Extraction of macrocyclic analytes

Abstract

The present invention relates to a method for extracting a macrocyclic analyte from a sample, said method comprising (a) producing a lysis admixture comprising said sample, a first organic solvent at a concentration of from 2.5% (v / v) to 18% (v / v), and an aqueous component at a concentration of at least 60% (v / v), and contacting said lysis admixture with a solid-phase extraction agent; (b) obtaining solid-phase extraction agent bound sample constituents; and (c) thereby extracting said macrocyclic analyte. The present invention also relates to systems, kits, extracts, and determining methods related thereto.

Description

[0001] Roche Diagnostics GmbH 04 September 2025

[0002] RD38635PC ST / NH

[0003] Extraction of macrocyclic analytes

[0004] The present invention relates to a method for extracting a macrocyclic analyte from a sample, said method comprising (a) producing a lysis admixture comprising said sample, a first organic solvent at a concentration of from 2.5% (v / v) to 18% (v / v), and an aqueous component at a concentration of at least 60% (v / v), and contacting said lysis admixture with a solid-phase extraction agent; (b) obtaining solid-phase extraction agent bound sample constituents; and (c) thereby extracting said macrocyclic analyte. The present invention also relates to systems, kits, extracts, and determining methods related thereto.

[0005] In vitro diagnostic (IVD) methods typically require a sample preparation step before performing the actual analysis. The sample preparation may imply several functions, like analyte release from the sample matrix, hemolysis in cases where analytes are entrapped within the erythrocytes (e.g. immunosuppressive drugs) and possibly analyte enrichment of low abundance analytes. Another important function of the sample preparation step is the removal or reduction of the sample matrix in order to avoid interferences; in particular, blood and blood- derived samples consist of a highly concentrated and complex mixture of proteins (albumins, immunoglobulins) and other biomolecules which may lead to problems in downstream analysis, e.g. obstruction or clogging of chromatography columns reducing their lifetime (Furey et al (2013) Taianta 115: 104), “drown out” or suppression of MS signals from the low-abundance analytes, and the like.

[0006] Mass spectrometry (MS), in particular liquid-chromatography tandem mass spectrometry (LC- MS / MS), has become the method of choice for analyte quantitation in in-vitro diagnostics. Especially in the case of the determination of small molecules having metabolites of similar structure, the specificity and accuracy of MS has become crucial for the generation of reliable results. Examples for such small molecules are vitamins like 25-hydroxy- vitamin D3, steroids like testosterone and immunosuppressive drugs like cyclosporine A and everolimus. In case of immunosuppressive drugs, the recommended matrix is whole blood, since these analytes are highly bound to erythrocytes (Al-Jenoobi, FI (2016), Austin Chromatography 3 : 1039; Sallustio, BC (2010), Bioanalysis 2: 1141), thus requiring a hemolysis step before their quantification. Conditions and reagents for hemolysis have been disclosed, e.g. by Ku et al., Journal of Pharmaceutical Sciences 63(l):60 (1974), by Reed & Yalkowsky, PDA J Pharm Sci and Tech 41 :37 (1987), in EP 4 154 010 Al, EP 2 032 979 Al, and EP 3 602 073 Al. For 25-hydroxy- vitamin D3, testosterone, cyclosporine A and everolimus it is also known that these analytes are bound to matrix proteins in the sample and require release from the matrix proteins before quantification: e.g. of cyclosporine A ("Personalized Immunosuppression in transplantation: role of biomarker monitoring and therapeutic drug monitoring", Oellerich & DasGupta (2016): Elsevier, Amsterdam) in blood, 50 - 70 % are bound to erythrocytes, and of the cyclosporine A found in plasma, 98% is bound to lipoproteins, whereas only 2% is found free in plasma. Similarly, of everolimus (Oellerich & DasGupta, loc. cit.) in blood, approx. 75% are bound to erythrocytes, and of the everolimus found in plasma, 75% is bound to plasma proteins, whereas 25% is found free in plasma. Extraction methods for immunosuppressive drugs have been proposed e.g. in US 2012 / 0134895 A.

[0007] The sample preparation step, including the sample pretreatment reagent, is the first and the most critical part of method development in LC-MS / MS analysis and other IVD quantitation methods / assays, as it directly impacts on the accuracy of the analytical method. The most popular pretreatment method from the state-of-the-art is the treatment of the samples (serum-, plasma- or whole blood) with organic solvents like methanol or acetonitrile, sometimes also with sulfate salts like ZnSCU as helping additive for precipitation, inducing an analyte release from the sample matrix and at the same time precipitation of matrix components (Sallustio, BC, loc. cit.; Gomes et al. (2013), Bioanalysis 5: 3063). However, these methods require removal of a precipitate and are, therefore, not compatible with some downstream applications, e.g. bead-enrichment for LC-MS / MS; moreover the organic solvent may inhibit analyte capture by the beads. Thus, bead enrichment LC-MS / MS methods typically require aqueous-based solutions for pre-analytics. Pretreatment reagents used for sample preparation in a bead enrichment workflow are ideally provided as stable and ready-to-use solutions. These solutions can be placed on-board of the instrument for a long period of time offering a workflow not needing user interactions.

[0008] Also, proteolytic sample treatment can be used for the removal of the sample matrix and the analyte release (US 7964363 B2; US 8221986 B2). However, proteolysis reagents produce a multitude of peptide fragments from matrix proteins, which may hinder a bead-enrichment workflow by occupying precious bead positions. As a consequence, analyte capture by the beads is not possible in a complete manner leading to analyte and sensitivity loss. Furthermore, proteolytic pretreatment methods require usually more than one pipetting step with different reagents, which leads to more complex and time consuming workflows on the analyzer.

[0009] Chaotropic reagents are also known in the literature as reagents for protein denaturation and cell solubilization (Peterson PA (1971), J Biol Chem 246, 7748; Marston FAO (1990), Methods Enzymol, 182:264) and are applied as release reagents in assays (17). However, typical formulations use highly dosed reagents, with 6-8 M concentrations of urea or guanidine HC1, which often cause interferences in LC-MS / MS measurements (Samskog J (2003), J Chromatography A, 998:83; Antignac JP (2005, Anal Chim Acta, 529: 129; Chiu ML (2010), JALA 15:233; Proc et al. (2010), J Proteome Res 9: 5422).

[0010] There is, thus, a need in the art for improved means and methods for releasing analytes such as macrocyclic analytes from samples, in particular for pretreatment reagents providing good extraction efficiency and not interfering with downstream applications such as solid-phase bead enrichment. This problem is solved by the means and methods disclosed herein.

[0011] In accordance, the present invention relates to a method for extracting a macrocyclic analyte from a sample, said method comprising

[0012] (a) producing a lysis admixture comprising said sample, a first organic solvent at a concentration of from 2.5% (v / v) to 18% (v / v), and an aqueous component at a concentration of at least 60% (v / v), and contacting said lysis admixture with a solid-phase extraction agent;

[0013] (b) obtaining solid-phase extraction agent bound sample constituents; and

[0014] (c) thereby extracting said macrocyclic analyte.

[0015] 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. identifying "a cell" relates to identifying at least one cell, in an embodiment to identifying a multitude of cells.

[0016] 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.

[0017] The methods specified herein below are in vitro methods, in an embodiment in vitro diagnostic methods or methods for providing extracts for diagnostic 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.

[0018] Thus, the methods may be or comprise steps aiding a medical practitioner in diagnosing a condition or disease and / or in deciding on further proceeding. As the skilled person will understand, the methods described herein may be, but in an embodiment are not, the only basis for said diagnosis and / or decision. In an embodiment, the decision takes into account further parameters obtained by further methods of obtaining information, such as medical examination of the subject, including e.g. diagnostic tests, and the like. Unless indicated otherwise herein, the methods may be performed under standard conditions, in particular SATP conditions; other conditions may, however, be envisaged, e.g. performing the methods or steps thereof under cold conditions, e.g. at of from 0°C to 10°C, in an embodiment of from 4°C to 8°C.

[0019] As used herein, the term "standard conditions", if not otherwise noted, relates to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e. in an embodiment, a temperature of 25°C and an absolute pressure of 100 kPa; also in an embodiment, standard conditions include a pH of 7. Moreover, 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).

[0020] The term "macrocyclic analyte", as used herein, relates a class of compounds comprising a macrocyclic structure. In an embodiment, the macrocyclic structure comprises of from 12 to 50 ring atoms, in an embodiment of from 12 to 40 ring atoms, in a further embodiment of from 15 to 35 ring atoms. In an embodiment, the macrocyclic analyte comprises 33 ring atoms, such as in ciclosporine, or the macrocyclic analyte comprises 23 ring atoms, such as in tacrolimus and in pimecrolimus; or the macrocyclic analyte comprises 31 ring atoms, such as in sirolimus, everolimus, and temsirolimus. In an embodiment, the macrocyclic analyte is a cyclic peptide, such as in ciclosporine, or is a macrolide, i.e. comprises a macrocyclic lactone structure. As is understood by the skilled person, the macrocyclic analyte may comprise further ring structures directly or indirectly attached to the macrocyclic ring. Moreover, the macrocyclic analyte may further comprise organic and / or inorganic side chains, including in particular methyl groups, ethyl groups, ethylene groups, keto groups, hydroxyl groups, cyclic alkyl groups, sugar residues, and the like. The macrocyclic analyte is, in an embodiment, an immunosuppressant, in a further embodiment an immunosuppressant in clinical use. Thus, in an embodiment, at least one macrocyclic analyte is cyclosporine (3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl- 33-[(lR,2R,4E)-l-hydroxy-2-methylhex-4-en-l-yl]-6,9,18,24-tetraisobutyl-3, 21 -diisopropyl- l,4,7,10,12,15,19,25,28-nonamethyl-l,4,7,10,13,16,19,22,25,28,31- undecaazacyclotritriacontane-2,5,8,11,14,17,20,23,26,29,32-undecone, CAS No. 59865-13-3; is everolimus ((lR,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-l,18- dihydroxy-12-[(2R)-l-[(lS,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]- 19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-l l,36-dioxa-4-azatri cyclo [30.3.1.04,9] hexatriaconta- 16,24,26,28-tetraene-2,3 , 10,14,20-pentone, C AS-number 159351 -69-6), is tacrolimus ((lR,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-l,14-dihydroxy-12- [(lE)-l-[(lR,3R,4R)-4-hydroxy-3-methoxycyclohexyl]prop-l-en-2-yl]-23,25-dimethoxy-

[0021] 13.19.21.27-tetramethyl- 17-(prop-2-en- 1 -yl)- 11 ,28-dioxa-4-azatricy clo [22.3.1.04,9] octacos-

[0022] 18-ene-2,3,10,16-tetrone, CAS number 104987-11-3), is sirolimus ((1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E, 30S,32S,35R)-l,18-dihydroxy-12-

[0023] {(2R)-l-[(lS,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-2-propanyl}-19,30-dimethoxy- 15,17,21,23,29,35-hexamethyl-l l,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-

[0024] 16.24.26.28-tetraene-2,3,10,14,20-pentone, CAS number 53123-88-9), is pimecrolimus

[0025] ((lR,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-12-[(lE)-l-[(lR,3R,4S)-4-chloro-3- m ethoxy cyclohexyl]prop- 1 -en-2-yl]- 17-ethyl- 1 , 14-dihydroxy-23 ,25-dimethoxy- 13 , 19,21 ,27- tetramethyl- 11 ,28-dioxa-4-azatricy clo[22.3.1.04,9]octacos- 18-ene-2,3 , 10,16-tetrone, CAS number 137071-32-0), or is temsirolimus ((lR,2R,4S)-4-[(2R)-2- [(lR,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-l,18-dihydroxy-19,30- dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-l l,36-dioxa-4- azatricy clo[30.3.1.04,9] hexatriaconta- 16, 24, 26, 28-tetraen-12-yl]propyl]-2-methoxy cyclohexyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate, CAS number 162635-04-3). In an embodiment, the analyte is selected from ciclosporin, everolimus, tacrolimus, and sirolimus, in an embodiment is selected from Cyclosporine A, everolimus, tacrolimus, and sirolimus.

[0026] The term "organic solvent" is understood by the skilled person; in an embodiment, the term relates to an organic compound, i.e. a compound comprising at least one carbon atom in its structure, having the capacity of dissolving solutes. In an embodiment, the organic solvent is an aliphatic solvent. In an embodiment, the organic solvent is a polar solvent, in an embodiment with a dielectric constant of from 15 F / m to 50 F / m. In an embodiment, the organic solvent comprises, in an embodiment is, isopropanol (2-propanol, CAS No. 67-63-0), dimethyl sulfoxide (DMSO, CAS No. 67-68-5), methanol (CAS No. 67-56-1) and / or acetonitrile (CAS No. 75-05-8). As the skilled person understands in view of the description herein, the organic solvent may be selected independently from the aforesaid list for the lysis solution, the wash solution(s), and the elution solution. Thus, the organic solvent in the lysis solution, which may also be referred to as first organic solvent, in an embodiment is isopropanol and / or dimethyl sulfoxide. In a further embodiment, the organic solvent in the wash solution, which may also be referred to as second organic solvent, is methanol and / or acetonitrile, in an embodiment is methanol, in a further embodiment is methanol in a first washing step, and / or is acetonitrile in any further washing steps. In a further embodiment, the organic solvent in the elution solution, which may also be referred to as third organic solvent, is acetonitrile.

[0027] The term "organic acid" is understood by the skilled person as well. In an embodiment, the term relates to a chemical compound comprising at least one -COOH group in its structure. In an embodiment, the organic acid is a Ci to C> organic acid, in an embodiment is a monocarbonic acid, in a further embodiment is a Ci to C> monocarbonic acid. In an embodiment, the organic acid is formic acid or acetic acid, in an embodiment is formic acid.

[0028] The term "base", as used herein, relates to a compound inducing an increase in pH in an aqueous solution. In an embodiment, the base is a Bronsted-Lowry base. In a further embodiment, the base is a compound comprising or generating hydroxide ions in an aqueous solution. In a further embodiment, the base is ammonia (NH3, CAS No. 7664-41-7), in a further embodiment an aqueous solution comprising ammonia.

[0029] The term "aqueous component", as referred to herein, includes each and every hyposmotic liquid composition comprising water, in an embodiment comprising at least 50% (v / v), in an embodiment at least 60% (v / v), in a further embodiment at least 70% (v / v), in a further embodiment at least 80% (v / v) water. In an embodiment, the aqueous component is water or a hyposmotic solution of at least one salt, in particular a buffer, in a further embodiment is water. In a further embodiment, the aforesaid hyposmotic solution is a solution with an osmolarity of at most 0.1 Osm / L, in an embodiment of at most 0.05 Osm / L, in a further embodiment at most 0.01 Osm / L. As the skilled person understands, the osmolarity of water as aqueous component is 0 Osm / L. In a further embodiment, the aqueous component is comprised in the lysis admixture at a concentration of from 60% (v / v) to 95% (v / v), in an embodiment of from 65% (v / v) to 87.5% (v / v), in a further embodiment of from 65% (v / v) to 80% (v / v). Since the aqueous component is hyposmotic, the lysis admixture is hyposmotic as well, in an embodiment with a maximal osmolarity as specified herein above for the hyposmotic solution. For the avoidance of doubt, as referred to herein, the aqueous component is a component in the lysis admixture in addition to the sample; thus, e.g. in case the aqueous component is water, water comprised in the sample is not taken into account when determining the concentration of the aqueous component.

[0030] The term "solid-phase extraction agent" is known to the skilled person and includes each and every agent allowing the macrocyclic analyte to bind, in an embodiment under the conditions of step (a). Thus, the solid-phase extraction agent in an embodiment is a hydrophobic solidphase extraction agent, i.e. in an embodiment consisting of a hydrophobic material and / or comprising a hydrophobic surface. In an embodiment, the solid-phase extraction agent has a high surface-to-volume ratio, thus, the solid-phase extraction agent may e.g. be a hydrophobic chromatography material, which may be or may not be packed in a chromatographic column. In an embodiment, the solid-phase extraction agent comprises beads, in a further embodiment magnetic beads, in a further embodiment with hydrophobic properties. Thus, the solid-phase extraction agent in an embodiment comprises solid phase extraction beads, in a further embodiment comprises magnetic solid phase extraction beads as described e.g. in WO 2019 / 141779 Al.

[0031] As is understood by the skilled person, the term "extracting" includes each and every process causing partial or complete separation of a compound of interest, in an embodiment an analyte, in a further embodiment a macrocyclic analyte as referred to herein, from a composition of matter, wherein said composition of matter may be known or suspected to comprise said compound of interest. In an embodiment, said extracting comprises binding the macrocyclic analyte to a solid-phase extraction agent, in an embodiment as specified in step (a), and elution of at least part of said macrocyclic analyte from said solid-phase extraction agent, in an embodiment as specified in step (b), and optionally at least one washing step preceding the elution. The washing step and / or the elution step may be repeated, e.g. may be performed twice, three times, or even four times. As the skilled person understands in view of the description herein, depending on the specific downstream application envisaged, it may be sufficient that the compound of interest, in particular the macrocyclic analyte, is concentrated relative to the sample, and / or is enriched relative to other sample constituents. Extraction may, however, also include purifying or essentially purifying the macrocyclic analyte. As is also understood by the skilled person, depending on the specific downstream application envisaged, extraction may be satisfactory already if non-analyte sample constituents (matrix compounds) were removed to an extent allowing the downstream application to be performed successfully. Also, it may be sufficient to increase analyte concentration compared to analyte concentration in the sample, irrespective of the fate of matrix components. Also depending on the specific downstream application envisaged, it may also be sufficient to extract a fragment of an analyte as specified herein below. In an embodiment, however, the analyte is extracted as such.

[0032] The term “sample”, as used herein, relates to a sample known or suspected to comprise a macrocyclic analyte as specified herein. In an embodiment, the sample is an aqueous sample, in a further embodiment is a biological sample, in a further embodiment 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. In an embodiment, samples of stool, urine, saliva, cerebrospinal fluid, blood, serum, plasma, or lacrimal fluid are encompassed as samples by the method of the present invention. 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. In an embodiment, samples are obtained from body fluids known to comprise macrocyclic analytes if these are present in the body of s subject, i.e., in an embodiment, blood, plasma, serum, saliva, or the like. It is to be understood that a sample may be further processed in order to carry out the method of the present invention. In an embodiment, the sample is a blood sample or a blood-derived sample comprising cells. In a further embodiment, the sample is a blood sample, in an embodiment a whole blood sample. In an embodiment, the sample comprises protein at a concentration of at least 1 mg / mL, in a further embodiment at least 5 mg / mL, in a further embodiment at least 10 mg / dL. As used herein, the term sample also includes subparts, in particular aliquots of a sample. Thus, producing a lysis admixture comprising said sample as specified herein may also be producing a lysis admixture comprising an aliquot of the sample originally provided. The aforesaid applies mutatis mutandis to "contacting a lysis admixture" with a solid-phase extraction agent; i.e. said expression includes contacting an aliquot of the lysis admixture with a solid-phase extraction agent. The term “contacting” is understood by the skilled person and in an embodiment relates to bringing a first composition of matter, e.g. a lysis solution, in physical contact with a second composition of matter, e.g. a sample, thereby allowing the first composition of matter and the second composition of matter to interact.

[0033] The term "lysis admixture", as referred to herein, relates to any admixture of a sample with a lysis solution as specified herein below. The lysis admixture comprises the sample, an organic solvent, which may also be referred to as first organic solvent, at a concentration of from 2.5% (v / v) to 18% (v / v), and an aqueous component at a concentration of at least 60% (v / v), all as specified herein above. As per convention in the art, a concentration indication of a compound relates to the respective solution comprising the compound. Thus, a concentration indication, e.g. X% (v / v), for the lysis admixture relates to the concentration of the indicated compound in the (complete) lysis admixture comprising the sample, the first organic solvent, and the aqueous component. Percentages not accounted for are in an embodiment sample, (additional) aqueous component, and / or water, in a further embodiment are sample. In conclusion, the lysis admixture produced in step (a) in an embodiment is a lysis admixture comprising a first organic solvent at a concentration of from 2.5% (v / v) to 18% (v / v), an aqueous component at a concentration of at least 60% (v / v), and said sample to 100%. Thus, in case the lysis admixture comprises 60% (v / v) aqueous component and 15% (v / v) organic solvent, the lysis admixture in an embodiment comprises 25% (v / v) sample, i.e. the sample is diluted fourfold in the lysis admixture compared to the starting concentration.

[0034] As indicated herein above, the organic solvent in the lysis solution and in the lysis admixture in an embodiment is isopropanol, dimethyl sulfoxide (DMSO), or a mixture thereof, in an embodiment is isopropanol or DMSO. The concentration of the organic solvent in the lysis admixture is of from 2.5% (v / v) to 18% (v / v), in an embodiment of from 2.7% (v / v) to 18% (v / v), in a further embodiment of from 4% (v / v) to 15% (v / v), in a further embodiment of from 5% (v / v) to 10% (v / v). As the skilled person understands in view of the description herein, the indicated composition of the lysis admixture can be produced by any proceeding deemed appropriate by the skilled person, provided that the composition as specified is achieved. Thus, on a strictly exemplary basis, the lysis admixture may be produced by admixing an aqueous lysis solution into a sample (or vice versa) to achieve the prescribed concentration of the first organic solvent and of the aqueous component; in such case, the lysis solution may e.g. be a two-fold concentrated lysis stock solution of which one part is admixed with one part of the sample, or a 4 / 3 fold lysis stock solution, of which three parts are diluted with one part of sample, or an 1.1 fold lysis stock solution, of which 9 parts are admixed with one part of sample. In a further embodiment, the lysis admixture may be produced by adding the first organic solvent and the aqueous component separately to the sample at amounts to achieve the indicated final concentrations; in such case, the aqueous component in an embodiment is added to the sample before the organic solvent, to avoid precipitation of proteins. The lysis admixture, in an embodiment, has the effect of lysing cells possibly present in a sample, in particular has the effect of hemolyzing red blood cells if present in the sample. In an embodiment, the lysis admixture does not cause precipitation of proteins, thus, in an embodiment, at most 25%, in a further embodiment at most 10%, in a further embodiment at most 5%, in a further embodiment at most 1%, of all protein present in a sample is precipitated in the lysis admixture. In an embodiment, the lysis admixture does not comprise precipitated protein. The lysis admixture may comprise further compounds as deemed appropriate by the skilled person, e.g. auxiliary components, such as a buffer and / or a salt. In an embodiment, however, the lysis admixture does not comprise a chaotropic agent and / or does not comprise a detergent. In an embodiment, the lysis admixture causes the macrocyclic analyte to become bound to the solid-phase extraction agent if the macrocyclic analyte and the solid-phase extraction agent are contacted in the presence of the lysis admixture in step (a).

[0035] In view of the description herein above, a "lysis solution", as the term is referred to herein, is any solution usable for producing a lysis admixture as specified herein above. Thus, the lysis solution in an embodiment has the activity of causing lysis of cells, such as red blood cells, in a sample, if present. Thus, the lysis solution typically may comprise the first organic solvent at a concentration higher than the intended concentration in the lysis admixture. Thus, concentration indications for the component(s) of the lysis admixture are target concentrations, and the lysis solution in an embodiment is the means to achieve this target concentration. In view of the description herein above, the lysis solution may e.g. be a 3 fold, 4 fold, 5 fold, or 10 fold, concentrated stock solution. In an embodiment, the concentration factor of the lysis solution is selected by the skilled person in an embodiment according to well-known, mostly practical parameters, e.g. such that a practically sensible volume of lysis solution can be pipetted.

[0036] The lysis solution comprises an organic solvent, which may also be referred to as first organic solvent, at a concentration of from 4% (v / v) to 100% (v / v). As the skilled person understands, in case the lysis solution comprises the first organic solvent at high concentration, addition of a second lysis solution comprising the aqueous component or additional aqueous component may be necessary. However, in an embodiment, the lysis solution comprises the first organic solvent at a concentration allowing to obtain the desired concentration of the first organic solvent and of the aqueous component in the lysis admixture by the addition of one lysis solution. Thus, the concentration of the first organic solvent in the lysis solution in an embodiment is of from 4% (v / v) to 20% (v / v), in the aqueous component, in an embodiment in water. In such case, the lysis solution may be 1.11 fold concentrated, such that diluting 1 part sample with 9 parts of lysis solution provides a lysis admixture comprising a tenfold diluted sample and organic solvent and aqueous component at the desired concentration is obtained. Similarly, a lysis solution which is 1.25fold, 1.33fold, or 1.5 fold concentrated may be used to provide 5fold, 4fold, or 3fold diluted samples, respectively, comprising the organic solvent and the aqueous component at the desired concentration. As indicated herein above, the organic solvent in the lysis solution in an embodiment is isopropanol, dimethyl sulfoxide (DMSO), or a mixture thereof, in an embodiment is isopropanol or DMSO. The lysis solution may comprise further compounds as deemed appropriate by the skilled person. In an embodiment, a further component of the lysis solution is the aqueous component, in particular water, thus, the lysis solution may be an aqueous solution comprising the first organic solvent at an appropriate concentration. In an embodiment, however, the lysis solution does not comprise a chaotropic agent and / or does not comprise a detergent.

[0037] The terms "standard" and "internal standard" are known to the skilled person. The internal standard may be added to the sample before, concomitant with, or after producing the lysis admixture. Thus, the internal standard may in particular be comprised in the lysis solution, or may be added after producing the lysis admixture, but in an embodiment before contacting said lysis admixture with a solid-phase extraction agent. The standard is selected by the skilled person in accordance with the intended downstream application, e.g. a method used for detection the macrocyclic analyte. E.g., in case the macrocyclic analyte is detected by a mass spectrometry method, the standard, in particular the internal standard, may be an isotopologue of the macrocyclic analyte.

[0038] The term "obtaining", such as in obtaining solid-phase bound sample constituents, is understood by the skilled person. In an embodiment, the term relates to making available, in an embodiment physically, a compound of interest, in particular one or more sample constituents, in an embodiment the macrocyclic analyte(s). As discussed herein above, depending on the intended downstream application, e.g. a detection method selected for detecting the macrocyclic analyte, it may not be necessary to (partially) purify the macrocyclic analyte, so obtaining solid-phase bound sample constituents as they are eluted from the solid-phase extraction agent may be sufficient to enable further analysis.

[0039] The solid-phase extraction agent obtained in step (a) may optionally be washed before step (b), using a wash solution. The term "wash solution" is used herein in its conventional meaning; in an embodiment, the term relates to any solution suitable for washing the solid-phase extraction agent, i.e. removing undesirable sample components while essentially retaining the macrocyclic analyte(s). In an embodiment, the wash solution comprises of from 10% (v / v) to 20% (v / v) of a second organic solvent as specified herein above, wherein said second organic solvent in the wash solution in an embodiment is methanol (CAS No. 67-56-1) and / or acetonitrile (CAS No. 75-05-8). In a further embodiment, the wash solution comprises of from 4% (v / v) to 10% (v / v) of a first organic acid as specified herein above. In an alternative embodiment, the wash solution comprises a base, in an embodiment ammonia, in an embodiment at a concentration of from 4% (v / v) to 10% (v / v), in an embodiment about 10% (v / v), in a further embodiment 10%.

[0040] The term "elution solution" is used herein in its conventional meaning; in an embodiment, the term relates to any solution suitable for eluting the macrocyclic analyte(s), if present, from the solid-phase extraction agent. Thus, after elution, the macrocyclic analyte(s) in an embodiment are dissolved in the elution agent. After elution, the solid-phase extraction agent may optionally be removed from the elution solution. In an embodiment, the elution solution comprises of from 80% (v / v) to 90% (v / v) of a third organic solvent, wherein said third organic solvent may in particular be acetonitrile. The elution solution may further comprise from 5% (v / v) to 10% (v / v) of a second organic acid, wherein said organic acid may in particular be acetic acid. In an alternative embodiment, the elution solution comprises a base as specified herein, in an embodiment ammonia, in an embodiment at a concentration of from 4% (v / v) to 15% (v / v), in an embodiment about 10% (v / v), in a further embodiment 10% (v / v).

[0041] The method comprises step (a) producing a lysis admixture comprising said sample, a first organic solvent at a concentration of from 2.5% (v / v) to 18% (v / v) and an aqueous component at a concentration of at least 60% (v / v), and contacting said lysis admixture with a solid-phase extraction agent. Means and methods for producing an admixture, in particular a lysis admixture, are known to the skilled person. In principle, each and every proceeding causing the indicated components of an admixture to become admixed may be used; thus, after or during adding a lysis solution, the admixture may be pipetted up and down, e.g. by an automated pipetting device, may be vortexed, the container comprising the lysis admixture may be inverted once or a multitude of times, and the like. The components of the lysis admixture may be combined in any container deemed appropriate by the skilled person, e.g. in a well of a multi-well plate, in a flask or tube, or in a pipet tip. The components of the lysis admixture are combined to achieve the indicated concentration in the lysis admixture. If optional further components are present in the lysis admixture, the indicated concentrations are the concentrations in the final lysis admixture. In particular, the first organic solvent is added to have a concentration of from 2.5% (v / v) to 18% (v / v) in the lysis admixture. As described herein above, said concentration may be achieved by adding a lysis solution to a sample or an aliquot thereof, or vice versa. As the skilled person understands, the specific volumes to be admixed depend in particular on the intended final volume of admixture and on the concentration of the first organic solvent in the lysis solution. Thus, depending on the concentration of the lysis solution, 1 part of sample may e.g. be admixed with three parts of lysis solution (if the lysis solution is 4 / 3 concentrated), with one part of lysis solution (if the lysis solution is twofold concentrated), or the like. The lysis admixture may be incubated for a certain period of time, e.g. up to 24h; in an embodiment, however, the lysis admixture is used without a dedicated incubation step, i.e. in an embodiment is maintained as such only for the time period required for the equipment and / or the operator to perform the next step. Thus, in an embodiment, the lysis admixture is produced in step (a) and is then directly, i.e. without dedicated incubation, used further, in an embodiment to optionally add an internal standard and to contacting with the solid-phase extraction agent. In an embodiment, the organic solvent at the indicated concentration causes the components in a sample, in particular biological macromolecules, to remain soluble during all of step (a), i.e. in particular during production of the lysis admixture, during optional incubation of the lysis admixture, and during contacting the lysis admixture with a solid-phase extraction agent. Thus, in an embodiment, no steps of removal of insoluble sample constituents, such as centrifugation and / or filtration, are required or performed during or after steps (a) and / or (b). For clarity, as the skilled person understands, the solid-phase extraction agent is not a sample constituent. Step (a) further comprises contacting the lysis admixture with a solid-phase extraction agent. Any proceeding deemed appropriate by the skilled person may be used to achieve said contacting. The amount of solid-phase extraction agent is selected by the skilled person as deemed appropriate in particular in dependence on the amount of sample constituents expected to bind to the solid-phase extraction agent, in particular in dependence on the expected maximum amount of macrocyclic analyte in the sample, and in dependence on the binding capacity of the solid-phase extraction agent. Typical amounts of solid-phase extraction agent per ml of blood sample are of from O. lmg to 50mg, in an embodiment of from Img to 30mg; however, depending on sample type and expected amounts of analyte, also higher or lower amounts may be used. The contacting of the lysis admixture with a solid-phase extraction agent in an embodiment comprises contacting of from 1 to 3 parts of lysis admixture with of from 0.5 to 3 parts of the suspension comprising the solid-phase extraction agent. In a further embodiment, step (a) comprises producing a lysis admixture as specified herein above and contacting 6 parts of said hemolysis admixture with 2 parts of a suspension comprising the solid-phase extraction agent. In a further embodiment, step (a) comprises producing a lysis admixture by admixing 1 part of sample with 4 parts of a lysis solution

[0042] As specified herein above, in the product of step (a) in an embodiment essentially all components except the solid-phase extraction agent, are in solution, i.e. are not precipitated. Thus, in case the solid-phase extraction agent comprises beads, the product of step (a) in an embodiment is an admixture comprising all sample constituents, the first organic solvent, and the solid-phase extraction agent dispersed in the admixture comprising said sample constituents and said first organic solvent.

[0043] The method further comprises step (b) obtaining solid-phase bound sample constituents. The term "obtaining" has been specified herein above. In an embodiment, the obtaining step comprises contacting the solid-phase extraction agent obtained in step (a) with an elution solution as specified elsewhere herein. The elution solution in an embodiment is selected to be hydrophobic in order to facilitate dissociation of the macrocyclic analyte from the solid-phase extraction agent. As the skilled person understands in view of the description herein, other sample constituents such as components of the sample matrix may, but do not have to be eluted from the solid-phase extraction agent as well. In an embodiment, however, the macrocyclic analyte is eluted from the solid-phase extraction agent. In accordance, step (b) may comprise an elution step, optionally preceded by one or more washing steps. Step (b) of the method optionally comprises step (bl) removing non-solid-phase bound sample constituents from the mixture obtained in step (a). Typically, step (bl) will comprise removing the first organic solvent and sample liquid together with non-solid-phase bound sample constituents. Thus, step (bl) may in particular be obtaining the solid-phase extraction agent from the mixture obtained in step (a); as is understood by the skilled person, said obtaining said solid-phase extraction agent typically comprises obtaining sample constituents possibly bound to the solid-phase extraction agent. The particular method used in step (bl) is selected by the skilled person in particular depending on the specific solid-phase extraction agent used; thus, in case the solid-phase extraction agent comprises magnetic material, such as magnetic beads, the solid-phase extraction agent may be obtained by applying magnetic force to the mixture obtained in step (a); in case the solid-phase extraction agent comprises beads having a higher density than the mixture obtained in step (a), the solid-phase extraction agent may be obtained by centrifugation; in case the solid-phase extraction agent comprises a solid-phase column material, the solid-phase extraction agent may be obtained by packing a column or by filtration; in case the solid-phase extraction agent is a multi-well plate, the solid-phase extraction agent may be obtained by withdrawing any liquid comprised in the mixture obtained in step (a). Removing non-solid-phase bound sample constituents from the mixture obtained in step (a) may be complete or essentially complete, said removal may, however also be partial; i.e., it may be sufficient to reduce the amount of sample matrix and other non-analyte compounds to an extent allowing determining the macrocyclic analyte.

[0044] Step (b) of the method optionally comprises step (b2) contacting said solid-phase bound sample constituents with a wash solution comprising of from 10% (v / v) to 20% (v / v) of a second organic solvent. The term wash solution has been specified herein above. The wash solution in an embodiment is selected to remove sample matrix or parts thereof, while preventing macrocyclic analytes from eluting from the solid-phase extraction agent. Thus, the wash solution comprises the aforesaid second organic solvent at the indicated concentration. In an embodiment, a wash solution comprises an organic acid or a base as specified herein above. Step (b2) may optionally be repeated at least once, wherein the same wash solution may be used in each performance of step (b2). In an embodiment, however, at least two different wash solutions are used in steps (b2), e.g., in an embodiment, two non-identical wash solutions may be used; thus, a first wash solution may be acidic, e.g. comprising of from 10% (v / v) to 20% (v / v) of a second organic solvent and of from 4% (v / v) to 10% (v / v) of a first organic acid, and a second wash solution comprising of from 10% (v / v) to 20% (v / v) of a second organic solvent and of from 4% (v / v) to 10% (v / v) of a base. In an embodiment, the second organic solvent in the first wash solution is non-identical to the second organic solvent in the second wash solution. Thus, the first wash solution may be e.g. 10% (v / v) methanol and 10% formic acid (v / v) in water, and the second wash solution may be 20% acetonitrile (v / v) and 10% ammonia (v / v) in water. In an embodiment, step (b2) is performed using a first wash solution such as 10% (v / v) methanol and 10% formic acid (v / v) in water, and thereafter step (b2) is performed using a second wash solution such as 20% acetonitrile (v / v) and 10% ammonia (v / v) in water. Thus, wash steps may be in particular performed in a manner such that the last wash step is performed with a wash solution comprising a base.

[0045] Step (b) of the method optionally comprises step (b3) removing said wash solution from the mixture obtained in step (b2). As the skilled person will understand, step (b3) may in particular be performed if step (b2) is performed. As the skilled person will also understand, removing a wash solution in step (b3) essentially corresponds to removing non-solid-phase bound sample constituents from the mixture obtained in step (a) in step (bl) Thus, the description of step (bl) herein above applies to step (b3) mutatis mutandis.

[0046] Step (b) of the method optionally comprises step (b4) contacting the solid-phase bound sample constituents with an elution solution comprising of from 80% (v / v) to 90% (v / v) of a third organic solvent and (i) from 4% (v / v) to 15% (v / v) of a base or (ii) of from 5% (v / v) to 10% (v / v) of a second organic acid. The elution solution has been described herein above. Contacting the solid-phase bound sample constituents with an elution solution may, as the skilled person understands, typically be accomplished by admixing the solid-phase extraction agent obtained from the preceding steps with an elution solution. If it is preferred to remove the preceding solution, e.g. a wash solution, only partially, e.g. to avoid loss of solid-phase extraction agent and / or any analyte bound thereto, the elution solution may also be applied from a concentrated stock solution. Thus, the concentrations indicated for the compounds used in step (b4) in an embodiment are final concentrations during the elution step. The elution may comprise incubation over extended periods of time, in an embodiment as specified herein for step (a). Elution step (b4) may also be repeated once or a multitude of times, wherein eluates obtained from steps (b4) in an embodiment in an embodiment are combined for further analysis. Advantageously, it was found in the work underlying the present invention that by performing extraction of macrocyclic metabolites as described herein, it is possible to perform the extraction in a fully automated manner, in particular avoiding manual steps such as centrifugation and withdrawal of supernatant. Thus, the method described herein lyses cells in manner which keeps cell constituents in solution and thus avoids the need for removal of a precipitate. Also, increase in viscosity, which is caused by some prior art extraction methods, is avoided. Acid wash of solid-phase extraction agent was found to improve removal of sample matrix and to avoid an increase of column back pressure in e.g. LC-MS. Moreover, in particular basic wash and elution steps were surprisingly found to provide clear supernatants after elution and to improve chromatography e.g. in LC-MS.

[0047] 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 method further comprises step (c) thereby extracting said macrocyclic analyte.

[0049] The present invention also relates to a system comprising

[0050] (i) a storage device comprising an organic solvent;

[0051] (ii) a liquid handling device configured to contact a sample with the organic solvent and a solidphase extraction agent;

[0052] (iii) a separation device configured to separate a solid-phase extraction agent from sample constituents;

[0053] (iv) a microprocessor; and

[0054] (v) a data storage device comprising stored software instructions for controlling the system, wherein the software instructions, when executed on the microprocessor, cause the system to perform the method for extracting a macrocyclic analyte described herein.

[0055] The term “system”, as used herein, relates to a system of means, such as units and devices, comprising at least the aforementioned means operatively linked to each other as to allow the determination. Typical means for determining particulate compounds, and means for carrying out the determination are disclosed above in connection with the methods of the invention. How to link the means in an operating manner will depend on the type of means included into the device. In an embodiment, the means are comprised by a single device. A "storage device" may be any device deemed appropriate by the skilled person for storing the indicated item(s). Thus, a storage device for an organic solvent may be a simple container suitable for enclosing and storing the organic solvent. The storage device for an organic solvent may be e.g. a bottle made of glass or an appropriate plastic material, optionally comprising a seal to prevent evaporation of the organic solvent. Also, the container and / or the seal may comprise tubing to allow the system to withdraw organic solvent from the storage device. The storage device may comprise further units, such a cooling unit, a container for storing an organic acid, a container for storing a base, and the like. In an embodiment, the storage device comprises a container for storing a lysis solution, at least one container for storing at least one wash solution, and / or a container for storing an elution solution, in an embodiment comprised in a cooling unit.

[0056] The term "liquid handling device" is used in its common meaning understood by the skilled person. Thus, the term includes in principle each and every device configured to handle a sample and an organic solvent. Thus, the liquid handling device may comprise units for transporting and / or storing liquid, such as a syringe, a manual or motor driven pipettor, or a motor controlled pump, in an embodiment fluidly connected via appropriate tubing. The pump may be a peristaltic pump, a syringe pump, or other similar device that allows small volumes of liquid samples to be aspirated and dispensed as specified herein elsewhere. The liquid handling device may further comprise a sample receiving unit, e.g. a conveyor transporting a multi-well plate into and / or within the system, a reaction container, e.g. a multi-well plate into which the sample and the lysis solution are pipetted, and the like. Appropriate devices are known in the art.

[0057] As used herein, the term "separation device" relates to any device configured to separate a solidphase extraction agent from sample constituents. Thus, the separation device may comprise any component(s) deemed appropriate by the skilled person for said separation. The specific choice of separation device may in particular depend on the solid-phase extraction agent selected. Thus, in case the solid-phase extraction agent is a surface of a multi-well plate, the separation device may e.g. be an aspirator removing a lysis admixture and / or a wash solution from a well of said multi-well plate. In case the solid-phase extraction agent comprises beads, the separation device may comprise a centrifuge and / or a filtration device. In case the solid-phase extraction agent comprises magnetic beads, the separation device may comprise a magnet, wherein said magnet may e.g. be a permanent magnet or an electromagnet.

[0058] The term "microprocessor" is understood by the skilled person. In an embodiment, the term includes each and every device or unit thereof comprising at least one integrated circuit, in an embodiment comprising the arithmetic, logic, and / or control circuitry required to perform the function(s) as indicated. Thus, the microprocessor may in particular be a microprocessor of a computer.

[0059] The system comprises or has access to a data storage device comprising stored software instructions for controlling the system, such as a memory. A "data storage device", as referred to herein, comprises a computer readable information storage medium, wherein appropriate media are known to the skilled person. The system may comprise or have access to a single storage device or multiple storage devices, located either locally with the computing device or accessible to the computing device across a network, for example. Computer-readable media may be any available media that can be accessed by the computing device and includes both volatile and non-volatile media. Further, computer readable-media may be one or both of removable and non-removable media. By way of example, and not limitation, computer- readable media may comprise computer storage media. Exemplary computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or any other memory technology, CD-ROM, Digital Versatile Disk (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used for storing a plurality of instructions capable of being accessed by the computing device and executed by the processor of the computing device.

[0060] The term "stored software instructions" is understood by the skilled person. The stored software instructions in an embodiment include any sequence of instructions configured to cause the system to perform at least the indicated steps. In an embodiment, the software instructions cause a liquid handling device comprised in the system to perform at least steps (a) and (b) of the method for extracting a macrocyclic analyte of the present description.

[0061] As the skilled person understands, the system described herein above is an extraction system, in an embodiment providing one or more extracted macrocyclic analyte(s) as product. The aforesaid extraction system may, however, also be comprised in a further system, such as an analysis system for said macrocyclic analyte, and / or a diagnostic system making use of determination of said macrocyclic analyte; such an analytic and / or diagnostic system may, in an embodiment, further comprise a detection device and optionally an analysis device.

[0062] The detection device is configured to detect a macrocyclic analyte and, typically, comprises at least one device for determining the macrocyclic analyte; means and methods for such determining have been described herein above and are, in principle, known in the art. In an embodiment, the detection device comprises at least one mass spectrometry device. The detector is in an embodiment adapted to allow determination of the amount of the macrocyclic analyte. The determined amount can be subsequently transmitted to the analysis device.

[0063] The term “determining” as used herein refers to semi quantitative or quantitative determination of a macrocyclic analyte referred to herein. Determining the amount of a macrocyclic analyte may be carried out by any technique which allows for establishing a measure of quantity of a macrocyclic analyte in a semi quantitative or quantitative manner. Suitable techniques depend on the molecular nature and the properties of the macrocyclic analytes and are discussed elsewhere herein in more detail.

[0064] Typically, the amount of a macrocyclic analyte can in principle be determined by determining a complex of the analyte with a detection compound, in particular an antibody or fragment thereof, i.e. in an immunoassay. Said determining of a complex of the analyte may be performed in any format deemed appropriate by the skilled person, in particular a sandwich, competition, or other assay format. Said assays will develop a signal which is indicative for the amount of a macrocyclic analyte.

[0065] In a further embodiment, the amount of a macrocyclic analyte may be determined by detecting the amount of molecular species of the macrocyclic analyte, or of fragments thereof. E.g., macrocyclic analytes may be detected as such or as their ions in mass spectrometry (MS). Methods for detecting the amount of molecular species of the macrocyclic analyte are available, including chromatographic separation techniques such as liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography, and / or size exclusion or affinity chromatography, coupled to appropriate detection devices. Such a detection device may e.g. be a photometer, e.g. an UV / VIS- photometer or an MS device. Appropriate devices and methods are known in the art. Further suitable methods comprise measuring a physical or chemical property specific for the macrocyclic analyte such as its precise molecular mass or NMR spectrum. Said methods comprise, preferably, biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass- spectrometers, NMR-analyzers, surface plasmon resonance measurement equipment or chromatography devices.

[0066] The macrocyclic analytes to be determined in accordance with the present invention are as such known in the art. Moreover, methods for the determination of the amount of the macrocyclic analytes are known to the skilled person as well. For example, the macrocyclic analytes can be measured as described in the Examples section.

[0067] The term "mass spectrometry device", abbreviated as "MS device", is understood by the skilled person. In an embodiment, the term relates to a device configured for performing a measurement via a mass spectrometer (MS); thus, the device, in an embodiment, comprises at least one MS unit. As used herein, the term “mass spectrometry unit”, in an embodiment, relates to a mass analyzer configured for detecting at least one analyte based on a mass to charge ratio of the analyte or a fragment thereof. In an embodiment, the MS unit is a tandem mass spectrometry (MS / MS) unit, in a further embodiment a triple quadrupole MS (QqQ-MS), in a further embodiment in Multiple Reaction Monitoring (MRM) mode. The MS device may further comprise at least one ionization source configured for generating molecular ions and for transferring the molecular ions into the gas phase. Ionization methods and appropriate ionization units are known in the art and include in particular electron ionization (El), chemical ionization (CI), electrospray ionization (ESI), atmospheric pressure ionization (APCI), atmospheric pressure photoionization (APPI), and matrix assisted laser desorption / ionization (MALDI).

[0068] In an embodiment, the MS device is a chromatography MS device, in particular a gas chromatography MS (GC-MS) device or a liquid chromatography MS (LC-MS) device, terms understood by the skilled person. Thus, in an embodiment, the device is configured for performing a combination of chromatography (e.g. LC or GC) with mass spectrometry (MS). Thus, the device, in an embodiment, comprises at least one LC and / or GC unit, and at least one MS unit, wherein the LC and / or GC unit(s) and the MS unit are coupled via at least one interface. As used herein, the term “liquid chromatography (LC) unit”, in an embodiment, relates to an analytical module configured to separate one or more analytes of interest of a sample from other components of the sample via liquid chromatography, in an embodiment for detection of the one or more analytes with the mass spectrometry device. The LC may be based on any separation principle deemed appropriate by the skilled person; in an embodiment, the LC is reverse phase chromatography, hydrophobic interaction chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, or chiral chromatography; in a further embodiment, the LC is reverse phase chromatography. The LC device may comprise at least one LC column. For example, the LC device may be a singlecolumn LC device or a multi-column LC device having a plurality of LC columns. The LC column may have a stationary phase through which a mobile phase is pumped in order to separate and / or elute and / or transfer the analyte(s) of interest. The LC unit may be or may comprise at least one high-performance liquid chromatography (HPLC) unit and / or at least one micro liquid chromatography (pLC) device. The term "gas chromatography" is understood by the skilled person; in an embodiment the same separation principles as for LC are applicable, however, the mobile phase being a gas in GC.

[0069] Data generated by the detection device in an embodiment are evaluated by an analysis device. The analysis device is configured to determine the macrocyclic analyte and comprises at least one data processor, which may also be referred to as a data processing unit, such as a microprocessor or computer, with an implemented algorithm for determining the amount of macrocyclic analyte present in the sample; said microprocessor may be the microprocessor comprised in the extraction system as specified herein above, or may be a separate microprocessor. Thus, the description of the microprocessor herein above applies to the microprocessor of the analysis device mutatis mutandis. The analysis device typically comprises or has access to a memory. Said memory may be a memory comprised in the extraction system as specified herein above, or may be a separate memory; thus the description of the memory herein above applies to the memory of the analysis device mutatis mutandis.

[0070] The analysis device may also comprise or have access to an output device. Exemplary output devices include displays, printers, files, and telecommunication devices, such as fax devices, data servers, and the like. According to some embodiments, a computing device may perform one or more steps of a method disclosed herein, and thereafter provide an output, via an output device, relating to a result of determination. Further disclosed and proposed herein is a computer program including computer-executable instructions for performing the 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 the method steps as indicated above may be performed by using a computer or a computer network, in an embodiment by using a computer program.

[0071] Also disclosed and proposed is a computer program product having program code means, in order to perform the 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.

[0072] Further disclosed and proposed is 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 the method according to one or more of the embodiments disclosed herein.

[0073] Moreover disclosed and proposed is a computer program product with program code means stored on a machine-readable carrier, in order to perform the 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.

[0074] In addition disclosed and proposed is a modulated data signal which contains instructions readable by a computer system or computer network, for performing the method according to one or more of the embodiments disclosed herein.

[0075] 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 the 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.

[0076] Specifically, the present invention further discloses:

[0077] - 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,

[0078] - 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,

[0079] - 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,

[0080] - 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,

[0081] - 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,

[0082] - 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

[0083] - 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.

[0084] The present invention also relates to a kit for macrocyclic analyte determination, the kit comprising

[0085] (I) a lysis solution comprising a first organic solvent at a concentration of from 4% (v / v) to 80% (v / v), in an embodiment 4% (v / v) to 20% (v / v), wherein said lysis solution does not comprise an organic acid; (II) a wash solution comprising (i) of from 10% (v / v) to 20% (v / v) of a second organic solvent and (i) from 4% (v / v) to 10% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a first organic acid; and

[0086] (III) an elution solution comprising of from 80% (v / v) to 90% (v / v) organic solvent and (i) from 4% (v / v) to 10% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a second organic acid.

[0087] The term “kit”, as used herein, refers to a collection of the aforementioned compounds, means or reagents which may or may not be packaged together, e.g. as a kit-of-parts. The components of the kit in an embodiment are comprised by separate containers (i.e. as a kit of separate parts) within the housing. As used herein, the term "housing" relates to a casing comprising the components as specified, in an embodiment enabling transport, in a further embodiment common translocation, of the components. Moreover, it is to be understood that the kit in an embodiment is to be used for practicing the methods referred to herein; thus, the kit in an embodiment is an analytic kit, in a further embodiment a diagnostic kit, in an embodiment for determining a macrocyclic analyte specified herein above. It is, in an embodiment, envisaged that all components are provided in a ready-to-use manner for practicing at least one of the methods referred to above. Further, the kit, in an embodiment, contains instructions for carrying out said methods. The instructions can be provided by a user's manual in paper or electronic form.

[0088] The present invention also relates to a use of a system as specified herein and / or a kit as specified herein for determining a macrocyclic analyte in a sample, said use preferably comprising the steps of a method described herein.

[0089] The present invention also relates to an extract of a macrocyclic analyte from a sample produced or producible by a method for extracting a macrocyclic analyte described herein.

[0090] The present invention also relates to a method for determining a macrocyclic analyte in a sample by a chromatography method, comprising

[0091] (A) providing an extract comprising the macrocyclic analyte bound to a solid-phase extraction agent, wherein said extract in an embodiment was obtained by contacting the sample with a lysis solution comprising a first organic solvent at a concentration of from 4% (v / v) to 20% (v / v) and with the solid-phase extraction agent; (B) washing said solid-phase extraction agent comprising bound macrocyclic analyte with a wash solution comprising of from 10% (v / v) to 20% (v / v) of a second organic solvent and (i) of from 4% (v / v) to 10% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a second organic acid;

[0092] (C) eluting said macrocyclic analyte from said solid-phase extraction agent with an elution solution comprising of from 80% (v / v) to 90% (v / v) of a third organic solvent and (i) of from 4% (v / v) to 10% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a second organic acid; and

[0093] (D) applying the macrocyclic analyte eluted in step (C) to a chromatography device, thereby determining said macrocyclic analyte.

[0094] Means and methods for performing the method for determining a macrocyclic analyte have been described herein above. In an embodiment, step (A) comprises step (a) of the method for extracting a macrocyclic analyte described herein above, and step (B) comprises step (b) of the method for extracting a macrocyclic analyte described herein above. Thus, the method for determining a macrocyclic analyte in an embodiment comprises steps (a) and (b) of the method for extracting a macrocyclic analyte described herein above, and step (C) eluting said macrocyclic analyte from said solid-phase extraction agent with an elution solution comprising of from 80% (v / v) to 90% (v / v) of a third organic solvent and (i) from 4% (v / v) to 10% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a first organic acid; and step (D) applying the macrocyclic analyte eluted in step (C) to an analysis device, thereby determining said macrocyclic analyte.

[0095] Summarizing the findings of the present invention, the following embodiments are particularly envisaged:

[0096] Embodiment 1 : A method for extracting a macrocyclic analyte from a sample, said method comprising

[0097] (a) producing a lysis admixture comprising said sample, a first organic solvent at a concentration of from 2.5% (v / v) to 18% (v / v), in an embodiment of from 2.7% v / v) to 18% (v / v), and an aqueous component at a concentration of at least 60% (v / v), and contacting said lysis admixture with a solid-phase extraction agent;

[0098] (b) obtaining solid-phase extraction agent bound sample constituents; and

[0099] (c) thereby extracting said macrocyclic analyte. Embodiment 2: The method of embodiment 1, wherein said macrocyclic analyte is an immunosuppressant analyte.

[0100] Embodiment 3: The method of embodiment 1 or 2, wherein said macrocyclic analyte is cyclosporine (CAS No. 79217-60-0), in an embodiment cyclosporine A (CAS No. 59865-13- 3), is everolimus (CAS No. 159351-69-6), is tacrolimus (CAS No. 104987-11-3), and / or is sirolimus (CAS No. 53123-88-9).

[0101] Embodiment 4: The method of any one of embodiments 1 to 3, wherein said sample is a liquid sample.

[0102] Embodiment s: The method of any one of embodiments 1 to 4, wherein said sample comprises protein at a concentration of at least Img / mL.

[0103] Embodiment 6: The method of any one of embodiments 1 to 5, wherein said sample is an aqueous sample.

[0104] Embodiment 7: The method of any one of embodiments 1 to 6, wherein said sample is a biological sample.

[0105] Embodiment 8: The method of any one of embodiments 1 to 7, wherein said sample is a blood sample or a blood-derived sample.

[0106] Embodiment 9: The method of any one of embodiments 1 to 8, wherein said sample is a whole blood sample.

[0107] Embodiment 10: The method of any one of embodiments 1 to 9, wherein said lysis solution, in an embodiment wherein the lysis admixture produced in step (a), does not comprise a chaotropic agent and / or does not comprise a detergent.

[0108] Embodiment 11 : The method of any one of embodiments 1 to 10, wherein said method does not comprise contacting the sample with a chaotropic agent and / or with a detergent before step (c).

[0109] Embodiment 12: The method of any one of embodiments 1 to 11, wherein said lysis admixture is produced by contacting the sample with a lysis solution.

[0110] Embodiment 13: The method of any one of embodiments 1 to 12, wherein the contacting of the lysis admixture with the solid-phase extraction agent comprises mixing the lysis admixture with a suspension comprising the solid-phase extraction agent.

[0111] Embodiment 14: The method of embodiment 13, wherein step (a) further comprises admixing an internal standard to the lysis admixture.

[0112] Embodiment 15: The method of any one of embodiments 1 to 14, wherein contacting said hemolysis admixture with a solid-phase extraction agent comprises contacting of from 1 to 3 parts of lysis admixture with of from 0.5 to 3 parts of the suspension comprising the solid-phase extraction agent.

[0113] Embodiment 16: The method of any one of embodiments 1 to 15, wherein step (a) comprises producing a lysis admixture comprising a first organic solvent at a concentration of from 2.5% (v / v) to 18% (v / v) and said sample and contacting 6 parts of said hemolysis admixture with 2 parts of the suspension comprising the solid-phase extraction agent.

[0114] Embodiment 17: The method of any one of embodiments 1 to 16, wherein step (a) comprises producing a lysis admixture by admixing 1 part of sample with 4 parts of a lysis solution comprising of from 4% (v / v) to 20% (v / v) of the first organic solvent, adding internal standard to produce a spiked lysis admixture, and contacting 6 parts of said spiked hemolysis admixture with 2 parts of the suspension comprising the solid-phase extraction agent.

[0115] Embodiment 18: The method of any one of embodiments 1 to 17, wherein the contacting of said lysis admixture with a solid-phase extraction agent is performed for at least Imin, in an embodiment at least 2 min, in a further embodiment at least 5 min.

[0116] Embodiment 19: The method of any one of embodiments 1 to 18, wherein the contacting of said lysis admixture with a solid-phase extraction agent is performed for at most 1 day, in an embodiment at most 12h, in a further embodiment at most 6 h.

[0117] Embodiment 20: The method of any one of embodiments 1 to 19, wherein the contacting of said lysis admixture with a solid-phase extraction agent is performed for of from 1 min to 1 day, in an embodiment of from 2 min to 12 h, in a further embodiment of from 3 min to 6 h, in a further embodiment of from 4 min to 3 h, in a further embodiment of from 5 min to 1 h, in a further embodiment about 5 min.

[0118] Embodiment 21 : The method of any one of embodiments 1 to 20, wherein the first organic solvent in the lysis solution in step (a) is isopropanol (2-propanol, CAS No. 67-63-0) and / or dimethylsulfoxide (DMSO, CAS No. 67-68-5).

[0119] Embodiment 22: The method of any one of embodiments 1 to 21, wherein said step (b) comprises step (bl) removing non-solid-phase bound sample constituents from the mixture obtained in step (a).

[0120] Embodiment 23 : The method of any one of embodiments 1 to 22, wherein said solid-phase extraction agent comprises solid phase extraction beads.

[0121] Embodiment 24: The method of any one of embodiments 1 to 23, wherein said solid-phase extraction agent comprises magnetic solid phase extraction beads.

[0122] Embodiment 25: The method of embodiment 24, wherein said removing non-solid phase bound sample constituent comprises applying a magnetic force to said magnetic solid phase extraction beads. Embodiment 26: The method of any one of embodiments 22 to 25, wherein said removing non-solid-phase bound sample constituents does not comprise centrifugation.

[0123] Embodiment 27: The method of any one of embodiments 1 to 26, wherein said step (b) comprises step (b2) contacting said solid-phase bound sample constituents with a wash solution comprising of from 10% (v / v) to 20% (v / v) of a second organic solvent and (i) of from 4% (v / v) to 15% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a first organic acid.

[0124] Embodiment 28: The method of embodiment 27, wherein said step (b) comprises step (b3) removing said wash solution from the mixture obtained in step (b2).

[0125] Embodiment 29: The method of embodiment 28, wherein said method comprises repeating steps (b2) and (b3) at least once.

[0126] Embodiment 30: The method of any one of embodiments 27 to 29, wherein said method comprises performing step (b2) a first time with a wash solution comprising said first organic acid, and comprises performing step (b2) a second time with a wash solution comprising said base.

[0127] Embodiment 31 : The method of any one of embodiments 27 to 30, wherein said step (b) comprises step (b3) removing said wash solution from the mixture obtained in step (b2).

[0128] Embodiment 32: The method of any one of embodiments 27 to 31, wherein said second organic solvent in the wash solution is methanol (CAS No. 67-56-1) and / or acetonitrile (CAS No. 75-05-8).

[0129] Embodiment 33: The method of any one of embodiments 27 to 32, wherein said second organic solvent in the wash solution is methanol in a first performance of step (b2), and is acetonitrile in the last performance of step (b2).

[0130] Embodiment 34: The method of any one of embodiments 27 to 33, wherein said wash solution is an aqueous solution.

[0131] Embodiment 35: The method of any one of embodiments 1 to 34, wherein said step (c) comprises step contacting the solid-phase bound sample constituents with an elution solution comprising of from 80% (v / v) to 90% (v / v) of a third organic solvent and (i) from 4% (v / v) to 10% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a second organic acid.

[0132] Embodiment 36: The method of embodiment 35, wherein the third organic solvent in the elution solution in step (c) is acetonitrile.

[0133] Embodiment 37: The method of any one of embodiments 27 to 36, wherein said first organic acid is a Ci to C> organic acid and / or said second organic acid is a Cl to C5 organic acid.

[0134] Embodiment 38: The method of any one of embodiments 27 to 37, wherein said first organic acid is the same as the second organic acid, in an embodiment are formic acid or acetic acid, in a further embodiment are formic acid.

[0135] Embodiment 39: The method of any one of embodiments 27 to 38, wherein said base is ammonia.

[0136] Embodiment 40: The method of any one of embodiments 1 to 39, wherein said aqueous component is water or a hyposmotic solution of at least one salt, in particular a buffer.

[0137] Embodiment 41 : The method of embodiment 40, wherein said hyposmotic solution is a solution with an osmolarity of at most 0.1 Osm / L, in an embodiment of at most 0.5 Osm / L, in a further embodiment at most 0.01 Osm / L.

[0138] Embodiment 42: The method of any one of embodiments 1 to 41, wherein said aqueous component is comprised in said lysis admixture at a concentration of from 60% (v / v) to 95% (v / v), in an embodiment of from 65% (v / v) to 87% (v / v), in a further embodiment of from 65% (v / v) to 80% (v / v).

[0139] Embodiment 43 : A system comprising

[0140] (i) a storage device comprising an organic solvent;

[0141] (ii) a liquid handling device configured to contact a sample with the organic solvent and a solidphase extraction agent;

[0142] (iii) a separation device configured to separate a solid-phase extraction agent from sample constituents;

[0143] (iv) a microprocessor; and

[0144] (v) a data storage device comprising stored software instructions for controlling the system, wherein the software instructions, when executed on the microprocessor, cause the system to perform the method according to any one of embodiments 1 to 42.

[0145] Embodiment 44: The system of embodiment 43, further comprising (vi) a detection device configured to detect a macrocyclic analyte.

[0146] Embodiment 45: The system of embodiment 44, further comprising (vii) an analysis device configured to determine the macrocyclic analyte.

[0147] Embodiment 46: The system any one of embodiments 43 to 45, wherein said separation device comprises at least one magnet.

[0148] Embodiment 47: A kit for macrocyclic analyte determination, the kit comprising

[0149] (I) a lysis solution comprising a first organic solvent at a concentration of from 4% (v / v) to 20% (v / v), wherein said lysis solution does not comprise an organic acid;

[0150] (II) a wash solution comprising (i) of from 10% (v / v) to 20% (v / v) of a second organic solvent and (i) from 4% (v / v) to 10% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a first organic acid; and

[0151] (III) an elution solution comprising of from 80% (v / v) to 90% (v / v) organic solvent and (i) from 4% (v / v) to 10% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a second organic acid.

[0152] Embodiment 48: The kit of embodiment 47, wherein said lysis solution is a 1.25fold concentrated solution and comprises of from 4% (v / v) to 20% (v / v) organic solvent.

[0153] Embodiment 49: The kit of embodiment 47 or 48, wherein the first organic solvent in the lysis solution is isopropanol and / or dimethylsulfoxide.

[0154] Embodiment 50: The kit of any one of embodiments 44 to 49, wherein said second organic solvent in the wash solution is methanol and / or acetonitrile.

[0155] Embodiment 51 : The kit of any one of embodiments 47 to 50, wherein the third organic solvent in the elution solution is acetonitrile.

[0156] Embodiment 52: The kit of any one of embodiments 47 to 51, wherein said first and second organic acids are formic acid or acetic acid.

[0157] Embodiment 53: The kit of any one of embodiments 47 to 52, wherein said base is ammonia.

[0158] Embodiment 54: The kit of any one of embodiments 47 to 53, further comprising a solidphase extraction agent.

[0159] Embodiment 55: Use of a system according to any one of embodiments 43 to 46 and / or a kit according to any one of embodiments 47 to 54 for determining a macrocyclic analyte in a sample.

[0160] Embodiment 56: The use of embodiment 52, wherein said determining comprises the steps of the method according to any one of embodiments 1 to 42.

[0161] Embodiment 57: An extract of a macrocyclic analyte from a sample produced or producible by a method according to any one of embodiments 1 to 42.

[0162] Embodiment 58: A method for determining a macrocyclic analyte in a sample by a chromatography method, comprising

[0163] (A) providing an extract comprising the macrocyclic analyte bound to a solid-phase extraction agent, wherein said extract in an embodiment was obtained by contacting the sample with a lysis solution comprising a first organic solvent at a concentration of from 4% (v / v) to 20% (v / v) and with the solid-phase extraction agent;

[0164] (B) washing said solid-phase extraction agent comprising bound macrocyclic analyte with a wash solution comprising of from 10% (v / v) to 20% (v / v) of a second organic solvent and of from 4% (v / v) to 10% (v / v) of a first organic acid; (C) eluting said macrocyclic analyte from said solid-phase extraction agent with an elution solution comprising of from 80% (v / v) to 90% (v / v) of a third organic solvent and (i) of from 4% (v / v) to 10% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a second organic acid; and

[0165] (D) applying the macrocyclic analyte eluted in step (C) to a chromatography device, thereby determining said macrocyclic analyte.

[0166] Embodiment 59: The method of embodiment 58, wherein said steps (A) to (C) have at least one further feature of any one of embodiments 1 to 57.

[0167] Embodiment 60: The method of embodiment 58 or 55 wherein step (D) comprises performing a chromatography method, in an embodiment a chromatography-mass spectrometry method, in a further embodiment a liquid chromatography-mass spectrometry (LC-MS) method.

[0168] Embodiment 61 : The method of any one of embodiments 1 to 42 and 58 to 60, wherein said method is an automated method.

[0169] Embodiment 62: The method of any one of embodiments 1 to 42 and 58 to 61, wherein said method is a fully automated method.

[0170] 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.

[0171] Figure Legends

[0172] Fig. 1 : a. Calibration curve for area ratio vs concentration with low to high concentration range for Cyclosporine on different days on one instrument. Linear calibration curve observed with R2values > 0.99 on different days; b. Calibration curve for area ratio vs concentration with low to high concentration range for Sirolimus on different days on one instrument. Linear calibration curve observed with R2values > 0.99 on different days; c. Calibration curve for area ratio vs concentration with low to high concentration range for Tacrolimus on different days on one instrument. Linear calibration curve observed with R2values > 0.99 on different days; d. Calibration curve for area ratio vs concentration with low to high concentration range for Cyclosporine on different days on one instrument. Linear calibration curve observed with R2values > 0.99 on different days. Fig. 2. %Accuracy calculated between target concentration and calculated concentration for Calibrators and QCs samples for different days and different instruments. %Accuracy observed between 85-115% for Calibrators and QCs for different days and different instruments, a. Cyclosporine b. Everolimus c. Sirolimus d. Tacrolimus.

[0173] Fig. 3: CV of area ratio calculated for Calibrators, QCs and spiked samples with range of low to high concentration for different days and different instruments. Inter and Intra-CV of area ratio observed within 15% from low to high concentration range, a. Cyclosporine b. Everolimus c. Sirolimus d. Tacrolimus.

[0174] Fig. 4: Passing-Bablok regression analysis for patient samples shows good correlation between reference method (ISD1.0) and hemolysis workflow method (ISD2.0) for a. Cyclosporine; b. Everolimus; c. Sirolimus; and d. Tacrolimus.

[0175] Fig. 5: Immunosuppressive drug (ISD) analytes are hydrophobic and known to be insoluble and instable in presence of high aqueous content in solution. Effect of lysis solution volume on analyte solubility and stability was determined using different dilution factors. Shown is signal correlation observed between theoretical and experiment signal based on dilution factors.

[0176] Fig. 6: Samples were injected with acidic bead wash and without acidic bead wash; shown is the maximal pressure per run over number of run; stable column pressure was observed when acidic bead wash used during sample preparation workflow.

[0177] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

[0178] 1.1 Sample Preparation

[0179] For sample preparation, 400 pL of lysis solution (4% (v / v) to 20% (v / v) organic solvent) were added in 100 pL of whole blood followed by short mixing to induce hemolysis. After that, 10 pL of internal standard (Everolimus-D4, 15ng / mL, Sirolimus-d3, 15ng / mL, Ascomycin, 15ng / mL and Cyclosporine A-(dlO), 300ng / mL) were added in 60 pL of hemolysed whole blood, vortex mixed and incubated 5 min to 15 min. In the next step, 20 pL of magnetic beads (40 mg / mL, inhouse-prepared) were added, vortex mixed and incubated 9.48 min to capture and immobilize the immunosuppressant onto the magnetic beads. After incubation, the magnetic beads were captured by using a magnet, the supernatant was removed and the beads were successively washed with 240 pL of 20% methanol with 10% IM formic acid and 20% acetonitrile with 10% 0.5M ammonia to minimize interfering matrix components. The immunosuppressant analytes and their internal standards were released from the magnetic beads using 100 pL of 80% acetonitrile with 10% 0.5 M ammonia. Finally 5 pL of this solution were injected into the LC injector.

[0180] 1.2 LC Separation

[0181] The separation of the immunosuppressants from other sample components was performed via liquid chromatography with the following parameters:

[0182] • HPLC column: HPLC C18 column, 2.6 pm, Dimensions: 1.0 x 50 mm

[0183] • Column temperature: 55 °C

[0184] • Flow rate: 440 pL / min

[0185] • Eluent A: 2 % 500 mM ammonium acetate in acetonitrile

[0186] • Eluent B: 85% acetonitrile with 15% methanol

[0187] • Injection volume: 5 pL (full-loop injection)

[0188] • Gradient: hold at 65% eluent B for equilibration for 36 s, LC injection, ramp from 65% to 98% eluent B in 36 s (flow diverted to MS only during this time), hold at 98% eluent B for cleaning for 36 s

[0189] • LC method total time: 108 s

[0190] 1.3 Mass spectrometry

[0191] MS was performed with the following parameters:

[0192] • ESI temperature: 500 °C

[0193] • MS polarity: positive-mode ionization

[0194] • ESI potential: 2.0 kV

[0195] • Nebulizer gas: 2.7 L / min

[0196] • Auxiliary (heating) gas: 5 L / min

[0197] • Curtain (counter) gas: 5 L / min

[0198] • Collision gas: 0.001 L / min

[0199] • Purge gas: 2 L / min

[0200] • MS resolution: unit resolution at both QI and Q3

[0201] MS settings specific for Traditional MRM (comparative example) o Cyclosporine : QI mass 1202.848 and Q3 mass 224.165 o Cyclosporine-dlO : QI mass 1212.911 and Q3 mass 224.165 o Everolimus : QI mass 975.615 and Q3 mass 908.552 o Everolimus-d4 : QI mass 979.646 and Q3 mass 912.583 o Sirolimus : QI mass 931.589 and Q3 mass 864.526 o Sirolimus-d3 : QI mass 934.608 and Q3 mass 864.526 o Tacrolimus : QI mass 821.516 and Q3 mass 768.468 o Ascomycin : QI mass 809.516 and Q3 mass 756.468

[0202] 1.4 Validation of workflow

[0203] To validate the hemolysis workflow, various experiments were performed. For linearity and precision five runs on five different days were performed. Thereby, the samples were prepared according to the described sample preparation procedure. Each run contained a blank sample (no analyte), calibrators from level 1 to 8 (containing known spiked analyte concentrations) and quality controls from QC1 to QC4 (containing known spiked analyte concentrations between low and high concentration) in 6 replicates. The accuracy of the method was evaluated by measuring % mean accuracy (difference between measured concentration and targeted concentration) at each concentration level of the calibration curve and QCs samples. The linearity was explored by determining the correlation co-efficient of the calibration curve (area ratio vs concentration), Thereby, a 1 / x2weighted least square regression analysis of standard plots associated with an eight-point standard curve was applied. A straight-line fit was applied to the data points by least square regression analysis where a constant proportionality was observed. All calibration curves analyzed were found linear for concentrations ranging from 7.58 to 1509.67 ng / mL for Cyclosporine A, 0.35 to 18.63 ng / mL for Everolimus, 0.39 to 32.25 ng / mL for Sirolimus and 0.46 to 30.36 ng / mL for Tacrolimus. For the correlation coefficient (r) values greater than 0.98 were observed during the course of validation (Figure 1). A mean accuracy between 85 to 115% was calculated for the calibration levels which was in line with the acceptance criteria (Figure 2).

[0204] 1.5 Reproducibility and precision

[0205] Reproducibility and precision were determined by preparing the calibrators level 1 to 8 (standardized samples having known concentration of analyte for calibration curve), quality controls QC 1 to 4 (standardized samples having known concentration between low to high concentration) and spiked concertation samples (SC) (known analyte concentration spiked in whole blood matrix covering the entire range of the calibration curve) according to the described sample preparation procedure. Subsequently, the samples were analyzed on different days and 3 different instruments. Both reproducibility and precision were determined by calculating the coefficients of variation (%CV) for the area ratios and the calculated concentration of QCs and SC samples for all replicates on all three instruments. All %CV values were observed within 15% for all QCs and SC samples on all three instruments (Figure 3).

[0206] 1.6 Method comparison

[0207] A method comparison was performed by preparing calibration samples from level 1 to 8 (standardized samples having known concentration of analyte for calibration curve), quality controls QC 1 and 2 (standardized samples having known concentration between low and high concentration) and patient samples (with known analyte concentrations determined by a reference method) according to the described sample preparation. The concentration values of the patient samples were calculated by using calibration curves for the respective analytes. Afterwards, the resulting concentration values were compared with the values determined via reference method. A Passing-Bablok regression analysis was performed to quantify the relation between two measurement methods. Slope, intercept and relative bias showed good correlation between the reference method and the established hemolysis workflow for all analytes (Figure 4).

[0208] 1.7 Effect of organic solvent concentration

[0209] Immunosuppressive drug (ISD) analytes are hydrophobic and known to be insoluble and instable in presence of high aqueous content in solution. The effect of the lysis solution and its volume on analyte solubility and stability was determined by using different dilution factors. Two samples with known low or high analyte concentration in whole blood matrix were prepared according to the described sample preparation procedure. The low concentrated samples contained 0.5 ng / mL of Sirolimus, Everolimus, Tacrolimus and lOng / mL of Cyclosporine whereas the high concentrated samples contained 30 ng / mL of Sirolimus, Everolimus, Tacrolimus and 1200 ng / mL of Cyclosporine. The samples were hemolysed with different dilution factors ranging from 3 fold dilution to 10 fold dilution. The theoretical signals which were calculated based on the dilution factors from known concentrations were compared with the experimental signals obtained after analysis in order to detect any signal loss due to insolubilities or degradation based on lysis solution volume (Figure 5). Similar results were obtained over a range of from 4% (v / v) to 20% (v / v) isopropanol or DMSO in the lysis solution (not shown). 1.8 LC column effects

[0210] Since whole blood contains a high concentration of protein, sample constituents can bind to the beads, re-elute during analyte elution and thus be injected to LC columns during analysis. To evaluate the extent of this effect, whole blood matrix samples were prepared according to the described sample preparation procedure. Afterwards, the samples were injected on LC columns with acidic bead wash (20% Methanol, 10% IM Formic acid) and without acidic bead wash (20% Methanol). The LC pressure was monitored during each injection and compared for the workflow with and without acidic bead wash. The LC pressure remained stable over more than 80 runs when acidic bead wash was used (Figure 6). Similar results were obtained for a variety of LC columns (not shown).

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[0231] - WO 2019 / 141779 Al

Claims

Roche Diagnostics GmbH 04 September 2024 RD38635PC ST / NHClaims1. A method for extracting a macrocyclic analyte from a sample, said method comprising(a) producing a lysis admixture comprising a first organic solvent at a concentration of from 2.5% (v / v) to 18% (v / v), an aqueous component at a concentration of at least 60% (v / v), and said sample to 100%, and contacting said lysis admixture with a solid-phase extraction agent;(b) obtaining solid-phase extraction agent bound sample constituents; and(c) thereby extracting said macrocyclic analyte.

2. The method of claim 1, wherein said macrocyclic analyte is an immunosuppressant analyte, in an embodiment is cyclosporine (CAS No. 59865-13-3), is everolimus (CAS No. 159351-69-6), is tacrolimus (CAS No. 104987-11-3), and / or is sirolimus (CAS No. 53123-88-9).

3. The method of claim 1 or 2, wherein said sample is a liquid sample, in an embodiment is a blood sample or a blood derived sample.

4. The method of any one of claims 1 to 3, wherein the contacting of said lysis admixture with a solid-phase extraction agent is for of from 1 min to 1 day, in an embodiment of from 2 min to 12 h, in a further embodiment of from 3 min to 6 h, in a further embodiment of from 4 min to 3 h, in a further embodiment of from 5 min to 1 h, in a further embodiment about 5 min.

5. The method of any one of claims 1 to 4, wherein the first organic solvent in the lysis solution in step (a) is isopropanol (2-propanol, CAS No. 67-63-0) and / or dimethyl sulfoxide (DMSO, CAS No. 67-68-5), and wherein said aqueous component is water.

6. The method of any one of claims 1 to 5, wherein said solid-phase extraction agent comprises magnetic solid phase extraction beads.

7. The method of any one of claims 1 to 6, wherein said step (b) comprises step (b2) contacting said solid-phase bound sample constituents with a wash solution comprising of from 10% (v / v) to 20% (v / v) of a second organic solvent and (i) of from 4% (v / v) to15% (v / v) of a base or (ii) of from 4% (v / v) to 10% (v / v) of a first organic acid; and optionally comprises step (b3) removing said wash solution from the mixture obtained in step (b2), in a further embodiment wherein said method comprises repeating steps (b2) and (b3) at least once, wherein in an embodiment the second organic solvent in the wash solution is methanol in a first performance of step (b2), and is acetonitrile in the last performance of step (b2).

8. The method of claim 7, wherein said method comprises performing step (b2) a first time with a wash solution comprising said first organic acid, and comprises performing step (b2) a second time with a wash solution comprising said base.

9. The method of any one of claims 1 to 8, wherein said step (b) comprises step (b4) contacting the solid-phase bound sample constituents with an elution solution comprising of from 80% (v / v) to 90% (v / v) of a third organic solvent and (i) from 4% (v / v) to 15% (v / v) of a base or (ii) of from 5% (v / v) to 10% (v / v) of a second organic acid, in an embodiment wherein the third organic solvent in the elution solution in step (b4) is acetonitrile.

10. The method of any one of claims 7 to 9, wherein said first organic acid is a Ci to C> organic acid and / or said second organic acid is a Cl to C5 organic acid and / or wherein said base is ammonia.

11. A system comprising(i) a storage device comprising an organic solvent;(ii) a liquid handling device configured to contact a sample with the organic solvent and a solid-phase extraction agent;(iii) a separation device configured to separate a solid-phase extraction agent from sample constituents;(iv) a microprocessor; and(v) a data storage device comprising stored software instructions for controlling the system, wherein the software instructions, when executed on the microprocessor, cause the system to perform the method according to any one of claims 1 to 10.

12. The system of claim 11, further comprising (vi) a detection device configured to detect a macrocyclic analyte and / or (vii) an analysis device configured to determine the macrocyclic analyte.

13. A kit for macrocyclic analyte determination, the kit comprising(I) a lysis solution comprising a first organic solvent at a concentration of from 4% (v / v) to 20% (v / v), wherein said lysis solution does not comprise an organic acid;(II) a wash solution comprising (i) of from 10% (v / v) to 20% (v / v) of a second organic solvent and (i) from 4% (v / v) to 15% (v / v) of a base or (ii) of from 5% (v / v) to 10% (v / v) of a first organic acid; and(III) an elution solution comprising of from 80% (v / v) to 90% (v / v) organic solvent and of from 4% (v / v) to 15% (v / v) of a base.

14. An extract of a macrocyclic analyte from a sample produced or producible by a method according to any one of claims 1 to 10.

15. A method for determining a macrocyclic analyte in a sample by a chromatography method, comprising(A) providing an extract comprising the macrocyclic analyte bound to a solid-phase extraction agent, wherein said extract was obtained by contacting the sample with a lysis solution comprising a first organic solvent at a concentration of from 4% (v / v) to 20% (v / v) and with the solid-phase extraction agent to produce a lysis admixture as specified in claim 1 ;(B) washing said solid-phase extraction agent comprising bound macrocyclic analyte with a wash solution comprising of from 10% (v / v) to 20% (v / v) of a second organic solvent and of from 5% (v / v) to 10% (v / v) of a first organic acid;(C) eluting said macrocyclic analyte from said solid-phase extraction agent with an elution solution comprising of from 80% (v / v) to 90% (v / v) of a third organic solvent and of from 4% (v / v) to 15% (v / v) of a base; and(D) applying the macrocyclic analyte eluted in step (C) to a chromatography device, thereby determining said macrocyclic analyte.

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