Valve leakage detection

Abstract

The present invention relates to a method for detecting an application valve leakage in an analytic system comprising an application valve fluidly connected to an eluent pump, to a trapping column, and to a detector unit, the method comprising (i) applying a sample to the trapping column via the application valve; (ii) applying the sample of step (i) to the detector unit; and (iii) determining at least one sample constituent in the dead time of the analytic system. The present invention also relates to an analytic system comprising an application valve fluidly connected to an eluent pump, a trapping column, and to a detector unit, configured to perform the method according to the present invention, and to methods for quality assurance and uses related thereto.

Description

[0001] Valve leakage detection

[0002] The present invention relates to a method for detecting an application valve leakage in an analytic system comprising an application valve fluidly connected to an eluent pump, to a trapping column, and to a detector unit, the method comprising (i) applying a sample to the trapping column via the application valve; (ii) applying the sample of step (i) to the detector unit; and (iii) determining at least one sample constituent in the dead time of the analytic system. The present invention also relates to an analytic system comprising an application valve fluidly connected to an eluent pump, a trapping column, and to a detector unit, configured to perform the method according to the present invention, and to methods for quality assurance and uses related thereto.

[0003] In chromatographic analysis systems, valves are used typically upstream of analytical columns. Specifically, the application valve (such as a rapid valve used in Rapid Liquid Chromatography (Rapid LC)) can be found in the injection system, which enables the switching between sample loading and elution modes. Usually the flow through the column is unidirectional, but e.g. in case of Rapid LC the sample is loaded in one direction and eluted in the opposite direction.

[0004] Valves, such as rapid valves, are spare parts in LC systems which need to be replaced regularly and which are critical for the correct function of the system, especially in e.g. fully integrated and automated chromatography and / or mass spectrometry clinical analyzers. Valves may connect an elution pump or a loading pump fluidically to the analytical column and further to the detector. In trap-and-elute settings, the loading pump is fluidically connected to the trapping column by the application valve in the loading position in frontflush mode. In the ejection position, the elution pump is fluidically connected to the trapping column in backflush mode and elutes the sample into the detector unit. The detector unit may be e.g. a mass spectrometer.

[0005] Valves are degrading over time due to frequent valve switching events, which cause wear-out of the stator and rotor, especially of the valve grooves on the rotor. Typically, the application valve is switched twice per injection. The wear-out of the valve may lead to leakage resulting in e.g. reduced back pressure, leakage of sample material, and / or leakage of mobile phase, and may incorrectly connect different instrument parts fluidically . In general, a broken valve may lead to decreased signal intensities up to missing peaks, fluidic leakages, and / or increased carry over. While a fluidic leakage may be rather easy to detect, decreased signal intensities are not. In case of Rapid LC, a broken valve may lead to an unwanted fluidic connection of loading pump, analytical column and the detector, leading to leakage of sample material directly into the detector unit during the sample loading step. This causes loss of sample material from the trapping column, and thus possibly loss of sensitivity and accuracy, i.e., potentially, to false results. Incorrect analytic results in turn may have a considerable negative impact on patient health if occurring in a clinical setting.

[0006] There is, thus, a need for improved methods for detecting application valve leakage, avoiding the drawbacks as referred to above. The technical problem underlying the present invention can be seen as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

[0007] In accordance, the present invention relates to a method for detecting an application valve leakage in an analytic system comprising an application valve fluidly connected to an eluent pump, to a trapping column, and to a detector unit, the method comprising

[0008] (i) applying a sample to the trapping column;

[0009] (ii) applying the sample of step (i) to the detector unit via the application valve; and

[0010] (iii) detecting at least one sample constituent in the dead time of the analytic system.

[0011] In general, terms used herein are to be given their ordinary and customary meaning to a person of ordinary skill in the art and, unless indicated otherwise, are not to be limited to a special or customized meaning. As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. Also, as is understood by the skilled person, the expressions "comprising a" and "comprising an" in an embodiment refer to "comprising one or more", i.e. are equivalent to "comprising at least one". In accordance, expressions relating to one item of a plurality, unless otherwise indicated, in an embodiment relate to at least one such item, in a further embodiment a plurality thereof; thus, e.g. performing "an analysis" relates to performing at least one analysis, in an embodiment to performing a multitude of analyses.

[0012] Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention.

[0013] The methods specified herein below are in vitro methods. The method steps may, in principle, be performed in any arbitrary sequence deemed suitable by the skilled person, but in an embodiment are performed in the indicated sequence; also, one or more, in an embodiment all, of said steps may be assisted or performed by automated equipment. Moreover, the methods may comprise steps in addition to those explicitly mentioned above.

[0014] As used herein, if not otherwise indicated, the term "about" relates to the indicated value with the commonly accepted technical precision in the relevant field, in an embodiment relates to the indicated value ± 20%, in a further embodiment ± 10%, in a further embodiment ± 5%. Further, the term "essentially" indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ± 20%, in a further embodiment ± 10%, in a further embodiment ± 5%. Thus, “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of’ encompasses any known acceptable additive, excipient, diluent, carrier, and the like. In an embodiment, a composition consisting essentially of a set of components will comprise less than 5% by weight, in a further embodiment less than 3% by weight, in a further embodiment less than 1% by weight, in a further embodiment less than 0.1% by weight of non-specified component(s).

[0015] The method of detecting an application valve leakage as described herein may be performed as a standalone method, to check valve tightness e.g. as part of a good practice protocol, such as a GLP protocol. The method may, however, also be integrated into methods of routine analyte measurements, in particular in highly automated settings. In such cases, the method described herein may be performed on the same sample used in an actual analyte measurement; in such case, the analyte measurement and the method of detecting an application valve leakage may be allocated to each other in the sense that the result of the latter has a potential impact on the former, i.e. in case application valve leakage is detected, the result of the analyte measurement may be deemed unreliable or invalid. Thus, a result of the analyte measurement and a result of the method of detecting an application valve leakage may be allocated to each other such that it is possible to identify that the two results were obtained using the same application valve. To that end, the method of detecting an application valve leakage may be performed after each at most 50000, in an embodiment after each at most 5000, in a further embodiment after each at most 1000, in a further embodiment after each at most 100, switching events of the application valve. As the skilled person understands, in such case, in case a valve leakage is detected by the method described herein, all analyte measurements following a preceding performance of the method of detecting an application valve leakage may have to be deemed unreliable or invalid, i.e. all analyte measurements following the preceding performance of the method of detecting an application valve leakage would be allocated to the most recent detecting. Thus, it is also envisaged that the method of detecting an application valve leakage is performed accompanying each analyte measurement, such that only one analyte measurement is allocated to a given performing of the method described herein. Also, the method of detecting an application valve leakage may be performed in case there is a suspicion of valve leakage; such suspicion may e.g. stem from detecting a pre-determined sample constituent at a nonpredetermined position during a preceding analytic run. E.g. an internal standard with a known elution time may be detected significantly before or after said known elution time. Suspicion of valve leakage may, however, also stem from a smaller than expected analyte and / or internal standard peak. In view of the description herein, the methods described can be performed on essentially any analytic system comprising the components indicated herein below, including analytic systems known in the art and being commercially available, such as trap-and-elute / MS devices, LC / MS or GC / MS devices, and the like.

[0016] The term “sample”, as used herein, relates to a sample known or suspected to comprise at least one analyte. Thus, the sample may e.g. be or comprise a sample of a subject, an environmental sample, or a technically produced composition of matter, such as an intermediate or final product of a production process, a calibration solution, or an external or internal standard. In an embodiment, the sample is or comprises a sample of a body fluid, a sample from a tissue or an organ, or a sample of wash / rinse fluid or a swab or smear obtained from an outer or inner body surface. The sample, in an embodiment, comprises at least one analyte as specified elsewhere herein. Samples of blood, plasma, serum, urine, saliva, or lacrimal fluid are encompassed as well. Samples can be obtained by use of brushes, (cotton) swabs, spatula, rinse / wash fluids, punch biopsy devices, puncture of cavities with needles or lancets, or by surgical instrumentation. However, samples obtained by well known techniques including, in an embodiment, scrapes, swabs or biopsies from the urogenital tract, perianal regions, anal canal, the oral cavity, the upper aerodigestive tract and the epidermis are also included as samples of the present invention. Cell-free fluids may be obtained from the body fluids or the tissues or organs by lysing techniques such as homogenization and / or by separating techniques such as filtration or centrifugation. In an embodiment, samples are obtained from body fluids known or suspected to comprise the analyte. Suitable sample types are selected by the skilled person depending in particular on the analyte of interest, accessibility of the sample material, and the like. In an embodiment, the sample is a blood sample or a blood-derived sample, such as a plasma or serum sample. It is to be understood that the sample may be processed, in particular pre-processed, in order to carry out the method of the present invention. Particularly, cells may be removed from the sample by methods and means known in the art. Moreover, at least one analyte may be extracted and / or purified from the sample by methods and means known in the art. Thus, the term sample also may relate to preparations comprising or suspected to comprise at least one analyte which were derived from a sample as specified herein above, e.g. which were diluted, enriched, purified and / or extracted from a sample as specified herein above.

[0017] The term “analyte”, as used herein, refers to any molecule which may or may not be present in a sample and the presence of which and / or amount of which shall be detected. In particular, an analyte may be a small molecule, a peptide, a protein, a oligonucleotide, a polynucleotide, such as an RNA or a DNA, a polymer or other macromolecule. Typically, however, analytes are small molecule compounds, in an embodiment with a molecular weight of less than 5000 Da, in a further embodiment less than 2000 Da. Thus, in an embodiment, the analyte has a molecular weight of from 1 Da to 5000 Da, in a further embodiment of from 10 Da to 3000 Da, in a further embodiment of from 25 Da to 2000 Da. In an embodiment, the analyte is comprised or suspected to be comprised in a sample as specified herein above, in particular a sample from a subject, in particular a bodily fluid sample. Thus, the analyte may be a chemical compound known or suspected to be present in the body of a subject or in a sample derived therefrom. Thus, the analyte may be a substrate for an enzyme of a metabolic pathway, an intermediate of such a pathway or a product obtained from a metabolic pathway. Thus, more typically, the analyte in accordance with the present invention may be a metabolite. Metabolic pathways are well known in the art and may vary between species. Preferably, said pathways include at least citric acid cycle, respiratory chain, photosynthesis, photorespiration, glycolysis, gluconeogenesis, hexose monophosphate pathway, oxidative pentose phosphate pathway, production and P-oxidation of fatty acids, urea cycle, amino acid biosynthesis pathways, protein degradation pathways such as proteasomal degradation, amino acid degrading pathways, biosynthesis or degradation of lipids, polyketides (including e.g. flavonoids and isoflavonoids), isoprenoids (including, e.g., terpenes, sterols, steroids, carotenoids, xanthophylls), carbohydrates, phenylpropanoids and derivatives, alcaloids, benzenoids, indoles, indole-sulfur compounds, porphyrines, anthocyans, hormones, vitamins, cofactors such as prosthetic groups or electron carriers, lignin, glucosinolates, purines, pyrimidines, nucleosides, nucleotides and related molecules such as tRNAs, microRNAs (miRNA) or mRNAs. Accordingly, small molecule compound metabolites are usually composed of the following classes of compounds: alcohols, alkanes, alkenes, alkines, aromatic compounds, ketones, aldehydes, carboxylic acids, esters, amines, imines, amides, cyanides, amino acids, peptides, thiols, thioesters, phosphate esters, sulfate esters, thioethers, sulfoxides, ethers, or combinations or derivatives of the aforementioned compounds. The small molecules among the metabolites may be primary metabolites which are required for normal cellular function, organ function or animal growth, development or health. Moreover, small molecule metabolites further comprise secondary metabolites having essential ecological function, e.g. metabolites which allow an organism to adapt to its environment. Furthermore, metabolites are not limited to said primary and secondary metabolites and further encompass artificial small molecule compounds. Said artificial small molecule compounds may be exogenously provided small molecules which are administered to or taken up by an organism but are not primary or secondary metabolites as defined above, or may be derived from such small molecules. For instance, artificial small molecule compounds may be metabolic products obtained from drugs by metabolic pathways of a subject, or may be the administered drugs as such. Also, the analyte may be a compound extraneously added to the sample, e.g. an internal standard. Moreover, a sample may comprise two or more analytes of interest, e.g. a compound administered to a subject and one or more of its metabolite(s). Also, an analyte present in the sample and its internal standard(s) may be analytes of interest within one sample.

[0018] In concurrence with the above, the term "sample constituent", as referred to herein, includes each and every molecule comprised in a sample; thus, an analyte, if present, is a sample constituent; also, compounds forming the sample matrix are sample constituents. In the context of the methods described herein, in principle any detectable sample constituent may be used; in an embodiment, a sample constituent present in high abundance in the sample is used; in a further embodiment, a sample constituent detectable by the same detection method as the analyte is used, i.e. in particular a sample constituent detectable by the detector unit as specified herein below. As the skilled person understands in view of the description herein, the sample constituent used in the methods described herein in an embodiment is a chemical molecule not present in detectable amounts in other compositions entering the detector unit, such as eluents. Thus, in an embodiment, the sample constituent is a compound specifically detectable in a sample. In an embodiment, the sample constituent used in the methods described herein is the analyte and / or an internal standard used in analyte determination. In case the detector unit comprises an analytic column, the sample constituent to be used according to the methods described herein may be selected to not be or only be negligibly retarded by the analytic column. Thus, in case the detector unit comprises an analytic column, the sample constituent to be used according to the methods described herein may be selected to not be retarded by the analytical column for more than the dead time of the analytical system.

[0019] The term "detecting", as used herein, is used in its common general meaning. In an embodiment, the term relates to bringing to attention, e.g. of an application valve leakage, in an embodiment to attention of an operator or a decision making algorithm. Thus, in an embodiment, the information that an application valve leakage was detected causes at least one of the following decisions to be taken: (i) that any analysis allocated to said detecting has a high probability of being incorrect and is flagged as unreliable, a result thereof is not output, and / or is repeated automatically on an analytic system not comprising said application valve; (ii) that no further analyses are performed on said analytic system unless the application valve was repaired and / or exchanged, and / or (iii) that the application valve must be repaired or exchanged.

[0020] The term “determining”, as used herein, refers to determining at least one characteristic feature of a sample constituent to be determined in a sample. Characteristic features in particular are features which characterize at least one physical, chemical, and / or biochemical property of a sample constituent. Such properties include, e.g., molecular weight, viscosity, density, electrical charge, spin, optical activity, color, fluorescence, chemiluminescence, elementary composition, chemical structure, capability to react with other compounds, and the like. Values for said properties may serve as characteristic features and can be determined by techniques well known in the art. Moreover, the characteristic feature may be any feature which is derived from a value of a physical and / or chemical property of a sample constituent by standard operations, e.g., mathematical calculations such as multiplication, division or logarithmic calculus. In an embodiment, the at least one characteristic feature allows the determination and / or chemical identification of the sample constituent and its amount. Accordingly, the characteristic value, in an embodiment, also comprises information relating to the abundance of the sample constituent from which the characteristic value is derived. For example, a characteristic value of a sample constituent may be a peak in a mass spectrum. Such a peak contains characteristic information of the sample constituent, i.e. the m / z information, as well as an intensity value being related to the abundance of the said sample constituent (i.e. its amount) in the sample. Moreover, determining as referred to in the methods of the present invention may, in an embodiment, include using a compound separation step prior to the determining step. In an embodiment, said compound separation step yields a time resolved separation of sample constituents. Suitable techniques for separation are described herein below and are known to the skilled person. A sample constituent comprised by a sample may be, in an embodiment, determined qualitatively, quantitatively, or semi-quantitatively. For quantitative determination, either the absolute or precise amount of the sample constituent will be determined or the relative amount of the sample constituent will be determined based on the value determined for the characteristic feature(s) referred to herein above. The relative amount may be determined in a case were the precise amount of a sample constituent can or shall not be determined. In such case, it can be determined whether the amount in which the sample constituent is present is increased or decreased with respect to a second sample comprising said sample constituent in a second amount, and / or with respect to a second sample constituent comprised in the sample in a second amount, which may be pre-determined; thus, the second sample constituent may e.g. be a sample constituent known to be present in the sample at a predetermined concentration, e.g. may be an internal standard added to the sample. Quantitatively analyzing a sample constituent, thus, also includes what is sometimes referred to as semi- quantitative analysis. Qualitative analysis, in an embodiment, is determining whether a sample constituent is present in a sample, in an embodiment above the detection threshold of the detection method. In view of the description herein below, the skilled person understands that in the context of the methods described herein, qualitative detection of a sample constituent in the dead time of the analytic system may frequently be sufficient to establish application valve leakage, although semi-quantitative or quantitative determination may be used as well, e.g. according to the methods proposed herein in the Examples.

[0021] The term “analytic system”, as used herein, relates to a system of means comprising at least the indicated means operatively linked to each other as to allow the result of the detection to be obtained. Thus, the analytic system comprises at least an application valve fluidly connected to an eluent pump, to a trapping column, and to a detector unit, all as specified herein below. Preferred means for applying a sample to a trapping column and to a detector unit, as well as for detecting at least one sample constituent, are in principle known to the skilled person and are discussed in more detail herein below. How to link the means in an operating manner will depend on the type of means included into the system. In an embodiment, the means are comprised by a single device. As referred to herein, the analytic system is configured to enable steps (i) to (iii) as indicated above, in an embodiment in the indicated order. Thus, in an embodiment, the analytic system comprises a controller unit configured to direct the steps as indicated to be performed. Also, the analytic system may comprise further components as deemed appropriate by the skilled person. The analytic system may in particular be a liquid analytic system or a gas analytic system.

[0022] In an embodiment, the analytic system comprises a sample treatment unit comprising a receptacle for a sample. The receptacle may directly contact the sample, or may be a receptacle for a further means receiving the sample, wherein the further means may be e.g. a multi-well plate, to which a sample or a plurality of samples may be applied. Moreover, the sample treatment unit, in an embodiment, comprises at least one reservoir for a sample pretreatment agent and a sample pretreatment agent, such as a diluent, a detergent, a precipitating agent, a buffer, an analyte binding agent, which may be bound to a solid surface, e.g. on a magnetic bead, and the like. In a further embodiment, the sample treatment unit comprises means for mixing and / or means for adjusting the temperature of a sample or a reaction mixture comprising a sample. The sample treatment unit may also comprise means for separation of sample constituents, such as a centrifuge, a magnet, a filter, and the like. In an embodiment, the analytic system comprises a waste container, which may be of any embodiment deemed appropriate by the skilled person.

[0023] In an embodiment, the analytic system comprises a sample loading unit comprising a loading pump, and optionally an injection valve for sample injection. The loading pump, in an embodiment, pumps a sample from a sample receptacle, in an embodiment from the sample treatment unit, or optionally from the injection valve, to the trapping column. The skilled person selects appropriate pumps in accordance with the requirements of the specific analytic system, in particular with the trapping column used and with relevant back pressure values, with requirements arising from sample type, diluent and / or liquid phase composition, and the like. The aforesaid pumping is via the application valve, in an embodiment via a sample loading port, a fluid connector, and an application port of the application valve in the loading position of the application valve. Thus, in an embodiment, the sample loading unit causes a loading pressure of from 1 MPa to 120 MPa, in an embodiment of from 2 MPa to 25 MPA, in a further embodiment of from 5 MPa to 15 MPa, in a further embodiment of about 10 MPa in said sample loading port, said fluid connector, said application port of the application valve, in the trapping column, and in interconnecting tubing.

[0024] In an embodiment, the analytic system comprises an input unit. The term "input unit", as used herein, relates to any arbitrary unit configured for a transfer of information from another entity to the system, in particular its data processing unit or a data storage medium, wherein another entity may be a further data processing device or a user. Thus, the input unit may comprise a user interface; the input unit may, however, also be a storage medium comprising a data collection, from which data, e.g. appropriate parameter values, may be retrieved.

[0025] In an embodiment, the analytic system comprises a data processing unit. The term “data processing unit” generally refers to an arbitrary unit adapted to perform or cause performing by the analytical system the method step(s) as described herein, in an embodiment by using at least one processor. Thus, as an example, the at least one data processing unit may comprise a software code stored thereon comprising a number of computer instructions. In an embodiment, the computer instructions, when executed on the microprocessor, cause the analytic system to perform a method as specified herein. The data processing unit may provide one or more hardware elements for performing one or more of the indicated operations and / or may provide one or more processors with software running thereon for performing one or more of the method steps.

[0026] In an embodiment, the analytic system further comprises a data output unit, in an embodiment operatively connected to a detector unit, an evaluation unit, and / or a data processing unit described herein below. The data output unit, in an embodiment, is adapted to output data obtained by the detector unit and, optionally, from other units of the analytic system. Suitable data output units are known to the skilled person and include simple output units such as an indicator signal or a display indicating that at least one sample constituent was detected in the dead time of the analytic system, in an embodiment above the detection threshold. An output unit may, however, also be an interface to an evaluation device, wherein said interface may be any kind of means of transferring data, including, e.g. cable connections like USB or LAN, wireless connections like wireless LAN, bluetooth, and the like, or indirect connections such as data transfer by instant messaging, email, or the like.

[0027] In an embodiment, the analytic system further comprises an evaluation device. As will be understood by the skilled person, the evaluation device may be comprised in the same housing as other components of the analytic system, e.g. as the detector unit, or may be a separate device. In an embodiment, the evaluation device comprises a microprocessor programmed to receive output data from an output unit and to perform logical operations providing an evaluation of said output data. Evaluation of output data may comprise, e.g., correcting data for values measured in at least one control, statistical calculations, e.g. calculating means of two or more parallel detection reactions, correcting data for dilution factors, comparing output data to reference values, peak finding and peak analysis, compiling data in a list, and the like. Evaluating data may, however, also comprise applying a decision making algorithm, e.g. as specified elsewhere herein, to the data obtained. In an embodiment, the evaluation device further comprises a data storage unit. In a further embodiment, said data storage unit comprises reference values, e.g. in a reference value data base. Moreover, in an embodiment, the data storage unit is adapted to store output data received from a system of the present invention. In an embodiment, in particular where means for automatically detecting at least one sample constituent are applied, the data obtained by said automatically determining can be processed by, e.g., a computer program in order to establish a result supporting a conclusion, e.g. detection of application valve leakage. Typical means for detection are disclosed in connection with embodiments relating to the methods of the invention herein elsewhere. The means may be operatively linked in that the result of the detection and the value thereof for the function of the application valve are provided to a user or to an automatic diagnostic unit. The results may be given as output of parametric diagnostic raw data, preferably, as absolute or relative amounts of at least one sample constituent detected. The output may, however, also be an information on the status of the application valve, or simply be a confirmation that an analysis allocated to the method for detecting an application valve leakage was or was not confounded by application valve leakage.

[0028] The analytic system comprises a trapping column. The term, "trapping column", is in principle known to the skilled person. In an embodiment, the term relates to each and every chromatographic column comprising at least one stationary phase and at least one mobile phase, configured to essentially immobilize at least one analyte from a sample, in an embodiment by binding to the stationary phase, e.g. by hydrophobic interaction, van der Waals forces, and / or ionic interaction(s). The skilled person selects appropriate stationary and mobile phases for a trapping column in dependence of well known parameters of an analyte of interest, such as hydrophobicity, charge, dipole moment, composition of eluent, and other known parameters. Appropriate trapping columns are known in the art. As the skilled person understands from the above, the trapping column may be used for trapping, i.e. binding, the analyte only, optionally in conjunction with non-binding of at least a part of the other sample constituents. The trapping column may, however, also additionally provide for chromatography of the analyte, e.g. in cases where a gradient of mobile phases is applied. The trapping column may be used in a trap-and- elute mode, i.e. the sample may be applied to the column in a first flow direction, while the analyte is eluted from the trapping column in a second, opposite, flow direction. In view of the description herein, it is irrelevant whether the sample constituent(s) used for determining in step (iii) are trapped, i.e. bound, by the trapping column, since in case of application valve leakage they enter the detector unit via a direct connection (leakage) between the application port and / or the sample loading port and the detector unit port in the loading position of the application valve; it will be appreciated that this is true also in case the sample constituent used in the method is the analyte and / or an internal standard. The analytic system also comprises an eluent pump. The term "eluent pump" is known to the skilled person and in an embodiment relates to a pump which pumps an eluent or a mixture of eluents from at least one reservoir to a detector unit. In an embodiment, the eluent pump is connected to an eluent pump port of the application valve. Also in an embodiment, in the loading position of the application valve, the elution pump port is connected via a fluid connector to the detector unit port of the application valve. Also in an embodiment, in the ejection position of the application valve, the elution pump port is connected via a fluid connector to the eluent port of the application valve; thus, in the ejection position, eluent may flow from the elution pump to the elution pump port, via a fluid connector to the eluent port, to the trapping column, then to the application port, then via a fluid connector to the detector unit port, and then to the detector unit. The skilled person selects appropriate elution pumps in accordance with the requirements of the specific analytic system, in particular of the trapping column used and with relevant back pressure values, with requirements arising from sample type, diluent and / or liquid phase composition, properties of the detector unit, and the like.

[0029] The term "detector unit" is known to the skilled person. In an embodiment, the term includes each and every means enabling determination of a compound desirable to be determined, wherein the compound desirable to be determined may be the sample constituent as specified herein above, the analyte, or both. Thus, the detector unit comprises at least one detector for detecting a compound desirable to be determined, the detector being adapted for performing a detection measurement detecting the compound desirable to be determined. The skilled person selects the detector according to the physical, chemical, and / or immunological properties of the compound desirable to be determined. The detector unit is configured to detect at least the sample constituent in step (iii); in case the detector of the detector unit cannot detect the analyte, the detector unit may comprise a second detector detecting the analyte. In an embodiment, however, the sample constituent and / or the detector are selected such that the detector detects the sample constituent and the analyte. Thus, in a further embodiment, the sample constituent is selected to be the analyte and / or an internal standard thereof. A light-absorbing sample constituent and / or analyte may be detected by optical means, e.g. by UV / VIS spectroscopy, a sample constituent and / or analyte having at least one immunologically detectable epitope may be detected by immunological means, and the like. In an embodiment, the detector unit comprises at least one mass spectrometry device. The detector unit may further comprise a means for enriching the analyte over other sample constituents, e.g. a chromatography unit comprising at least one chromatography column, in an embodiment as specified herein below in the context of chromatography / mass spectrometry. In an embodiment, however, the analyte is transferred from the trapping column, via the application valve, directly to the detector, in particular a mass spectrometry device. As the skilled person will understand in view of the description herein, any compound separation step, e.g. on a chromatographic unit, may be relevant for detecting the at least one sample constituent in step (iii) only in as far as may increase the time required for the sample constituent to become detectable. However, since sample leakage typically is a continuous process, there may be no defined start time of applying the sample constituent to the chromatography unit, in particular in case of long-duration (e.g. >10s) sample application, so it may not be possible to determine a retention time of sample constituents in the chromatography unit. The detector unit may be connected to the analytic system for the purpose of detecting application valve leakage; i.e. the detector unit may be different from the detector unit used for determining an analyte. In an embodiment, however, the detector unit used for detecting the at least one sample constituent is the same which is used for detecting the analyte and / or the internal standard, in particular in case the methods described herein are integrated into analytic methods, as described herein above. Thus, the detector unit may in particular be adapted for determining the analyte and the sample constituent. Thus, the detector unit in an embodiment is selected from the list consisting of a mass spectrometry (MS) device, a diode array detector (DAD), a UV / VIS detector, a conductivity monitor, a fluorescence detector, a refractive index detector, a radio flow detector, a chiral detector, an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD). In a further embodiment, the detector unit comprises, in a further embodiment is, a mass spectrometry unit.

[0030] The term "mass spectrometry", which may be abbreviated as "MS", is known to the skilled person. In mass spectrometry, analytes in a sample are ionized in order to generate charged molecules or molecule fragments. Afterwards, the mass-to-charge value of the ionized analyte or fragments thereof is measured. Ionization of molecules can be carried out by any method deemed appropriate, in particular by electron impact ionization, fast atom bombardment, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or matrix assisted laser desorption ionization (MALDI). Mass spectrometry as referred to herein encompasses all techniques which allow for the determination of the molecular weight (i.e. the mass) or a mass variable corresponding to a compound to be determined or a fragment thereof. In an embodiment, mass spectrometry is used in combination with a chromatographic separation step, in particular as gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS), direct infusion mass spectrometry or Fourier transform ion-cyclotrone-resonance mass spectrometry (FT-ICR-MS), capillary electrophoresis mass spectrometry (CE-MS), high-performance liquid chromatography coupled mass spectrometry (HPLC-MS), quadrupole mass spectrometry, any sequentially coupled mass spectrometry, such as MS-MS or MS-MS-MS, inductively coupled plasma mass spectrometry (ICP-MS), pyrolysis mass spectrometry (Py-MS), ion mobility mass spectrometry, or time of flight mass spectrometry (TOF). How to apply these techniques is well known to the person skilled in the art. Moreover, suitable devices are commercially available.

[0031] The analytic system also comprises an application valve. The term "valve" is understood by the skilled person; in an embodiment, the term relates to a device controlling flow rate and / or flow direction of a liquid or gas, in an embodiment in a closed system. In an embodiment, the valve is an automatic valve, i.e. in an embodiment is adapted for switching without interaction with an operator; thus, the automatic valve may be switched based on a status of the analytic system, such as e.g. start of sample application and / or start of sample ejection.

[0032] As is known to the skilled person, an analytic system typically has at least one valve, in an embodiment has a multitude of valves. As referred to herein, the "application valve" is the valve of an analytic system adapted for sample application to a trapping column in a loading position and to sample ejection to a detector unit in an ejection position. Thus, in an embodiment the application valve has at least two switching positions being a sample loading position and a sample ejection position. As the skilled person understands, the terms "sample loading" and "sample ejection" are used herein using the trapping column as a point of reference, i.e. sample loading relates to loading of a sample onto the trapping column, and sample ejection relates to eluting the sample from the trapping column, in an embodiment, into the detector unit. A trapping column typically causes a back pressure against sample application; thus, the analytic system may additionally comprise a sample loading unit comprising a sample loading pump, as specified herein above. In an embodiment, the sample is loaded through the application valve in the loading position with a pressure of at least 1 MPA, in an embodiment at least 2 MPa, in a further embodiment at least 3 MPa. In an embodiment, the sample is loaded through the application valve in the loading position with a pressure of at most 120 MPA, in an embodiment at most 50 MPa, in a further embodiment at most 20 MPa. In an embodiment, the sample is loaded through the application valve in the loading position with a pressure of from 1 MPa to 120 MPa, in an embodiment of from 3 MPa to 25 MPA, in a further embodiment of from 5 MPa to 15 MPa. In an embodiment, the application valve is a rapid application valve, i.e. an application valve of a rapid chromatography system, in particular of a rapid liquid chromatography system.

[0033] In the analytic system, the application valve is fluidly connected to an eluent pump, to a trapping column, and to a detector unit. Thus, the application valve comprises a multitude of ports, in an embodiment at least three, in an embodiment at least four, in a further embodiment at least five, in a further embodiment at least six ports, the term "port", in concurrence with common general use, relating to any means adapted for providing a fluid connection to the indicated unit. In an embodiment, the application valve comprises of from three to ten, in an embodiment of from four to eight, in a further embodiment of from five to seven, in a further embodiment six, ports. As referred to herein, ports may be referred to by designations indicating the unit or device they connect to; thus, the port connecting the application valve to the elution pump is the elution pump port; the port connecting the application valve to the detector unit is the detector unit port, and any port connecting the trapping column to the application valve is a trapping column port. Since the trapping column may be connected to the application valve on both ends, there may be two trapping column ports; in the case of trap-and-elute systems, the trapping column port connected to the loading pump in the loading position may also be referred to as the application port, and the trapping column port connected to the elution pump in the sample ejection position may be referred to as the eluent port. In an embodiment, the application port and the detector unit port are directly adjacent on the valve, i.e. there is no port intervening the application port and the detector unit port. In a further embodiment, the sample loading port and the detector unit port are directly adjacent on the valve.

[0034] In a further embodiment, in a non-leaking application valve, neither the application port nor the sample loading port are fluidly connected to the detector unit port in the sample loading position, however, the application port is fluidly connected to the detector unit port in the sample ejection position of the application valve. Also in an embodiment, the sample loading port, i.e. the port through which the sample is applied to the analytic valve, and the application port, are pressurized during sample loading, in an embodiment with a pressure of at least 1 MPa, in an embodiment at least 1 MPa, in a further embodiment at lest 3 MPa. In an embodiment, during sample loading, the pressure applied to the application port and the sample loading port is higher than the pressure applied to the detector unit port. Thus, in case a loading pump is used for sample loading, the pressure of the sample loading pump in an embodiment is higher than the pressure of the elution pump during sample loading.

[0035] The application valve further comprises at least one fluid connector, the term "fluid connector", as referred to herein, relating to any means providing a fluid connection between at least two ports of the application valve, in an embodiment between two ports of the application valve. Thus, the fluid connector, in an embodiment, is an internal structure of the application valve. The fluid connector may in particular be a groove or cavity in the application valve, in an embodiment having a geometry to connect a port to a first neighboring port in a first valve position, and to a second neighboring port in a second valve position, wherein said first neighboring port is non-identical to said second neighboring port. In an embodiment, the application valve has at least two, in a further embodiment at least three fluid connectors. The fluid connectors may be arranged such that each of the fluid connectors independently connects two ports; thus, the fluid connectors of the application valve are not mutually fluidly interconnected except for potential connections via elements exterior to the application valve, such as e.g. a tubing connecting a port connected to a first fluid connector to a second port connected to a second fluid connector. I.e., in an embodiment, the application valve comprises a multitude of fluid connectors arranged such that in a switching position each port is fluidly connected to one of its neighboring ports, but not to its other neighboring port. As regards exemplary port / fluid connector combinations and geometries, express reference is made to the Examples provided herein below. Iterations of the allocations specifically shown in the Examples which provide for the same functionality are accessible to the skilled person without further ado.

[0036] In an embodiment, the application valve comprises two mutually movable elements, in particular a rotor and a stator. Appropriate embodiments are known in the art. Movement of the rotor and the stator of the application valve against each other may cause abrasion and other damage, which may result in a new, undesirable fluid connection from the application port and / or the sample loading port to the detector unit port, i.e. may result in leakage. In an embodiment, application valve leakage is caused by a malfunction of the element separating the application port and the detector unit port, and or the element separating the sample loading port and the detector unit port. The term "leakage" is used herein in its meaning known to the skilled person; thus, in an embodiment, the term leakage relates to a spatially and / or timely inappropriate transfer of a liquid and / or a gas. In an embodiment, a leakage is loss of a liquid or a gas from an analytic system or a component thereof, in particular a valve thereof. More specifically, leakage of an application valve permits transfer of sample from an application port or from a loading unit port of an application valve to a detector unit port in a loading position, wherein said transfer in an embodiment does not include contacting the sample with the trapping column. As will be understood by the skilled person, said leakage, which is detectable with the methods described herein, may be seen to be indicative of the specific leakage detected, but may also be taken to be indicative of an application valve being unfit for further use and of the risk that other leakage may occur, e.g. from the elution pump port and / or the detector unit port to other ports of the application valve and / or to the exterior of the application valve, which in turn may cause a decrease of an analyte peak. Structurally, leakage may be caused by abrasion of components of the application valve, e.g. caused by repeated switching events, by an by inappropriate change in dimension of an element of the application valve, e.g. an inappropriate dilation of the outer enclosure of an application valve, by a fracture within the valve creating a connection between the application port or the loading unit port and the detector unit port, or the like.

[0037] As the skilled person is aware of, every analytic system has a, however small, internal volume between the sample, e.g. as bound to a trapping column, and the detector. Since a sample constituent, even if not interacting at all with components of the analytic system, cannot reach the detector before this internal volume has been transported through the analytic system, this internal volume is also referred to as the "dead volume" of the system. As the skilled person also understands, for a given analytic system and given flow rates, the dead volume of an analytic system can also be described as a "dead time" of the analytic system, i.e. the time required to transport the dead volume through the analytic system. Thus, the dead time is the minimum time required for any sample constituent to be transported from the trapping column to the detector. Thus, any sample constituent reaching the detector before the end of the dead time cannot have been transported via the regular sample transport process within the analytic system. As referred to herein, dead volume and dead time start with switching the application from the loading position to the ejection position.

[0038] In the context of the analytic system described herein, the term "connected" relates to a connection allowing for essentially lossless transport of a liquid or gas, in an embodiment under a pressure as indicated elsewhere herein. Thus, in such cases being connected typically is being fluidly connected or being gaseously connected.

[0039] The method comprises step (i) applying a sample to the trapping column via the application valve. Samples, as well as means and methods of applying a sample to a trapping column, have been described herein above. As referred to herein, the application valve is in the loading position during step (i). Specifically, a sample may be applied to a trapping column via an application port by a means suitable to overcome the back pressure of the trapping column. Such a means may be a syringe operated by an operator; in an embodiment, however, a loading pump is used, in an embodiment in conjunction with an injection valve. During sample application, at least for a fraction of the time required to apply the sample, the pressure in the application port and the sample loading port of the application valve is higher than the pressure at the detector unit port. Thus, sample leaking from the application port and / or the sample loading port of the application valve may be transferred into the detector unit port and, eventually, the detector unit.

[0040] The method further comprises step (ii) applying the sample of step (i) to the detector unit. The application valve is in the ejection position during step (ii); in an embodiment, step (ii) starts with switching the application valve to the ejection position. As described herein above, each analytic system has a dead time during which sample constituents cannot reach the detector by the regular connections of the analytical system. Nonetheless, applying the sample to the detector unit requires transporting eluent over the trapping column and the application valve into the detector. Thus, any sample material having entered the detector unit port already while the application valve was in the loading position will be transported to the detector, in an embodiment before the end of the dead time. Methods for applying a sample from a trapping column to a detector unit via an application valve are in principle known in the art and have been described herein above. In an embodiment, step (ii) comprises, the application valve being in the ejection position, pumping eluent over the analytic system, via the trapping column, the application valve, into the detector unit.

[0041] The method further comprises step (iii) determining at least one sample constituent in the dead time of the analytic system. The terms sample constituent, dead volume, and analytic system have been described herein above. The at least one sample constituent may be determined by any means deemed appropriate by the skilled person. In an embodiment, the method described herein is integrated into an analytic method and the at least one sample constituent is determined by the same detector, in an embodiment by the same method, as the analyte. As discussed herein above, the method may be or may be comprised in an analytic method for determining an analyte; in such case, the sample compound detected in step (iii) in an embodiment is the analyte and / or an internal standard. As will be understood, step (iii) does not necessarily have to comprise identifying the at least one sample constituent; in the context of the methods described herein, it may be sufficient to qualitatively determine that a sample constituent was determined. In an embodiment, however, quantitative methods of determining a sample constituent are applied.

[0042] Determining of a sample constituent in a dead time of an analytical system may be performed by any method deemed appropriate by the skilled person. In an embodiment, a mean signal, e.g. a mean intensity of an MS signal at a predetermined m / z range or value is calculated. This may be accomplished e.g. as shown herein in the Examples, i.e. as the average signal intensity over a predetermined time interval. This mean signal may be compared to a predetermined reference value. Such a reference value may e.g. be an averaged mean signal determined with a multitude of analytical systems with known non-leaking application valves. In such case, e.g. a ratio of the mean ratio of the analytical system of interest may be divided by the aforesaid averaged mean signal, wherein a ratio of essentially 1 in an embodiment would be deemed indicative of absence of application valve leakage, and a ratio of significantly higher than 1 could be deemed indicative of application valve leakage. Since application valve leakage tends to increase with increasing wear and tear, said ratio may also be used to categorize valve leakage, e.g. by assigning a ratio value of essentially 1 to absence of leakage, a ratio value of from 2 to 5 to beginning leakage, with possible automatic recommendation to the user to contact a service technician, and a ratio of higher than 5 being indicative of substantial leakage with possible automatic halting of all measurements on the affected analytic system and information of the user on substantial degradation with possible detrimental effects on measurement results.

[0043] Determining of a sample constituent in a dead time of an analytical system may also be performed by determining the ratio of the amount of sample constituent eluting in the dead time to the amount of sample constituent eluting at the expected runtime. As the skilled person will understand, this method may be used e.g. in case the sample constituent to be determined is the analyte and / or an internal standard thereof. The amount of sample constituent eluting in the dead time and the amount of sample constituent eluting at the expected runtime may be determined by performing standard peak detection measures, followed by peak height determination and / or peak integration to determine the respective amounts. As the skilled person will understand, it may not be necessary to determine amounts as absolute amounts or concentrations in such case, since also relative amounts or arbitrary units can be used. In an embodiment, a value of said ratio of at most 0.1 is deemed acceptable, i.e. indicative of no valve leakage, while a value of from more than 0.1 to 1 may be indicative of beginning valve leakage, while a value of more than 1, in an embodiment more than 2, is indicative of severe valve leakage which in an embodiment is not tolerable. As the skilled person understands in view of the description herein, since sample application may extend over substantial portions of the loading phase, a sample constituent peak in the dead time of the analytical system may have an unusual form including a plateau, so peak recognition algorithms may need adaptation to that end.

[0044] Specific Examples of methods for determining a sample constituent are provided herein in the Examples.

[0045] As the skilled person understands in view of the description herein, determining at least one sample constituent in the dead time of the analytic system is indicative of application valve leakage. Thus, the method may further comprise step (iv) detecting presence of application valve leakage in case at least one sample constituent is determined in the dead time of the analytic system, and detecting absence of application valve leakage in case no sample constituent is determined in the dead time of the analytic system.

[0046] In view of the above, the method may comprise further steps, e.g. step (oa), preceding step (i), switching the application valve into the loading position, and / or step (ia), preceding step (ii), switching the application valve into the ejection position. Further steps may e.g. relate to washing and / or regenerating the analytical system, in particular the trapping column and, optionally, any analytical column(s).

[0047] Advantageously, it was found in the work underlying the present invention that application valve leakage can be detected by detecting sample constituents in the dead time of an analytic system. The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.

[0048] The present invention further relates to an analytic system comprising an application valve fluidly connected to an eluent pump, a trapping column, and to a detector unit, configured to perform a method as specified herein.

[0049] The analytic system, its components, and optional and auxiliary units have been described herein above. The analytic system may be an analytic system comprising an MS unit as detector unit, in particular an LC / triple quadrupole mass spectrometry (LC / TQMS) system.

[0050] The terms “device” and "unit" are used essentially interchangeably herein, and relate to a collection of means which are operatively linked to each other to provide the indicated function, in an embodiment as part of the analytic system. Said means may be implemented in a single physical unit or in physically separated units which are operatively linked to each other. Suitable components and their properties are described elsewhere herein and also herein above in the context of the methods. Consequently, one or more methods of the present invention can be implemented by the analytic system specified herein. Thus, in an embodiment, the analytic system is configured to perform at least one method as specified elsewhere herein. The analytic system may comprise further units, in an embodiment as specified herein above, and / or any other units deemed appropriate by the skilled person.

[0051] The present invention also relates to a method for quality assurance in sample analysis, said method comprising

[0052] (a) determining at least one analyte in said sample,

[0053] (b) performing the method for detecting an application valve leakage described herein; and

[0054] (c) flagging the determination of step (a) as invalid in case a sample constituent is detected in step (b).

[0055] The method for quality assurance (QA) may be assisted or performed by automated equipment and may, in particular, be performed on an analytic system described herein elsewhere. Said method may be included in a QA process and / or in a method of analyte determination. The term "quality assurance" is used in its meaning known to the skilled person and, in an embodiment, relates to any measure taken to ensure and / or verify that a process, in particular an analyte measurement, fulfills pre-defined quality criteria. Such quality criteria may in particular be ensuring correctness of measurement within pre-defined error limits, prevention of sample cross-contamination, and the like. As the skilled person is aware of, the method for QA proposed herein may be one of a multitude of QA measures, which may relate to the same or different quality criteria.

[0056] The term "flagging a determination" is used herein in a broad sense including any measure allocating a QA relevant evaluation to at least one measurement and / or modifying a proceeding as a consequence thereof, if necessary. The flagging may be specific for the result of the aforesaid method, or may be generic, e.g. indicating that at least one QA criterion was not fulfilled. Thus, flagging a determination may be allocating an information to a result of step (a) that the result is invalid, has not passed QA, or the like. Flagging may, however, also be automatically repeating the measurement of step (a) on a different analytic system and / or on the same analytic system after exchanging or repairing the application valve, or may be not outputting a result of step (a) on an output unit.

[0057] The method comprises step (a) determining at least one analyte in the sample. Appropriate means and measures for performing step (a) are determined by the skilled person without further ado taking into account the analyte of interest, sample type, and other parameters.

[0058] The method comprises step (b) performing the method for detecting an application valve leakage, which has been described herein above. Step (b) may be performed concomitant to step (a), i.e., a sample constituent may be determined in the dead time while ejection of the analyte from a trapping column has started. As the skilled person understands in view of the description herein above, step (b) may be performed each time a step (a) is performed; step (b) may, however, also be performed after a multitude of steps (a) have been performed on the same or on different analyte(s) and / or on the same or different samples. Thus, in the method for QA, step (b) may be performed after step (a) was performed at most 50000 times, in an embodiment at most 5000 times, in a further embodiment at most 1000 times, in a further embodiment at most 100 times. The method comprises further step(s) (c) and / or (d), wherein step (c) is flagging the determination of step (a) as invalid in case a sample constituent is detected in step (b). As discussed herein above, flagging a determination is to be understood as a broad term. Also, in case a sample constituent is detected in step (b), alternatively or in addition the further measure of step (d) may be elicited, wherein step (d) is causing the analytic system to prevent any further sample application to said application valve. This may be achieved by any means deemed appropriate by the skilled person, e.g. by preventing switching to the sample loading position, by depressurizing the affected analytic system, e.g. by inactivating the loading pump, or the like. Thus, in an embodiment, further analyses are prevented from being performed on the analytic system. Moreover, repair or exchange of the application valve may be initiated. Also, in case no sample constituent is detected in step (b), the sample may be flagged as well, e.g. as having passed this specific QA method, by releasing the result from step (a), e.g. to an output unit, and the like.

[0059] The present invention also relates to a method of surveilling performance of an application valve comprised in an analytic system, said method comprising performing the method for detecting an application valve leakage described herein after at most 500000 application valve switching events.

[0060] Also, the present invention relates to a method of management of at least one application valve in an analytic system, comprising the steps of the method of surveilling performance of an application valve and the further step of replacing said application valve in case application valve performance is classified as non- satisfactory; wherein application valve performance is classified as non- satisfactory in particular in case application valve leakage is detected.

[0061] Furthermore, the present invention relates to a use of determining a sample constituent in a dead time of an analytic system for detecting application valve leakage.

[0062] The invention further discloses and proposes a computer program including computerexecutable instructions for performing a method according to the present invention in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network. Specifically, the computer program may be stored on a computer-readable data carrier. Thus, specifically, one, more than one or even all of method steps a) to d) as indicated above may be performed by using a computer or a computer network, in an embodiment by using a computer program.

[0063] The invention further discloses and proposes a computer program product having program code means, in order to perform a method according to the present invention in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network. Specifically, the program code means may be stored on a computer-readable data carrier.

[0064] Further, the invention discloses and proposes a data carrier having a data structure stored thereon, which, after loading into a computer or computer network, such as into a working memory or main memory of the computer or computer network, may execute a method according to one or more of the embodiments disclosed herein.

[0065] The invention further proposes and discloses a computer program product with program code means stored on a machine-readable carrier, in order to perform a method according to one or more of the embodiments disclosed herein, when the program is executed on a computer or computer network. As used herein, a computer program product refers to the program as a tradable product. The product may generally exist in an arbitrary format, such as in a paper format, or on a computer-readable data carrier. Specifically, the computer program product may be distributed over a data network.

[0066] Finally, the invention proposes and discloses a modulated data signal which contains instructions readable by a computer system or computer network, for performing a method according to one or more of the embodiments disclosed herein.

[0067] In an embodiment, referring to the computer-implemented aspects of the invention, one or more of the method steps or even all of the method steps of a method according to one or more of the embodiments disclosed herein may be performed by using a computer or computer network. Thus, generally, any of the method steps including provision and / or manipulation of data may be performed by using a computer or computer network. Generally, these method steps may include any of the method steps, typically except for method steps requiring manual work, such as providing the samples and / or certain aspects of performing the actual measurements. Specifically, the present invention further discloses:

[0068] A computer or computer network comprising at least one processor, wherein the processor is adapted to perform the method according to one of the embodiments described in this description, a computer loadable data structure that is adapted to perform the method according to one of the embodiments described in this description while the data structure is being executed on a computer, a computer program, wherein the computer program is adapted to perform the method according to one of the embodiments described in this description while the program is being executed on a computer, a computer program comprising program means for performing the method according to one of the embodiments described in this description while the computer program is being executed on a computer or on a computer network, a computer program comprising program means according to the preceding embodiment, wherein the program means are stored on a storage medium readable to a computer, a storage medium, wherein a data structure is stored on the storage medium and wherein the data structure is adapted to perform the method according to one of the embodiments described in this description after having been loaded into a main and / or working storage of a computer or of a computer network, and a computer program product having program code means, wherein the program code means can be stored or are stored on a storage medium, for performing the method according to one of the embodiments described in this description, if the program code means are executed on a computer or on a computer network.

[0069] In view of the above, the following embodiments are particularly envisaged:

[0070] Embodiment 1 : A method for detecting an application valve leakage in an analytic system comprising an application valve fluidly connected to an eluent pump, to a trapping column, and to a detector unit, the method comprising

[0071] (i) applying a sample to the trapping column via the application valve ;

[0072] (ii) applying the sample of step (i) to the detector unit; and

[0073] (iii) determining at least one sample constituent in the dead time of the analytic system. Embodiment 2: The method of embodiment 1, wherein said analytic system is an automated analytic system.

[0074] Embodiment 3 : The method of embodiment 1 or 2, wherein said analytic system is a liquid analytic system or a gas analytic system.

[0075] Embodiment 4: The method of any one of embodiments 1 to 3, wherein said application valve is an automatic application valve.

[0076] Embodiment 5: The method of any one of embodiments 1 to 4, wherein said application valve is a rapid application valve.

[0077] Embodiment 6: The method of any one of embodiments 1 to 5, wherein said application valve has at least two switching positions being a sample loading position and a sample ejection position.

[0078] Embodiment ?: The method of any one of embodiments 1 to 6, wherein the sample is loaded through the application valve in the loading position with a pressure of at least 3 MPa. Embodiment 8: The method of any one of embodiments 1 to 7, wherein said application valve comprises two mutually movable elements.

[0079] Embodiment 9: The method of any one of embodiments 1 to 8, wherein said application valve comprises a multitude of ports and at least one fluid connector.

[0080] Embodiment 10: The method of any one of embodiments 1 to 9, wherein said application valve comprises detector unit port and (i) an application port and / or (ii) a sample loading port, wherein said application port and / or sample loading port are not fluidly connected to said detector unit port in the sample loading position.

[0081] Embodiment 11 : The method of embodiment 10, wherein said application port and said detector unit port are fluidly connected in the sample ejection position.

[0082] Embodiment 12: The method 10 or 11, wherein said application port and / or said sample loading port is / are pressurized during sample loading.

[0083] Embodiment 13: The method of any one of embodiments 10 to 12, wherein said detector unit port is a port fluidly connected to the detector unit.

[0084] Embodiment 14: The method of any one of embodiments 10 to 13, wherein said application valve comprises at least six ports and wherein said application port and said detector unit port are two of said at least six ports.

[0085] Embodiment 15: The method of any one of embodiments 10 to 14, wherein the application port or the sample loading port is directly neighboring the detector unit port on the application valve. Embodiment 16: The method of any one of embodiments 9 to 15, wherein said application valve comprises a multitude of fluid connectors arranged such that in a switching position each port is fluidly connected to one of its neighboring ports, but not to its other neighboring port. Embodiment 17: The method of any one of embodiments 9 to 16, wherein each of said fluid connectors independently connects two of said ports.

[0086] Embodiment 18: The method of any one of embodiments 1 to 17, wherein said application valve comprises a rotor and a stator.

[0087] Embodiment 19: The method of embodiment 18, wherein said application valve is a six- port application valve, wherein the stator comprises six ports and the rotor comprises three fluid connectors, or wherein the rotor comprises six ports and the stator comprises three fluid connectors.

[0088] Embodiment 20: The method of embodiment 19, wherein a first application valve port is connected to a sample loading unit, a second application valve port is connected to the trapping column, a third application valve port is connected to the detector unit, a fourth application valve port is connected to an eluent pump, a fifth application valve port is connected to the trapping column, and a sixth application valve port is connected to an outlet, in an embodiment a waste outlet.

[0089] Embodiment 21 : The method of embodiment 20, wherein, in the sample loading position of the application valve, a first fluid connector connects the first and the second application valve port, a second fluid connector connects the third and the fourth application valve port, and a third fluid connector connect the fifth and the sixth application valve port.

[0090] Embodiment 22: The method of embodiment of embodiment 20 or 21, wherein, in the sample ejection position of the application valve, a first fluid connector connects the second and the third application valve port, a second fluid connector connects the fourth and the fifth application valve port, and a third fluid connector connects the sixth and the first application valve port.

[0091] Embodiment 23 : The method of any one of embodiments 1 to 22, wherein the system further comprises a chromatography column.

[0092] Embodiment 24: The method of any one of embodiments 1 to 23, wherein said sample constituent is determined by the detector unit, in an embodiment selected from the list consisting of a mass spectrometry (MS) device, a diode array detector (DAD), a UV / VIS detector, a conductivity monitor, a fluorescence detector, a refractive index detector, a radio flow detector, a chiral detector, an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD). Embodiment 25: The method of any one of embodiments 1 to 24, wherein said method is an analytic method for determining an analyte, and wherein the sample compound detected in step (iii) is the analyte and / or an internal standard.

[0093] Embodiment 26: The method of any one of embodiments 1 to 25, wherein said application valve leakage is associated with transfer of sample material onto the detector unit while the application valve is in the sample loading position.

[0094] Embodiment 27: The method of any one of embodiments 1 to 26, wherein said application valve leakage is caused by an increase in space between the application valve rotor and the application valve stator.

[0095] Embodiment 28: The method of any one of embodiments 10 to 26, wherein said application valve leakage is caused by a malfunction of the element separating the application port and the detector unit port and / or the element separating the sample loading port and the detector unit port.

[0096] Embodiment 29: The method of any one of embodiments 1 to 28, wherein said application valve leakage is caused by abrasion of at least part of the application valve rotor and / or the application valve stator.

[0097] Embodiment 30: The method of any one of embodiments 10 to 29, wherein said application valve leakage is caused by formation of a fluid connection from the application port to the detector unit port and / or from the sample loading port to the detector unit port in the sample loading position.

[0098] Embodiment 31 : The method of any one of embodiments 1 to 30, wherein said method is performed after at least one pre-determined sample constituent was detected at a nonpredetermined position during a preceding analytic run.

[0099] Embodiment 32: An analytic system comprising an application valve fluidly connected to an eluent pump, a trapping column, and to a detector unit, configured to perform the method according to any one of embodiments 1 to 31.

[0100] Embodiment 33: The analytic system of embodiment 32, wherein said detector unit is selected from the list consisting of a mass spectrometry (MS) device, a diode array detector (DAD), a UV / VIS detector, a conductivity monitor, a fluorescence detector, a refractive index detector, a radio flow detector, a chiral detector, an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD).

[0101] Embodiment 34: The analytic system of embodiment 32 or 33, wherein said analytic system is comprised in a chromatography-MS system, in an embodiment an LC / MS or GC / MS system. Embodiment 35: The analytic system of any one of embodiments 32 to 34, wherein said analytic system is comprised in an LC / triple quadrupole mass spectrometry (LC / TQMS) system.

[0102] Embodiment 36: A method for quality assurance in sample analysis, said method comprising

[0103] (a) determining at least one analyte in said sample,

[0104] (b) performing the method according to any one of embodiments 1 to 31; and

[0105] (c) flagging the determination of step (a) as invalid in case a sample constituent is detected in step (b) and / or

[0106] (d) causing the analytic system to prevent any further sample application to said application valve.

[0107] Embodiment 37: The method of embodiment 36, wherein the sample constituent in step

[0108] (b) is the analyte of step (a) or an internal standard.

[0109] Embodiment 38: The method of embodiment 36 or 37, wherein step (b) is performed concomitantly to step (a).

[0110] Embodiment 39: A method of surveilling performance of an application valve comprised in an analytic system, said method comprising performing the method according to any one of embodiments 1 to 31 after at most 500000 application valve switching events.

[0111] Embodiment 40: The method of embodiment 39, wherein said method is performed after at most 1000, in an embodiment 100, switching events.

[0112] Embodiment 41 : The method of embodiment 39 or 40, wherein said method comprises performing the method according to any one of embodiments 1 to 31 for each chromatographic run.

[0113] Embodiment 42: The method of any one of embodiments 39 to 41, wherein application valve performance is classified as non-satisfactory in case at least one sample constituent is determined in step (c) of said method.

[0114] Embodiment 43 : A method of management of at least one application valve in an analytic system, comprising the steps of the method according to any one of embodiments 39 to 42 and the further step of replacing said application valve in case application valve performance is classified as non-satisfactory.

[0115] Embodiment 44: Use of determining a sample constituent in a dead time of an analytic system for detecting application valve leakage. All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0116] Figure Legends

[0117] Fig. 1 : Schematic representation of an exemplary six-port application valve (rapid valve) in a trap-and-elute setup in the (A) loading and (B) ejection position; tubing connectors between units are depicted as solid lines, flow direction is indicated by arrows position.

[0118] Fig. 2: As in Fig. 1, but with an injection valve 30 and a loading pump 40.

[0119] Fig. 3: As in Fig. 1(A), but case of application valve leakage; sample leakage direction is indicated by a dashed arrow.

[0120] Fig. 4: Exemplary photographs of worn-out and leaking application valve units, (A) application valve rotor, (B) application valve stator; damaged sections are indicated by arrowheads.

[0121] Fig. 5: Chromatograms obtained with a defective (A) and a new (B) application valve in the experimental setup of Example 6. (C) Pressure graphs of loading and elution pumps of the application valves of (A) and (B).

[0122] Fig. 6: Exemplary schematic representation of a valve leakage detection algorithm.

[0123] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

[0124] Example 1 : Non-leaking six-port rapid valve (Fig. 1 and 2))

[0125] As a non-limiting exemplary embodiment, a six-port application valve 20 in an analytical system 10 is discussed: the stator may comprise six ports 110, 120, 140, 150, 160, 170 and the rotor may comprise three fluid connectors 210 (Fig. 1), or the rotor may comprise six ports 110, 120, 140, 150, 160, 170 and the stator may comprise three fluid connectors 210. In such case, in an embodiment, a first valve port (the sample loading port 160) is connected to sample loading unit 360, a second valve port (the first trapping column port 130, which may also be referred to as application port 140) is connected to trapping column 330, a third valve port (the detector unit port 120) is connected to detector unit 320, a fourth valve port (the elution pump port 110) is connected to eluent pump 310, a fifth valve port (the second trapping column port 130, which may also be referred to as eluent port 150) is connected to trapping column 330, and a sixth valve port (the waste port 170) is connected to waste 370. In such case, in the sample loading position of the application valve (Fig. 1(A) and 2(A)), a first fluid connector 210 connects sample loading port 160 and application port 140, a second fluid connector 210 connects detector unit port 120 and elution pump port 110, and a third fluid connector 210 connects eluent port 130 and waste port 170. Sample entering the analytic system via the sample loading port 160, e.g. from sample loading unit 360, flows through sample loading port 160, a fluid connector 210, application port 140, to trapping column 330, where the analyte and possibly other sample constituents are bound (trapped). Waste flows from trapping column 330 to eluent port 150, a further fluid connector 210, waste port 170, and into waste 370. As will be appreciated, there is no fluid connection between sample application port 160 and detector unit port 120 or between application port 140 and detector unit port 120 in the loading position of application valve 20.

[0126] In the sample ejection position of application valve 20 (Fig. 1(B) and 2(B)), a first fluid connector 210 connects elution pump port 110 and eluent port 150, a second fluid connector 210 connects application port 140 and detector unit port 120, and a third fluid connector 210 connects the sample loading port 160 and waste port 170. Eluent flows from eluent pump 310 to elution pump port 110, a fluid connector 210, eluent port 150, trapping column 30, application port 140, a second fluid connector 210, detector unit port 120, and to the detector unit. It will be appreciated that any sample material having entered detector unit port 120 will be transported to detector unit 320 in the sample ejection position of application valve 20.

[0127] Example 2: Leaking six-port rapid valve

[0128] As shown in Fig. 4, repeated switching of application valves 20 may lead to abrasions which create grooves in the valve material which can create undesirable fluid connections between ports 100 and possibly the exterior of the application valve. Fig. 3 shows schematically how leakage in the loading position of an application valve can lead to transfer of sample material from the application port 140 to the detector unit port 120. Possible flow of sample material is indicated by a dashed arrow.

[0129] It will be appreciated that there are other configurations which may lead to the same type of leakage; e.g. if sample application port 160 and application port 140 were swapped in Fig. 1 to

[0130] 3, sample application port 160 would be neighboring detector unit port 120, and sample material could be transferred from sample loading port 160 into detector unit port 120.

[0131] Also, abrasions as shown in Fig. 4 do not necessarily have to be restricted to form a connection between two neighboring ports. Thus, it is also envisaged that sample transfer may occur directly from sample loading port 160 into detector unit port 120.

[0132] Example 3 Acetaminophene was determined with a system setup essentially as shown in Fig. 2, with parameters shown in Table 1. Using a leaking application valve, the chromatogram of Fig. 5(A) was obtained, while with a new application valve, the chromatogram shown in Fig. 5(B) was obtained.

[0133] Table 1 : Sample composition and chromatographic method parameters

[0134] Results in Figure 5 show that an internal leakage in the rapid valve lead to an early eluting peak (about 2 sec after injection) as well as reduced peak height and area at the expected elution time (about 24 sec). By replacing in the defective rapid valve by a new one, the early eluting peak disappears and the initial and expected peak height is restored.

[0135] Example 4: Leakage detection via an average mean signal (reference)

[0136] The mean signal is calculated over an interval A in the dead time of the analytic system, i.e. within the loading phase, interval A:= [0, T / 2], where T is the total number of measured data points within the cycle of length T.

[0137] (Formula (1)), wherein < SA> is the mean signal measured during interval A and Si is the signal of the i-th data point within that interval. The obtained value is then normalized to the average background signal < SA>normalwhich is calculated in the same way but based on multiple measurements on different systems with known normal valve function. A failure is detected if this quantity is large:

[0138] (Formula (2)): Equation used to assess degradation of valve n.

[0139] This method is simple and straightforward but may not be able to differentiate from other errors, e.g. carry-over with increased signal levels in the interval A, which may also result in n »1 but the underlying problem is not valve failure. As a non-limiting example, when using the method according to this Example a valve is considered to be in the initial phase of the process of degradation, i.e. in a state where degradation does not influence results in a significant manner, if n ~1. A valve is considered defect if n»l; e.g. if n = 5 then substantial degradation has occurred or relevant degradation is imminent. If the n = 2-5 then the user is automatically informed and contacting a service technician is recommended. If n > 5 all pending measurements are halted and the user informed about substantial degradation that may significantly affect reliability of the obtained results.

[0140] Example 5: Leakage detection via peak analysis

[0141] As a further method to detect leakage, a peak detection algorithm was applied within the time interval of the loading phase, i.e. in the dead time of the analytic system, and the elution phase and the height H (alternatively area) of the detected peaks is determined, Subsequently, the ratio r of the height of the peaks in the loading and elution phase is computed:

[0142] Similar to Example 4, a threshold for r was defined to distinguish between a normal and abnormal situation. As a non-limiting Example, r < 0.1 was defined as normal and r > 0.1 was defined as abnormal. This means that roughly 10% penetration is tolerated. In the above example of Fig. 5(A) it was found that r=l .7, which is indicative of total valve failure with need for immediate exchange. We speculate that valve aging is a creeping process and that the evolvement of r can be tracked over time to predict the time point of total valve failure. Notably, with the aforesaid example application valve of Fig. 5(A), no change in pressure, neither at the loading pump nor at the elution pump was detected (Fig. 5(C)).

[0143] The approach of peak detection in Example 5 is not affected by an increased background signal. Various peak picking algorithms are readily available and can be used for the above purpose. For example, the Scipy signal processing package offers the function “peak find”. This function takes a 1-D array and finds all local maxima by simple comparison of neighboring values. A peak or local maximum is defined as any value whose two direct neighbors have a smaller amplitude and the position and height can be retrieved as an output. The input parameter “peak prominence” can be used to tune the sensitivity of the function in peak detection. The parameter is a measure of how much a peak stands out from the surrounding baseline of the signal and is defined as the vertical distance between the peak and its lowest contour line. In Example 5, a value of 2000 was effective to detect the main peaks and ignore irrelevant peaks. In general the value can be fixed or made assay dependent for increased specificity.

[0144] In conclusion, an algorithm for performing the method of Example 4 on several analytical measurements can be represented as shown in Fig. 4.

[0145] Example 6: Valve leakage detection algorithm

[0146] An exemplary, non-limiting algorithm for valve leakage detection based on a measurement as described herein above is shown schematically in Fig. 6:

[0147] Input data of a measurement are input to algorithm 600. To the input data preprocessing 610, such as filtering or smoothing to remove noise which could interfere with the peak picking process, is applied. Peak picking 620 is performed to determine peak height(s) and / or peak area(s) in the loading and elution phases. If several peaks are found in the respective phases, e.g. the highest peak may be selected for further steps. From the identified peaks, r, i.e. a peak height or peak area ratio may be calculated 630. Depending on the resulting value of r, an alarm may be issued 640, e.g. in case r is not < 0.01 ; in such case, the stream to the affected application valve may be interrupted to prevent any further operation. If the judgement criterion is passed, e.g. in case r is < 0.01, no warning is issued 650. In both cases, i.e. in case a warning is issued or not the method may optionally continue by loading the next incoming data set.

[0148] Reference signs

[0149] 10 analytic system

[0150] 20 application valve

[0151] 30 injection valve

[0152] 40 loading pump

[0153] 50 application valve rotor

[0154] 60 application valve stator

[0155] 100 application valve port

[0156] 110 elution pump port

[0157] 120 detector unit port

[0158] 130 trapping column port

[0159] 140 application port

[0160] 150 eluent port

[0161] 160 sample loading port

[0162] 170 waste port

[0163] 210 fluid connector

[0164] 310 elution pump

[0165] 320 detector unit

[0166] 330 trapping column

[0167] 360 sample loading unit

[0168] 370 waste

[0169] 510 Dead time peak

[0170] 520 Expected peak

[0171] 600 Valve detection method

[0172] 610 Data preprocessing

[0173] 620 Peak picking

[0174] 630 Peak ratio (r) computing

[0175] 640 Issuing alarm and / or preventing further application valve use

[0176] 650 Issuing no alarm

Claims

Claims1. A method for detecting an application valve leakage in an analytic system comprising an application valve fluidly connected to an eluent pump, to a trapping column, and to a detector unit, the method comprising(i) applying a sample to the trapping column via the application valve ;(ii) applying the sample of step (i) to the detector unit; and(iii) determining at least one sample constituent in the dead time of the analytic system wherein determining at least one sample constituent in the dead time of the analytic system is indicative of application valve leakage.

2. The method of claim 1, wherein said application valve is an automatic application valve, preferably is a rapid application valve.

3. The method of claim 1 or 2, wherein said application valve has at least two switching positions being a sample loading position and a sample ejection position.

4. The method of any one of claims 1 to 3, wherein the sample is loaded through the application valve in the loading position with a pressure of at least 3 MPa.

5. The method of any one of claims 1 to 4, wherein said application valve comprises a detector unit port and (i) an application port and / or (ii) a sample loading port, wherein said application port and / or sample loading port are not fluidly connected to said detector unit port in a sample loading position.

6. The method of claim 5, wherein said application port and said detector unit port are fluidly connected in the sample ejection position.

7. The method of claim 5 or 6, wherein said detector unit port is a port fluidly connected to the detector unit.

8. The method of any one of claims 5 to 7, wherein the application port or the sample loading port is directly adjacent to the detector unit port on the application valve.

9. The method of any one of claims 5 to 8, wherein said application valve comprises a multitude of fluid connectors arranged such that in a switching position each port is fluidly connected to one of its neighboring ports, but not to its other neighboring port, preferably wherein each of said fluid connectors independently connects two of said ports.

10. The method of any one of claims 1 to 9, wherein said sample constituent is determined by the detector unit, in an embodiment selected from the list consisting of a mass spectrometry (MS) device, a diode array detector (DAD), a UV / VIS detector, a conductivity monitor, a fluorescence detector, a refractive index detector, a radio flow detector, a chiral detector, an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD).

11. The method of any one of claims 1 to 10, wherein said method is an analytic method for determining an analyte, and wherein the sample constituent detected in step (iii) is the analyte and / or an internal standard.

12. The method of any one of claims 1 to 11, wherein said method is performed after at least one pre-determined sample constituent was detected at a non-predetermined position during a preceding analytic run or after at most 50000, in an embodiment at most 5000, in a further embodiment at most 1000, in a further embodiment at most 100 switching events of the application valve.

13. An analytic system comprising an application valve fluidly connected to an eluent pump, a trapping column, and to a detector unit, wherein said detector unit preferably is selected from the list consisting of a mass spectrometry (MS) device, a diode array detector (DAD), a UV / VIS detector, a conductivity monitor, a fluorescence detector, a refractive index detector, a radio flow detector, a chiral detector, an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD), wherein said analytic system further comprises a data processing unit adapted to perform or cause performing by the analytical system the method according to any one of claims 1 to 12.

14. A method for quality assurance in sample analysis, said method comprising(a) determining at least one analyte in said sample,(b) performing the method according to any one of claims 1 to 12; and(c) flagging the determination of step (a) as invalid in case a sample constituent is detected in step (b); and / or (d) causing the analytic system to prevent any further sample application to said application valve.

15. Use of determining a sample constituent in a dead time of an analytic system for detecting application valve leakage.

Images