Key information

Title
Sample preparation method
Application number
20240093
Case type
National
Status
23.06.2025 Patent meddelt (B1)
Filed
01.02.2024
Effective date
01.02.2024
Publicly available
23.06.2025
Next annual fee due
28.02.2027
Applicant
FISHLAB AS (NO)
Owner
FISHLAB AS (NO)
Inventor
Cato Brede (NO)
Agent
HÅMSØ PATENTBYRÅ AS (NO)
Granted
23.06.2025
Patent number
348856
Expiry date
01.02.2044

Abstract and drawing

Disclaimer: This text has been machine-scanned and may contain errors – please refer to "Publications" for legally binding content.
The invention relates to a sample preparation method comprising the steps of: a) obtaining a biological sample, the biological sample comprising an aqueous solution and at least one analyte dissolved in the aqueous solution; b) adding a water-miscible solvent to the biological sample to obtain a sample solution; c) adding a salting-out reagent or a sugaring-out reagent to the sample solution of step b) to obtain a phase separated solution, wherein the aqueous solution forms a water phase, and the water-miscible solvent forms a solvent phase separated from the aqueous phase, and wherein the at least one analyte is dissolved in the solvent phase; and d) adding a derivatisation reagent to the solvent phase to obtain a derivative of the at least one analyte.

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 02.01.2026 12-01 Saksbehandling 20240093(348856) UT PT Varsel om betaling av første årsavgift (3317) (PT20240093) HÅMSØ PATENTBYRÅ AS
 27.06.2025 11-01 Saksbehandling 20240093(348856) UT PT Registreringsbrev nasjonal patent (15) (PT20240093)
08.04.2025 10-01 Saksbehandling 20240093 UT Intention to grant HÅMSØ PATENTBYRÅ AS
 28.02.2025 09-05 Saksbehandling 20240093 INN P31147NO00 Response_Office_Action HÅMSØ PATENTBYRÅ AS
 28.02.2025 09-04 Saksbehandling 20240093 INN P31147NO00 Oversatte krav HÅMSØ PATENTBYRÅ AS
 28.02.2025 09-03 Saksbehandling 20240093 INN P31147NO00 Description_and_claims_mark-up HÅMSØ PATENTBYRÅ AS
 28.02.2025 09-02 Saksbehandling 20240093 INN P31147NO00 Description_and_claims HÅMSØ PATENTBYRÅ AS
 28.02.2025 09-01 Saksbehandling 20240093 INN Korrespondanse (Hovedbrev inn) HÅMSØ PATENTBYRÅ AS
 10.12.2024 08-01 Saksbehandling 20240093 UT Substantive examination HÅMSØ PATENTBYRÅ AS
 18.10.2024 07-04 Saksbehandling 20240093 INN P31147NO00_Response_Office_Action HÅMSØ PATENTBYRÅ AS
 18.10.2024 07-03 Saksbehandling 20240093 INN P31147NO00 Amended_claims_Mark-up HÅMSØ PATENTBYRÅ AS
 18.10.2024 07-02 Saksbehandling 20240093 INN P31147NO00 Amended_claims HÅMSØ PATENTBYRÅ AS
 18.10.2024 07-01 Saksbehandling 20240093 INN Korrespondanse (Hovedbrev inn) HÅMSØ PATENTBYRÅ AS
 18.04.2024 06-02 Saksbehandling 20240093 UT PT report 12:37:55
18.04.2024 06-01 Saksbehandling 20240093 UT 20240093_1840847_PT 08 Eng HÅMSØ PATENTBYRÅ AS
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29.02.2024 04-02 Saksbehandling 20240093 INN P31147NO00 - Signert generalfullmakt HÅMSØ PATENTBYRÅ AS
 29.02.2024 04-01 Saksbehandling 20240093 INN Korrespondanse (Hovedbrev inn) HÅMSØ PATENTBYRÅ AS
 02.02.2024 03-01 Saksbehandling 20240093 UT Formal Examination 1 HÅMSØ PATENTBYRÅ AS
 02.02.2024 02-01 Saksbehandling 20240093 UT Informasjon til oppfinner Cato Brede
 01.02.2024 01-03 Saksbehandling 20240093 INN P31147NO00_Figures HÅMSØ PATENTBYRÅ AS
 01.02.2024 01-02 Saksbehandling 20240093 INN P31147NO00_Description_and_claims HÅMSØ PATENTBYRÅ AS
 01.02.2024 01-01 Saksbehandling 20240093 INN Søknadsskjema Patent HÅMSØ PATENTBYRÅ AS

Description, claims and drawings

Disclaimer: This text has been machine-scanned and may contain errors – please refer to "Publications" for legally binding content. Description
SAMPLE PREPARATION METHODTECHNICAL FIELDThe invention relates to a sample preparation method and a kit for performing said sample preparation method. More specifically the invention relates to a sample preparation method for a combined extraction and derivatisation of an analyte from a biological sample.BACKGROUNDBioanalysis refers to the quantitative measurement of drugs, metabolites, and other substances in biological samples such as blood, urine, or tissues. This field is particularly important in the development of pharmaceuticals, where bioanalytical data is used to determine the pharmacokinetics, efficacy, and safety of new drug candidates. Bioanalytical techniques include mass spectrometry, chromatography, and immunoassays, which are used to detect and quantify small molecules, peptides, and large molecules like proteins or nucleic acids. Bioanalysis is also used in clinical diagnostics and research and plays a critical role in ensuring the safety and efficacy of drugs and other medical treatments. Sample preparation is a critical step in bioanalysis, as it involves the extraction, purification, and concentration of analytes from biological samples prior to analysis. The quality and reproducibility of bioanalytical data crucially depend on sample preparation, which affects the sensitivity, selectivity, and accuracy of the analytical method. The choice of sample preparation technique depends on the type of biological samples, the physicochemical properties of the analyte, and the analytical method used.Common sample preparation techniques in bioanalysis include liquid-liquid extraction (LLE), solid-phase extraction (SPE), protein precipitation (PPT), and derivatisation. Other recent techniques that have gained attention include microextraction methods, such as liquid-phase microextraction, solid-phase microextraction, and magnetic-solid phase extraction, as well as nanomaterial-assisted sample preparation. Good sample preparation practices ensure that the final sample is free from interferences from the biological samples and contaminants and is compatible with the analytical method used.Derivatisation is a sample preparation technique commonly used in bioanalysis to enhance the detection and quantitation of small molecules and biomolecules. The technique involves modifying the chemical structure of the analyte by attaching a derivatizing reagent, typically through a chemical reaction, to form a more stable and/or more chromatographically favourable derivative of the analyte.Liquid chromatography coupled with mass spectrometry (LC-MS) or with tandem mass spectrometry (LC-MS/MS) have become an important tool in clinical laboratories for detecting and quantifying small molecules and biomolecules in various biological samples such as blood, serum, plasma, and urine. LC-MS/MS offers high selectivity, sensitivity, and specificity for bioanalysis, allowing for accurate and precise diagnoses of various diseases and disorders. In clinical laboratories, LC-MS/MS is used for a wide range of applications, including therapeutic drug monitoring, toxicology, endocrinology, and clinical chemistry. Typically, LC-MS/MS is widely used for the quantification of immunosuppressants, drugs, hormones, metabolites, and vitamins. Despite its advantages, LC-MS/MS use in clinical laboratories can have significant challenges, such as the need for highly specialized staff, expensive equipment, extensive method development, complex sample preparation procedures, and long analysis times.Derivatisation can increase sensitivity, selectivity, and chromatographic resolution in analytical methods such as LC-MS or LC-MS/MS. When using LC-MS/MS, derivatisation is particularly useful for improving detection of molecules that have low ionization efficiency. Common analytical derivatisation reagents can react with carboxylic acids, amines, alcohols, ketones, phenols, and thiols, and these reagents can introduce a variety of functional groups onto the analyte.Derivatisation can be combined with SPE, LLE, or even PPT to enhance the detection and quantification of small molecules and biomolecules in biological samples. In SPE, analytes are selectively retained on a solid-phase sorbent, while interfering substances are removed. The analytes are subsequently eluted with an appropriate extraction solvent and the derivatisation reagent is added to the extraction solvent comprising the analytes. A useful derivatisation reagent for vitamin D3 and its metabolites is 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD). It reacts with the diene functional group common to vitamin D molecules and results in products that increases the signal in the LC-MS/MS analysis to such an extent that even traces of 1,2-dihydroxyvitamin D3 (calcitriol) can be measured in human serum samples.Hydrazines and hydrazides are useful derivatisation reagents for steroid hormones which contain a ketone functional group. The reaction results in formation of hydrazone functional groups on the molecules. The hydrazones produce a significantly higher signal in the LC-MS/MS analysis, which allows steroid hormones to be quantified at low levels in biological samples, such as human saliva and fish feces.Sample preparation can be a bottleneck for the use of LC-MS/MS in the clinical laboratory because it is a complex and time-consuming process. For instance, the sample preparation using LLE of 25-hydroxyvitamin D3 in serum, typically involves extraction with a nonpolar extraction solvent such as hexane or heptane. This procedure involves transfer of the extraction solvent from the LLE container to another container where the extraction solvent is removed by evaporation resulting in a dry extract. A water-miscible solvent is then added to the dry extract to ensure compatibility with the reversed phase LC separation, which uses an aqueous mobile phase. For improvement of detectability in the analysis, it is possible to add derivatisation reagent in this final step of the sample preparation. Similarly, sample preparation using SPE, comprises the retention of analytes on a solidphase sorbent, while interfering substances are removed. The analytes are subsequently eluted with an appropriate extraction solvent. Derivatives of the analytes can be formed by adding a derivatizing reagent either before or after performing the SPE procedure. The main disadvantages of SPE are the high cost of the SPE cartridges, the lengthy procedure, as well as the need for special equipment such as a vacuum station on the pipetting robot. PPT involves adding an organic solvent or a salt to the biological sample, which destabilizes the protein-protein and protein-solvent interactions and leads to protein denaturation and precipitation. Once the proteins have been precipitated, they can be removed from the sample by centrifugation, leaving behind the small molecules and other biomolecules of interest in the supernatant. Derivatisation reagent can be added either before or after the extraction procedure to improve the sensitivity and selectivity of the method by converting the analyte into a more detectable and/or chromatographically favourable form prior to the instrumental analysis. However, PPT is a crude type of sample preparation which is often associated with matrix effects that lowers the analytical sensitivity.Salting-out assisted liquid-liquid extraction (SALLE) is a sample preparation technique that uses the salting-out effect to separate water-miscible organic solvents from aqueous solutions. The technique typically involves adding a water-miscible organic solvent to a biological sample, followed by adding a high concentration of salt, such as sodium chloride or ammonium sulphate, to the solution. This reduces the water miscibility of the organic solvent and causes it to separate from the aqueous phase. Another similar technique as SALLE is so-called sugaring-out assisted liquid-liquid extraction (SULLE), where high concentration of sugar is applied instead of salt. The separated organic solvent comprising the analytes of interest is removed from the aqueous solution and subjected to further preparation or analysis.In a paper by Francisco et al. (Francisco, K.C.A.; Brandão, P.F.; Ramos, R.M.; Gonçalves, L.M.; Cardoso, A.A.; Rodrigues, J.A. Salting-out Assisted Liquid–Liquid Extraction with Dansyl Chloride for the Determination of Biogenic Amines in Food. International Journal of Food Science & Technology 2020, 55, 248–258), SALLE was combined with derivatisation of the analyte. The derivatisation reagent, dansyl chloride, was added to the biological sample together with an extraction solvent (acetonitrile) and followed by addition of salt (sodium chloride and sodium hydroxide) at the initial step of the SALLE procedure. In this SALLE procedure, the derivatisation reagent may be mixed with aqueous solutions, and so the dansyl chloride could be added directly to the aqueous sample solutions. Other derivatisation reagents may not tolerate water or will perform poorly in the presence of water. It is therefore a need to simplify sample preparation for derivatisation using derivatisation reagents that are not compatible with water.In a paper by Alshabrawy et.al. (Alshabrawy, A.K.; Bergamin, A; Sharma, D.K.; Hickey, S.M.; Brooks D.A.; O’Loughlin, P.; Wiese, M.D.; Anderson, P.H. LC-MS/MS analysis of vitamin D3 metabolites in human serum using a salting-out based liquid-liquid extraction and DAPTAD derivatization, J. Chromatography B, 2021, 122654), the use of SALLE followed by DAPTAD derivatization is described. The serum sample is mixed with internal standard and mixed with acetonitrile solvent in an Eppendorf tube, followed by addition of ammonium acetate to induce solvent phase separation (salting out effect). After this SALLE operation, a volume of the acetonitrile supernatant is removed from the Eppendorf tube and transferred into a new clean Eppendorf tube where the solvent is evaporated by using a vacuum centrifuge. Derivatization is conducted by adding a solution of DAPTAD to the dry extract. After quenching of the reaction with ethanol, the derivatized sample extract is evaporated, again using a vacuum centrifuge. The dried and derivatized sample extract was finally re-constituted in 90% acetonitrile in an autosampler vial.In a paper by Schorr et.al. (Schorr, P.; Stokes, C.S.; Volmer, D.A. Improved quantitative LC-MS/MS analysis of vitamin D metabolites in serum after one-pot double derivatization, J. Pharmaceutical and Biomedical Analysis, 2023, 234, 115522), it is disclosed protein precipitation with acetonitrile, followed by liquid-liquid extraction (LLE) with ethyl acetate, and finally derivatization of vitamin D metabolites by using two different reagents (PTAD and acetic acid anhydride) in a one-pot derivatization reaction. Here, serum is mixed with internal standard before protein precipitation with acetonitrile. The supernatant was then transferred to a 1.5 mL reaction vial followed by evaporation in a vacuum centrifuge. Water and ethyl acetate were added for liquid-liquid extraction (LLE) carried out twice, followed by transfer of both ethyl acetate solvent extracts to another 1.5 mL reaction tube. The ethyl acetate was then evaporated in a vacuum centrifuge, followed by one-pot derivatization using PTAD and acetic acid anhydride. After derivatization and drying, the residue was re-dissolved in 90% methanol and transferred to autosampler vials. In a paper by Ying Xue et.al. (Xue, Y.; He, H.; Li, H.-D.; Deng, Y.; Yan, M.; Cai, H.-L.; Tang, M.-M.; Dang, R.L.; Jiang, P. Simultaneous quantification of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in rats shows strong correlations between serum and brain tissue levels, Int. J. Endocrinology, 2015, ID 296531), it is disclosed an LC-MS/MS method for measurement of vitamin D metabolites in rat serum and brain samples. Sample preparation was by SALLE followed by derivatization. The brain sample was mixed with acetonitrile and internal standard, followed by homogenization, vortex mixing, and centrifugation. Supernatant was then transferred into another Eppendorf tube and dried with nitrogen gas and followed by PTAD derivatization. A similar procedure was applied for the serum sample, also here using two different containers for extraction and derivatization. For analysis samples were kept in the autosampler in vials at 4 °C.In a paper by Sa'aleek et.al. (Sa'aleek, A. A.; Alshishani, A.; Shaghlil, L.; Alhesan, J.S.A.; Alebibi, Y. Determination of vitamin D3 in pharmaceutical products using salting-out assisted liquid-liquid extraction coupled with reversed phase liquid chromatography, Microchemical Journal, 2023, 109077), it is disclosed measurement of vitamin D in pharmaceuticals by HPLC-UV and with SALLE as sample preparation. Sample powder was mixed with water in a centrifuge tube and shaken. Then, acetonitrile was added to the vial, followed by sonication and addition of sodium chloride for phase separation by salting-out effect. The mixture was vortexed and centrifuged. Finally, 1.0 mL of the clear supernatant solution was diluted using MeOH before injection in the HPLC instrument. To do the dilution with MeOH the authors would need to transfer the supernatant to another vial.In a paper by Yang et.al. (Yang, M.-Y.; Huang, C.-Y.; Chiu, T.H.T.: Chang, K.C.; Lin, M.N.; Chen, L.Y.; Hu, A. Using gas chromatography and mass spectrometry to determine 25-hydroxyvitamin D levels for clinical assessment of vitamin D deficiency, J. Food and Drug analysis, 2019, 27, 494-501), it is disclosed the measurement of vitamin D metabolites in blood by LC-MS and GC-MS. Sample preparation was protein precipitation (PPT), liquidliquid extraction (LLE), and derivatization. In an unspecified container the plasma sample was mixed first with acetonitrile for PPT and then with hexane for LLE. After vortex mixing and centrifugation, the organic solvent layer was collected into an unspecified container. Yang et al. further discloses two different derivatization procedures, for LC and GC analy sis respectively. For LC-MS/MS analysis, the solvent extract was dried by rotavapor, followed re-suspending in methanol before adding derivatization reagent (Amplifex™) and finally water. For GC-MS/MS analysis, the solvent extract was dried by rotavapor, followed re-suspending in a mixture of pyridine and TMS-reagent.SUMMARYThe invention has for its object to remedy or to reduce at least one of the drawbacks of the prior art, or at least provide a useful alternative to prior art.The object is achieved through features, which are specified in the description below and in the claims that follow.The invention is defined by the independent patent claims. The dependent claims define advantageous embodiments of the invention.In a first aspect, the invention relates to a sample preparation method comprising the steps of:a) obtaining a biological sample, the biological sample comprising an aqueous solution and at least one analyte dissolved in the aqueous solution;b) adding a water-miscible solvent to the biological sample to obtain a sample solution;c) adding a salting-out reagent or a sugaring-out reagent to the sample solution of step b) to obtain a phase separated solution, wherein the aqueous solution forms a water phase, and the water-miscible solvent forms a solvent phase separated from the aqueous phase, and wherein the at least one analyte is dissolved in the solvent phase; andd) adding a derivatisation reagent to the solvent phase to obtain a derivative of the at least one analyte.The steps a) to d) are performed in one container.The sample preparation method according to the first aspect of the invention is a so called salting-out assisted liquid-liquid extraction (SALLE) or sugaring-out assisted liquidliquid extraction (SULLE), combined with derivatization of the analyte. The phase separation may be achieved by waiting for a period of time after the addition of the salting-out reagent or the sugaring-out reagent. The period of time may depend on the watermiscible solvent and the salting-out reagent or the sugaring-out reagent.The addition of the salting-out reagent or the sugaring-out reagent to the sample solution in step c) may result in the sample solution separating into two distinct phases; one aqueous phase and one solvent phase that comprises the at least one analyte. The solvent phase may typically be on top of the aqueous phase due to the water-miscible solvent typically having a lower density compared with the aqueous solution.The salting-out reagent or the sugaring-out reagent is typically added in a high concentration, such that the sample solution is highly or completely saturated with the salting-out reagent or with the sugaring-out reagent. The salting-out reagent or the sugaring-out reagent may reduce the water-miscibility of the water-miscible solvent and may cause it to separate from the aqueous solution. All, or most of the salting-out reagent or the sugaring-out reagent may be dissolved in the aqueous phase, such that the solvent phase is substantially free of the salting-out reagent or the sugaring-out reagent. The distribution of the salting-out reagent or the sugaring-out reagent between the aqueous phase and the solvent phase may be dependent on the type of biological sample, the type of watermiscible solvent and the type of salting-out reagent or sugaring-out reagent, and the combination thereof.The sample preparation method according to the first aspect of the invention has several advantages. Firstly, it is a simple an efficient method for separating and concentrating analytes from biological samples. It may not require the use of hazardous organic solvents or expensive equipment. Furthermore, the water-miscible solvent may be compatible with the reversed phase liquid-chromatography separation column, meaning that the water-miscible solvent comprising the derivative of the at least one analyte may be directly analysed using an instrument for detecting the derivative of the at least one analyte, such as an instrument for liquid-chromatography coupled with mass spectrometry (LC-MS), an instrument for liquid-chromatography with tandem mass spectrometry (LC-MS/MS), an instrument for gas-chromatography coupled with mass spectrometry (GC-MS) or an instrument for gas-chromatography with tandem mass spectrometry (GCMS/MS). There may be no need to transfer the water-miscible solvent to another vial for evaporation because the water-miscible solvent may be used directly for analysis. Furthermore, the sample preparation method may also be easy to automate, which may streamline the sample preparation process and reduce variability between samples.Some derivatisation reagents, such as 4-phenyl-1,2,4-triazole-3,5-dione (PTAD), may not be compatible with water. The derivatisation reagents may be unstable in protic solvents such as water and methanol, in which they may be degraded. For performing derivatisation reactions with derivatisation reagents which are not compatible with water, the reactions must be performed in solutions that are substantially free of water.The inventors discovered that the sample preparation method according to the first aspect of the invention, resulted in a solvent phase separated from the aqueous phase such that the solvent phase was substantially free of water. This was evidenced by the possibility of adding the PTAD derivatisation reagent directly to the solvent phase, without first removing the solvent phase to another container. The PTAD derivatisation reagent was added directly to the solvent phase of the phase separated solution, while the solvent phase was in the same container as the aqueous phase. The PTAD derivatisation reagent was observed to completely mix with the solvent phase while no PTAD derivatisation reagent was seen entering the aqueous phase. The PTAD derivatisation reagent has a pink colour which turns yellow upon degradation in water, so inspection was straightforward. No change of colour of the PTAD derivatisation reagent was observed.The sample preparation method according to the first aspect of the invention may therefore be less time-consuming and more cost efficient. There may be no need for removing the solvent phase and adding it to another container for evaporating and changing the solvent to a solvent that is compatible with the reversed phase liquid-chromatography separation column, as the water-miscible solvent forming the solvent phase is already compatible with the separation column. Furthermore, there may be no need for removing the solvent phase before adding the derivatisation reagent. There may be no need for adding the solvent phase to another container for evaporating and changing the solvent to a solvent that is compatible with derivatisation reagents. The solvent phase may be substantially free of water so that the derivatisation reagent may be added directly to the solvent phase. The derivatisation reagent may therefore be added directly to the solvent phase without removing the solvent phase from the container in which the phase separation occurred. The sample preparation method according to the first aspect of the invention is therefore a sample preparation for a combined extraction and derivatisation of the at least one analyte from a biological sample.The sample preparation method may also be used with derivatisation reagents that are compatible with water, i.e. derivatisation reagents that do not degrade in the presence of water.In an embodiment of the invention, step d) may comprise heating the phase separated solution comprising the derivatisation reagent in the solvent phase to speed up the reaction time of the derivatisation reagent with the at least one analyte.It may be advantageous that the derivatisation reaction is fast so that the derivatisation reaction may occur before some of the derivatisation reagent may enter the aqueous phase.In an embodiment of the invention, the sample preparation method may comprise, before step c), the step of adding an internal standard molecule solution to the biological sample or to the sample solution. The internal standard molecule solution may comprise at least one type of internal standard molecule.In another embodiment of the invention, the water miscible solvent of step b) may comprise at least one type of internal standard molecule.In another embodiment of the invention, the step of adding a salting-out reagent or a sugaring-out reagent to the sample solution of step b) comprises the step of adding a salting-out solution comprising the salting-out reagent and at least one type of internal standard molecule, or adding a sugaring-out solution comprising the sugaring-out reagent and at least one type of internal standard molecule. Addition of a type of internal standard molecule may be used to create an internal standard calibration curve. The internal standard calibration curve may then be used to calculate the concentration of the analyte in the biological sample.In an embodiment of the invention, obtaining the phase separated solution in step c) may comprise mixing the sample solution comprising the salting-out reagent or the sugaringout reagent. The salting-out reagent or the sugaring-out reagent may be added to the sample solution in a container, and then the sample solution may be mixed by for example shaking the container, by vortex mixing, or by pipetting the sample solution up and down.In an embodiment of the invention, obtaining the phase separated solution may comprise centrifuging the sample solution comprising the salting-out reagent or the sugaring-out reagent. The salting-out reagent or the sugaring-out reagent may be added to the sample solution in a container, and then the sample solution may be centrifuged for some time, for example for around 15 min, and by using a certain centrifugal force, for example a centrifugal force of around 2000xG.In an embodiment of the invention, obtaining the phase separated solution may comprise cooling the sample solution comprising the salting-out reagent or the sugaring-out reagent. The salting-out reagent or the sugaring-out reagent may be added to the sample solution in a container, and then the sample solution may be cooled down to a temperature of for example -20 ⁰C or lower for a suitable amount of time to obtain the phase separated solution.The container may typically be a container compatible with an instrument for detecting the derivative of the at least one analyte in the water-miscible solvent. The container may for example be a vial, or a well in a microplate. It will be understood that the microplate may have different shapes, number of wells and sizes of wells. The container may be closed with a pierceable cap before introduction to an instrument for detecting the derivative of the at least one analyte. Since there may be no need to transfer the water-miscible solvent to another container after the phase separation, all the steps of the sample preparation method may be performed in the same container. The invention presented herein is therefore a highly efficient sample preparation method. It is a combined sample preparation method wherein extraction of at least one analyte from a biological sample with a water-miscible solvent, inducing phase separation by adding a salting-out reagent or sugaring-out reagent, and performing analytical derivatisation may be performed in the same container. The sample preparation method may therefore easily be automated.In an embodiment of the invention, the sample preparation method may further comprise the step of:e) aspiring a portion of the solvent phase for analysis of the derivative of the at least one analyte.Aspiring a portion of the solvent phase for analysis of the derivative of the at least one analyte may be performed from a same container in which the phase separation occurred. The solvent phase may typically be on top of the aqueous phase, such that aspiring a portion of the solvent phase may be performed without coming into contact with the aqueous phase. Aspiring a portion of the solvent phase may also be performed if the aqueous phase is on top of the solvent phase.The sample preparation method may further comprise the step of:f) directly injecting the portion of the solvent phase from step e) into an instrument for detecting the derivative of the at least one analyte.The solvent phase, comprising the water-miscible solvent and the derivative of the at least one analyte may be analysed using an instrument, such as a LC-MS, a LC-MS/MS, a GC-MS, or a GC-MS/MS without further processing of the water-miscible solvent. A portion of the solvent phase may therefore be aspired and directly injected into an instrument for detecting and/or analysing the derivative of the at least one analyte. By directly injected, it is meant that no further processing of the aspired portion is performed before injection into the instrument. The aspiration and injection may be performed manually. The steps e) and f) may be performed by an autosampler. The autosampler may be a part of an instrument for detecting and/or analysing the derivative of the at least one analyte. The autosampler may comprise an autosampler needle for the aspiration and injection of the portion of the solvent phase.The autosampler may increase the efficiency and the reliability of the analysis of the analyte from the biological sample, compared to manual aspiration and injection. Using an autosampler may be advantageous for high-throughput analysis. It is understood that different kinds of autosampler needles may be used, depending on the analysis to be performed.The autosampler may be configured for positioning a tip of the autosampler needle in the solvent phase, such that a portion of the water-miscible solvent only is aspired. This may avoid aspiration of the aqueous phase by the autosampler needle.In an embodiment of the invention, the sample preparation method may comprise, after step d), the step of placing the container in an autosampler compartment of an instrument for detecting the derivative of the at least one analyte. The autosampler compartment of the instrument may comprise an autosampler.Placing the container in the autosampler compartment after the derivatisation reaction may further increase the efficiency and accuracy of the sample preparation method according to the first aspect of the invention. A further step of transferring a portion of the solvent phase, comprising the derivative of the at least one analyte, to a container compatible with an autosampler compartment may be avoided.The instrument for detecting the derivative of the at least one analyte may be an instrument for liquid-chromatography coupled with mass spectrometry or an instrument for liquid-chromatography with tandem mass spectrometry.The instrument for detecting the derivative of the at least one analyte may be an instrument for gas-chromatography coupled with mass spectrometry or an instrument for gaschromatography with tandem mass spectrometry. The sample preparation method according to the invention may be particularly advantageous for preparing samples for LC-MS, LC-MS/MS, GC-MS, or GC-MS/MS, because the method is simple, effective and may easily be standardised and automated.The biological sample may comprise whole blood, serum plasma, urine, saliva, or cerebrospinal fluid.The water miscible solvent of step b) may comprise one or more of acetonitrile, acetone, methanol, ethanol, 1-propanol, or 2-propanol.The water miscible solvent may preferably comprise acetonitrile. Acetonitrile may be inert and may not degrade derivatisation reagents such as PTAD. Using acetonitrile may lead to low interference of the subsequent analysis of the analyte due to the small amount of phospholipids that may be extracted. The acetonitrile may preferably be anhydrous. The anhydrous acetonitrile may typically be provided from a standard provider, wherein the anhydrous acetonitrile comprises less than 0.001% water or less than 0.005% water.The water miscible solvent of step b) may comprise a mixture of two or more of the following: acetonitrile, acetone, methanol, ethanol, 1-propanol, and 2-propanol.The salting-out reagent of step c) may comprises one of ammonium sulphate, ammonium acetate, or sodium chloride. The salting-out reagent may be any reagent suitable for a salting-out assisted liquid-liquid extraction, i.e. a reagent causing a phase separation between the water-miscible solvent and the aqueous solution.The sugaring-out reagent may be any reagent suitable for a sugaring-out assisted liquidliquid extraction, i.e. a reagent causing a phase separation between the water-miscible solvent and the aqueous solution. The sugaring-out reagent may for example comprise fructose.In an embodiment of the invention, the at least one analyte may comprise a diene functional group, and the derivatisation reagent may react with said diene functional group in a Diels-Alder reaction. The derivatisation reagent may be 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD).The at least one analyte may be one of vitamin D3, 25-hydroxyvitamin D3, or 1,25-dihydroxyvitamin D3. Each of vitamin D3, 25-hydroxyvitamin D3, or 1,25-dihydroxyvitamin D3 comprises a diene functional group.In an embodiment of the invention, the at least one analyte may comprise a phenol functional group, and the derivatisation reagent may react with said phenol functional group. The at least one analyte may be a steroid hormone comprising a phenol functional group, such as estradiol.The derivatisation reagent may be one of nicotinoyl chloride, isonicotinoyl chloride, or dansyl chloride.The at least one analyte may comprise a ketone functional group, and the derivatisation reagent may react with said ketone functional group. The at least one analyte may be a steroid hormone comprising a ketone functional group, such as testosterone.The derivatisation reagent may be one of hydrazine, hydrazide or hydrazinopyridine. In an embodiment of the invention, step d) of the sample preparation method comprises adding two or more derivatisation reagents, wherein the two or more derivatisation reagents can react with different functional groups of the at least one analyte.Adding two or more derivatisation reagents in step d) may be advantageous for detecting and analysing two or more analytes in the same biological sample. For example, one analyte may comprise a diene functional group, and another analyte may comprise a phenol functional group. Then a first derivatisation reagent that can react with the diene functional group can be added in step d) together with a second derivatisation reagent that can react with the phenol functional group. In another example, a first derivatisation reagent that can react with a ketone functional group may be added in step d) together with a second derivatisation reagent that can react with a diene functional group or that can react with a phenol functional group. In a second aspect, the invention relates to a kit for performing the sample preparation method according to the first aspect of the invention, the kit comprising:- a water-miscible solvent;- a salting-out reagent or a sugaring-out reagent; and- a derivatization reagent; and- a container for performing the sample preparation method in one container, wherein the container is suitable for placing in an autosampler, and wherein the container consists of a vial, or a well in a microplate.The kit may further comprise one or more internal standard molecules.The kit may comprise a solution comprising the one or more type of internal standard molecules. The water-miscible solvent may comprise the one or more type of internal standard molecules. The salting-out reagent may be comprised in a solution, which may also comprise the one or more type of internal standard molecules.The sugaring-out reagent may be comprised in a solution, which may also comprise the one or more type of internal standard molecules.The kit may comprise a microplate.BRIEF INTRODUCTION OF THE FIGURESIn the following is described an example of a preferred embodiment illustrated in the accompanying figures, wherein:Figure 1 shows a sample preparation method according to the invention, performed in one vial.Figure 2A shows a multiple reaction monitoring-chromatogram for detection of PTAD derivative of 25-hydroxycholecalciferol in human serum;Figure 2B shows a multiple reaction monitoring-chromatogram for detection of PTAD derivative of an isotope labelled internal standard of 25-hydroxycholecalciferol in human serum; Figure 2C shows a multiple reaction monitoring-chromatogram for detection of PTAD derivative of 25-hydroxycholecalciferol in fish plasma;Figure 2D shows a multiple reaction monitoring-chromatogram for detection of PTAD derivative of an isotope labelled internal standard of 25-hydroxycholecalciferol in fish plasma;Figure 2E shows a multiple reaction monitoring-chromatogram for detection of PTAD derivative of 1,25-dihydroxycholecalciferol in fish plasma;Figure 2F shows a multiple reaction monitoring-chromatogram for detection of PTAD derivative of an isotope labelled internal standard of 1,25-dihydroxycholecalciferol in fish plasma;Figure 1G shows a multiple reaction monitoring-chromatogram for detection of PTAD derivative of cholecalciferol in fish plasma;Figure 2H shows a multiple reaction monitoring-chromatogram for detection of PTAD derivative of an isotope labelled internal standard of cholecalciferol in fish plasma;Figure 3A shows an internal standard calibration curve for quantification of 1,25-dihydroxycholecalciferol;Figure 3B shows an internal standard calibration curve for quantification of 25-hydroxycholecalciferol; andFigure 3C shows an internal standard calibration curve for quantification of cholecalciferol.DETAILED DESCRIPTIONFigure 1 shows a sample preparation method according to the invention, performed in one container, here shown as a vial 1. In a step 100, a biological sample 10, an internal standard solution 12, and a water-miscible solvent 13 is mixed together in the vial 1 to obtain a sample solution 2. The biological sample 1 comprises an aqueous solution (not shown) and at least one analyte (not shown) dissolved in the aqueous solution. In a step 110, a solution of salting-out or sugaring-out reagent 13 is added to the vial 1. For assisting the phase separation, the vial 1 is centrifuged in a step 120 to obtain a phase separated solution 3. The phase separated solution 3 comprises an aqueous phase 4 at the bottom of the vial 1, formed by the aqueous solution, and a solvent phase 5 on top of the aqueous phase 4, wherein the solvent phase 5 is formed of the water-miscible solvent 13 comprising at least the analyte. In a step 130, a derivatisation reagent 14 is added to the vial 1, into the solvent phase 5 only. After the derivatisation reagent has reacted with the at least one analyte to obtain a derivative of the at least one analyte (not shown), a portion of the solvent phase 5 only is aspired by an autosampler needle 15. The portion of the solvent phase 5 comprises the derivative of the at least one analyte. The portion may then be injected directly into an instrument for detecting and analysing the derivative of the at least one analyte.In an example of the invention, the sample preparation method was used to determine vitamin D3 metabolites in human serum and in fish plasma. The vitamin D3 metabolites analysed were 25-hydroxycholecalciferol (25-hydroxyvitamin D3, calcifediol), 1,25-dihydroxycholecalciferol (calcitriol) and Vitamin D3 (cholecalciferol).In this example, calibration standard solutions were prepared in calf serum with concentrations of the vitamin D3 metabolites in the range of 5 – 80 nmol/L.All samples of fish plasma and human serum, as well as calibration standard solutions and blank water samples were prepared using the sample preparation method. The prepared samples were then analysed with LC-MS/MS using multiple reaction monitoring (MRM), also known as selected reaction monitoring (SRM), for detection of PTAD derivatives of the vitamin D3 metabolites.In a first step, 200 µL of each sample was added to a well of a 96 deep-well microplate with well volume of 1 mL. The samples being fish plasma, human serum, calibration standard solutions and blank water. In a second step, 200 µL of a water-miscible solvent was added to each sample in the deep-well microplate. The water-miscible solvent used in this example was acetonitrile. The acetonitrile comprised an isotope labelled internal standard of each vitamin D3 metabolite.In a third step, 200 µL of a salting-out solution was added for inducing phase separation of the water-miscible solvent and the aqueous solution in the samples. The salting-out solution used in this example was saturated ammonium sulphate.In a fourth step, the deep-well microplate was capped by a washable and re-usable cap mat and then shaken for 30 seconds and centrifuged at 2000 x G for 15 minutes. A phase separation in the samples were obtained, wherein the aqueous solution formed an aqueous phase, and the water-miscible solvent formed a solvent phase separated from the aqueous phase, and wherein the solvent phase was on top of the aqueous phase. The vitamin D3 metabolites was dissolved in the solvent phase.In a fifth step, the cap mat was removed from the plate and 20 μL of anhydrous acetonitrile containing 15 mg/mL PTAD was added to solvent phase. The deep-well microplate was left for between 5 and 15 min for the derivatisation reaction between the PTAD and the vitamin D3 metabolite to take place. The derivatisation reaction may typically be complete after around 5 minutes.PTAD, having a pink colour, was observed to completely mix with the solvent phase, while no PTAD was seen entering the aqueous phase. PTAD turns yellow upon degradation in water. No change of colour of PTAD was observed during the derivatisation reaction. In a sixth step, the plate was capped with a pierceable microplate seal tape and placed in an autosampler compartment of a LC-MS/MS instrument for detecting the derivative of the vitamin D3 metabolite. The autosampler needle was programmed to aspire 10 μL from the solvent phase, by setting the needle tip distance 18 mm from the bottom of each well containing a sample to be analysed. In this example, the sample preparation method was conducted in room temperature. It will be understood that different steps of the sample preparation method may be performed at different temperature to optimise reaction conditions.It will be understood that the volumes of the samples, the water-miscible solvent, the salting-out solution, or the sugaring-out solution, and the amount and concentration of the derivatisation reaction may be varied to optimise the sample preparation method. For example, it may be advantageous to add a different amount of water-miscible solvent, depending on the type of biological sample.It will also be understood that different combinations of the type of biological sample, the type of water-miscible solvent, the type of salting-out reagent or the type of sugaring-out reagent, and the type of derivatisation reagent may be used to optimise the sample preparation method. For example, a derivatisation reagent that may be compatible with water may be added to a solvent phase that may not be substantially free of water, but may comprise some water.The results from the analysis of human serum and fish plasma are shown in Figures 1A to 1H. Using MRM, each of the PTAD derivatives of the vitamin D3 metabolites and the PTAD derivatives of the isotope labelled internal standards were detected. The area under the peaks were calculated for each PTAD derivatives of the vitamin D3 metabolite and for each PTAD derivatives of the isotope labelled internal standard. The ratio of the areas of the vitamin D3 metabolite peak and the respective isotope labelled internal standard peak was calculated and compared with the respective internal standard calibration curve (see Figures 2A to 2C). The MRM-parameters used for each sample is summarised in Table 1. The results are shown in Table 2.Table 2) Overview of samples and MRM-parameters 1Calcifediol: 25-hydroxycholecalciferol (25-hydroxyvitamin D3). 2D6 Calcifediol: Isotope (6 deuterium) labelled 25-hydroxyvitamin D3 as internal standard. 3Calcitriol: 1,25-dihydroxycholecalciferol. 4D6 Calcitriol: Isotope (6 deuterium) labelled 1,25-dihydroxycholecalciferol as internal standard. 5Cholecalciferol: Vitamin D3. 6D3 Ergocholecalciferol: Isotope (3 deuterium) labelled ergocholecalciferol (vitamin D2) as internal standard.Table 2) Amount of vitamin D3 metabolites in the biological samplesThe internal standard calibration curves are shown in Figures 2A to 2C. For each calibration standard solution sample, wherein a known concentration of each of the vitamin D3 metabolite had been added, the area under the peaks were calculated for each PTAD derivatives of the vitamin D3 metabolite and for each PTAD derivatives of the isotope labelled internal standard. The ratio of the areas of the vitamin D3 metabolite peak and the respective isotope labelled internal standard peak was calculated and plotted against the known concentration of the vitamin D3 metabolite, to obtain the internal standard calibration curves shown in Figures 2A to 2C.It should be noted that other combinations of water-miscible solvents and salting-out reagents or sugaring-out reagents may be used to achieve a similar result. The derivatisation reagent may not enter the aqueous phase due to a better solubility in the solvent phase. The derivatisation reagent may not enter the aqueous phase due to a layer of fat and/or proteins forming between the aqueous phase and the solvent phase. The layer may also for example comprise triglycerides, which may not have a good solubility in neither the aqueous phase, nor the solvent phase. The salting-out reagent in the aqueous phase may lead to precipitation of proteins, and the precipitated proteins may form a layer between the aqueous phase and the solvent phase.It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. Use of the verb “comprise”, and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article “a” or “an” preceding an element does not exclude the presence of a plurality of such elements.The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.
Claims
C l a i m s1. A sample preparation method comprising the steps of:a) obtaining a biological sample, the biological sample comprising an aqueous solution and at least one analyte dissolved in the aqueous solution;b) adding a water-miscible solvent to the biological sample to obtain a sample solution;c) adding a salting-out reagent or a sugaring-out reagent to the sample solution of step b) to obtain a phase separated solution, wherein the aqueous solution forms an aqueous phase, and the water-miscible solvent forms a solvent phase separated from the aqueous phase, and wherein the at least one analyte is dissolved in the solvent phase; andd) adding a derivatisation reagent to the solvent phase to obtain a derivative of the at least one analyte,wherein the steps a) to d) are performed in one container.2. The sample preparation method according to claim 1, wherein the method comprises, before step c), the step of adding an internal standard molecule solution to the biological sample or to the sample solution.3. The sample preparation method according to claim 1, wherein the water miscible solvent of step b) comprises at least one type of internal standard molecule.4. The sample preparation method according to claim 1, wherein the step of adding a salting-out reagent or a sugaring-out reagent to the sample solution of step b) comprises the step of adding a salting-out solution comprising the salting-out reagent and at least one type of internal standard molecule, or adding a sugaring-out solution comprising the sugaring-out reagent and at least one type of internal standard molecule.5. The sample preparation method according to any one of the preceding claims, wherein obtaining the phase separated solution comprises mixing the sample solution comprising the salting-out reagent or the sugaring-out reagent.6. The sample preparation method according to any one of the preceding claims, wherein obtaining the phase separated solution comprises centrifuging the sample solution comprising the salting-out reagent or the sugaring-out reagent.7. The sample preparation method according to any one of the preceding claims, wherein obtaining the phase separated solution comprises cooling the sample solution comprising the salting-out reagent or the sugaring-out reagent.8. The sample preparation method according to any one of the preceding claims, wherein the method further comprises, after step d), the step of:e) aspirating a portion of the solvent phase for analysis of the derivative of the at least one analyte.9. The sample preparation method according to claim 8, wherein the method comprises, after step e), the step of:f) directly injecting the portion of the solvent phase from step e) into an instrument for detecting the derivative of the at least one analyte.10. The sample preparation method according to claim 8 and 9, wherein step e) and f) are performed by an autosampler needle.11. The sample preparation method according to any one of claims 1 to 7, wherein the method comprises, after step d), the step of placing the container in an autosampler compartment of an instrument for detecting the derivative of the at least one analyte.12. The sample preparation method according to any one of claims 9 to 11, wherein the instrument for detecting the derivative of the at least one analyte is an in strument for liquid-chromatography coupled with mass spectrometry or an instrument for liquid-chromatography with tandem mass spectrometry.13. The sample preparation method according to any one of claims 9 to 11, wherein the instrument for detecting the derivative of the at least one analyte is an instrument for gas-chromatography coupled with mass spectrometry or an instrument for gas-chromatography with tandem mass spectrometry.14. The sample preparation method according to any one of the preceding claims, wherein the biological sample comprises whole blood, serum plasma, urine, saliva, or cerebrospinal fluid.15. The sample preparation method according to any one of the preceding claims, wherein the water miscible solvent of step b) comprises one of acetonitrile, acetone, methanol, ethanol, 1-propanol, or 2-propanol.16. The sample preparation method according to any one of the preceding claims, wherein the salting-out reagent comprises one or more of ammonium sulphate, ammonium acetate, or sodium chloride.17. The sample preparation method according to any one of the preceding claims, wherein the sugaring-out reagent comprises fructose.18. The sample preparation method according to any one of the preceding claims, wherein the at least one analyte comprises a diene functional group, and wherein the derivatisation reagent can react with said diene functional group.19. The sample preparation method according to claim 18, wherein the derivatisation reagent is 4-phenyl-1,2,4-triazoline-3,5-dione.20. The sample preparation method according to claim 18 or 19, wherein the at least one analyte is one of vitamin D3, 25-hydroxyvitamin D3, or 1,25-dihydroxyvitamin D3.21. The sample preparation method according to any one of claims 1 to 20, wherein the at least one analyte comprises a phenol functional group, and wherein the derivatisation reagent can react with said phenol functional group.22. The sample preparation method according to any one of claims 1 to 21, wherein the at least one analyte comprises a ketone functional group, and wherein the derivatisation reagent can react with said ketone functional group.23. A kit for performing the sample preparation method according to any one of the preceding claims, the kit comprising:- a water-miscible solvent;- a salting-out reagent or a sugaring-out reagent; and- a derivatization reagent; and- a container for performing the sample preparation method in one container, wherein the container is suitable for placing in an autosampler, and wherein the container consists of a vial, or a well in a microplate.24. The kit according to claim 23, wherein the kit further comprises one or more type of internal standard molecule.25. The kit according to claim 23, wherein the kit further comprises a container for performing the sample preparation method.O v e r s a t t e p a t e n t k r a v1. En fremgangsmåte for klargjøring av en prøve, omfattende trinnene å:a) fremskaffe en biologisk prøve, hvor den biologiske prøven omfatter en vandig løsning og minst én analytt oppløst i den vandige løsningen;b) tilsette et vannblandbart løsemiddel til den biologiske prøven, for å fremskaffe en prøveløsning;c) tilsette en utsaltingsreagens eller en utsukringsreagens til prøveløsningen i trinn b), for å fremskaffe en faseseparert løsning, hvori den vandige løsningen danner en vandig fase, og det vannblandbare løsemiddelet danner en løsemiddelfase separert fra den vandige fasen, og hvori den minst ene analytten er oppløst i løsemiddelfasen; ogd) tilsette en derivatiseringsreagens til løsemiddelfasen, for å fremskaffe et derivat av den minst ene analytten,hvori trinnene a) til d) utføres i én beholder.2. Fremgangsmåten for klargjøring av en prøve ifølge krav 1, hvori fremgangsmåten omfatter, før trinn c), trinnet å tilsette en internstandard-substansløsning til den biologiske prøven eller til prøveløsningen.3. Fremgangsmåten for klargjøring av en prøve ifølge krav 1, hvori det vannblandbare løsemiddelet i trinn b) omfatter minst én type internstandard-substans.4. Fremgangsmåten for klargjøring av en prøve ifølge krav 1, hvori trinnet tilsetting av en utsaltingsreagens eller en utsukringsreagens til prøveløsningen i trinn b) omfatter trinnet tilsetting av en utsaltingsløsning omfattende utsaltingsreagensen og minst én type internstandard-substans, eller tilsetting av en utsukringsløsning omfattende utsukringsreagensen og minst én type internstandard-substans.5. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av de foregående kravene, hvori fremskaffelse av den faseseparerte løsningen omfatter blanding av prøveløsningen omfattende utsaltingsreagensen eller utsukringsreagensen.6. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av de foregående kravene, hvori fremskaffelse av den faseseparerte løsningen omfatter sentrifugering av prøveløsningen omfattende utsaltingsreagensen eller utsukringsreagensen.7. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av de foregående kravene, hvori fremskaffelse av den faseseparerte løsningen omfatter avkjøling av prøveløsningen omfattende utsaltingsreagensen eller utsukringsreagensen.8. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av de foregående kravene, hvori fremgangsmåten videre omfatter, etter trinn d), trinnet å:e) trekke ut en andel av løsemiddelfasen for analyse av derivatet av den minst ene analytten.9. Fremgangsmåten for klargjøring av en prøve ifølge krav 8, hvori fremgangsmåten omfatter, etter trinn e), trinnet å:f) direkte injisere andelen av løsemiddelfasen fra trinn e) i et instrument for detektering av derivatet av den minst ene analytten.10. Fremgangsmåten for klargjøring av en prøve ifølge krav 8 og 9, hvori trinn e) og f) utføres med en autosamplernål.11. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av kravene 1 til 7, hvori fremgangsmåten omfatter, etter trinn d), trinnet plassering av beholderen i et autosamplerrom i et instrument for detektering av derivatet av den minst ene analytten.12. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av kravene 9 til 11, hvori instrumentet for detektering av derivatet av den minst ene analytten er et instrument for væskekromatografi koblet med massespektrometri, eller et instrument for væskekromatografi med tandem massespektrometri.13. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av kravene 9 til 11, hvori instrumentet for detektering av derivatet av den minst ene analytten er et instrument for gasskromatografi koblet med massespektrometri, eller et instrument for gasskromatografi med tandem massespektrometri.14. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av de foregående kravene, hvori den biologiske prøven omfatter helblod, serumplasma, urin, spytt eller cerebrospinalfluid.15. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av de foregående kravene, hvori det vannblandbare løsemiddelet i trinn b) omfatter én av acetonitril, aceton, metanol, etanol, 1-propanol eller 2-propanol.16. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av de foregående kravene, hvori utsaltingsreagensen omfatter én eller flere av ammoniumsulfat, ammoniumacetat eller natriumklorid.17. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av de foregående kravene, hvori utsukringsreagensen omfatter fruktose.18. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av de foregående kravene, hvori den minst ene analytten omfatter en dienfunksjonell gruppe, og hvori derivatiseringsreagensen kan reagere med nevnte dienfunksjonelle gruppe.19. Fremgangsmåten for klargjøring av en prøve ifølge krav 18, hvori derivatiseringsreagensen er 4-fenyl-1,2,4-triazolin-3,5-dion.20. Fremgangsmåten for klargjøring av en prøve ifølge krav 18 eller 19, hvori den minst ene analytten er én av vitamin D3, 25-hydroksyvitamin D3, eller 1,25-dihydroksyvitamin D3.21. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av kravene 1 til 20, hvori den minst ene analytten omfatter en fenolfunksjonell gruppe, og hvori derivatiseringsreagensen kan reagere med nevnte fenolfunksjonelle gruppe.22. Fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av kravene 1 til 21, hvori den minst ene analytten omfatter en ketonfunksjonell gruppe, og hvori derivatiseringsreagensen kan reagere med nevnte ketonfunksjonelle gruppe.23. Et sett for utføring av fremgangsmåten for klargjøring av en prøve ifølge hvilket som helst av de foregående kravene, hvilket sett omfatter:- et vannblandbart løsemiddel;- en utsaltingsreagens eller en utsukringsreagens;- en derivatiseringsreagens; og- en beholder for utføring av fremgangsmåten for klargjøring av en prøve i én beholder, hvori beholderen er egnet for plassering i en autosampler, og hvori beholderen består av en liten flaske, eller en brønn i en mikroplate.24. Settet ifølge krav 23, hvori settet videre omfatter én eller flere typer internstandardsubstans.25. Settet ifølge krav 23, hvori settet omfatter en mikroplate.
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IPC classesG01N 33/82G01N 33/00G01N 33/487G01N 33/48CPC classesG01N 33/82G01N 33/00G01N 33/487G01N 33/48

Citations

ALSHABRAWY, ALI K., et al., "LC-MS/MS analysis of vitamin D3 metabolites in human serum using a salting-out based liquid-liquid extraction and DAPTAD derivatization", July 2000, Journal of Chromatography B 1173 (2021) 122654 ()SCHORR, PASCAL et al., "Improved quantitative LC-MS/MS analysis of vitamin D metabolites in serum after one-pot double derivatization", Improved quantitative LC-MS/MS analysis of vitamin D metabolites in serum after one-pot double derivatization ()XUE, YING et al., et al., "Simultaneous Quantification of 25-Hydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3 in Rats Shows Strong Correlations between Serum and Brain Tissue Levels", International Journal of Endocrinology, Volume 2015, Article ID 296531, 10 pages ()ABU SA`ALEEK, ASMAA "Determination of vitamin D3 in pharmaceutical products using salting-out assisted liquid–liquid extraction coupled with reversed phase liquid chromatography" Microchemical Journal; Volume 193, October 2023, 109077. ()YANG, MING-YEH et al., "Using gas chromatography and mass spectrometry to determine 25-hydroxyvitamin D levels for clinical assessment of vitamin D deficiency" journal of food and drug analysis 27 (2019) 494-501. ()ZHANG, JUN, et. Al., "Sugaring-out assisted liquid/liquid extraction with acetonitrile for bioanalysis using liquid chromatography–mass spectrometry", Microchemical Journal 108 (2013) 198–202. ()WO 2023155000 A1 (A1)CN 113552229 A (A)

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FISHLAB AS
c/o Sven David Lausten Knudsen, Storhaug Alle 8 4015 STAVANGER NO (STAVANGER Municipality, Rogaland county)Org. number: 922878692Inventors
Cato Brede
Åsvegen 5 4120 TAU NO (STRAND Municipality, Rogaland county)

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HÅMSØ PATENTBYRÅ AS
HÅMSØ PATENTBYRÅ AS

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