratio depending, for example, on the LC conditions, the type of sample and / or analytes flowing therebetween and the desired washing effect. According to one embodiment, at least one diluent 42, 43 for diluting the at least one concentrated composition 44 can be used as a washing liquid and / or mixed with another washing liquid 41.

[0102] In this case, the downstream pump 40 may be controlled by the controller 90 to pump the cleaning fluid through the conduit 30 at a flow rate higher than the flow rate 15 of the fluid streams 11 , 12 , 13 .

[0103] FIG. 4 shows, in a schematic form, yet another variation of the analytical system 100 and analytical method of FIGS. 1 and 3, an analytical system 100' in which the downstream pump 40 has an additional function. In particular, the analytical fluid system 10' includes at least one fluid stream 11, 12, 13, including an HPLC column, as in the embodiment of FIG. 3, and at least one fluid container includes a washing liquid 41. The controller 90 is now further configured to control the downstream pump 40 and the downstream valve 20 to connect to the at least one fluid stream 11, 12, 13 in order to backwash the at least one HPLC column with the washing liquid 41, thereby washing the at least one HPLC column with the washing liquid 41. More particularly, the downstream pump 40 is connected to the downstream valve 20 via a washing selection valve 70. The washing selection valve 70 is configured to alternately connect the downstream pump 40 to either of the fluid streams 11, 12, 13, and to the conduit 30 via the downstream valve 20. In particular, the wash selection valve 70 is connectable to the fluid streams 11, 12, 13 via respective fluid stream wash ports 71, 72, 73 which lead to respective three-way valves 16, 17, 18 which are fluidly connected to the downstream valve 20 via downstream pump inlet ports 21′, 22′, 23′ which are also waste ports 21′, 22′, 23′, each of the three-way valves 16, 17, 18 comprising a wash selection valve inlet port, a downstream valve outlet port, and a waste outlet port leading to waste 50. The wash selection valve 70 further comprises a valve-conduit wash port 74 connected to the downstream valve 20 via the downstream pump inlet port 24 for connection to the conduit 30. In the example shown in FIG. 4, the wash selection valve 70, downstream valve 20 and three-way valve 16 are switched so that the downstream pump 40 is connected to the fluid stream 11 via the fluid stream wash port 71, the three-way valve 16, the downstream pump inlet port 21′ and the fluid stream port 21 in order to backwash the fluid stream 11 with at least one wash liquid 41, thereby washing the fluid stream 11 with at least one wash liquid 41. At the same time, the fluid stream 12 is connected to the conduit 30 via the fluid stream port 22 and the valve-ionization source port 25, and the fluid stream 13 is directed to the waste 50 via the fluid stream port 23, the waste port 23′ and the three-way valve 18, respectively. It should be noted that the direction of flow 15 of the fluid stream 11 is reversed with respect to the normal flow direction, as shown in FIGS. 1 and 3.The analytical system 100' may comprise other valves (not shown) upstream of the fluid flows 11, 12, 13, for example one or more upstream valves, for example to allow the outflow and disposal of washing fluids in a backwash mode.

[0104] The controller 90 may be configured to automatically backwash the HPLC columns of each fluid stream 11, 12, 13 at regular intervals and / or when a pressure increase above a predetermined threshold is detected in at least one of the fluid streams and / or a decrease in the performance of the HPLC column below a predetermined threshold is detected.

[0105] The controller 90 may be configured to manage a fluid flow-mass spectrometer connection time, which is the connection time between at least one fluid flow 11, 12, 13 and the ionization source 61, a downstream pump-mass spectrometer connection time, which is the connection time between the downstream pump 40 and the ionization source 61, and a downstream pump-fluid flow connection time, which is the connection time between at least one fluid flow 11, 12, 13 and the downstream pump 40, by controlling the switching of any one or more valves, including the downstream valve 20, the cleaning selection valve 70 and the three-way valves 16, 17, 18.

[0106] In the foregoing specification, numerous specific details are described to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that the specific details are not necessary to practice the present teachings. In other instances, well-known materials or methods have not been described in detail to avoid obscuring the present disclosure.

[0107] In particular, modifications and variations of the disclosed embodiments are certainly possible in light of the above description, and it is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically devised in the above examples.

[0108] Throughout the foregoing specification, references to "one embodiment," "embodiment," "one example," or "example," "one aspect," or "aspect" mean that a particular feature, structure, or characteristic described in connection with an embodiment or example or aspect is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment," "in one embodiment," "one example," or "example," "one aspect," or "aspect" in various places throughout the specification are not necessarily all referring to the same embodiment or example or aspect.

[0109] Furthermore, the particular features, structures or characteristics may be combined in any suitable combinations and / or subcombinations in one or more embodiments or examples or aspects.

Claims

1. a mass spectrometer (60) and an ionization source (61) coupled to said mass spectrometer (60); an analytical fluid system (10, 10') connectable to the ionization source (61) via a downstream valve (20) for injecting a sample into the mass spectrometer (60) via the ionization source (61), the downstream valve (20) being located downstream relative to the analytical fluid system (10, 10') in a normal flow direction through a fluid stream towards the ionization source (61); a downstream pump (40) fluidly connectable to the ionization source (61) via the downstream valve (20), the downstream pump (40) being fluidly connected to a plurality of fluid containers containing respective fluids (41, 42, 43, 44), the fluids including at least one concentrated composition (44) for calibrating the mass spectrometer (60) and at least one diluent (42, 43) for diluting the at least one concentrated composition (44); a controller (90) configured to control the downstream pump (40) to obtain at least one diluted composition (45) by automatically mixing at least one concentrated composition (44) with at least one diluent (42, 43) at a predetermined dilution ratio, to inject the at least one diluted composition (45) into the ionization source (61), to acquire a mass spectrum (62) of the at least one diluted composition (45), and to perform calibration (63) of the mass spectrometer (60) based on evaluation (64) of the mass spectrum (62); An analysis system (100, 100') comprising:

2. The analytical system (100, 100') of claim 1, wherein the calibration comprises a mass axis check and / or a mass axis adjustment.

3. In the event of a failure or predicted failure of the mass axis check and / or mass axis adjustment based on the evaluation (64) of the mass spectrum (62) of the at least one diluted composition (45), the controller (90) may be further configured to perform any one or more actions selected from adapting the dilution factor of the at least one concentrated composition, adjusting one or more mass analysis acquisition parameters, and performing a maintenance procedure before repeating the mass axis check and / or mass axis adjustment.

4. 4. The analytical system of claim 1, wherein the at least one concentrated composition comprises (i) cesium iodide, (ii) ethylamine and / or formic acid, (iii) methanol and / or water, and (iv) cyclosporin A and / or 5-(4-hydroxyphenyl)-5-phenylhydantoin and / or ammonium formate, and the at least one diluent is any of methanol, acetonitrile, ethanol, propanol, isopropanol, or any combination thereof.

5. In said at least one concentrated composition (44), (i) the cesium iodide has a concentration of 0.1 μg / mL to 100 mg / mL; (ii) ethylamine, when present, has a concentration of 0.01 μg / mL to 1 mg / mL, and formic acid, when present, has a concentration of 0.001% (v / v) to 10% (v / v); (iii) cyclosporin A, if present, has a concentration of 0.01 μg / mL to 1000 μg / mL; 5-(4-hydroxyphenyl)-5-phenylhydantoin, if present, has a concentration of 0.01 μg / mL to 1000 μg / mL; and ammonium formate, if present, has a concentration of 0.01 mM to 1 M; (iv) Methanol, water, or a mixture thereof is added up to 100%; The analytical system (100, 100') according to claim 4.

6. 2. The analytical system (100, 100') of claim 1, wherein the at least one diluent (42, 43) is any of methanol, acetonitrile, ethanol, propanol, isopropanol, or any combination thereof.

7. 2. The analytical system (100) of claim 1, wherein the analytical fluid system (10) includes a plurality of fluid streams (11, 12, 13), at least one fluid container containing a cleaning liquid (41), and the controller (90) is further configured to control the downstream pump (40) and the downstream valve (20) to connect to the ionization source (61) between two successive fluid streams to flush liquid from a previous fluid stream from the conduit (30) between the downstream valve (20) and the ionization source (61) with the cleaning liquid (41) before liquid from a subsequent fluid stream enters the conduit (30).

8. 2. The analytical system (100′) of claim 1, wherein the analytical fluid system (10′) includes at least one fluid stream (11, 12, 13) including an HPLC column, and at least one fluid container includes a cleaning solution (41), and the controller (90) is further configured to control the downstream pump (40) and the downstream valve (20) to connect to the at least one fluid stream (11, 12, 13) to backwash the at least one HPLC column with the cleaning solution (41), thereby cleaning the at least one HPLC column with the cleaning solution (41).

9. 1. An automated analytical method comprising the use of a mass spectrometer (60), connecting a downstream pump (40) to an ionization source (61) coupled to the mass spectrometer (60) via a downstream valve (20) disposed downstream of an analytical fluid system (10), the downstream valve (20) being disposed downstream with respect to the analytical fluid system (10, 10′) in a general flow direction through a fluid stream toward the ionization source (61), the downstream pump (40) being fluidly connected to a plurality of fluid containers containing respective fluids (41, 42, 43, 44), the fluids including at least one concentrated composition (44) for calibrating the mass spectrometer (60) and at least one diluent (42, 43) for diluting the at least one concentrated composition (44); controlling said downstream pump (40) to obtain at least one diluted composition (45) by automatically mixing at least one concentrated composition (44) with at least one diluent (42, 43) at a predetermined dilution ratio; injecting said at least one diluent composition (45) into said ionization source (61); obtaining a mass spectrum (62) of said at least one diluted composition (45); performing a calibration (63) of the mass spectrometer (60) based on an evaluation (64) of the mass spectrum (62); An automated analytical method comprising:

10. 10. The automated analytical method of claim 9, wherein performing the calibration (63) comprises checking and / or adjusting the mass axis.

11. 11. The automated analytical method of claim 10, wherein in the event of a failure or predicted failure of the mass axis check and / or mass axis adjustment based on the evaluation (64) of the mass spectrum (62) of the at least one diluted composition (45), the automated analytical method comprises performing one or more actions selected from adapting the dilution factor of the at least one concentrated composition, adjusting one or more mass spectrometry acquisition parameters, and performing a maintenance procedure before repeating the mass axis check and / or mass axis adjustment.

12. 12. The automated analytical method of any one of claims 9 to 11, wherein the at least one concentrated composition (44) comprises (i) cesium iodide, (ii) ethylamine and / or formic acid, (iii) methanol and / or water, and (iv) cyclosporin A and / or 5-(4-hydroxyphenyl)-5-phenylhydantoin and / or ammonium formate, and the at least one diluent is any of methanol, acetonitrile, ethanol, propanol, isopropanol, or any combination thereof.

13. In said at least one concentrated composition (44), (i) the cesium iodide has a concentration of 0.1 μg / mL to 100 mg / mL; (ii) ethylamine, when present, has a concentration of 0.01 μg / mL to 1 mg / mL, and formic acid, when present, has a concentration of 0.001% (v / v) to 10% (v / v); (iii) cyclosporin A, if present, has a concentration of 0.01 μg / mL to 1000 μg / mL; 5-(4-hydroxyphenyl)-5-phenylhydantoin, if present, has a concentration of 0.01 μg / mL to 1000 μg / mL; and ammonium formate, if present, has a concentration of 0.01 mM to 1 M; (iv) Methanol, water or a mixture thereof is added up to 100%; The automated analytical method according to claim 12.

14. 10. The automated analytical method of claim 9, further comprising controlling the downstream pump (40) and the downstream valve (20) to connect the ionization source (61) between two successive fluid streams of the analytical fluid system (10) to flush liquid from a previous fluid stream from the conduit (30) between the downstream valve (20) and the ionization source (61) with a washing liquid (41) before liquid from the subsequent fluid stream enters the conduit (30).

15. 10. The automated analytical method of claim 9, comprising controlling the downstream pump (40) and any one or more valves, including the downstream valve (20), to connect at least one fluid stream (11, 12, 13) of the analytical fluid system (10) including an HPLC column to backwash the HPLC column with a wash solution (41), thereby washing the HPLC column with the wash solution (41).

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