WO2016019037A1 - Free hormone and hormone metabolite workup and analysis by mass spectrometry - Google Patents

Abstract

Method for performing ultrafiltration on an unbound non-polar analyte in a sample and analysis of same is described. The sample can be a hormone, such as cholecalciferol (Vitamin D). The method comprises preinserting a solution comprising an organic solvent and an internal standard for the unbound analyte, into the filtrate recovery chamber prior to filtration.

Description

FREE HORMONE AND HORMONE METABOLITE WORKUP AND ANALYSIS BY MASS SPECTROMETRY

RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Application No.

62/030,459, filed on July 29, 2014 and US Provisional Application No. 62/139,435, filed on March 27, 2015, the entire contents of both of which are herein incorporated by reference.

GOVERNMENT INTEREST

[0002] This invention was made with U.S. Government support under grant numbers

1UL1RR031975-01, 1R01AG033867-01 and 5M01RR13297, awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

FIELD

[0003] 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.

BACKGROUND

[0004] 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.

[0005] 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.

[0006] An important class of hormone is the thyroid hormones. Examples of thyroid hormones are thyroxine (T4), free thryoxine (FT4), triiodothyronine (T3) and free

triiodothyronine (FT3). 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]. 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.

[0007] 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.

[0008] Vitamin D is an essential nutrient with important physiological roles in the positive regulation of calcium (Ca²⁺) 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 D₂ (ergocalciferol) and vitamin D₃ (cholecalciferol). Vitamin D₃ 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 D₃ must undergo metabolic activation to generate bioactive metabolites. In humans, the initial step of vitamin D₃ activation occurs primarily in the liver and involves hydroxylation to form the intermediate metabolite 25-hydroxyvitamin D₃ (25 -hydroxy cholecalciferol; calcifediol;

250HD₃). Calcifediol is the major form of vitamin D₃ in the circulation. Circulating 250HD₃ is then converted by the kidney to 1,25-dihydroxyvitamin D₃ (calcitriol; l,25(OH)₂D₃), which is generally believed to be the metabolite of vitamin D₃ with the highest biological activity.

[0009] Vitamin D₂ is derived from fungal and plant sources. Some over-the-counter dietary supplements contain ergocalciferol (vitamin D₂) rather than cholecalciferol (vitamin D₃). Drisdol, the only high-potency prescription form of vitamin D available in the United States, is formulated with ergocalciferol. Vitamin D₂ undergoes a similar pathway of metabolic activation in humans as vitamin D₃, forming the metabolites 25-hydroxyvitamin D₂ (250HD₂) and 1,25- dihydroxyvitamin D₂ (l,25(OH)₂D₂). Vitamin D₂ and vitamin D₃ 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 D₂ and vitamin D₃ 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 D₂ and vitamin D₃ and that methods described for the analysis of one type of vitamin D can be utilized on the other type with appropriate modification. For better clarity, as used herein, reference to metabolites of 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. In certain embodiments the

hydroxyvitamin D metabolite is hydroxylated at the 1 and 25 position. In certain embodiments the vitamin D metabolite is l ,25-dihydroxyvitamin D3 (la,25(OH)₂D₃) or la,25- dihydroxyvitamin D₂ (la,25(OH)₂D₂). In certain embodiments the dihydroxyvitamin D metabolites are naturally present in a body fluid of a mammal (such as a human). In certain embodiments the methods as described herein selectively detect l ,25 -dihydroxyvitamin D₃ (la,25(OH)₂D₃) and/or 1 a,25 -dihydroxyvitamin D₂ (la,25(OH)₂D₂).

[00010] Measurement of vitamin D, the inactive vitamin D precursor, is rare in clinical settings and has little diagnostic value. Rather, serum levels of 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ (total 25-hydroxyvitamin D; "250HD") are a useful index of vitamin D nutritional status and the efficacy of certain vitamin D analogs. Therefore, the measurement of 250HD is commonly used in the diagnosis and management of disorders of calcium metabolism. In this respect, low levels of 250HD are indicative of vitamin D deficiency associated with diseases such as hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, elevated alkaline phosphatase, osteomalacia in adults and rickets in children. In patients suspected of vitamin D intoxication, elevated levels of 250HD distinguishes this disorder from other disorders that cause hypercalcemia.

[00011] Measurement of l,25(OH)₂D is also used in clinical settings. For example certain disease states such as kidney failure can be diagnosed by reduced levels of circulating l,25(OH)₂D and elevated levels of l,25(OH)₂2D may be indicative of excess parathyroid hormone or may be indicative of certain diseases such as sarcoidosis or certain types of

lymphoma.

[00012] 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.

[00013] The accurate analysis and quantification of hormones is becoming more important. For example, estrogen and estrogen-like compounds are playing an ever-increasing role in today's society through hormone replacement therapy. Also, the analysis and

quantification of estrogen and estrogen-like compounds helps in the management of estrogenic) related diseases, like breast cancer. In addition, the accurate analysis and quantification of T4 and T3 is an issue recognized by those skilled in the art. The presence of circulating

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. However, technically, it is difficult to measure the free hormone concentrations since these are so low. It is easier to measure the total (free and protein-bound) thyroid hormone concentrations; total hormone concentrations are measured at nanomolar levels whereas free hormone concentrations are

20 measured in the picomole range and to be valid, must be free from interference by the much

higher total hormone concentrations.

[00014] In another example, 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

25 measurement of free vitamin D and total vitamin D measured in a subject sample (See FIG. 15) and that variability in a healthy patient population of free Vitamin D is less so than that of the total vitamin D which may provide an indicator of the relative importance in the use of the measuring of free vitamin D compared to the total as an indicator of possible disease states. Relatively low levels of free vitamin D in a subject when compared to a population of healthy

30 subjects can be an indicator of a vitamin D deficiency. A vitamin D deficiency can indicate an increased risk of a condition such as those referred above. Likewise, it is technically difficult to measure 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.

[00015] Presently, the common methods of hormone analysis use immunoassay techniques. Table 1 lists the common hormones and the current methods for their analysis.

[00016] For example, 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.

[00017] 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.

[00018] 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.

[00019] However, the current immunoassays are disadvantageous for the following reasons:

(1) Immunoassays are specific to one hormone, therefore every hormone must be analyzed separately.

(2) Numerous kits must be purchased and procedures must be learned for each hormone being analyzed.

(3) Various instruments to read the results from the immunoassays must be purchased.

[00020] For example, the analysis of estriol and progesterone from a sample requires both a gamma counter and a photon counter.

[00021] (4) The kits for the assays can be expensive.

[00022] (5) The current immunoassays lack specificity and may show approximately 15 fold difference in results using kits from different manufacturers [5].

[00023] (6) The procedures involve many steps and can take a significant amount of time.

[00024] (7) In the case of a radioimmunoassay, precautions are necessary because of the radioisotopes involved. [00025] Detection of vitamin D metabolites has been accomplished by radioimmunoassay with antibodies co-specific for 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2. Because the current immuno logically-based assays do not separately resolve 25-hydroxyvitamin D3 and 25- hydroxyvitamin D2, the source of a deficiency in vitamin D nutrition cannot be determined without resorting to other tests.

[00026] Immunoassays are notoriously unreliable with more and more literature published supporting their lack of specificity [6-13]. Table 2 shows the major differences reported by the College of American Pathologists program for proficiency testing of thyroid hormones that clearly illustrates the difference in specificity of the various antibodies used. For example, Table 2 shows mean results between different methods reported in the College of American

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].

[00027] More recently, hormones have been analyzed and quantified by mass

spectrometry. However, there are several disadvantages to these methods.

[00028] For example, 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. 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.

[00029] 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.

[00030] A method for analysis of 17-hydroxyprogesterone by HPLC electrospray ionization tandem mass spectrometry from dried blood spots has also been described [25].

However, this method analyses only one analyte at a time, and requires liquid-liquid extraction, which is laborious and time consuming, with sample extraction alone taking 50 minutes to complete.

[00031] A gas chromatography mass spectrometry method to analyze the production rates of testosterone and dihydrosterone has been disclosed [26].

[00032] More recently, reports have been published that disclose methods for detecting specific vitamin D metabolites using mass spectrometry. For example Yeung B. et al., J.

Chromatogr. 1993, 645(1): 115-23; Higashi T, et al, Steroids. 2000, 65(5):281-94; Higashi T, et al, Biol Pharm Bull. 2001, 24(7):738-43; and Higashi T, et al, J Pharm Biomed Anal. 2002, 29(5):947-55 disclose methods for detecting various vitamin D metabolites using liquid chromatography and mass spectrometry. These methods require that the metabolites be derivatized prior to detection by mass-spectrometry. Methods to detect underivatized l,25(OH)2D3 by liquid chromatography/mass-spectrometry are disclosed in Kissmeyer and Sonne, J Chromatogr A. 2001, 935(l-2):93-103. Detection of vitamin D metabolites by mass spectrometry is also discussed in U.S. Patent Application Publication No. 2009/0137056 to B. Holmquist et al.

[00033] Finally, there is no known method of analyzing free thyroxine (FT4) or free triiodothyronine (FT3) by mass spectrometry. Most laboratories perform FT4 testing routinely employing the analogue (direct) immunoassay approach on one of the major clinical chemistry platforms. This approach is not universally accepted and has been the subject of criticism (29). There are frequent occasions when the validity of the FT4 result generated in this manner is questioned. For this reason a "reflex" testing for all direct ΡΤ4'8<2.5^Λ percentile is often done to diagnose hypothyroidism. These are sent out for FT4 measurements employing the current gold standard of equilibrium dialysis. This is also done for samples when the direct FT4 is >97.5^Λ percentile and the TSH is normal. Approximately 50% of these FT4 send-outs have results within the normal range when measured by equilibrium dialysis and are therefore false positives by the direct FT4 method. However, the equilibrium dialysis procedures are time-consuming and expensive. Similarly, FT3 is also currently measured by immunoassay.

[00034] Similarly, attempts to measure free vitamin D from physiological samples, even using high sensitivity instruments such as mass spectrometry has been difficult due to the extremely low concentrations of free vitamin D that are being measured and the very non-polar and water insoluble nature of vitamin D and its metabolites.

[00035] Up until now, current methods of analyzing free vitamin D and related metabolites from physiological fluids have been inadequate.

TABLE 1: METHODS AND INSTRUMENTS FOR STEROID AND THYROID

HORMONES [1]

RIA: Radioimmunoassay

EIA: Enzyme Linked Immunoassay

FPIA: Fluorescence Polarization Immunoassay

TABLE 2: PROBLEMS WITH IMMUNOASSAYS: DATA ACQUIRED FROM CAP

PT PROGRAM 2003

Table 2: Problems with Immunoassays: Data acquired for samples from the CAP PT

Program 2003.

SUMMARY

[00036] 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.

[00037] 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.

[00038] The applicant's teaching permits the analysis of hormones in a number of complex matrices as they might be found in nature, e.g. the human body. For, example, hormone analysis can be performed on samples of blood, saliva, serum, plasma and urine.

[00039] There are several features to this teaching: (1) It provides a total and specific analysis for hormones in a sample. The present method allows for the analysis of many hormones simultaneously or sequentially. (2) The procedure does not require an

immunoprecipitation reaction. The majority of other methods for hormone analysis required an immunoassay. Immunoassays are expensive, specific to a particular analyte and involve several steps. (3) 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 μί. 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. (7) 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.

[00040] Accordingly, there is provided 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.

[00041] Accordingly, there is provided 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.

[00042] Accordingly, there is provided 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.

[00043] There is also provided a method of instructing an analysis of a sample that comprises or is suspected of comprising FT4 and/or FT3 hormone. The method comprises providing instructions to prepare and analyze the sample, as described above.

[00044] Accordingly, there is provided 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. [00045] Accordingly, there is provided 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.

[00046] Accordingly there is provided a kit 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.

[00047] Accordingly there is provided a kit 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.

[00048] There is also provided a kit 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.

[00049] Accordingly there is provided use of a mass spectrometer for analyzing a sample containing or suspected of containing FT4, FT3 or both.

[00050] Accordingly, there is provided 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.

[00051] In some embodiments, 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. In some embodiments, the internal standard is a deuterated version of the unbound analyte. In some embodiments, the analyte is vitamin D or a metabolite thereof. In some embodiments, the analyte is cholesterol. In some embodiments, the organic solvent is methanol.

[00052] In some embodiments, the method further comprises performing a quantitative analysis on the filtrate. In some embodiments, the quantitative analysis is performed by mass spectrometry. In some embodiments, the mass spectrometry is performed using MRM. In some embodiments, the analyte is 25 OH vitamin D3 and the MRM transitions monitored are about 383/229.

[00053] Accordingly, there is provided 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.

[00054] In some embodiments, the analyzing is performed using a mass spectrometry technique. In some embodiments, the mass spectrometry technique includes an MRM analysis. In some embodiments, the MRM analysis includes a quantitative analysis. In some

embodiments, the quantitative analysis determines the concentration of 25-hydroxyvitamin D3 and/or 25-hydroxyvitamin D2. In some embodiments, the organic solvent is methanol.

[00055] Accordingly, there is provided 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 an organic solvent and an internal standard for the free non-polar biological analyte or a metabolite thereof, into the filtrate recovery chamber prior to filtering.

[00056] In some embodiments, 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. In some embodiments, the analyzing is performed using a mass spectrometry technique. In some embodiments, the mass spectrometry technique includes an MRM analysis. In some embodiments, the MRM analysis includes a quantitative analysis. In some

embodiments, the organic solvent is methanol.

[00057] Accordingly, there is provided a kit 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. In some embodiments, the organic solvent can be methanol.

[00058] These and other features of the applicant's teachings are set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[00059] The skilled person in the art will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the applicant's teachings in any way.

[00060] The methods, including the best approaches known to the inventors, can be better understood with reference to the following description taken in combination with the following drawings, in which:

[00061] FIG. 1 is a mass spectrum of a blank plasma sample containing T4 and T3.

[00062] FIG. 2 is a mass spectrum of a globulin standard containing T4 and T3.

[00063] FIG. 3 is a typical tandem mass spectrometric chromatogram obtained for T4 and

T3 for a plasma sample. T4 m/z (776/127); D₂T4 m/z (778/127); T3 m/z (650/127)

[00064] FIG. 4 is a graph showing T3 measured by Isotope Dilution Tandem Mass

Spectrometry vs. Immunoassay. IA=075 MS+0.21; r=0.848; S_y,_x=0.1956; n=49. [00065] FIG. 5 is a graph showing T4 measured by Isotope Dilution Tandem Mass

Spectrometry vs. Immunoassay. IA=1.13 MS-8.99; r=0.931; S_y,_x=9.54; n=50

[00066] FIG. 6 is a graph showing a typical chromatogram for free T4 (11.2 pg/mL) and deuterated internal standard.

[00067] FIG. 7 is a graph showing the effect of temperature on FT4 by tandem mass spectrometry and ultrafiltration.

[00068] FIG. 8 is graph showing the comparison of the tandem mass spectrometric method with the equilibrium dialysis method for the measurement of free T4.

[00069] 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.

[00070] FIGS. 10a, b, and c are a series of mass spectrums showing the analysis of FT4

(a), FT3 (b), and FT4-d2 (c) using an API 5000™.

FIG. 11 is a depiction of the components of an ultrafiltration tube to be used in a centrifuge device for sample concentration.

[00071] 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.

[00072] FIG. 13 depicts a chart showing the relationship between concentrations of parathyroid hormones and total 25 hydroxyvitamin D3

[00073] FIG. 14 depicts a chart showing the relationship between concentrations of parathyroid hormones and free 25 hydroxyvitamin D3

[00074] FIG. 15 depicts a chart showing the relationship between the concentrations of free 25 -hydroxyvitamin D3 and total 25 -hydroxyvitamin D3

[00075] FIG. 16 depicts a calibration curve generated for free 25 -hydroxy vitamin D3 using the within teachings.

[00076] FIG. 17 depicts the stability profile of free vitamin D at various temperatures over time.

[00077] FIG. 18 depicts chromatograms for the analysis of Free 25-OH Vitamin D3 and deuterated Free 25-OH Vitamin D3-d₆ internal standard.

[00078] FIG. 19 depicts chromatograms for the analysis of Free 25-OH Vitamin D3 and deuterated Free 25-OH Vitamin D3-d₆ internal standard.

[00079] FIG. 20 depicts measured PTH concentrations for African-Americans vs. Others. [00080] FIG. 21 depicts a chromatogram obtained from a 10 pg/mL standard obtained when using the within teachings

[00081] FIG. 22 depicts a chromatogram obtained from a patient sample when using the within teachings.

DESCRIPTION OF VARIOUS EMBODIMENTS

[00082] The applicant's teaching provides methods of analysis for hormones. The hormones may include:

[00083] Dehydroepiandrosterone (DHEA)

Dehydroepiandrosterone sulphate (DHEAS)

Aldosterone

Cortisol

Corticosterone

11-Deoxycortisol

Androstenedione

Testosterone

Estradiol

17-OH Progesterone

Progesterone

Allopregnanolone

160H Estrone

2-OH Estrone

Estrone

Estriol

Vitamin D, and its metabolites 25hydroxyvitamin D and 1,25 dihydroxyvitamm D

Free vitamin D and its metabolites, free 25hydroxyvitamin D and free 1,25 dihydroxyvitamm D thyroxine

free thyroxine

triiodothyronine

free triiodothyronine

catecholamines

metanephrines other steroid hormones

other thyroid hormones

other small peptide hormones

other amines

[00084] While this list specifically recites the analysis of free thyroxine and free vitamin

D, amongst others, it should be appreciated that 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. In particular, the biological analytes that may be metabolites of or precursors to the hormones specified above. For example, 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.

Sample

[00085] 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 3000™, or 200 to 500 μΐ, for steroid hormones when using the API 4000™ or API 5000™, can be used. A sample size of 500 to 600 μΙ_, for FT4 and FT3 can be used when using the API 4000™ or API 5000™. A sample size of 900 μί can be used for vitamin D or metabolites thereof.

Deproteinization

[00086] 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. Separation of Hormones from the Sample

[00087] The hormones are separated by methods known to those skilled in the art. For example, the hormones may be separated by liquid chromatography through a column. Many different columns can be used. For example, 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. As is known to those skilled in the art, the shorter the carbon chain, the shorter the retention time. The hormones are subsequently eluted from the column.

[00088] The hormones may also be separated by centrifugation. For example, 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. Alternatively, the hormones may be separated by equilibrium dialysis or other methods known to those skilled in the art.

[00089] When using ultrafiltration, for non-polar hormones such as for example vitamin D or metabolites of vitamin D, 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. This method is most useful when performing ultrafiltration by the use of ultrafiltration tubes used in centrifuge devices such as for example, Vivaspin tubes from Sartorius. In such cases, 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. Preferably, 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. However, other types of internal standards may also be used. In this manner, 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. In some embodiments, 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. Particularly preferred, 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. Optionally 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. Generally, 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. In operation as depicted in FIG 12 A, 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. 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. As a result, after the filter force is removed from tube 100, for example, by removal from a centrifuge, as depicted in FIG. 12B, 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.

Introduction of Hormones into a Mass Spectrometer

[00091] The hormones are then introduced into a mass spectrometer. Optionally, 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.

Isotope Dilution Tandem Mass Spectrometry

[00092] The methods employ isotope dilution mass spectrometry.

Instrumentation and Ionization Techniques

[00093] 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.

[00094] 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 3000™ mass spectrometer and the API 4000™ mass spectrometer, described in for example U.S. Pat. Nos. 4,121,099; 4,137,750; 4,328,420;

4,963,736; 5,179,278; 5,248,875; 5,412,208; and 5,847,386 (SCIEX, Foster City, Calif./Concord Ontario, Canada). When analyzing thyroid hormones, a spectrometer with a turbo spray ion source, such as the API 2000™ and API 3000™ mass spectrometers can be used. When analyzing FT4, the API 4000™ mass spectrometer can be used. When analyzing FT3, the API 5000™ mass spectrometer can be used. When analyzing FT3 and FT4 simultaneously the API 5000™ mass spectrometer can be used. When analyzing free vitamin D the SCIEX Triple Quad 6500 mass spectrometer system can be used. [00095] 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. Typically, for thyroid hormones, the spectrometer is employed in the negative mode.

[00096] 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.

[00097] 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

[00098] 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.

[00099] The 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. EXAMPLES

[000100] Aspects of the applicant's teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.

[000101] Analysis of a Sample for Thyroid Hormones

[000102] A sample of 100 μΙ_, of plasma was used. Proteins were precipitated with 150 μΙ_, of acetonitrile, capped and vortexed. The sample was then centrifuged, and 200 μΙ_, of the supernatant was injected onto a Supelco LC-18-DB™ 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.

[000103] The eluant was introduced into an ion- spray ionization chamber and analyzed by API 2000™ 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.

[000104] This demonstrates a simple method of preparing a complex biological matrix for analysis of hormone content, and a sensitive analytical method that permits the simultaneous analysis of two hormones, T3 and T4.

[000105] 2. Analysis of Thyroid Hormones using a Methanol Gradient to Elute the

Hormones

[000106] A sample of 100 μΙ_, of plasma was used. Proteins were precipitated with 150 μΙ_, of acetonitrile, containing an internal standard of deuterated T4 and vortexed. The sample was centrifuged, and 200 μΙ_, 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.

[000107] A sample of the eluant was introduced into an ion-spray ionization chamber and analyzed by an API 3000™ mass spectrometer using the negative mode. The ionization may be by electrospray using a turboionspray chamber. FIG. 1 and FIG. 2 shows the mass spectrums generated for T3 and T4.

[000108] This demonstrates a simple method of preparing a complex biological matrix for analysis of thyroid hormone content, and a sensitive analytical method that permits the simultaneous analysis of multiple hormones.

[000109] 3. Analysis of Thyroid Hormones using Isotope Dilution Tandem Mass

Spectrometry [000110] 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.

Chemicals and Reagents

[000111] Standards of T4 and T3 were purchased from Sigma (St. Louis, Mo., USA). A stable deuterium-labeled internal standard, L-thyroxin-d2 was synthesized according to procedures described in the literature [16], [17] 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 purchased from Sigma.

Solutions and Standards

[000112] Stock solutions of T3, T4 and internal standard (IS) were prepared separately to obtain a concentration of 1 mg/mL for each. 40% ammonium hydroxide (v/v) in methanol was used as a solvent. The analyte stock solutions were diluted with methanol to obtain the spiking solutions. The solutions were stored at 4°C. and could be used for several months. Standards for the calibration curve in the range of 0.325 to 5 ng/mL for T3 and 12.5 to 200 ng/mL for T4 were prepared by adding the analyses to 3% human γ-globulin (volume of spiking solution<2% of fmal volume). Quality control (QC) samples (Diagnostic Product Corp., Los Angeles, USA) at low, medium and high levels were used. A solution of 50-ng/mL d₂-T4 in methanol was used as the internal standard.

Sample Preparation

[000113] 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 μΐ of supernatant was injected into the LC-MS-MS system. LC/MS/MS Conditions

[000114] An API 3000™ tandem mass-spectrometer (SCIEX, Toronto, Canada) equipped with TurbolonSpray and Shimadzu HPLC system was used to perform the analysis. Negative ion multiple reaction-monitoring (MRM) mode was used. The transitions to monitor were selected at m/z 650^ 127 for T3, m/z 776^ 127 for T4, m/z 778^ 127 for d₂-T4. Nitrogen served as auxiliary, curtain and collision gas. Gas flow rates, source temperature, Ion Spray voltages and collision energies were optimized for every compound by infusion of 1 μg/mL of the standard solutions in methanol at 20 μί/ηιίη and by flow-injection analysis (FIA) at LC flow rate. The main working parameters for the mass spectrometer are summarized in Table 3. Data processing was performed on Analyst 1.2 software package.

LC-MS-MS Procedure

[000115] 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. 100 μΐ of the sample was injected onto a Supelco LC-18-DB (3.3 cm x 3.0 mm, 3.0 μιη ID) chromatographic column equipped with a Supelco Discovery C-18 (3.0 mm) Guard column, where it underwent cleaning with 20% (v/v) methanol in 5 mM ammonium acetate pH=4.0 at flow rate 0.8 mL/minute. After 3.5 minutes of cleaning 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.

[000116] Immunoassays for T4 and T3

[000117] T4 was measured by the Dade RxL Dimension™ (Dade-Behring Diagnostics, Glasgow, Del.) and T3 by the DPC Immulite™ (Diagnostic Product Corporation, Los Angeles, Calif.) according to the manufacturer's specifications.

Results

[000118] The mass spectrometer working parameters used are shown in Tables 3 and 4.

[000119] Replicate sera were assayed both within-day and between-day at several concentrations. The within-day and between-day precision data is provided in Tables 5 and 6.

[000120] Recovery studies for T4 and T3 are shown in Tables 7 and 8. All results shown are the means of 8 replicates. [000121] FIG. 3 shows a typical tandem mass spectrometric chromatogram obtained for T3 and T4 (T4 m/z (776/127); D₂T4 m/z (778/127); T3 m/z (650/127)).

[000122] Specimens were tested for T3 and T4 by both immunoassay (T3 DPC Immulite, T4 Dade Behring Dimension™ RxL) and by tandem mass spectrometry. Linear regression correlations (Prism) are shown in FIGS. 4 and 5.

[000123] 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.

Discussion

[000124] Evidence initially gleaned from both the CAP PT Program and pediatric reference ranges employing different immunoassays indicated the probability of lack of specificity for T4 and T3 immunoassay tests. To adequately assess this phenomenon, the isotope dilution tandem mass spectrometric method was developed as described in this example. Serum T4 and T3 detection methods have evolved through a variety of technologies since the 1950s.

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]. 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]. The displacement of hormone binding from serum proteins by such agents, together with the large sample dilution employed in modern assays, facilitates the binding of hormone to the antibody reagent.

[000125] Since T3 is ten-fold lower in concentration compared with T4 in blood it therefore presents both a technical sensitivity and precision challenge despite the use of a higher specimen volume. Although a reliable high-range T3 measurement is critical for diagnosing

hyperthyroidism, a reliable normal-range measurement is also important for adjusting antithyroid drug dosage and detecting hyperthyroidism in sick hospitalized patients, in whom a

paradoxically normal T3 value may indicate hyperthyroidism.

[000126] The correlation coefficient for the T4 comparisons (0.931) is significantly better than for the T3 comparisons (0.848) (FIGS. 4 and 5). T3 by tandem mass spectrometry gave slightly higher results than those obtained by the DPC Immulite (FIG. 4). While this is true for children, preliminary data for non-pregnant and pregnant women indicates a very poor correlation for T3 in both groups (r between 0.407-0.574) (i.e. there is a poor correlation between DPC Immulite and the method of the present teaching in both non-pregnant and pregnant women).

[000127] The reasons for this are not clear but could include standardization issues, heterophilic antibodies, etc. Of importance, reverse T3, which lacks a daughter ion of 127 m/z, does not interfere in the tandem mass spectrometry methods. Applying the tandem mass spectrometric method to CAP PT samples in the K/K (thyroid) general ligand program again revealed that around 85% of the immunoassay methods for T3 gave means on samples which were lower than the means obtained by the tandem mass spectrometry methods of this applicant's teaching while 15% had higher means. For T4, the tandem mass spectrometry method resulted in lower means than those of the immunoassay methods.

[000128] In conclusion, correlations between immunoassays and tandem mass spectrometry for T4 and T3 have been demonstrated. The correlation is better for T4 than for T3. Further, the correlation is less impressive during pregnancy. Recovery studies from several different sera using deuterated T4 as internal standard showed consistent (90-109%>) recoveries for both T4 and T3 (Tables 7 and 8). The recovery differences found between samples were surprisingly larger for T4 than for T3. This indicates a lack of need to use deuterated T3 as the T3 internal standard. 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.

TABLE 3: TANDEM MASS-SPECTROMETER WORKING

PARAMETERS

TABLE 4: GRADIENT PARAMETERS

TABLE 5: WITHIN DAY PRECISION (N=10)

T4 24.1 0.437 1.81 81.2 1.502 1.85 TABLE 6: BETWEEN DAY PRECISION (N=20, 1 RUN PER DAY FOR 20 DAYS)

TABLE 7: RECOVERY OF ADDED THYROXINE (T4)

*NA - not applicable TABLE 8: RECOVERY OF ADDED TRIIODOTHYRONINE (T3)

*NA - not applicable

4. Analysis of Free Thyroxine (FT4)

[000129] Most routine clinical chemistry service laboratories provide for the measurement of free thyroxine (FT4) by an analogue (direct) method with 24 hours and 7 day per week availability. Nevertheless, the validity of analogue FT4 immunoassays has long been questioned and patient's results using this approach frequently do not fit in with the clinical picture. Because of this, direct free T4's that are below the 2.5^th percentile and many that are above the 97.5^th percentile are often sent for further measurement by the current "gold standard" method for FT4, equilibrium dialysis. In approximately 50% of these cases the FT4 by equilibrium dialysis has been found to be normal. 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. Methods:

Chemicals and Reagents

[000130] 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.

Solutions and Standards

[000131] Stock solutions of T4 and internal standard (IS) 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 d₂-T4 in methanol was used as internal standard.

Sample Preparation

[000132] 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. This 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. LC/MS/MS Setup

[000133] An API 4000™ tandem mass-spectrometer (SCIEX, Toronto, Canada) equipped with TurboIonSpray and Agilent 1100 HPLC system was used to perform the analysis. Negative ion multiple reaction-monitoring (MRM) mode was used. The transitions to monitor were selected and are m/z 775.9 -> 126.9 for T4, m/z 777.9-M26.9 for d₂-T4. Nitrogen served as auxiliary, curtain and collision gas. Gas flow rates, source t°, Ion Spray voltages and collision energies were optimized for every compound by infusion of 1 μg/mL standards solutions in methanol at 20 μί/ηιίη and by flow-injection analysis (FIA) at LC flow rate. The main working parameters of mass spectrometer used are summarized in Table 9. Data processing was performed on Analyst 1.4.1 software package. Although the negative mode was used in this example, a positive mode can be used but is less sensitive. LC-MS-MS Procedure

[000134] 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. 400 μΙ_, of the sample was injected onto the Supelco LC-18-DB (3.3 mm x 3.0 mm, 3.0 μιη ED) chromatographic column equipped with a Supelco Discovery C-18 (3.0 mm) guard column, where it underwent cleaning with 20% (v/v) methanol in 5 mM ammonium acetate pH 4.0 at flow rate 0.8 mL/min. After 4 minutes of cleaning 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.

Equilibrium Dialysis

[000135] The Nichols free T4 kit (Nichols Institute Diagnostics, Catalogue # 30-0652, San Clemente, Calif.) was used according to the directions provided by the manufacturer. A comparison between the equilibrium dialysis and the tandem mass spectrometric method were performed on patient samples (n=68).

Analogue/Direct Free T4

[000136] The Dade RxL Dimension was used for the direct free T4 method. (Dade-Behring Diagnostics, Glasgow, Del). Results on patient samples were compared with values obtained using tandem mass spectrometry (n=-154). Between-Day and Within-Day Precision

[000137] The between-day and within-day precision was assessed at 3 different

concentrations (Table 12). Results and Discussion

[000138] 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

FT4 is temperature dependent (31). If the centrifugation of the Amicon Centrifree tubes occurs at 25°C. (see FIG. 7 and Table 11) the results obtained by the tandem mass spectrometric method closely correlate with those obtained by equilibrium dialysis, which employs a temperature of 37°C. This 12°C. temperature difference is probably the result of different membranes being employed in the equilibrium dialysis and ultrafiltration methods. The correlation between the new isotope dilution tandem mass spectrometric method and the conventional gold standard equilibrium dialysis method was excellent. Equilibrium dialysis=0.971 Mass

Spectrometry+0.041, n=68, Syx=1.381, r=0.954 (FIG. 8). In contrast a poor correlation was found with the analogue (direct) FT4 method (Immunoassay=0.326 Mass Spectrometry+6.27, n=154, Syx=l .96, r=0.459, FIG. 9). The between-day and within-day precision shows all concentrations tested gave coefficient of variations (Cvs) of less than 7.1% (Table 12). This performance is superior to that obtained using the difficult equilibrium dialysis method. The lower limit of detection (a reading greater than three standard deviations over the baseline noise) is 2.5 pg/mL .

[000139] These studies confirm that the analogue procedures give poor results for free T4 which is further supported when reflex testing for all FT4s below the 2.5^th percentile and all FT4s above the 97.5^th percentile which also have normal thyroid-stimulating hormone (TSH) values is done. Approximately, 50% of these free T4s run on either the Dade RxL Dimension or the DPC Immulite™ give normal results when run by equilibrium dialysis. Finally in the present study, 80% of FT4s greater than the 96.7^th percentile by tandem MS are associated with TSHs of less than 1.0 uIU/mL (the latter measured by the Dade RxL Dimension™) while in the same cohort of patients, only 40% of FT4s greater than the 96.7^th percentile measured by direct IA had TSHs of less than 1.0 uIU/mL.

[000140] It should also be noted that prior to using tandem mass spectrometry on the plasma ultrafiltrate, attempts were made to measure FT4 on the ultrafiltrate by IA using several approaches which included an RIA kit (Nichols), the Dade RxL™ and DPC IMMULITE™ platforms. In all cases results were exceedingly low indicating that this was not a viable alternative.

[000141] In conclusion, a new isotope dilution tandem mass spectrometric method for the measurement of FT4 employing ultrafiltration has been developed. The procedure has excellent precision, compares well with the gold standard. Based on these attractive characteristics this method of FT4 measurement will have a wide applicability in the clinical setting.

TABLE 9. TANDEM MASS-SPECTROMETER WORKING PARAMETERS

TABLE 10. GRADIENT PARAMETERS

TABLE 11: EFFECT OF TEMPERATURE ON FREE T4 AND ON FT4/TT4

RATIOS FREE T4 (PG/ML)

Dial - samples running on MS after dialysis

Free T4/ Total T4*

* Free T4 (pg/mL), Total T4 (ng/mL)

TABLE 12: WITHIN-DAY AND BETWEEN-DAY PRECISION

[000138] This demonstrates a simple method for preparing and detecting FT4 by mass spectrometry.

[000139] 5. Analysis of Thyroid Hormones and Steroid Hormones

[000140] A sample of 500 to 1000 of plasma is used. Proteins are precipitated with 150 μΐ_^ of acetonitrile and vortexed. The sample is centrifuged, and 200 μΐ_^ 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.

[000141] A sample of the eluant is introduced into an ion- spray ionization chamber and analyzed by API 3000™ 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.

[000142] This demonstrates a simple method of preparing a complex biological matrix for analysis of possible steroid and thyroid hormone content. Steroid hormones which are run in the negative mode can be run simultaneously with the thyroid hormones.

[000143] The results indicate that this technique, allows for the identification and characterization of low levels of thyroid hormone in human plasma and saliva.

[000144] 6. Analysis of FT3 Hormone

[000145] 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 5000™ mass spectrometer.

[000146] 7. Simultaneous Analysis of FT4 and FT3

[000147] Patients with either hyperthyroidism or hypothyroidism require frequent assessment of thyroid function through measurement of their FT4 and FT3 concentrations.

Further, people with thyroid ablation require thyroid replacement therapy, such as synthroid. Measurement of their FT4 and FT3 concentrations is important when assessing their dosage regimen. Accordingly, an efficient assay method for the simultaneous analysis of FT3 and FT4 is beneficial.

[000148] FT4 and FT3 were analyzed simultaneously by a similar method of Example 4 except using the API 5000™ mass spectrometer. ΙΟΟμί 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.

[000149] 8. Analysis of free Vitamin D

[000150] A SCIEX Triple Quad 6500 tandem mass spectrometer with operating parameters described in Table 13, equipped with an Atmospheric Pressure Chemical Ionization (APCI) source and Shimadzu High-Performance Liquid Chromatography (HPLC) system was employed. MRM's were analyzed in positive mode. Blood expecting to contain one or more of free vitamin D2, D3 or metabolites thereof was collected in red top tubes without serum separator and 0.5 mL of human serum/plasma was placed in the top of a 10 KDa ultrafiltration device (VIVASPIN 2 Hydrosart, Sartorius Stedim) with 0.6 mL methanol containing deuterated internal standard placed in the bottom section of ultrafiltration device and centrifuged in an Eppendorf

temperature controlled centrifuge with a fixed angle rotor at 2200 g and a temperature of 37 °C for 8.5min. The tube was capped, vortex mixed briefly for 15 seconds, and centrifuged for 3 min at 13,000 rpm and 4C. After centrifugation, 300 of supernatant was injected onto a Poroshell 120 SB-C8 2.7 μιη column (4.6 x 5mm). Mass spectrometer, source working parameters, and binary gradient are listed in Tables 13 and 14. Preliminary studies suggest that the reference intervals are in the range of 1.5-15.5 pg/mL. While 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.

[000151] To determine a quantitative amount of free vitamin D3 in the blood sample, a calibration curve was created by utilizing 400 of calibration standards in 60% methanol. To each of these were added an internal standard of 400 μΐ_^ of a deuterated IS of vitamin D in 100% methanol and a further 400 of 41% methanol. Calibration preparation is discussed below.

[000152] MRM analysis of the patient sample after it has been processed by the method was performed by focusing on the transitions 383/229 for free vitamin D3 and 389/211 for the internal standard. Other transitions include those shown in Table 15 TABLE 13: TANDEM MASS-SPECTROMETER WORKING PARAMETERS

Parameters Value

Curtain Gas N₂ (CUR) 35

Nubilzer current 3

Source Temp. (°C) 350

Gas 1 N₂ ion source(GSl) 60

Gas 2 N₂ ion source (GS2) 60

Collision Gas N₂(CAD) 7

Declustering Potential 70

Entrance Potential (EP) 10 V

Collision Cell Exit Potential(CXP) 19 V

Dwell time 100 msec

Column Temp. (°C) 40

TABLE 14: HPLC WORKING PARAMETERS

Gradient Timetable Time (min) Solvent A (%) Solvent B (%)

Cleaning (0.70 mL/min) 0.00 80 20

Elution (0.70 mL/min) 2.00 80 20

4.50 7 93

8.0 2 98

8.5 0 100

8.6 80 20

10.0 80 20

Solvent A: 2% methanol containing 0.1% formic acid

Solvent B: 98% methanol containing 0.1% formic acid.

Flow rate for Table 14 is 0.7 mL/min TABLE 15: MRM MASS SPECTROMETER PARAMETERS

MRM Collision Energy Retention Time

Analyte Transition (V) (min)

25 OH Vitamin D3 - Qualitative 383.3/211.2 34 7.50

25 OH Vitamin D3 - Quantitative 383.3/229.2 25 7.50

25 OH Vitamin D3 - d6 389.3/211.2 34 7.49

Additional FREE Vitamin D3 Analysis

[000153] All solvents (methanol, water and formic acid) are Optima LC/MS grade.

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.

TABLE 16

Sample Preparations

[000154] 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. [000155] For each ultrafiltration, 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 μΐ, 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

(Eppendorf, model #5702R, AG, Hamburg). The samples were spun for 8.5 minutes at 37°C at 2200 g.

[000156] 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 μί were additional spun in 90 second intervals at the same conditions until each ultrafiltration device had indicated that at least 300 μί 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)). The stored sample can be vortexed and centrifuged at 4°C for 3 minutes prior to analysis using LC-MS/MS. [000157] Sample Analysis

[000158] Samples were analyzed by LC-MS/MS in a SCIEX Triple Quad 6500 Tandem Mass Spectrometer equipped with an Atmospheric Pressure Chemical Ionization (APCI) Source and Shimadzu Nexera XR LC20AD HPLC system.

[000159] 300 μΐ_^ of each aliquot from the ultrafiltration was injected into an Agilent Poroshell 120 SB-C8 column (4.6 x 50mm, 2.7 μιη) where both 25-OH Vitamin D₃ and internal standard undergo an on-line extraction, gradient chromatographic separation and elution.

Retention Time of 8.0 min. Mobile phase A: 2% (v/v) methanol-water solution 0.1% formic acid added and B: 98%> (v/v) methanol-water solution with 0.1%> formic acid added. Needle Wash 90/10 H₂0/ACN, 0.1% Formic Acid. Time(min B%

0.0 20

2.0 20

4.5 93

8.0 98

8.5 100

8.6 20

10.0 20

TABLE 17: ELUTION TIME IS AROUND 8.0 MIN AT FLOW RATE OF 0.7 ML/MIN

[000160] Quantitation was performed using Multiple Reaction Monitoring (MRM) analysis in positive mode using transitions of mass-to-charge (m/z) 383.3^229.2 for 25-OH Vitamin D3 and 389.3-»211.2 for 25-OH Vitamin D3-d6. MRM was also performed at 389.3^229.1 and 389.3->211.1 for 25-OH Vitamin D3 and 25-OH Vitamin D3-d6, respectively. Examples of Chromatograms are shown in FIGS. 18 and 19. Nitrogen served as curtain and collision gas. The main working parameters of the mass spectrometer were: Collision gas 7, curtain gas 35, auxiliary gas (GSl) 60, nebulizer current 3, probe temperature 350°C, entrance potential 10V, and dwell time 50 msec.

Results

[000161] The between-day coefficients of variation (CVs) were below 10% for free 25-OH Vitamin D3 at all concentrations tested (Table 18).

TABLE 18 [000162] Accuracy ranged between 90% and 110%. Good linearity was also obtained within the concentration range of 1-25 pg/mL for free 25-OH Vitamin D3 (r > 0.995) as depicted in FIG. 16. The range of results from 34 healthy volunteers was 1.5 to 17.9 pg/mL. This cohort with supplement of 8 patients with elevated parathyroid hormone (PTH). Plots of PTH versus free and total 25-OH Vitamin D3 (FIGS. 13 and 14) showed good correlation for the free and poor correlation for the total. Finally, plotting free versus total showed poor correlation (FIG. 15).

[000163] A poor correlation was observed between total 25-OH Vitamin D3 and PTH. The free concentration of 25-OH Vitamin D3 may be a possible indicator for both bone diseases and/or patients with a variety of cancers.

Measured Concentrations

[000164] Mean Normal African American = 7.6 pg/mL, n=17

[000165] Mean Normal, remaining subjects = 6.2 pg/mL, n=37

[000166] S/N = 14 at 2.5 pg/mL

Statistics

TABLE 19

[000167] PTH concentrations were also determined for African- American and Non- African-Americans and mean concentrations were determined to be 46.4 pg/mL,n=17 and 37.1 pg/mL, n= 29, respectively (FIG. 20).

Other Embodiments

[000168] In other embodiments, when operating the HPLC, solvent "B" can be 100% methanol or a mixture of 90 to 95% methanol with the remaining mixture comprising ethanol. [000169] As an example, 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.

[000170]

TABLE 20

[000171] Analysis was performed on various standards and the following data points obtained which result in a substantially straight calibration line utilizing 100% methanol.

TABLE 21

[000172] 25-OH Vitamin D3 Chromatograms obtained for an analysis of a 10 pg/mL standard and a patient sample found to contain 5.2 pg/mL are depicted in Figs. 21 and 22, respectively.

[000173] While the applicant's teachings are described in conjunction with various embodiments, it is not intended that the applicant's teachings be limited to such embodiments. On the contrary, the applicant's teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

REFERENCES

[000174] All references listed herein are incorporated by reference in their entirety.

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Claims

1. 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 ,

inserting a sample containing said unbound analyte into said filtration chamber, 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.

2. The method of claim 1 wherein said filter force is centrifugation.

3. The method of claim 1 wherein the internal standard is a deuterated version of the unbound analyte.

4. The method of claim 1 wherein the analyte is vitamin D or a metabolite thereof.

5. The method of claim 1 wherein the analyte is cholesterol.

6. The method of claim 1 further comprising performing a quantitative analysis on the filtrate.

7. The method of claim 6 wherein the quantitative analysis is performed by mass spectrometry.

8. The method of claim 7 wherein the mass spectrometry is performed using MRM.

9. The method of claim 8 wherein the analyte is 25 OH vitamin D3 and the MRM transitions monitored are about 383/229. 10. The method of claim 1 wherein the organic solvent is methanol.

11. A method for analyzing 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 performing an ultrafiltration of said sample, said ultrafiltration being performed by utilizing an ultrafiltration device comprising a filtration chamber for containing a 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 prior to performing said ultrafiltration, a solution comprising an organic solvent and an internal standard for the free vitamin D or metabolites thereof, is inserted into the filtrate recovery chamber.

12. The method of claim 11 wherein said analyzing is performed using a mass spectrometry technique. 13. The method of claim 12 wherein said mass spectrometry technique includes an MRM analysis.

14. The method of claim 13 wherein said MRM analysis includes a quantitative analysis. 15. The method of claim 14 wherein said quantitative analysis determines the concentration of 25-hydroxyvitamin D3 and/or 25-hydroxyvitamin D2. The method of claim 11 wherein the organic solvent is methanol.

17. A kit 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.