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WO2016019037A1 - Traitement et analyse d'hormone libre et de métabolite hormonal par spectrométrie de masse - Google Patents

Traitement et analyse d'hormone libre et de métabolite hormonal par spectrométrie de masse Download PDF

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Publication number
WO2016019037A1
WO2016019037A1 PCT/US2015/042689 US2015042689W WO2016019037A1 WO 2016019037 A1 WO2016019037 A1 WO 2016019037A1 US 2015042689 W US2015042689 W US 2015042689W WO 2016019037 A1 WO2016019037 A1 WO 2016019037A1
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vitamin
sample
free
analysis
ultrafiltration
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PCT/US2015/042689
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Steven J. Soldin
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Georgetown University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/18—Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/06—Tubular membrane modules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/16—Rotary, reciprocated or vibrated modules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/10—Devices for withdrawing samples in the liquid or fluent state
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2676—Centrifugal separation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/02—Rotation or turning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration

Definitions

  • the invention relates to methods and kits for analyzing and workup of free hormone and free hormone analysis by use of ultrafiltration and mass spectrometry.
  • Hormones are biological messengers. They are synthesized by specific tissues (glands) and are secreted into the blood. The blood carries them to target cells where they act to alter the activities of the target cells.
  • Hormones are chemically diverse, and are generally categorized into three main groups: (1) small molecules derived from amino acids, for example thyroxine, (2) polypeptides or proteins, for example insulin and thyroid-stimulating hormone, and (3) molecules derived from cholesterol, for example steroids and vitamin D.
  • thyroid hormones An important class of hormone is the thyroid hormones.
  • thyroid hormones are thyroxine (T4), free thryoxine (FT4), triiodothyronine (T3) and free
  • T4 and T3 enter cells and bind to intracellular receptors where they increase the metabolic capabilities of the cell by increasing mitochondria and mitochondrial enzymes.
  • T4 and T3 are important in regulating a number of biological processes, including growth and development, carbohydrate metabolism, oxygen consumption, protein synthesis and fetal neurodevelopment. Synthesis of all circulating T4 and a small percentage of circulating T3 occurs on thyroglobulin molecules located within the thyroid. The bulk of the T3 present in the blood is produced enzymatically via monodeiodination of T4 by specific intracellular deiodinases—enzymes present in the follicular cells and the cells of target tissues [1].
  • T4 In serum drawn from healthy human subjects, total T4 is present at about 60-fold higher concentration than total T3. T4 acts as a prohormone, as the reservoir for the production of T3, the active hormone. The metabolic activity associated with thyroid hormone (TH) is initiated by T3 binding to specific nuclear receptors within target cells. Thyroid hormone concentrations in blood are essential tests for the assessment of thyroid function.
  • Steroids make up another important class of hormones.
  • examples of steroid hormones include estrogens, progesterone and testosterone.
  • Estrogen is the name of a group of hormones of which there are three principle forms, estrone, estradiol and estriol.
  • Estrogens and progesterone cause the development of the female secondary sexual characteristics and develop and maintain the reproductive function.
  • Testosterone develops and maintains the male secondary sex characteristics, promotes growth and formation of sperm.
  • Steroids enter target cells and bind to intracellular receptors and then cause the production of mRNA coding for proteins that manifest the changes induced by steroids.
  • Vitamin D is an essential nutrient with important physiological roles in the positive regulation of calcium (Ca 2+ ) homeostasis. Vitamin D can be made de novo in the skin by exposure to sunlight or it can be absorbed from the diet. There are two forms of vitamin D; vitamin D 2 (ergocalciferol) and vitamin D 3 (cholecalciferol). Vitamin D 3 is the form synthesized de novo by animals. It is also a common supplement added to milk products and certain food products produced in the United States. Both dietary and intrinsically synthesized vitamin D 3 must undergo metabolic activation to generate bioactive metabolites. In humans, the initial step of vitamin D 3 activation occurs primarily in the liver and involves hydroxylation to form the intermediate metabolite 25-hydroxyvitamin D 3 (25 -hydroxy cholecalciferol; calcifediol;
  • 250HD 3 Calcifediol is the major form of vitamin D 3 in the circulation. Circulating 250HD 3 is then converted by the kidney to 1,25-dihydroxyvitamin D 3 (calcitriol; l,25(OH) 2 D 3 ), which is generally believed to be the metabolite of vitamin D 3 with the highest biological activity.
  • Vitamin D 2 is derived from fungal and plant sources. Some over-the-counter dietary supplements contain ergocalciferol (vitamin D 2 ) rather than cholecalciferol (vitamin D 3 ). Drisdol, the only high-potency prescription form of vitamin D available in the United States, is formulated with ergocalciferol. Vitamin D 2 undergoes a similar pathway of metabolic activation in humans as vitamin D 3 , forming the metabolites 25-hydroxyvitamin D 2 (250HD 2 ) and 1,25- dihydroxyvitamin D 2 (l,25(OH) 2 D 2 ).
  • Vitamin D 2 and vitamin D 3 have long been assumed to be biologically equivalent in humans, however recent reports suggest that there may be differences in the bioactivity and bioavailability of these two forms of vitamin D (Arras et. al, (2004) J. Clin. Endocrinol. Metab. 89:5387-5391). While vitamin D 2 and vitamin D 3 may be different and referred to as being distinct in the within teachings, a reference herein to vitamin D or metabolites thereof should be understood to refer to either one of, or both of vitamin D 2 and vitamin D 3 and that methods described for the analysis of one type of vitamin D can be utilized on the other type with appropriate modification.
  • vitamin D can include any hydroxylated vitamin D species (either D2 or D3) that may be found in the circulation of an animal which is formed by a biosynthetic or metabolic pathway for vitamin D or a synthetic vitamin D analog.
  • hydroxylated vitamin D species either D2 or D3
  • hydroxyvitamin D metabolite is hydroxylated at the 1 and 25 position.
  • the vitamin D metabolite is l ,25-dihydroxyvitamin D3 (la,25(OH) 2 D 3 ) or la,25- dihydroxyvitamin D 2 (la,25(OH) 2 D 2 ).
  • the dihydroxyvitamin D metabolites are naturally present in a body fluid of a mammal (such as a human).
  • the methods as described herein selectively detect l ,25 -dihydroxyvitamin D 3 (la,25(OH) 2 D 3 ) and/or 1 a,25 -dihydroxyvitamin D 2 (la,25(OH) 2 D 2 ).
  • Measurement of l,25(OH) 2 D is also used in clinical settings.
  • certain disease states such as kidney failure can be diagnosed by reduced levels of circulating l,25(OH) 2 D and elevated levels of l,25(OH) 2 2D may be indicative of excess parathyroid hormone or may be indicative of certain diseases such as sarcoidosis or certain types of
  • lymphoma lymphoma
  • vitamin D3 Likewise other areas of interest relating to free concentrations of vitamin D3 include Depressive illness, Seizure disorders, Oncology, Bone diseases, Alzheimer's disease, 5 Schizophrenia, Autism, Parkinson's disease, Multiple Sclerosis and pregnancy.
  • iodothyronine-binding autoantibodies that interfere with total T4 and T3 immunoassays (“IAs") is a known phenomenon [2], [3], [4]. These autoantibodies may give falsely high, or falsely low values of thyroid hormone measurements depending on the assay separation method used, and 15 are often in discordance with the clinical features [2], [3], [4]. Serum free T4 and T3 (FT4 and
  • FT3 measurements are a way to compensate for such abnormal binding.
  • the measurement of free vitamin D or metabolites thereof in bodily fluids such as for example blood plasma is representative of the amount of bioavailable vitamin D in a subject. It has been found that there is a relatively poor correlation between the
  • a vitamin D deficiency can indicate an increased risk of a condition such as those referred above.
  • free vitamin D concentrations since these are so small and are particularly exacerbated by the non-polar and non- water soluble nature of vitamin D. It is much easier to measure the total (free/unbound + protein bound) vitamin D concentrations since these concentrations can be several orders of magnitude higher than the concentration of free vitamin D alone in a given subject.
  • estriol is analyzed by a radioimmunoassay utilizing radiolabeled antigen (iodine 125) in competition with unlabelled estriol in the sample, for a known amount of antibody.
  • the assay is read using a gamma counter.
  • Androstenedione is analyzed using an enzyme immunoassay comprising horseradish peroxidase. Unlabeled antigen in the sample is in competition with enzyme labeled antigen for a fixed number of antibody binding sites. The assay is read using a microtitre plate enzyme immunoassay reader.
  • chemiluminescent immunoassay Several hormones are currently analyzed using a chemiluminescent immunoassay. For example, progesterone, testosterone, Cortisol and T3 are analyzed using this method.
  • the assay utilizes an assay-specific antibody-coated bead.
  • the assay is read using a photon counter.
  • estriol and progesterone from a sample requires both a gamma counter and a photon counter.
  • kits for the assays can be expensive.
  • the current immunoassays lack specificity and may show approximately 15 fold difference in results using kits from different manufacturers [5].
  • Pathologists Proficiency Testing (CAP PT) Program can vary by a factor of approximately 2. Some factors such as pregnancy, estrogen therapy or genetic abnormalities in protein binding have also reportedly made immunoassay methods for T4 and T3 diagnostically unreliable [2], [3], [14], [15]. Currently serum total free T4 (FT4) and free T3 (FT3) concentrations are most commonly measured by immunoassay methods. Recently some reports of quantitative measurement of T4 and T3 by high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC- MS) or tandem mass spectrometry (LC-MS/MS) were published [16-20]. All those methods required extraction, derivatization and even prior chromatographic separation that are very time- consuming [21], [22].
  • HPLC high performance liquid chromatography
  • GC-MS gas chromatography mass spectrometry
  • LC- MS liquid chromatography mass spectrometry
  • LC-MS/MS tandem mass spectrometry
  • a method of analyzing urinary testosterone and dihydrotestosterone glucuronides using electrospray tandem mass spectrometry has been described [23].
  • the method involves a complex system employing high performance liquid chromatography (HPLC) and a three-column two-switching valve.
  • HPLC high performance liquid chromatography
  • the shortcomings include the following: (i) the hormone glucuronides were analyzed, not the hormones, (ii) the method is applicable to urine only and (iii) only two analytes were analyzed simultaneously, (iv) the limit of detection (LOD) was 200 pg ml "1 for testosterone and the limit of quantification was 10 ug L "1 for dihydrotestosterone and (v) the method is complex.
  • LOD limit of detection
  • Another publication discloses a method for the determination of estradiol in bovine plasma by an ion trap gas chromatography-tandem mass spectrometry technique [24].
  • the shortcomings include the following: (i) only one analyte was analyzed, (ii) 4 ml of plasma was required for the analysis of one analyte, (iii) the limit of detection was 5 pg ml "1 , and (iv) derivation was required because the method employs gas chromatography.
  • FPIA Fluorescence Polarization Immunoassay
  • the applicant's teaching provides a fast and accurate method of free vitamin D and metabolites of free vitamin D analysis and other biological analytes and kits for use thereby.
  • a plurality of hormones can be analyzed simultaneously or sequentially. The procedure allows for as little as 100 of a sample to be analyzed. In addition, minimal sample preparation time is required.
  • hormone analysis permits the analysis of hormones in a number of complex matrices as they might be found in nature, e.g. the human body.
  • hormone analysis can be performed on samples of blood, saliva, serum, plasma and urine.
  • the present teaching requires minimal sample preparation time. For example, preparing a sample for hormone analysis can be done within 6 minutes. (4) The procedure does not require a large sample size. A plasma or serum sample can be as small as 100 for thyroid hormones. For FT4 and FT3 the sample can be between 500 and 600 ⁇ . The current methods for hormone analysis that utilize mass spectrometry require 4-15 mL of plasma. (5) The methods use simple preparation techniques that are easy to use and highly reproducible. (6) The methods permit analysis to be performed on a variety of sample types.
  • the methods permit the analysis of hormones in a sample of saliva or urine which permits simple sample acquisition and the remote submission of samples to a clinic for analysis. In previous other clinical methods, samples are taken by invasive means directly from the patient in a clinic. (8) The analysis by mass spectrometry is highly accurate. In addition, the procedure of the present methods are highly reproducible. (9) The methods permit the analysis of a wide range of hormone concentrations. In addition, the limit of detection can be fairly low.
  • a method for mass spectrometric analysis of a sample containing or suspected of containing free thyroxine (FT4) hormone comprising the steps (a) providing a sample containing or suspected of containing FT4 hormone, (b) separating FT4 hormone from the sample, (c) collecting FT4 hormone, and (d) analyzing FT4 hormone using a mass spectrometer.
  • a method for mass spectrometric analysis of a sample containing or suspected of containing free triiodothyronine (FT3) hormone comprising the steps (a) providing a sample containing or suspected of containing FT3 hormone, (b) separating FT3 hormone from the sample, (c) collecting FT3 hormone, and (d) analyzing FT3 hormone using a mass spectrometer.
  • a method for mass spectrometric analysis of a sample containing or suspected of containing free thyroxine (FT4) and free triiodothyronine (FT3) hormone comprising the steps (a) providing a sample containing or suspected of containing FT4 and FT3 hormone, (b) separating FT4 and FT3 hormone from the sample, (c) collecting FT4 and FT3 hormone, and (d) analyzing FT4 and FT3 hormone using a mass spectrometer.
  • the method comprises providing instructions to prepare and analyze the sample, as described above.
  • a system for the mass spectrometric analysis of a sample containing or suspected of containing FT4 comprising (a) reagents for separating FT4 from the sample, including internal standards, (b) reagents for analyzing FT4 hormone using a mass spectrometer, and (c) a mass spectrometer.
  • a system for the mass spectrometric analysis of a sample containing or suspected of containing FT3 comprising (a) reagents for separating FT3 from the sample, including internal standards, (b) reagents for analyzing FT3 hormone using a mass spectrometer, and (c) a mass spectrometer.
  • kits for use in mass spectrometric analysis of a sample containing or suspected of containing FT4 comprising (a) reagents for separating FT4 from the sample, (b) reagents for analyzing the FT4 using a mass spectrometer, (c) a solution of FT4, and (d) instructions for analyzing the FT4 using a mass spectrometer.
  • kits for use in mass spectrometric analysis of a sample containing or suspected of containing FT3, comprising (a) reagents for separating FT3 from the sample, (b) reagents for analyzing the FT3 using a mass spectrometer, (c) a solution of FT3, and (d) instructions for analyzing the FT3 using a mass spectrometer.
  • kits for use in mass spectrometric analysis of a sample containing or suspected of containing FT4 and FT3, comprising (a) reagents for separating FT4 and FT3 from the sample, (b) reagents for analyzing the FT4 and FT3 using a mass spectrometer, (c) a solution of FT4 and FT3, and (d) instructions for analyzing the FT4 and FT3 using a mass spectrometer.
  • an ultrafiltration method of an unbound analyte comprising: providing an ultrafiltration tube having a filtration chamber for containing a sample, a filtrate recovery chamber for receiving filtrate from said ultrafiltration and a filter medium disposed between and fluidly coupled with said filtration chamber and filtrate recovery chamber, inserting a solution comprising an organic solvent and an internal standard for the unbound analyte into the filtrate recovery chamber of said ultrafiltration tube , and applying a filter force to said sample to force one or more components of said sample through said filter medium thereby forming a filtrate of said unbound analyte.
  • the filter force is centrifugation. In other embodiments, the filter force can be achieved through use of vacuum or positive pressure when the tube is configured in an appropriate manner.
  • the internal standard is a deuterated version of the unbound analyte.
  • the analyte is vitamin D or a metabolite thereof.
  • the analyte is cholesterol.
  • the organic solvent is methanol.
  • the method further comprises performing a quantitative analysis on the filtrate.
  • the quantitative analysis is performed by mass spectrometry.
  • the mass spectrometry is performed using MRM.
  • the analyte is 25 OH vitamin D3 and the MRM transitions monitored are about 383/229.
  • a method for analyzing of a sample for the presence of free vitamin D or a metabolite thereof comprising: a) separating free vitamin D from bound vitamin D or from a metabolite thereof by filtrating by providing an ultrafiltration device comprising a filtration chamber having a solution, the sample, a filtrate recovery chamber for receiving filtrate and a filter medium disposed between and fluidly coupled to said filtration chamber and filtrate recovery chamber; b) collecting free vitamin D or metabolites thereof separated from bound vitamin D or metabolites thereof in said filtrate recovery chamber; and c) analyzing free vitamin D or metabolites thereof separated from bound vitamin D or metabolites thereof; wherein said separating comprises preinserting a solution comprising an organic solvent and an internal standard for the free vitamin D or metabolites thereof, into the filtrate recovery chamber prior to filtering.
  • the analyzing is performed using a mass spectrometry technique.
  • the mass spectrometry technique includes an MRM analysis.
  • the MRM analysis includes a quantitative analysis.
  • the quantitative analysis determines the concentration of 25-hydroxyvitamin D3 and/or 25-hydroxyvitamin D2.
  • the organic solvent is methanol.
  • a method for analysis of a sample containing or suspected of containing a free non-polar biological analyte or a metabolite thereof comprising: a) separating the free non-polar biological analyte or metabolite thereof from a bound non-polar biological analyte or a metabolite thereof by filtering by providing an ultrafiltration device comprising a filtration chamber having a solution, the sample, a filtrate recovery chamber for receiving filtrate and a filter medium disposed between and fluidly coupled to said filtration chamber and filtrate recovery chamber; b) collecting the free non-polar biological analyte or metabolite thereof separated from the bound non-polar biological analyte or metabolite thereof in said filtrate recovery chamber; and c) analyzing the free non-polar biological analyte or metabolite thereof separated from the bound non-polar biological analyte or metabolite thereof; wherein said separating comprises preinserting a solution comprising a solution comprising a solution compris
  • the free non-polar biological analyte is selected from the group consisting of free vitamin D, free Cortisol, free testosterone, free cholesterol and free estradiol.
  • the analyzing is performed using a mass spectrometry technique.
  • the mass spectrometry technique includes an MRM analysis.
  • the MRM analysis includes a quantitative analysis.
  • the organic solvent is methanol.
  • kits for the analysis of vitamin D or a metabolite thereof comprising: an ultrafiltration tube comprising a filtration chamber, a filtration medium and a filtrate recovery chamber; an organic solvent; an internal standard comprising a deuterated version of vitamin D or a metabolite thereof; two or more calibration standards, each of the calibration standards comprising a different concentration of vitamin D or a metabolite thereof; and instructions for use.
  • the organic solvent can be methanol.
  • FIG. 1 is a mass spectrum of a blank plasma sample containing T4 and T3.
  • FIG. 2 is a mass spectrum of a globulin standard containing T4 and T3.
  • FIG. 3 is a typical tandem mass spectrometric chromatogram obtained for T4 and
  • T3 for a plasma sample.
  • FIG. 4 is a graph showing T3 measured by Isotope Dilution Tandem Mass
  • FIG. 5 is a graph showing T4 measured by Isotope Dilution Tandem Mass
  • FIG. 6 is a graph showing a typical chromatogram for free T4 (11.2 pg/mL) and deuterated internal standard.
  • FIG. 7 is a graph showing the effect of temperature on FT4 by tandem mass spectrometry and ultrafiltration.
  • FIG. 8 is graph showing the comparison of the tandem mass spectrometric method with the equilibrium dialysis method for the measurement of free T4.
  • FIG. 9 is a graph showing the comparison of the tandem mass spectrometric method with the direct immunoassay method on the Dade RxL Dimension for the measurement of free T4.
  • FIGS. 10a, b, and c are a series of mass spectrums showing the analysis of FT4
  • FIG. 11 is a depiction of the components of an ultrafiltration tube to be used in a centrifuge device for sample concentration.
  • FIG. 12A and B is a depiction of an assembled ultrafiltration tube to be used in a centrifuge device prior to filtration and after filtration.
  • FIG. 13 depicts a chart showing the relationship between concentrations of parathyroid hormones and total 25 hydroxyvitamin D3
  • FIG. 14 depicts a chart showing the relationship between concentrations of parathyroid hormones and free 25 hydroxyvitamin D3
  • FIG. 15 depicts a chart showing the relationship between the concentrations of free 25 -hydroxyvitamin D3 and total 25 -hydroxyvitamin D3
  • FIG. 16 depicts a calibration curve generated for free 25 -hydroxy vitamin D3 using the within teachings.
  • FIG. 17 depicts the stability profile of free vitamin D at various temperatures over time.
  • FIG. 18 depicts chromatograms for the analysis of Free 25-OH Vitamin D3 and deuterated Free 25-OH Vitamin D3-d 6 internal standard.
  • FIG. 19 depicts chromatograms for the analysis of Free 25-OH Vitamin D3 and deuterated Free 25-OH Vitamin D3-d 6 internal standard.
  • FIG. 20 depicts measured PTH concentrations for African-Americans vs. Others.
  • FIG. 21 depicts a chromatogram obtained from a 10 pg/mL standard obtained when using the within teachings
  • FIG. 22 depicts a chromatogram obtained from a patient sample when using the within teachings.
  • the applicant's teaching provides methods of analysis for hormones.
  • the hormones may include:
  • DHEA Dehydroepiandrosterone
  • DHEAS Dehydroepiandrosterone sulphate
  • Vitamin D and its metabolites 25hydroxyvitamin D and 1,25 dihydroxyvitamm D
  • the methods may also include the measure of free unbound analytes of the other hormones listed.
  • the applicant's teaching may also be utilized with other biological analytes and to specifically those biological analytes that are non-polar.
  • cholesterol and free cholesterol are known to be precursors to the production of vitamin D in the body. The within methods may therefore also be utilized for the analysis of cholesterol and free cholesterol.
  • any sample containing or suspected of containing a hormone can be used, including physiological fluids such as a sample of blood, plasma, serum, urine or saliva.
  • the sample may contain both free and conjugated or bound hormones.
  • a sample size of at least about 100 for hormones generally, or at least about 700 for steroid hormones when using API 3000TM, or 200 to 500 ⁇ , for steroid hormones when using the API 4000TM or API 5000TM, can be used.
  • a sample size of 500 to 600 ⁇ _, for FT4 and FT3 can be used when using the API 4000TM or API 5000TM.
  • a sample size of 900 ⁇ can be used for vitamin D or metabolites thereof.
  • the sample may be de-proteinated. This can be done by conventional techniques known to those skilled in the art. For example, a sample can be de-proteinated with acetonitrile, containing internal standard, followed by vortexing and centrifugation.
  • the internal standard may be, for example, the deuterated hormone.
  • the hormones are separated by methods known to those skilled in the art.
  • the hormones may be separated by liquid chromatography through a column.
  • Many different columns can be used.
  • the column may be a C- 18 column or, for example, a C-8 column.
  • the column may also be a C6, C4, C2 or similar column.
  • the shorter the carbon chain the shorter the retention time.
  • the hormones are subsequently eluted from the column.
  • the hormones may also be separated by centrifugation.
  • FT4 may be separated from other compounds, including bound T4 by centrifugation using an ultrafiltration device. After centrifugation, the ultrafiltrate will contain FT4, while the bound T4 and other compounds will be unable to pass through the filter.
  • the hormones may be separated by equilibrium dialysis or other methods known to those skilled in the art.
  • the ultrafiltration step can be performed using an ultrafiltration device in which the filtrate collector is prefilled prior to the filtration with a solution comprising an organic solvent, such as methanol and an internal standard.
  • an organic solvent containing an internal standard is placed into the bottom of the tube and the filtrate from the sample is deposited during filtration into the organic solvent containing internal standard.
  • the internal standard is a deuterated version of the analyte that is being analyzed and therefore would differ in mass by the number of deuterium atoms present.
  • other types of internal standards may also be used.
  • free vitamin D and metabolites thereof can be separated from bound vitamin D and metabolites thereof in a manner that allows it to be measured using various analytical techniques, including for example, mass spectrometry. While this step is particularly useful for the preparing a sample of vitamin D or metabolites thereof, such a method is potentially useful for other analytes described herein.
  • the organic solvent can be selected from the group of alcohol solvents, which can include, for example, methanol, ethanol, n-propanol, isopropanol, etc. and the like.
  • the organic solvent can also comprise more than one type of solvent and can include a mixture of such solvents, such as for example a mixture of 90 or 95% methanol with the remaining solvent comprising ethanol.
  • the organic solvent is methanol.
  • the filtration medium can be typically selected to exclude molecules that are protein bound. When referring to bound proteins, it is intended to mean that Vitamin D being bound to proteins in serum/plasma. [00090] Referring to FIG. 11, an example of the components of a typical ultrafiltration tube 100 is depicted.
  • the ultrafiltration tube 100 comprises a filtration chamber 101 and filtrate recovery tube 106.
  • the filtration chamber 101 comprises sidewalls 102 that define the chamber and a bottom portion that comprises an ultrafilter medium 103.
  • the bottom portion may also include a porous support such as sintered glass that provides backing to the filter medium 103.
  • the sidewalls 102 may taper towards the bottom portion to assist in directing fluid toward the filter medium.
  • the top portion of the filtration chamber 101 comprises an opening 105 that is defined by a lip 104. Generally the diameter of the lip 104 is wider than that of the sidewalls and the sidewalls 102 taper towards the lip 104 to provide a continuous wall.
  • the filtration chamber may have a cap or plug mechanism.
  • the filtrate recovery tube 106 comprises sidewalls 107 and a bottom wall 108.
  • the sidewalls 107 may taper towards the bottom wall 108.
  • the sidewalls 107 define an opening 111 at the top that forms a lip 112.
  • the diameter of the lip 112 is smaller than that of the lip 104 of the filtration chamber 101, but is larger than that of the diameter of the sidewalls 102 of the filtration chamber 101.
  • a cap 110 is attached via a flexible connector 109 to the sidewalls 107 to allow the capping of the filtration chamber.
  • the filtration chamber 101 is filled with a sample to be filtered containing an appropriate analyte, such as for example a sample 113 containing, or suspected of containing both bound and unbound/free vitamin D or related metabolites.
  • an appropriate analyte such as for example a sample 113 containing, or suspected of containing both bound and unbound/free vitamin D or related metabolites.
  • a solution 114 comprising methanol and a Vitamin D or related metabolites internal standard is preinserted into the filtrate recovery tube 106.
  • the filtration chamber 111 is then inserted into the filtrate recovery tube 106 and the cap 110 is closed.
  • the lip 104 of the filtration chamber 101 or the taper portions of the sidewalls 102 generally rests on that of the lip 112 of the filtrate recovery tube 106 and is suspended over the bottom.
  • the filtrate recovery tube 106 containing the filtration chamber 101 is then inserted into an appropriate device that will generate a force that will drive components of the sample 113 through the ultrafiltration medium 103.
  • This can be achieved by use of a centrifuge.
  • the ultrafiltration medium generally allows the passage of free/unbound vitamin D and related metabolites, but prevents the passage of larger protein bound vitamin D and related metabolites.
  • the remaining sample 115 will be comprised of an increased concentration of protein bound analytes whereas the remaining liquid 116 located in the bottom of the filtrate recovery tube 106 will comprise methanol, an internal standard, free vitamin D and related metabolites and other molecules with sufficiently small size to traverse the filter. While depicted here as having a horizontal filter medium, such a method can also be utilized with vertically mounted filter mediums such as those for example, depicted in US Patent No. 5,647,990. Other types of ultrafiltration tube may also be utilized as a result of the within teachings.
  • the hormones are then introduced into a mass spectrometer.
  • the separation step and step of introducing the hormones into a mass spectrometer can be combined using a combined liquid chromatography spectrometry apparatus (LC/MS). This procedure is based on an online extraction of the injected sample with subsequent introduction into the mass spectrometer using a built-in switching valve.
  • LC/MS liquid chromatography spectrometry apparatus
  • the methods employ isotope dilution mass spectrometry.
  • the hormones are subjected to ionization.
  • Various ionization techniques can be used. For example, photoionization, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI) and electron capture ionization may be used. Electrospray ionization can be utilized when analyzing thyroid hormones.
  • the following mass spectrometers can be used: any tandem-mass spectrometer, including hybrid quadrupole-linear ion trap mass spectrometers and liquid chromatography- tandem mass spectrometers such as the API 3000TM mass spectrometer and the API 4000TM mass spectrometer, described in for example U.S. Pat. Nos. 4,121,099; 4,137,750; 4,328,420;
  • a spectrometer with a turbo spray ion source such as the API 2000TM and API 3000TM mass spectrometers can be used.
  • the API 4000TM mass spectrometer can be used.
  • the API 5000TM mass spectrometer can be used.
  • the API 5000TM mass spectrometer can be used.
  • the SCIEX Triple Quad 6500 mass spectrometer system can be used.
  • Ionization may be performed by utilizing the mass spectrometer in the negative or the positive mode, depending on a particular analyte's tendency to give rise to a particular ion form, as is known to those skilled in the art.
  • the spectrometer is employed in the negative mode.
  • Hormones are identified on the basis of the mass to charge ratio of their molecular ions and fragment ions, as is known to those skilled in the art. When the hormones are purified by liquid chromatography, they can also be identified by their retention times.
  • Hormones are quantified by their intensity as determined in the mass spectrometer in counts per second. Calibration curves for known concentrations of the hormones are established for comparison.
  • Kits for use in mass spectrometric analysis of a sample comprising or suspected of comprising FT4, FT3 or both are also provided.
  • the kits are assembled as is known to those skilled in the art.
  • the kits can comprise, for example, reagents for separating the hormone from the sample, reagents for analyzing the hormone using a mass spectrometer, a solution of the hormone, and instructions.
  • kits may also include an ultrafiltration tube comprising a filtration chamber, a filtration medium and a filtrate recovery chamber, the filtration medium being sized to prevent passage of molecules greater that are bound; an organic solvent, such as for example, methanol; an internal standard comprising a deuterated version of vitamin D or a metabolite thereof; two or more calibration standards, each of the calibration standards comprising a different concentration of vitamin D or a metabolite thereof; and instructions for use.
  • an ultrafiltration tube comprising a filtration chamber, a filtration medium and a filtrate recovery chamber, the filtration medium being sized to prevent passage of molecules greater that are bound
  • an organic solvent such as for example, methanol
  • an internal standard comprising a deuterated version of vitamin D or a metabolite thereof
  • two or more calibration standards each of the calibration standards comprising a different concentration of vitamin D or a metabolite thereof
  • a sample of 100 ⁇ _, of plasma was used. Proteins were precipitated with 150 ⁇ _, of acetonitrile, capped and vortexed. The sample was then centrifuged, and 200 ⁇ _, of the supernatant was injected onto a Supelco LC-18-DBTM chromatographic column equipped with Supelco Discovery C-18 guard column, coupled to a tandem mass spectrometer (LC/MS/MS). The column was washed with 20% methanol in 5 mM ammonium acetate for 3 minutes. The valve was switched and the sample was eluted in 75% to 95% methanol. The total run time was 6 minutes. Slight adjustments to the volumes, concentrations and times described can be made, as is known to those skilled in the art.
  • the eluant was introduced into an ion- spray ionization chamber and analyzed by API 2000TM mass spectrometer using the negative mode.
  • the mass/charge ratios for T4 and T3 ions is 775.8 and 650 respectively.
  • the ionization may be by electrospray using a turboionspray chamber.
  • a sample of 100 ⁇ _, of plasma was used. Proteins were precipitated with 150 ⁇ _, of acetonitrile, containing an internal standard of deuterated T4 and vortexed. The sample was centrifuged, and 200 ⁇ _, of the supernatant was injected onto a C-18 column coupled to a tandem mass spectrometer (LC/MS/MS). The column was washed with 20% methanol in 5 mM ammonium acetate for 3 minutes. The valve on the column was switched and the sample was eluted in a methanol gradient of 20 to 100%. The total run time was 7 minutes. Slight adjustments to the volumes, concentrations and times described can be made by those skilled in the art.
  • FIG. 1 and FIG. 2 shows the mass spectrums generated for T3 and T4.
  • This example describes an isotope dilution tandem mass spectrometry method for the simultaneous determination of T4 and T3 in serum.
  • the method is accurate, specific, precise (% CVs between 3.5 and 9.0), simple—requiring no extraction and only protein precipitation, and fast. For example it can be done in less than seven minutes.
  • HPLC grade methanol was purchased from VWR Scientific. All other chemicals were of analytical grade and purchased from Sigma.
  • Serum or plasma samples were thawed at room temperature. 150 of IS solution was added to aliquots of 100 of the serum or plasma sample. After 30 seconds of vortex mixing, the samples were stored for 10 minutes at room temperature to allow complete protein precipitation. The samples were centrifuged for 10 minutes at 15,000 rpm and 100 ⁇ of supernatant was injected into the LC-MS-MS system. LC/MS/MS Conditions
  • the procedure used is based on an online extraction/cleaning of the injected samples with subsequent introduction into the mass-spectrometer by using a built-in Valco switching valve.
  • the switching valve was activated, the column was flushed with water/methanol gradient at flow rate 0.5 mL/min and the samples were introduced into the mass-spectrometer.
  • the gradient parameters used are shown in Table 4.
  • T4 was measured by the Dade RxL DimensionTM (Dade-Behring Diagnostics, Glasgow, Del.) and T3 by the DPC ImmuliteTM (Diagnostic Product Corporation, Los Angeles, Calif.) according to the manufacturer's specifications.
  • FIG. 3 shows a typical tandem mass spectrometric chromatogram obtained for T3 and T4 (T4 m/z (776/127); D 2 T4 m/z (778/127); T3 m/z (650/127)).
  • the lower limit of quantitation of the mass spectrometry method was found to be 0.15 ng/mL for both T3 and T4. Detection limit was around 0.062 ng/niL.
  • Radioimmunoassay (RIA) methods to detect thyroid hormones were developed in the 1970s.
  • Serum T4 and T3 concentrations are currently measured by competitive immunoassay methods (IAs) that are mostly non-isotopic and use enzymes, fluorescence or chemiluminescence molecules as signals [27].
  • IAs competitive immunoassay methods
  • Table 2 clearly indicates that current IAs for T4 and T3 lack specificity and give mean results differing by a factor of approximately 2 in the College of American Pathologists Proficiency Testing (CAP PT) programs.
  • Total hormone assays necessitate the inclusion of a displacing agent (such as salicylate) to release the hormone from its binding proteins [28].
  • a displacing agent such as salicylate
  • hyperthyroidism a reliable normal-range measurement is also important for adjusting antithyroid drug dosage and detecting hyperthyroidism in sick hospitalized patients, in whom a
  • the isotope dilution tandem mass spectrometric method of the applicant's teaching is rapid (less than 7 minutes), accurate (provides the true result as has been assessed by recovery studies), specific (measures only the analyte it purports to measure), precise (low % CV) and easy to perform.
  • the present methods teach a rapid, reliable free T4 method employing isotope dilution tandem mass spectrometry and compares results obtained by this method with both the analogue (direct) free T4 and the time-consuming and relatively expensive equilibrium dialysis procedures.
  • Thyroxine (T4) was purchased from Sigma (St Louis, Mo.).
  • a stable deuterium- labeled internal standard, L-thyroxin-d2 was synthesized according to procedures described in the literature (29, 30) by Dr Tomas Class from the Chemistry Department at Georgetown University.
  • HPLC grade methanol was purchased from VWR Scientific. All other chemicals were of analytical grade and were purchased from Sigma.
  • T4 and internal standard were prepared separately to obtain concentration of 10 mg/mL for each using 40% ammonium hydroxide (v/v) in methanol as a solvent.
  • the analyte stock solutions were diluted with methanol to obtain the spiking solutions.
  • the solutions were stored at -20°C. and could be used for several months.
  • Standards for the T4 calibration curve in the range of 2.5-50 pg/mL were prepared by adding the analytes to water.
  • a solution of 0.05 ng/mL d 2 -T4 in methanol was used as internal standard.
  • Serum or plasma samples were obtained from greater than 42 healthy pregnant and 29 non-pregnant women in a study approved by the Institutional Review Board (IRB) and were thawed at room temperature.
  • 0.6 ml samples were filtered through Centrifree YM-30 ultrafiltration devices (30,000 MW cut-off, Millipore, Bedford, Mass.) by centrifugation employing the Eppendorf temperature controlled centrifuge (model # 5702 R, Eppendorf, AG, Hamburg) and using a fixed angle rotor at 2900 rpm and a temperature of 25° for 1 hour.
  • 180 IS [0.05 ng/mL] was added to 360 ultrafiltrate and 400 was injected onto the C-18 column of the LC/MS/MS system.
  • the ultrafiltration process replaces the dialysis step of the classic equilibrium dialysis method.
  • the ultrafiltration step includes removal of all proteins having a molecular weight of greater than 30,000.
  • the liquid chromatography step can be used to further separate and purify the hormone.
  • the switching valve was activated, the column was flushed with a water/methanol gradient at flow rate of 0.6 mL/min and the samples were introduced into the mass-spectrometer.
  • the gradient parameters that were used are shown in Table 10.
  • the free T4 chromatogram is shown in FIG. 6.
  • Tables 9 and 10 provide the analytical parameters employed for the tandem mass spectrometric method.
  • FIG. 6 shows a typical chromatogram for free T4 measured by tandem mass spectrometry using the method described. The time per analysis is approximately 8.0 minutes although a steeper gradient could shorten this to about 6 minutes.
  • the Eppendorf centrifuge allows for the centrifugation of 30 tubes simultaneously so that the total run time for 30 patient samples at the 25°C. temperature used is 1 hour plus 3 hours and 15 minutes, or 4 hours and 15 minutes.
  • This ultrafiltration plus LC/MS/MS assay is considerably quicker than the time consuming equilibrium dialysis method. The latter requires 16-18 hour dialysis at 37°C. followed by an immunoassay and therefore the turn-around-time is several days. Also, very few laboratories in North America provide the equilibrium dialysis approach. The concentration of
  • a sample of 500 to 1000 of plasma is used. Proteins are precipitated with 150 ⁇ ⁇ of acetonitrile and vortexed. The sample is centrifuged, and 200 ⁇ ⁇ of the supernatant is injected onto a C-18 column coupled to a tandem mass spectrometer (LC/MS/MS). The column is washed with 20% methanol in 5 mM ammonium acetate for 3 minutes. The valve on the column is switched and the sample is eluted in a methanol gradient of 20 to 100%. The total run time is 10 minutes. Slight adjustments to the volumes, concentrations and times described can be made, as is known to those skilled in the art.
  • a sample of the eluant is introduced into an ion- spray ionization chamber and analyzed by API 3000TM mass spectrometer using the negative mode for thyroid hormones in the sample.
  • Steroid hormones in the sample are ionized by photoionization, with the spectrometer in the negative or positive mode.
  • Analysis in the positive mode is typically made for DHEA, Aldosterone, Cortisol, 11-Deoxycortisol, Androstenedione, Testosterone, Estradiol, 17-OH Progesterone, Progesterone, AUopregnalone, Vitamin D, 25,hydroxyl Vitamin D, 1,25 dihydroxy Vitamin D, corticosterone and aldosterone, whereas analysis in the negative mode is typically made for 16-OH Estrone, 2-OH Estrone, Estriol and DHEAS. However, it is possible to analyze any of the hormones in either positive or negative mode.
  • FT3 was analyzed by the same method as FT4 (Example 4), except for the analysis of the same transition ions for total T3 and using the API 5000TM mass spectrometer.
  • FT4 and FT3 concentrations are important when assessing their dosage regimen. Accordingly, an efficient assay method for the simultaneous analysis of FT3 and FT4 is beneficial.
  • FT4 and FT3 were analyzed simultaneously by a similar method of Example 4 except using the API 5000TM mass spectrometer. ⁇ mixture of T3 (25 pg/mL) and T4 (1 ng/mL) with internal standard T4-d2 were injected onto the column by autosampler, and the column was washed by 20% MeOH buffer for 2 minutes. Gradient elution started from 20% MeOH to 100% MeOH in 2 minutes after the Valco valve was activated at 2 minutes, and then kept at 100% for another 2 minutes. The retention times were: T3, 4.34 minutes, T4, 4.60 minutes, and T4-d2, 4.61 minutes.
  • FIG. 10 shows the mass spectrums of the analytes. Standard curves for FT3 (1-25 pg/ml) and FT4 (5-50 pg/ml) can be run with the analysis of the samples.
  • VIVASPIN 2 Hydrosart are specifically exemplified, other types of ultrafiltration centrifuge type filter units such as the Ultrafree 30 KDa cutoff membranes from Miilipore described above for free T3 and free T4 may be also used provided that an organic solvent containing an internal standard is placed in the filtrate capturing portion of the device before performing the ultrafiltration.
  • Solvent A 2% methanol containing 0.1% formic acid
  • Solvent B 98% methanol containing 0.1% formic acid.
  • Eppendorf LoBind safe-lock tubes (1.5 mL, clear, catalog # 022431081) were utilized for standard, QC and sample preparations. Dilute a 10 ng/mL 25-OH Vitamin D3 sub-stock solution in MeOH to 1 ng/mL with 60% (v/v) methanol-water solution. The 1 ng/ml 25-OH Vitamin D3 solution was further diluted to 100 pg/ml with 60%> (v/v) methanol-water solution. Standard solutions were prepared in 60%> MeOH according to Table 16.
  • Serum samples were obtained from whole blood drawn into red top tubes without serum separator followed by centrifugation for 10 min at 13000 rpm Serawere inspected for clots and the clots removed if present.
  • Deuterium labeled internal standard was prepared using 25-OH Vitamin D3-d6.
  • 600 of 20 pg/mL internal standard in MeOH was pipetted into the bottom of individual filtrate collection cups of the VIVASPIN 2 ultrafiltration devices (10,000 MW cut-off, Sartorius, Goettingen). 500 ⁇ , of each serum sample was pipetted into the top of the individual VIVASPIN 2 ultrafiltration devices and each filled ultrafiltration device was inserted into a temperature controlled centrifuge that was set at 40°C initially
  • the ultrafiltration devices were then inspected to determine if 300 of sample had been filtered (ie, sample level in the ultrafiltration device was at or below the 200 line). Any remaining ultrafiltration devices that had not filtered 300 ⁇ were additional spun in 90 second intervals at the same conditions until each ultrafiltration device had indicated that at least 300 ⁇ of sample had been filtered.
  • the combination ultrafiltrate of the samples and internal standards were then pipetted into Eppendorf LoBind safe-lock tubes (1.5 mL, clear, catalog # 022431081) tubes and vortexed and centrifuged for 3 minutes at 13,000 rpm and 4°C.
  • the ultrafiltrate containing the free vitamin D3 and/or internal standard in 66% methanol is stable for at least one week when stored at -80C in a suitable container such as for example a protein LoBind tube EPPENDORF (cat # 022431081 (1.5mL)).
  • a suitable container such as for example a protein LoBind tube EPPENDORF (cat # 022431081 (1.5mL)).
  • the stored sample can be vortexed and centrifuged at 4°C for 3 minutes prior to analysis using LC-MS/MS.
  • solvent "B” when operating the HPLC, can be 100% methanol or a mixture of 90 to 95% methanol with the remaining mixture comprising ethanol.
  • 25 -OH Vitamin D3 and internal standard undergo an on-line extraction, gradient chromatographic separation and elution. Retention Time of approximately 5 minutes.
  • Mobile phase A 2% (v/v) methanol-water solution 0.1% formic acid added and B: 100% methanol added. The gradient utilized is show in Table 20.
  • Lum SM Nicoloff JT, Spencer CA, Kaptein EM. Peripheral tissue mechanism for maintenance of serum triiodothyronine values in a thyroxine-deficient state in man. J Clin Invest 1984; 73(2):570-575.
  • Thienpont LM De Brabandere VI, Stockl D, De Leenheer AP. Development of a new method for the determination of thyroxine in serum based on isotope dilution gas chromatography mass spectrometry. Biol Mass Spectrom 1994; 23(8): 475-482.
  • Thienpont LM Fierens C, De Leenheer AP, Przywara L. Isotope dilution-gas chromatography/mass spectrometry and liquid chromatography/electrospray ionization- tandem mass spectrometry for the determination of triiodo-L-thyronine in serum. Rapid Commun Mass Spectrom 1999; 13(19): 1924-1931.

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Abstract

L'invention concerne un procédé pour la réalisation d'une ultrafiltration sur un analyte non polaire non lié dans un échantillon et l'analyse de celui-ci. L'échantillon peut être une hormone, telle que le cholécalciférol (vitamine D). Le procédé comprend l'introduction préalable d'une solution comprenant un solvant organique et un étalon interne destiné à l'analyte non lié, dans la chambre de récupération du filtrat avant la filtration.
PCT/US2015/042689 2014-07-29 2015-07-29 Traitement et analyse d'hormone libre et de métabolite hormonal par spectrométrie de masse WO2016019037A1 (fr)

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CN113009006A (zh) * 2021-01-20 2021-06-22 泊迈生物医学检测(苏州)有限公司 唾液中脱氢表雄酮含量检测方法及试剂盒
WO2023179804A1 (fr) * 2022-03-22 2023-09-28 合肥歆智医疗器械有限公司 Méthode de détermination de la teneur en substance libre à l'aide d'une conversion ultrafiltration-dialyse à équilibre
CN117147740A (zh) * 2023-10-31 2023-12-01 合肥歆智医疗器械有限公司 测定血中游离孕激素的超滤-质谱法及试剂盒
KR102802882B1 (ko) * 2022-04-05 2025-05-08 한국과학기술연구원 혈액 내 유리 스테로이드 측정을 위한 희석용액 및 이를 이용한 스테로이드 측정방법

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110208442A (zh) * 2019-05-24 2019-09-06 国科卓越(北京)医药科技研究有限公司 一种检测生物样品中黄体酮的药物浓度的方法
CN113009006A (zh) * 2021-01-20 2021-06-22 泊迈生物医学检测(苏州)有限公司 唾液中脱氢表雄酮含量检测方法及试剂盒
WO2023179804A1 (fr) * 2022-03-22 2023-09-28 合肥歆智医疗器械有限公司 Méthode de détermination de la teneur en substance libre à l'aide d'une conversion ultrafiltration-dialyse à équilibre
KR102802882B1 (ko) * 2022-04-05 2025-05-08 한국과학기술연구원 혈액 내 유리 스테로이드 측정을 위한 희석용액 및 이를 이용한 스테로이드 측정방법
CN117147740A (zh) * 2023-10-31 2023-12-01 合肥歆智医疗器械有限公司 测定血中游离孕激素的超滤-质谱法及试剂盒

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