Methods for determining levels of vitamin D and its metabolites - Patent Application 20070122997

Abstract

The present invention relates to a method for determining the level of vitamin D and its metabolites and uses thereof. Furthermore, it is an object of the present invention to provide a kit for determining the level of vitamin D and its metabolites and uses thereof.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for determining the level of vitamin D and its metabolites and uses thereof. Furthermore, it is an object of the present invention to provide a kit for determining the level of vitamin D and its metabolites and uses thereof. [Background technology]

[0002] Background of the Invention The majority (85%) of vitamin D metabolites in serum and plasma samples are tightly bound to vitamin D binding protein. To release vitamin D metabolites from vitamin D binding protein for subsequent purification of the analyte and quantification by LC-MS / MS, protein precipitation via, for example, acetonitrile is applied or salicylic acid sodium salt is used as a releasing reagent. Vitamin D metabolites, preferably 25-OH-D3, 25-OH-D2, 24R,25(OH)2-D3, in human serum or plasma are quantified using, for example: (1) Protein precipitation followed by online sample preparation and LC-MS / MS (see Clinical Biochemistry 45(2012)1491-1496) or (2) An analyte release step using salicylic acid sodium salt solution followed by immunobead capture and chemiluminescence detection (see US Pat. No. 7,087,395).

[0003] Salicylate displaces vitamin D as a protein ligand (Varshney et al.: Ligand Binding Strategies of Human Serum Albumin. Chirality, 2010, 22, pp 80-81) and prevents vitamin D from binding back to proteins (US Patent Application Publication No. 2010 / 0068725).

[0004] Both vitamin D and salicylic acid are part of an MS analyte portfolio, for example the Cobas MS analyte portfolio, which can be measured automatically one after the other.

[0005] Therefore, it is strongly recommended to avoid salicylic acid sodium salt (sodium salicylate) as a potential analyte-releasing reagent (pretreatment) for iVitD assays (iVitD assays refer to vitamin D assays for use with mass spectrometers, preferably LC-MS-based vitamin D assays), as long as salicylic acid is also part of the analyte portfolio, as there is a risk that residues of the iVitD (pre)treatment reagent could falsify the results of subsequent salicylic acid measurements.

[0006] A further disadvantage of salicylic acid sodium salt (sodium salicylate) is that liquid handling (pipetting process) is difficult due to its high kinematic viscosity (23 mPas at 6 °C for a 5.6 M solution in 0.01 M PBS / methanol 9 / 1), and high consumption is required, creating a carryover risk, as the salt may remain on the instrument after the vitamin D workflow and falsify subsequent salicylic acid measurements.A further disadvantage of salicylic acid sodium salt (sodium salicylate) is that this salt must be applied as a highly concentrated solution to reach a satisfactory serum:pretreatment volume ratio, preferably in the range of 145:45 to 193:36.

[0007] Therefore, there is an urgent need in the art to overcome the above problems.

[0008] An object of the present invention is to provide a method for determining the level of vitamin D and its metabolites and use thereof. Furthermore, an object of the present invention is to provide a kit for determining the level of vitamin D and its metabolites and use thereof.

[0009] This object(s) is / are solved by the subject matter of the independent claims. Further embodiments are the subject matter of the dependent claims. Summary of the Invention

[0010] Summary of the Invention In the following, the present invention relates to the following aspects: In a first aspect, the present invention provides a method for determining the level of vitamin D and its metabolites in a sample, comprising the steps of: a) treating the sample with a releasing reagent, the releasing reagent being provided at a level effective to release vitamin D and its metabolites from proteins or lipids present in the sample; The method does not include the addition of sodium salicylate as a releasing agent, The releasing reagent is benzoate anion, one or two hydroxyl groups attached to the phenyl group of the benzoate anion, Optionally, at least one residue having a molar mass of at least 15 g / mol, which is attached to a phenyl group of the benzoate anion, Sodium or ammonium cation or a salt comprising The releasing agent is 3-hydroxybenzoic acid or 2,4-dihydroxybenzoic acid. treating with a releasing reagent; b) optionally purifying the sample obtained from step a), and c) determining the levels of vitamin D and its metabolites using mass spectrometry.

[0011] In a second aspect, the present invention relates to the use of the method of the first aspect of the invention for determining the level of vitamin D and its metabolites in a sample.

[0012] In a third aspect, the present invention provides a kit for determining the level of vitamin D and its metabolites in a sample, the kit being suitable for carrying out a method according to the first aspect of the invention, comprising: a releasing reagent provided at a level effective to release vitamin D and its metabolites from proteins or lipids present in the sample, the kit not including sodium salicylate as the releasing reagent; The releasing reagent is benzoate anion, one or two hydroxyl groups attached to the phenyl group of the benzoate anion, Optionally, at least one residue having a molar mass of at least 15 g / mol, which is attached to a phenyl group of the benzoate anion, Sodium or ammonium cation or a salt comprising The release reagent is 3-hydroxybenzoic acid or 2,4-dihydroxybenzoic acid.

[0013] In a fourth aspect, the present invention relates to the use of a kit according to the third aspect of the invention in a method according to the first aspect of the invention. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the calibration curve (area ratio vs. concentration ratio): Calibration is performed by LC-MS / MS of vitamin D standards in a solvent (60% MeOH) containing the internal standard concentrated as in the treated samples. [Figure 2] FIG. 2 shows the recovery of candidate release agents for 25-OH Vitamin D3 (FIG. 2). [Figure 3] FIG. 3 shows the recovery of candidate release agents for 25-OH Vitamin D2 (FIG. 3). [Figure 4] FIG. 4 shows the recovery of candidate release agents for 24R25-di-OH vitamin D3 (FIG. 4). [Figure 5] FIG. 5 shows the recovery of potential release agents for 24R25-di-OH Vitamin D2 (FIG. 5). [Figure 6]Figure 6 shows the area ratio of 25-OH vitamin D3. The area ratio of 25-OH vitamin D3 is shown for four different sample types depending on the exact pretreatment composition. Highlighted settings are no pH adjustment (0% FA), 2.275 M 3-methylsalicylic acid sodium salt, and 35% (v / v) methanol in the solvent. [Figure 7] Figures 7A and 7B show the intensity of 25-OH vitamin D3 as a function of time in patient serum (15PPTA5144) treated with 2.3 M sodium 3-methylsalicylate. Chromograms are shown to compare the effects of sample treatment with PT1 (Figure 7A) and the newly developed (pre)treatment (Figure 7B). The mass trace of 25-OH vitamin D3 is applied. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention Before describing the present invention in detail below, it is to be understood that the present invention is not limited to the specific embodiments and examples described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0016] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching cited herein, the body of the present specification shall control.

[0017] Each element of the present invention is described below. While these elements are listed with specific embodiments, it is understood that they can be combined in any way and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context dictates otherwise.

[0018] definition It will be understood that the word "comprise", and variations such as "comprises" and "comprising", imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0019] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0020] Ratios, concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range." It is understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​expressly recited as boundaries of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly recited. By way of example, a numerical range of "4% to 20%" should be interpreted not only to include the explicitly recited value 4% to 20%, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, etc., and subranges such as 4-10%, 5-15%, 10-20%, etc. This same principle applies to ranges reciting minimum or maximum values. Moreover, such interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0021] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0022] As used herein, the term "determining" the level of vitamin D and its metabolites refers to determining or measuring the level of vitamin D and its metabolites in a sample using a suitable detection method, e.g., as described elsewhere herein, to quantify vitamin D and its metabolites.

[0023] In this context, "level" or "level value" encompasses absolute amount, relative amount or concentration, as well as any value or parameter that correlates thereto or can be derived therefrom.

[0024] As used herein, the term "sample" or "patient sample" refers to a biological sample obtained for the purpose of in vitro evaluation. In the methods of the present invention, the sample or patient sample may preferably include any bodily fluid. Samples may include blood, serum, plasma, urine, saliva, and synovial fluid. Preferred samples are whole blood, serum, or plasma. As will be understood by those skilled in the art, any such evaluation is performed in vitro. The patient sample is then discarded. The patient sample is used only in the in vitro methods of the present invention, and material from the patient sample is not returned to the patient's body.

[0025] The term "vitamin D and its metabolites" in this context refers to vitamin D, which is used as a general term for several secosteroids found in the human body and essentially consists of or includes two basic families: vitamin D2 and vitamin D3. Vitamin D2 and its metabolites are not produced by the body but are produced with food, either medicinal or fungal. Vitamin D3 is synthesized in large quantities in the skin from the precursor molecule 7-dehydrocholesterol under sunlight / UVB irradiation, and is therefore in its physiological form. Further UV-B irradiation again degrades vitamin D3 into inactive metabolites, which self-limit the synthesis process in the skin. Synonyms for vitamin D2 are ergocalciferol, and synonyms for vitamin D3 are cholecalciferol and calciferol. Both vitamin D2 and vitamin D3 and their metabolites are highly lipophilic and bind to carrier molecules, vitamin D-binding proteins (DBP, VDBP), in plasma, which are essential for transport. Both molecules are transported to the liver, where they are hydroxylated at the 25-position to produce the vitamin D3 family: calcidiol / calcifediol or 25-OH-vitamin D / 25-hydroxycholecalciferol. This is then hydroxylated in the kidney at the 1-position to form the biologically active 1,25-vitamin D / 1,25-dihydroxycholecalciferol, calcitriol. In the vitamin D2 family, the corresponding metabolites are called ercalcidiol / 25-hydroxyergocalciferol and ercalcitriol / 1,25-dihydroxyergocalciferol. Preferably, the metabolites of vitamin D include: 25-OH vitamin D3, 25-OH vitamin D2, 24R,25(OH)2-vitamin D3, 24R,25(OH)2-vitamin D2, 1,25(OH)2 vitamin D2, and / or 1,25(OH)2 vitamin D3. [ka] [ka]

[0026] Vitamin D and its metabolites are analytes of interest. Additionally, salicylic acid and its salts, such as sodium salicylate, can be analytes of interest. In the context of this disclosure, the terms "analyte," "analyte molecule," or "analyte of interest" are used interchangeably to refer to chemical species that are analyzed by mass spectrometry. Chemical species, or analytes, suitable for analysis by mass spectrometry can be any type of molecule present in a living organism, including, but not limited to, nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, peptides, proteins (e.g., cell surface receptors, cytoplasmic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g., vitamin D), molecules characterized by a particular modification of another molecule (e.g., a sugar moiety or phosphoryl residue on a protein, a methyl residue on genomic DNA), or substances internalized by the organism (e.g., therapeutic drugs, drugs of abuse, toxins, etc.), or metabolites of such substances. Such analytes may serve as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance in a biological system that is used as an indicator of the biological state of that system.

[0027] In the context of the present disclosure, the term "treating a sample with a releasing reagent" means that the sample and the releasing reagent come together to have the potential to interact, e.g., react with each other. This may mean that the releasing reagent may be added to the sample, or vice versa.

[0028] The term "releasing reagent" in this context means a chemical substance capable of releasing vitamin D and its metabolites from proteins or lipids. For example, the releasing reagent is suitable for reducing the interaction and / or interference of proteins or lipids with vitamin D and its metabolites.

[0029] The term "effective level of a releasing reagent" in this context means an effective absolute amount, an effective relative amount or an effective concentration suitable for releasing vitamin D and its metabolites from proteins or lipids, as well as any effective value or effective parameter correlated thereto or derivable therefrom. Preferably, at least 10% of the bound vitamin D and its metabolites are released from proteins and / or lipids.

[0030] The term "the method does not add sodium salicylate as a releasing reagent" in this context means that the method does not add sodium salicylate as a releasing reagent. Optionally, it can also mean that the method and / or sample does not contain sodium salicylate as a releasing reagent.

[0031] The term "the releasing reagent is a salt" in this context means that the releasing reagent comprises or consists of two ions - a positively charged ion, sodium or ammonium cation, and a negatively charged ion, benzoate anion. Additionally or alternatively, the term "the releasing reagent is a salt" in this context means that the releasing reagent is an aqueous salt solution.

[0032] The term "benzoate anion" in this context means a negatively charged ion having the formula: [ka]

[0033] The benzoate anion comprises a phenyl group and a carboxylate anion covalently bonded to the phenyl group of the benzoate anion. One or two hydroxyl groups can be further linked or attached to the phenyl group.

[0034] The term "protein" in this context means any of a class of nitrogenous organic compounds having large molecules made up of long chains of one or more amino acids and which are essential parts of all living organisms, especially as structural components of body tissues such as muscles, hair, etc., as well as enzymes and antibodies. Proteins are, for example, vitamin D-binding protein or serum albumin.

[0035] The term "lipid" in this context means any of a class of organic compounds that are fatty acids or derivatives thereof, which are insoluble in water but soluble in organic solvents. They include many natural oils, waxes, and steroids. The term "lipid" includes free fatty acids.

[0036] The term "mass spectrometry" ("Mass Spec" or "MS") or "mass spectrometric determination" or "mass spectrometry" refers to an analytical technique used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." MS techniques generally involve (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating their mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ion source and an ion detector. Generally, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrometry instrument, where a combination of magnetic and electric fields causes the ions to follow a path in space depending on their mass ("m") and charge ("z"). The term "ionization" or "ionizing" refers to the process of generating ions of an analyte having a net charge equal to one or more units. Anions have a net negative charge of one or more units, while cations have a net positive charge of one or more units. MS methods can be performed in either "negative ion mode," in which negative ions are generated and detected, or "positive ion mode," in which positive ions are generated and detected. "Following fragmentation as determined by mass spectrometry" can mean, for example, that a compound, composition, or complex has passed through a mass spectrometer and been fragmented.

[0037] "Tandem mass spectrometry" or "MS / MS" involves multiple steps of selective mass analysis, with analyte fragmentation occurring between steps. In a tandem mass spectrometer, ions are generated in an ion source and separated by mass-to-charge ratio in the first stage of mass analysis (MS1). Ions of specific mass-to-charge ratios (precursor ions or parent ions) are selected, and fragment ions (daughter ions) are generated by collision-induced dissociation, ion-molecule reactions, or photodissociation. The resulting ions are then separated and detected in the second stage of mass analysis (MS2).

[0038] Mass spectrometers separate and detect ions of slightly different masses, easily distinguishing between different isotopes of a given element. Mass spectrometry is therefore an important method for accurate mass determination and characterization of analytes, including, but not limited to, low molecular weight analytes, peptides, polypeptides, or proteins. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits, and functional interactions, and the total measurement of proteins in proteomics. De novo sequencing of peptides or proteins by mass spectrometry can usually be performed without prior knowledge of the amino acid sequence.

[0039] Most sample workflows in MS further include a sample preparation and / or enrichment step, where, for example, the analyte of interest is separated from the matrix using gas or liquid chromatography. Typically, for a mass spectrometry measurement, the following three steps are performed: 1. A sample containing an analyte of interest is ionized, usually by complexation with a cation, often by protonation to a cation. Ionization sources include, but are not limited to, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). 2. The ions are sorted and separated according to their mass and charge. High field asymmetric waveform ion mobility spectrometry (FAIMS) can be used as an ion filter. 3. The separated ions are detected, for example, in multiple reaction mode (MRM), and the results are displayed on a chart.

[0040] The term "electrospray ionization" or "ESI" refers to a method in which a solution is passed down a short length of capillary tube and a high positive or negative potential is applied to the end of the capillary tube. The solution that reaches the end of the tube is vaporized (atomized) into a jet or spray of very small droplets in solvent vapor. This mist of droplets passes through an evaporation chamber, which is slightly heated to prevent condensation and evaporate the solvent. As the droplets become smaller, the electrical surface charge density increases until natural repulsion between like charges causes ions and neutral molecules to be released.

[0041] The term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry method similar to ESI. However, APCI generates ions through ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is sustained by an electrical discharge between the spray capillary and a counter electrode. The ions are then typically extracted into a mass analyzer using a set of differentially pumped skimmer stages. A counterflow of dry, preheated Ni gas may be used to improve solvent removal. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar entities.

[0042] "High-field asymmetric waveform ion mobility spectrometry (FAIMS)" is an atmospheric pressure ion mobility technique that separates gas-phase ions according to their behavior in strong and weak electric fields.

[0043] "Multiple reaction mode" or "MRM" is a detection mode of an MS instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0044] Mass spectrometric determination may be combined with additional analytical methods, including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and / or ion mobility-based separation techniques.

[0045] In the context of this disclosure, a sample may be derived from an "individual" or "subject." Typically, the subject is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0046] Prior to analysis by mass spectrometry, samples may be treated or pretreated in a manner specific to the sample and / or analyte. In the context of the present disclosure, the terms "treatment" or "pretreatment" refer to any measures necessary to enable subsequent analysis of the desired analyte by mass spectrometry. (Pre)treatment measures typically include, but are not limited to, elution of solid samples (e.g., elution of dried blood spots), addition of a hemolyzing reagent (HR) to whole blood samples, and addition of an enzymatic reagent to urine samples. Similarly, addition of an internal standard (ISTD) is considered sample pretreatment.

[0047] The term "hemolytic reagent (HR)" refers to a reagent that lyses cells present in a sample, and in the context of the present invention, hemolytic reagent specifically refers to a reagent that lyses cells present in a blood sample, including but not limited to red blood cells present in a whole blood sample. A well-known hemolytic reagent is water (HO). Further examples of hemolytic reagents include, but are not limited to, deionized water, hypertonic liquids (e.g., 8M urea), ionic liquids, and different surfactants.

[0048] Typically, an internal standard (ISTD) is a known quantity of a substance that exhibits similar properties to the analyte of interest when subjected to a mass spectrometric detection workflow (i.e., including any pretreatment, enrichment, and actual detection steps). The ISTD exhibits similar properties to the analyte of interest, but is clearly distinguishable from it. By way of example, during a chromatographic separation, such as gas or liquid chromatography, the ISTD has approximately the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. The ISTD, however, exhibits a different molecular weight than the analyte of interest from the sample. This allows ions from the ISTD and the analyte to be distinguished in mass spectrometry using their different mass-to-charge (m / z) ratios. Both are subjected to fragmentation to produce daughter ions. These daughter ions can be distinguished by their m / z ratios and their respective parent ions. As a result, the signals from the ISTD and the analyte can be determined and quantified separately. Because the ISTD is added in a known amount, the signal intensity of an analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of the ISTD allows for relative comparison of the amount of analyte detected, allowing for unambiguous identification and quantification of the analyte of interest present in the sample when the analyte(s) reach the mass spectrometer. Typically, although not necessarily, the ISTD is an isotopically labeled variant of the analyte of interest (e.g., 2 H, 13 C, or 15 (including labels such as N).

[0049] In addition to pretreatment, the sample may also be subjected to one or more enrichment steps. In the context of the present disclosure, the term "first enrichment process" or "first enrichment workflow" refers to an enrichment step that occurs following sample (pre)treatment and provides a sample containing enriched analytes compared to the initial sample. The first enrichment workflow may involve chemical precipitation (e.g., using acetonitrile) or the use of a solid phase. Suitable solid phases include, but are not limited to, solid phase extraction (SPE) cartridges and beads. The beads may be nonmagnetic, magnetic, paramagnetic, or supermagnetic. The beads may be coated differently to be specific for the analyte of interest. The coating may vary depending on the intended application, i.e., the intended capture molecule. Those skilled in the art will be familiar with which coating is suitable for which analyte. The beads can be made of a variety of different materials. The beads may have different sizes and may include porous or non-porous surfaces.

[0050] In the context of the present disclosure, the term "second enrichment process" or "second enrichment workflow" refers to an enrichment process that occurs following sample (pre)treatment and a first enrichment process, and provides a sample containing enriched analytes compared to the initial sample and the sample after the first enrichment process.

[0051] The term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of the differential distribution of the chemical components as they flow around or over a stationary liquid or solid phase.

[0052] The terms "liquid chromatography" or "LC" refer to the process of selectively retarding one or more components of a fluid solution as the fluid permeates uniformly through a column or capillary passage of finely divided material. Retardation results from the distribution of mixture components between one or more stationary phase(s) and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal phase liquid chromatography (NPLC), while methods in which the stationary phase is less polar than the mobile phase (e.g., a water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) are called reversed phase liquid chromatography (RPLC).

[0053] "High-performance liquid chromatography" or "HPLC" refers to a method of liquid chromatography in which the degree of separation is increased by passing a mobile phase under pressure through a stationary phase, typically a densely packed column. Typically, the column is packed with a stationary phase composed of irregular or spherical particles, a porous monolithic layer, or a porous membrane. HPLC has historically been divided into two distinct subclasses based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal-phase liquid chromatography (NPLC), while the opposite (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is called reverse-phase liquid chromatography (RPLC). Micro-LC refers to HPLC methods using columns with narrow internal diameters, typically less than 1 mm, e.g., about 0.5 mm. "Ultra-high performance liquid chromatography" or "UHPLC" refers to an HPLC method using a pressure of 120 MPa (17,405 lbf / in2), or approximately 1200 atmospheres. Rapid LC refers to an LC method using a short column with an internal diameter as described above and a length of less than 2 cm, e.g., 1 cm, at the above flow rate and pressure (micro LC, UHPLC). A short rapid LC protocol involves trapping / washing / elution steps using a single analytical column, achieving LC in a very short time of less than 1 minute.

[0054] Additionally, "hydrophilic interaction chromatography" (HILIC), size-exclusion LC, ion-exchange LC, and affinity LC are well known.

[0055] LC separations may be single-channel LC or multi-channel LC, comprising multiple LC channels arranged in parallel, in which analytes may be separated according to their polarity or log P value, size, or affinity, as is commonly known to those skilled in the art.

[0056] A "kit" is any article of manufacture (e.g., a package or container) containing at least one reagent, such as a drug for treating a disorder or a probe for specifically detecting a biomarker gene or protein of the present invention. The kit is preferably promoted, distributed, or sold as a unit for carrying out the method of the present invention. Typically, the kit can further include carrier means compartmentalized to receive in close confinement one or more container means, such as vials, tubes, etc. In particular, each of the container means comprises one of the separate elements used in the method of the first aspect. The kit can further include one or more other reagents, including, but not limited to, a reaction catalyst. The kit may further include one or more other containers containing additional materials, including, but not limited to, buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. Tablets can be displayed on the container to indicate that the composition is to be used for a particular application, and can also indicate instructions for either in vivo or in vitro use. The computer program code can be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or directly to a computer or data processing device. Additionally, the kit may contain standard amounts of acids as described elsewhere herein for calibration purposes.

[0057] Embodiment In a first aspect, the present invention provides a method for determining the level of vitamin D and its metabolites in a sample, comprising the steps of: a) treating the sample with a releasing reagent, the releasing reagent being provided at a level effective to release vitamin D and its metabolites from proteins or lipids present in the sample; The method does not include the addition of sodium salicylate as a releasing agent, The releasing reagent is benzoate anion, one or two hydroxyl groups attached to the phenyl group of the benzoate anion, Optionally, at least one residue having a molar mass of at least 15 g / mol, which is attached to a phenyl group of the benzoate anion, Sodium or ammonium cation or a salt comprising The releasing agent is 3-hydroxybenzoic acid or 2,4-dihydroxybenzoic acid. treating with a releasing reagent; b) optionally purifying the sample obtained from step a), and c) determining the levels of vitamin D and its metabolites using mass spectrometry.

[0058] According to step (a), the sample is treated with a releasing reagent, provided at a level effective to release vitamin D and its metabolites from proteins or lipids present in the sample. The releasing reagent is a salt comprising a benzoate anion, one or two hydroxyl groups attached to the phenyl group of the benzoate anion, optionally at least one residue having a molar mass of at least 15 g / mol attached to the phenyl group of the benzoate anion, sodium cation or ammonium cation, or the releasing reagent is 3-hydroxybenzoic acid or 2,4-dihydroxybenzoic acid.

[0059] In an embodiment of the first aspect of the present invention, the release reagent is selected from the group consisting of sodium 3-methylsalicylate, ammonium salicylate, sodium 3-hydroxybenzoate, 3-hydroxybenzoic acid and 2,4-dihydroxybenzoic acid.

[0060] In an embodiment of the first aspect of the present invention, the release reagent is a salt selected from the group consisting of sodium 3-methylsalicylate, ammonium salicylate and sodium 3-hydroxybenzoate. Preferably, the release reagent is sodium 3-methylsalicylate.

[0061] In an embodiment of the first aspect of the invention, the release reagent has the formula: [ka]

[0062] In an embodiment of the first aspect of the present invention, the releasing reagent is selected from the following compounds having the following formula: [ka]

[0063] In an embodiment of the first aspect of the present invention, the releasing reagent is sodium 3-methylsalicylate having a concentration in the range of 0.7M to 2.8M, preferably in the range of 1.5M to 2.5M, for example 2.0M.

[0064] In an embodiment of the first aspect of the present invention, the releasing reagent is ammonium salicylate, preferably having a concentration of 5.6M.

[0065] In an embodiment of the first aspect of the present invention, the releasing reagent is sodium 3-hydroxybenzoate, preferably having a concentration of 2.8M.

[0066] In an embodiment of the first aspect of the present invention, the releasing reagent is 3-hydroxybenzoic acid having a concentration of 0.05M.

[0067] In an embodiment of the first aspect of the present invention, the releasing reagent is 2,4-dihydroxybenzoic acid having a concentration of 0.05M.

[0068] In an embodiment of the first aspect of the invention, the releasing reagent is formulated as an ammonium or sodium salt, thus allowing for increased solubility in a sample, for example a serum sample.

[0069] In an embodiment of the first aspect of the present invention, the release reagent is sodium methylsalicylate, such as sodium 3-methylsalicylate.

[0070] In an embodiment of the first aspect of the present invention, the releasing reagent is sodium 4-methylsalicylate.

[0071] In an embodiment of the first aspect of the present invention, the releasing reagent is sodium 5-methylsalicylate.

[0072] In an embodiment of the first aspect of the present invention, the releasing reagent is sodium 6-methylsalicylate.

[0073] In an embodiment of the first aspect of the present invention, the releasing reagent is ammonium methyl salicylate.

[0074] In an embodiment of the first aspect of the present invention, the releasing reagent is ammonium 3-methylsalicylate.

[0075] In an embodiment of the first aspect of the present invention, the releasing reagent is ammonium 4-methylsalicylate.

[0076] In an embodiment of the first aspect of the present invention, the releasing reagent is ammonium 5-methylsalicylate.

[0077] In an embodiment of the first aspect of the present invention, the releasing reagent is ammonium 6-methylsalicylate.

[0078] In an embodiment of the first aspect of the present invention, the release reagent is ammonium hydroxybenzoate or sodium hydroxybenzoate.

[0079] In an embodiment of the first aspect of the present invention, the pH value is adjusted by adding an acid, such as formic acid. For example, to release vitamin D and its metabolites from proteins or lipids and adjust the pH value, 145 μl of serum sample, 45 μl of release reagent, and 10 μl of acid are mixed. Preferably, formic acid is used at a concentration range of 0 to 1.8% (450 mM).

[0080] In an embodiment of the first aspect of the present invention, the formic acid has a concentration in the range of 25 to 450 mM.

[0081] In an embodiment of the first aspect of the present invention, an acid, such as formic acid, is added in step a). Therefore, the amount of release reagent (concentration of the salt solution) required can be reduced by adding an acid (e.g., formic acid) without impairing performance. The release reagent and the acid interact synergistically. For example, the following two combinations result in equivalent vitamin D release in patient serum samples: 450 mM formic acid / 0.7 M sodium 3-methylsalicylate and 0 mM formic acid / 2.3 M sodium 3-methylsalicylate.

[0082] In an embodiment of the first aspect of the invention, the method comprises, after or before step a), at least one further step a1) or a2) or both: a1) coupling vitamin D and its metabolites obtained from step a) to a solid phase; a2) adding an internal standard to the sample. Preferably, the internal standard is added to the sample before step (a). Preferably, the coupling of vitamin D and its metabolites obtained from step (a) to a solid phase is performed after step (a).

[0083] Suitable solid phases include, but are not limited to, Solid Phase Extraction (SPE) cartridges and beads.

[0084] In an embodiment of the first aspect of the present invention, one or more beads can be non-magnetic, magnetic, paramagnetic, or supermagnetic. The beads can be coated differently to be specific for the analyte of interest. The coating can vary depending on the intended use, i.e., the intended capture molecule. Those skilled in the art will be familiar with which coating is suitable for which analyte. The beads can be made of a variety of different materials. The beads can have various sizes and can include porous or non-porous surfaces. For example, Elecsys beads (purchased from Roche Diagnostics) coated with analyte-specific antibodies can be used as the solid phase in step (a1). Alternatively, other magnetic beads, for example, covalently bound to antibodies, can be used as the solid phase in step (a1).

[0085] In an embodiment of the first aspect of the present invention, after step (a) and before step (b), the sample is washed to remove or at least reduce the concentration of undesired sample components (proteins, lipids), salts (e.g., sodium chloride or 3-methylsalicylic acid sodium salt), or preservatives (e.g., sodium azide, sodium benzoate, oxypyrion, methylisothiazolinone). The washing step can be performed by using water or phosphate-buffered saline, for example, 0.01 M PBS. After the described washing step, a further washing step can be performed to elute the magnetic beads from the analyte of interest. The further washing step can be performed by using a solvent, for example, methanol.

[0086] Optionally, the sample obtained from step a) is purified according to step (b). The following purification methods or their combinations can be used: liquid chromatography, high-performance liquid chromatography, hydrophilic interaction chromatography (HILIC), size-exclusion LC, ion-exchange LC, affinity LC. In principle, other purification methods are known to those skilled in the art and can be used to purify the sample. Therefore, other known purification methods, such as extraction, will not be described in detail.

[0087] According to step (c), the levels of vitamin D and its metabolites are determined using mass spectrometry.

[0088] In an embodiment of the first aspect of the invention, the level of an analyte of interest other than vitamin D and its metabolites may be determined using mass spectrometry, preferably after step (c) or before step (a) of the method.

[0089] In an embodiment of the first aspect of the present invention, the level of salicylic acid and its salts is determined using mass spectrometry. Preferably, the level of salicylic acid is determined using mass spectrometry. The salicylic acid can be treated in a suitable manner before determining the level of salicylic acid.

[0090] In an embodiment of the first aspect of the present invention, at least one residue is alkyl, preferably methyl (15 g / mol), ethyl (29 g / mol) or propyl (43 g / mol). In particular, exactly one residue is attached to the phenyl group of the benzoate anion. More preferably, the residue is attached to the 3-, 4- and / or 5-position of the phenyl group of the benzoate anion.

[0091] In an embodiment of the first aspect of the present invention, step (a) includes an additive, and the additive is a buffer, water, and / or alcohol. The buffer can be selected from the following group: phosphate buffered saline (PBS), ammonium acetate solution, ammonium formate solution. The alcohol can be selected from the following group: methanol, ethanol, 1-propanol, 2-propanol.

[0092] Preferably, the buffer is phosphate buffered saline (PBS).

[0093] In an embodiment of the first aspect of the present invention, the concentration of PBS is 0.01M.

[0094] Preferably, the alcohol is methanol, for example, in the range of 5% to 50% (v / v).

[0095] In an embodiment of the first aspect of the present invention, the ratio of alcohol to buffer is 5:95 to 40:60 (v / v), preferably 20:80 to 50:50 (v / v), more preferably 35:65 to 40:60 (v / v).

[0096] In an embodiment of the first aspect of the present invention, the sample is a serum, plasma or whole blood sample. For example, if the sample is a whole blood sample, an additional separation step, such as centrifugation, may be performed.

[0097] In an embodiment of the first aspect of the invention, the sample is a human sample.

[0098] In an embodiment of the first aspect of the present invention, vitamin D and its metabolites are selected from the group consisting of 25-OH vitamin D3, 25-OH vitamin D2, 24R,25(OH)2-vitamin D3, 1,25(OH)2 vitamin D2, 1,25(OH)2 vitamin D3 and 24R,25(OH)2-vitamin D2.

[0099] In an embodiment of the first aspect of the present invention the protein is vitamin D binding protein or albumin, preferably vitamin D binding protein.

[0100] In an embodiment of the first aspect of the present invention, the determined levels of vitamin D and its metabolites are in the range of 2 to 150 ng / ml for 25-OH vitamin D3 or 25-OH vitamin D2.

[0101] In an embodiment of the first aspect of the present invention, the determined levels of vitamin D and its metabolites are in the range of 0.2 to 15 ng / ml for 24R,25(OH)2-vitamin D3 or 24R,25(OH)2-vitamin D2.

[0102] In an embodiment of the first aspect of the present invention, the determined levels of vitamin D and its metabolites are in the range of 7 to 150 pg / ml for 1,25(OH)2 vitamin D2 or in the range of 7 to 150 pg / ml for 1,25(OH)2 vitamin D3.

[0103] In an embodiment of the first aspect of the present invention, the method is performed automatically. The term "automatically" or "automated" as used herein is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. The term can specifically, but is not limited to, refer to a process that is performed entirely by at least one computer and / or computer network and / or machine, particularly without manual action and / or user interaction.

[0104] In an embodiment of the first aspect of the present invention, step b) is carried out by chromatography, preferably by liquid chromatography (LC) and / or high performance liquid chromatography (HPLC).

[0105] In an embodiment of the first aspect of the present invention, step c) is carried out by using triple quadrupole mass spectrometry.

[0106] In an embodiment of the first aspect of the present invention, vitamin D and its metabolites are ionized by using electrospray ionization (ESI).

[0107] In a second aspect, the present invention relates to the use of the method of the first aspect of the invention for determining the level of vitamin D and its metabolites in a sample. All embodiments mentioned for the first aspect of the invention apply to the second aspect of the invention and vice versa.

[0108] In a third aspect, the present invention provides a kit for determining the level of vitamin D and its metabolites in a sample, the kit being suitable for carrying out a method according to the first aspect of the invention, comprising: a releasing reagent provided at a level effective to release vitamin D and its metabolites from proteins or lipids present in the sample, the kit not including sodium salicylate as the releasing reagent; The releasing reagent is benzoate anion, one or two hydroxyl groups attached to the phenyl group of the benzoate anion, Optionally, at least one residue having a molar mass of at least 15 g / mol, which is attached to a phenyl group of the benzoate anion, Sodium or ammonium cation or a salt comprising The release reagent is 3-hydroxybenzoic acid or 2,4-dihydroxybenzoic acid.

[0109] All embodiments described for the first aspect of the invention and / or the second aspect of the invention apply to the third aspect of the invention and vice versa.

[0110] In a fourth aspect, the present invention relates to the use of a kit according to the third aspect of the invention in a method according to the first aspect of the invention.

[0111] All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention apply to the fourth aspect of the invention and vice versa.

[0112] In an embodiment of at least one or all aspects of the invention, the method of the first aspect of the invention and / or the kit of the third aspect of the invention is used in a device. Preferably, the device is a clinical diagnostic system.

[0113] A "clinical diagnostic system" is a laboratory automation device dedicated to analyzing samples for in vitro diagnostics. Clinical diagnostic systems can have different configurations depending on the need and / or desired laboratory workflow. Further configurations can be achieved by combining multiple devices and / or modules together. A "module" is a work cell, typically smaller in size than the entire clinical diagnostic system, with a dedicated function. This function can be analytical, but can also be pre-analytical or post-analytical, or the function can be a support function for any of the pre-analytical, analytical, or post-analytical functions. Specifically, a module can be configured to cooperate with one or more other modules to perform a dedicated task in a sample processing workflow, for example, by performing one or more pre-analytical and / or post-analytical steps. In particular, a clinical diagnostic system can include one or more analytical devices designed to perform respective workflows optimized for specific types of analysis, such as clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separations, mass spectrometry, etc. Thus, a clinical diagnostic system can include one analytical device or any combination of such analytical devices with their respective workflows, and pre-analysis and / or post-analysis modules can be coupled to individual analytical devices or shared by multiple analytical devices. Alternatively, pre-analysis and / or post-analysis functions can be performed by units integrated into the analytical devices. A clinical diagnostic system can include functional units such as a liquid handling unit for pipetting, pumping, and / or mixing samples and / or reagents and / or system fluids, as well as functional units for sorting, storing, transporting, identifying, separating, and detecting. A clinical diagnostic system can include a sample preparation station for automated preparation of samples containing analytes of interest, a liquid chromatography (LC) separation station optionally with multiple LC channels, and / or a sample preparation / LC interface for inputting the optionally prepared sample into any one of the LC channels.The clinical diagnostic system may further include a controller programmed to assign samples to predefined sample preparation workflows, each of which includes a predefined series of sample preparation steps and requires a predefined time for completion depending on the analyte of interest. The clinical diagnostic system may further include a mass spectrometer (MS) and an LC / MS interface for connecting the LC separation station to the mass spectrometer. The terms "automatically" and "automated" as used herein are broad terms and should be given their ordinary and customary meaning to those skilled in the art and should not be limited to a specific or customized meaning. The terms may specifically, but are not limited to, refer to a process performed entirely by at least one computer and / or computer network and / or machine, particularly without manual action and / or user interaction.

[0114] In an embodiment of at least one or all aspects of the present invention, the clinical diagnostic system includes a sample preparation station.

[0115] A "sample preparation station" may be a pre-analytical module coupled to one or more analytical devices or units within an analytical device, designed to perform a series of sample processing steps aimed at removing or at least reducing interfering matrix components in the sample and / or concentrating the analyte of interest in the sample. Such processing steps may include one or more of the following processing operations performed sequentially, in parallel, or in a staggered manner on a sample or multiple samples: pipetting (aspiration and / or delivery) of fluids, pumping of fluids, mixing with reagents, incubation at a specific temperature, heating or cooling, centrifugation, separation, filtering, sieving, drying, washing, resuspension, aliquoting, transport, storage, etc.

[0116] The clinical diagnostic system, e.g., a sample preparation station, may also include a buffer unit for receiving multiple samples before a new sample preparation initiation sequence is initiated, and the samples may be individually randomly accessible, the preparation of each of which may be initiated according to the sample preparation initiation sequence.

[0117] Clinical diagnostic systems utilize mass spectrometry in a more convenient and reliable manner, and are therefore suitable for clinical diagnosis. In particular, random access sample preparation and LC separation can be used to obtain high throughput, e.g., up to 100 samples / hour or more, while allowing online connection to mass spectrometry. Furthermore, the process can be fully automated, increasing walk-away time and reducing the level of skill required.

[0118] The inventors have surprisingly found that the method can be performed on a fully automated device, such as the cobas i601 analyzer (serum working area solution), which may mean that there is a soft analyte release step followed by immunobead capture and detection by LC-MS / MS.

[0119] In an extensive study, we were able to demonstrate that 22 reagents were evaluated for their applicability to replace salicylic acid sodium salt as a pretreatment in the cobas MS workflow. Results from the initial screening study indicated that most reagents lacked significant release potential, and only five had potential release potential. 3-methylsalicylic acid sodium salt solution appeared to be the best choice, as it yielded the highest analyte recovery. In a subsequent comprehensive experimental design, 3-methylsalicylic acid sodium salt solution was optimized and evaluated in detail. It is the most promising pretreatment. When this reagent was applied, high analyte recoveries comparable to those achieved with salicylic acid sodium salt were achieved. Furthermore, the advantage of 3-methylsalicylic acid sodium salt compared to salicylic acid sodium salt is that this reagent does not interfere with the associated MRM transitions, which could falsify the salicylic acid results.

[0120] In further embodiments, the present invention relates to the following aspects: 1. A method for determining the level of vitamin D and its metabolites in a sample, comprising: a) treating the sample with a releasing reagent, the releasing reagent being provided at a level effective to release vitamin D and its metabolites from proteins or lipids present in the sample; The method does not include the addition of sodium salicylate as a releasing agent, The releasing reagent is benzoate anion, one or two hydroxyl groups attached to the phenyl group of the benzoate anion, Optionally, at least one residue having a molar mass of at least 15 g / mol, which is attached to a phenyl group of the benzoate anion, Sodium or ammonium cation or a salt comprising The releasing agent is 3-hydroxybenzoic acid or 2,4-dihydroxybenzoic acid. treating with a releasing reagent; b) optionally purifying the sample obtained from step a), and c) determining the levels of vitamin D and its metabolites using mass spectrometry.

[0121] 2. The method of aspect 1, wherein the releasing reagent is selected from the group consisting of sodium 3-methylsalicylate, ammonium salicylate, sodium 3-hydroxybenzoate, 3-hydroxybenzoic acid, and 2,4-dihydroxybenzoic acid.

[0122] 3. The method of aspect 1 or 2, wherein the releasing reagent is a salt selected from the group consisting of sodium 3-methylsalicylate, ammonium salicylate, and sodium 3-hydroxybenzoate.

[0123] 4. The method of any one of aspects 1-3, wherein the releasing reagent is sodium 3-methylsalicylate.

[0124] 5. The method of any one of aspects 1 to 4, wherein at least one residue is alkyl, preferably methyl or ethyl.

[0125] 6. The method of any one of aspects 1-5, wherein the releasing reagent is sodium 3-methylsalicylate having a concentration in the range of 0.7M to 2.8M.

[0126] 7. The method of any one of aspects 1-6, wherein the releasing reagent is ammonium salicylate, preferably having a concentration of 5.6 M.

[0127] 8. The method of any one of aspects 1 to 7, wherein the releasing reagent is sodium 3-hydroxybenzoate, preferably having a concentration of 2.8 M.

[0128] 9. The method of any one of aspects 1-8, wherein the releasing reagent is 3-hydroxybenzoic acid having a concentration of 0.05M.

[0129] 10. The method of any one of aspects 1-9, wherein the releasing reagent is 2,4-dihydroxybenzoic acid having a concentration of 0.05M.

[0130] 11. The method of any one of aspects 1-10, wherein the release reagent is formulated as an ammonium salt or a sodium salt.

[0131] 12. Step c) is followed by at least one further step d): 12. The method of any one of aspects 1-11, comprising: d) determining the level of salicylic acid and salts thereof using mass spectrometry.

[0132] 13. The method of any one of aspects 1-12, wherein the releasing reagent is sodium methylsalicylate, such as sodium 3-methylsalicylate.

[0133] 14. The method of any one of aspects 1-13, wherein the releasing reagent is sodium 4-methylsalicylate.

[0134] 15. The method of any one of aspects 1-14, wherein the releasing reagent is sodium 5-methylsalicylate.

[0135] 16. The method of any one of aspects 1-15, wherein the releasing reagent is sodium 6-methylsalicylate.

[0136] 17. The method of any one of aspects 1-16, wherein the releasing reagent is ammonium salicylate.

[0137] 18. The method of any one of aspects 1-17, wherein the releasing reagent is ammonium 3-salicylate.

[0138] 19. The method of any one of aspects 1-18, wherein the releasing reagent is 4-ammonium salicylate.

[0139] 20. The method of any one of aspects 1-19, wherein the releasing reagent is ammonium 5-salicylate.

[0140] 21. The method of any one of aspects 1-20, wherein the releasing reagent is ammonium 6-salicylate.

[0141] 22. The method of any one of aspects 1-21, wherein the releasing reagent is ammonium hydroxybenzoate or sodium hydroxybenzoate.

[0142] 23. The method according to any one of aspects 1 to 22, wherein the pH value is adjusted, for example, by adding an acid, for example formic acid.

[0143] 24. The method of any one of aspects 1-23, wherein the formic acid has a concentration in the range of 25 to 450 mM.

[0144] 25. After or before step a), at least one further step a1) or a2) or both: a1) coupling vitamin D and its metabolites obtained from step a) to a solid phase; a2) adding an internal standard to the sample; 25. The method of any one of aspects 1 to 24, comprising:

[0145] 26. The method of any one of aspects 1 to 25, wherein step a) comprises additives, said additives being a buffer and an alcohol.

[0146] 27. The method of any one of aspects 1-26, wherein the buffer is phosphate buffered saline (PBS).

[0147] 28. The method of any one of aspects 1-27, wherein the concentration of PBS is 0.01M.

[0148] 29. The method of any one of aspects 1-28, wherein the alcohol is methanol.

[0149] 30. The method according to any one of aspects 1 to 29, wherein the ratio of alcohol to buffer is 5:95 to 40:60 (v / v), preferably 35:65 to 40:60 (v / v).

[0150] 31. The method of any one of aspects 1 to 30, wherein the sample is a serum, plasma or whole blood sample.

[0151] 32. The method of any one of aspects 1-31, wherein the sample is a human sample.

[0152] 33. The method of any one of aspects 1-32, wherein vitamin D and its metabolites are selected from the group consisting of 25-OH vitamin D3, 25-OH vitamin D2, 24R,25(OH)2-vitamin D3, 1,25(OH)2 vitamin D2, 1,25(OH)2 vitamin D3 and 24R,25(OH)2-vitamin D2.

[0153] 34. The method of any one of aspects 1-33, wherein the protein is vitamin D binding protein or albumin.

[0154] 35. The method of any one of aspects 1-34, wherein the determined levels of vitamin D and its metabolites are in the range of 2 to 150 ng / ml for 25-OH vitamin D3 or 25-OH vitamin D2.

[0155] 36. The method of any one of aspects 1-35, wherein the determined levels of vitamin D and its metabolites are in the range of 0.2 to 15 ng / ml for 24R,25(OH)2-vitamin D3 or 24R,25(OH)2-vitamin D2.

[0156] 37. The method of any one of aspects 1-36, wherein the determined levels of vitamin D and its metabolites are in the range of 7 to 150 pg / ml for 1,25(OH)2 vitamin D2 or in the range of 7 to 150 pg / ml for 1,25(OH)2 vitamin D3.

[0157] 38. The method of any one of aspects 1-37, wherein the method is performed automatically.

[0158] 39. The method according to any one of aspects 1 to 38, wherein step b) is carried out by chromatography, preferably by liquid chromatography (LC) and / or high performance liquid chromatography (HPLC).

[0159] 40. The method of any one of embodiments 1-39, wherein step c) is carried out by using triple quadrupole mass spectrometry.

[0160] 41. The method of any one of aspects 1-40, wherein vitamin D and its metabolites are ionized by using electrospray ionization (ESI).

[0161] 42. Use of a method according to any one of aspects 1 to 41 for determining the level of vitamin D and its metabolites in a sample.

[0162] 43. A kit for determining the level of vitamin D and its metabolites in a sample, the kit being suitable for carrying out the method according to any one of aspects 1 to 41, a releasing reagent provided at a level effective to release vitamin D and its metabolites from proteins or lipids present in the sample, the kit not including sodium salicylate as the releasing reagent; The releasing reagent is benzoate anion, one or two hydroxyl groups attached to the phenyl group of the benzoate anion, Optionally, at least one residue having a molar mass of at least 15 g / mol, which is attached to a phenyl group of the benzoate anion, Sodium or ammonium cation or a salt comprising The kit, wherein the releasing reagent is 3-hydroxybenzoic acid or 2,4-dihydroxybenzoic acid.

[0163] 44. Use of the kit of embodiment 43 in a method according to any one of embodiments 1 to 41. [Example]

[0164] The following examples are offered to illustrate, but not to limit, the invention claimed herein.

[0165] Analytes (vitamin D and its metabolites) and internal standards, respectively: ·24R,25(OH)2-D2 13C5(Endotherm Life Science Molecules,Saarbrucken) ·24R,25(OH)2-D3 13C5(Endotherm Life Science Molecules,Saarbrucken) ·25-OH-D2 13C5(Endotherm Life Science Molecules,Saarbrucken) ·25-OH-D3 13C5(Endotherm Life Science Molecules,Saarbrucken) ·24R,25(OH)2-D2(Endotherm Life Science Molecules,Saarbrucken) ·24R,25(OH)2-D3(Sigma, Schnelldorf) 25-OH-D2 (Sigma, Schnelldorf) 25-OH-D3 (Sigma, Schnelldorf)

[0166] Sample matrix: Natural serum pool (Roche, Penzberg) Vitamin D-free serum (Golden West Diagnostics, Temecula, USA) Patient serum and plasma

[0167] Solvents, reagent additives and HPLC elution additives: Methanol (Biosolve, Valkenswaard / Netherlands) Formic acid (FA) (VWR, Radnor / USA) MilliQ water (Merck, Darmstadt)

[0168] ImmunoBeads suspension components: Pre-coated Elecsys bead suspension (Roche, Penzberg) ·Antibody solution (Roche, Penzberg) 0.1M PBS (Roche, Mannheim)

[0169] Chemicals being evaluated as potential release agents: Sodium salicylate (Sigma, Schnelldorf) Phenyl salicylate (Sigma, Schnelldorf) Diflunisal (Sigma, Schnelldorf) Ethyl-5-ethoxy-2-hydroxybenzoate (Sigma, Schnelldorf) Sulfasalazine (Sigma, Schnelldorf) Methyl salicylate (Sigma, Schnelldorf) Aspirin (Sigma, Schnelldorf) Methyl-4-hydroxybenzoate (Sigma, Schnelldorf) 3-Hydroxybenzoic acid (Sigma, Schnelldorf) 3-Methylsalicylic acid (Sigma, Schnelldorf) 4-Methylsalicylic acid (Sigma, Schnelldorf) Ethylene glycol monosalicylate (Sigma, Schnelldorf) Salicylic acid (Sigma, Schnelldorf) 2-Hydroxy-5-methylbenzoic acid (Sigma, Schnelldorf) 2-Methoxy-benzoic acid (Sigma, Schnelldorf) 4-Hydroxyisophthalic acid (Sigma, Schnelldorf) Methylparaben sodium (Carbosynth, Newbury / UK) 2,4-Dihydroxybenzoic acid (ABCR, Karlsruhe) 3-Methylsalicylic acid sodium salt (ABCR, Karlsruhe) 3-Hydroxybenzoic acid sodium salt (ABCR, Karlsruhe) 2-Hydroxy-4-trifluoromethylbenzoic acid (ABCR, Karlsruhe) Ammonium salicylate (ABCR, Karlsruhe) Sodium chloride (Merck, Darmstadt)

[0170] sample: 0.30 ml of di-OH vitamin D stock solution (0.74 μg / ml 24R,25-di-OH vitamin D3 and 2.00 μg / ml 24R,25-di-OH vitamin D2 in methanol) and 0.55 ml of mono-OH vitamin D stock solution (10.00 μg / ml 25-OH vitamin D3 and 10.00 μg / ml 25-OH vitamin D2 in methanol) are added to 99.15 ml of the naive serum pool and homogenized. The resulting concentrations in the spiked pool are therefore: 3.9ng / ml of 24R,25-di-OH vitamin D3 6.0ng / ml of 24R,25-di-OH vitamin D2 55.0ng / ml of 25-OH vitamin D2 84.9ng / ml of 25-OH vitamin D3 Taking into account the endogenous amount in the original natural serum pool (according to internal LC-MS / MS results, the endogenous amount of 25-OH vitamin D3 is 19.9 ng / ml; the corresponding value for 24R,25-di-OH vitamin D3 is approximately 1.9 ng / ml).

[0171] (Pre)treatment preparation: All potential release agents were dissolved (as nearly concentrated as possible) in methanol / 0.01 M PBS 1 / 9 (v / v). The molar concentrations of the chemicals evaluated were as follows: Concentration Potential Release Agent Abbreviation 5.6M Sodium Salicylate 'PT1' 0.0009M Phenyl salicylate 'PT17' 0.0004M Diflunisal 'PT18' 0.004M Ethyl-5-ethoxy-2-hydroxybenzoate 'PT19' 0.009M sulfasalazine 'PT11' 0.01M Methyl salicylate 'PT16' 0.01M Aspirin 'PT10' 0.03M Methyl-4-hydroxybenzoate 'PT14' 0.05M 3-hydroxybenzoic acid 'PT5' 0.01M 3-methylsalicylic acid 'PT15' 0.02M 4-methylsalicylic acid 'PT6' 0.05M Ethylene glycol monosalicylate 'PT3' 0.03M Salicylic Acid 'PT12' 0.01M 2-hydroxy-5-methylbenzoic acid 'PT13' 0.03M 2-Methoxy-benzoic acid 'PT9' 0.001M 4-hydroxyisophthalic acid 'PT4' 1.4M Sodium Methylparaben 'PT8' 0.05M 2,4-dihydroxybenzoic acid 'PT7' 1.4M 3-Methylsalicylic acid sodium salt 'PT21' 2.8M 3-hydroxybenzoic acid sodium salt 'PT20' 0.006M 2-hydroxy-4-trifluoromethylbenzoic acid 'PT22' 5.6M Ammonium Salicylate 'PT2' 2.9M Sodium Chloride 'PT23'

[0172] The molar concentration of each reagent is chosen according to its solubility, particularly for sodium and ammonium salts, where high molar concentrations of 1.4 M or more are possible.

[0173] Internal standard stock and working solutions: Prepare internal standard stock solutions (11 μg / ml 24R,25-di-OH vitamin D3 13C5; 44 μg / ml 25-OH vitamin D3 13C5; 44 μg / ml 25-OH vitamin D2 13C5) in methanol. Prepare internal standard working solutions (73 ng / ml 24R,25-di-OH vitamin D3 13C5; 291 ng / ml 25-OH vitamin D3 13C5; 291 ng / ml 25-OH vitamin D2 13C5) in water / methanol 6 / 4 (v / v).

[0174] Immunobead suspension: 1507 μl of pre-coated Elecsys bead suspension (21.24 mg / ml) is mixed with 5732 μl of 0.01 M PBS and 762 μl of antibody solution (1.05 mg / ml). After an incubation period of 2 hours at room temperature, the immunobead suspension is ready to use.

[0175] Sample preparation: Sample preparation is performed with a calibrated automated liquid handling robot (Hamilton, Bonaduz / Switzerland).

[0176] Ten microliters of vitamin D-free serum (representing the ISTD dummy) is pipetted into 145 μl of sample in a plastic container. After incubation at 37°C for 231 seconds with periodic shaking, 45 μl of pretreatment reagent is added. Again, incubation is continued at 37°C for 640 seconds with periodic shaking. Thus, vitamin D should or will be gently released from the vitamin D-binding protein. In the next step, 50 μl of immunobead suspension is pipetted into the mixture. Again, incubation is continued at 37°C for 393 seconds with periodic shaking. After two bead washes with 0.01 M PBS and magnetic separation, 60 μl of elution reagent water / methanol 2 / 8 (v / v) is added and a 40-second incubation period is performed. 40 μl of the supernatant can be transferred to an HPLC glass vial, while 20 μl must remain in the container to avoid carryover of beads into the supernatant. In the final step, 40 μl of internal standard working solution is added to the supernatant and homogenized by spit-and-pipetting technique.

[0177] LC-MS / MS: HPLC-MS / MS analysis was performed using a 1290 Infinity Multisampler and 1290 Inifinity LC System (Agilent Technologies, Santa Clara, USA) coupled to a Triple Quad 6500+MS (Sciex, Darmstadt).

[0178] To separate the analytes, 40 μl of each sample is injected onto an analytical C18 column (50 × 2.1 mm, 2.6 μm, Hitachi, Tokyo, Japan). This is achieved at a flow rate of 1.0 ml / min and a column temperature of 50 °C. Separation of the analytes is achieved with the following gradient of 50 mM formic acid (A) and methanol (B): 0.00 minutes:80% B 0.70 minutes: 90% B 0.75 minutes: 98% B 1.70 minutes: 98% B 1.80 minutes: 80% B 2.40 minutes:80% B

[0179] The coupled MS detector was operated in positive electrospray ionization (ESI) mode. Vitamin D analytes were detected using multiple reaction monitoring (MRM) techniques. Two time periods were applied: 0.00-0.60 min for the measurement of 24R,25-di-OH vitamin D2 / D3 and 0.60-2.40 min for the detection of 25-OH vitamin D2 / D3. The ion source settings and MRM parameters were as follows: First period: 0.00~0.60 minutes Source temperature: 500°C Nebulizer gas: 40 units Heating gas: 70 units IonSpray voltage: 3000V Curtain gas: 35 units Collision gas: 10 units [Table 1] Second period: 0.60~2.40 minutes Source temperature: 500°C Nebulizer gas: 60 units Heating gas: 70 units IonSpray voltage: 3000 V Curtain gas: 35 units Collision gas: 10 units [Table 2]

[0180] Calibration was performed by LC-MS / MS of vitamin D standards in a solvent (60% MeOH) containing an internal standard concentrated like the treated sample. For example, the calibration levels of 25-OH vitamin D3 were 2, 5, 10, 25, 50, and 100 ng / ml, and the concentration of 25-OH vitamin D3 13C5 was set at 30 ng / ml. The results are shown in Figure 1.

[0181] Therefore, the correction factor

number

[0182] Due to the fact that the internal standard concentration is the same in both the calibrators and the processed samples, it is possible to calculate the recovery of each analyte using the following formula:

number

number

[0183] The results are shown in Figures 2 to 5, which show the recovery rates of the candidate release reagents: 25-OH vitamin D3 (Figure 2), 25-OH vitamin D2 (Figure 3), 24R25-di-OH vitamin D3 (Figure 4), and 24R25-di-OH vitamin D2 (Figure 5).

[0184] Each error bar in Figures 2 and 5 is constructed using the min and max of the data. PT2~23: 2 consecutive PT1: 4 consecutive

[0185] The lowest recoveries are achieved by PT8 (1.4 M methylparaben sodium salt) and PT14 (0.03 M methylparaben). In general, the group of benzoic acid methyl esters does not appear to be suitable for releasing vitamin D and its metabolites.

[0186] The highest recovery is achieved by PT1 (5.6 M sodium salicylate), followed by PT21 (1.4 M 3-methylsalicylic acid sodium salt), PT2 (5.6 M ammonium salicylate), PT20 (2.8 M 3-hydroxybenzoic acid sodium salt), PT5 (0.05 M 3-hydroxybenzoic acid) and PT7 (0.05 M 2,4-dihydroxybenzoic acid).

[0187] PT21 (1.4 M 3-methylsalicylic acid sodium salt) exhibited the highest analyte recovery, relatively low salt consumption, and suitable parent and product ions, and is therefore the best option for replacing salicylic acid in vitamin D pretreatment.

[0188] Further pre-processing optimization within the framework of a "Design of Experiments" setup (DoE) Preliminary studies have shown that the following factors have the highest level of impact and will be evaluated in detail on analyte recovery: [Table 3]

[0189] Figure 6 shows the area ratio of 25-OH vitamin D3. As can be seen from Figure 6, Acidic pH adjustment has a negative effect on analyte recovery when analyzing spiked serum without vitamin D. Therefore, the addition of formic acid (FA) is not recommended as this matrix will be used in the preparation of calibrator solutions. Optimal recovery results are obtained for all matrices (serum, plasma, vitamin D-free serum) using the following formulation: 0% FA (this means no pH adjustment is necessary) 2.3M 3-methylsalicylic acid sodium salt 35 / 65 (v / v) methanol / 0.01M PBS The optimized pretreatment with 2.3 M 3-methylsalicylic acid sodium salt results in similar analyte recovery as a pretreatment containing 5.6 M salicylic acid sodium salt (PT1). For example, both chromatograms are shown for a patient serum containing 3.8 ng / ml 25-OH vitamin D3 (see Figures 7A and 7B).

[0190] As can be seen from the examples, 2.3 M 3-methylsalicylic acid sodium salt dissolved in 35 / 65 MeOH / 0.01 M PBS (v / v) is the best candidate release reagent. The improvements compared to the (pre)treatment containing 5.6 M salicylic acid sodium salt (PT1) as the release reagent are as follows: Low dynamic viscosity (8.3 mPa·s at 6°C for 3-methylsalicylic acid sodium salt and 23 mPa·s at 6°C for salicylic acid sodium salt) for improved pipetting Less salt was consumed, but performance was comparable (vitamin D release: approximately 18 mg of 3-methylsalicylic acid sodium salt per sample vs. approximately 26 mg of salicylic acid sodium salt per sample). No carryover risk: salicylate measurements are independent of preceding vitamin D post-treatment

[0191] This patent application claims priority to European patent application 20199000.9, the contents of which are incorporated herein by reference.

Claims

1. 1. A method for determining the level of vitamin D and its metabolites in a sample, comprising: a) treating the sample with a releasing reagent, the releasing reagent being provided at a level effective to release vitamin D and its metabolites from proteins or lipids present in the sample; The method does not include the addition of sodium salicylate as the releasing reagent; the releasing reagent is 3-methylsalicylic acid sodium salt, ammonium salicylate, 3-hydroxybenzoic acid sodium salt, 3-hydroxybenzoic acid, or 2,4-dihydroxybenzoic acid; treating said sample with a releasing reagent; and c) determining the levels of vitamin D and its metabolites using mass spectrometry; A method comprising:

2. Before step c), b) purifying the sample obtained from step a); The method of claim 1 , comprising:

3. 3. The method of claim 1, wherein the releasing reagent is sodium 3-methylsalicylate having a concentration ranging from 0.7M to 2.8M.

4. Step c) is followed by at least one further step d): d) determining the levels of salicylic acid and its salts using mass spectrometry; The method according to any one of claims 1 to 3, comprising:

5. After or before step a), at least one further step a1) or a2) or both: a1) coupling vitamin D and its metabolites obtained from step a) to a solid phase; a2) adding an internal standard to said sample; The method according to any one of claims 1 to 4, comprising:

6. 6. The method of any one of claims 1 to 5, wherein step a) comprises additives, said additives being a buffer and an alcohol.

7. 7. The method according to any one of claims 1 to 6, wherein vitamin D and its metabolites are selected from the group consisting of 25-OH vitamin D3, 25-OH vitamin D2, 24R,25(OH)2-vitamin D3, 1,25(OH)2 vitamin D2, 1,25(OH)2 vitamin D3 and 24R,25(OH)2-vitamin D2.

8. The method according to any one of claims 1 to 7, wherein the protein is vitamin D binding protein or albumin.

9. The method according to any one of claims 1 to 8, wherein the method is carried out automatically.

10. The method according to any one of claims 1 to 9, wherein step b) is carried out by chromatography and step c) is carried out by using triple quadrupole mass spectrometry.

11. 1. A kit for determining the level of vitamin D and its metabolites in a sample, comprising:

10. The kit of claim 1, wherein the releasing reagent is provided at a level effective to release vitamin D and its metabolites from proteins or lipids present in the sample, and the kit does not include sodium salicylate as the releasing reagent. kit.

12. Use of the kit according to claim 11 in the method according to any one of claims 1 to 10.

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