WO2011068654A1

WO2011068654A1 - Vitamin d detection by mass spectrometry with laser diode thermal desorption

Info

Publication number: WO2011068654A1
Application number: PCT/US2010/056461
Authority: WO
Inventors: Nigel J. Clarke
Original assignee: Quest Diagnostics Investments LLC
Current assignee: Quest Diagnostics Investments LLC
Priority date: 2009-12-03
Filing date: 2010-11-12
Publication date: 2011-06-09
Anticipated expiration: 2012-06-03

Abstract

Methods are provided for determining the amount of one or more vitamin D metabolites in a sample using mass spectrometry. The methods generally comprise volatilizing a sample by heating with a laser, ionizing one or more vitamin D metabolites in the volatilized sample, and detecting the amount of one or more ions by mass spectrometry. The methods may be used to detect two or more vitamin D metabolites in a single assay.

Description

VITAMIN D DETECTION BY MASS SPECTROMETRY WITH LASER DIODE

THERMAL DESORPTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial Number 61/266,488 (filed 12/03/2009), which is incorporated herein by reference in its entirety including all figures and tables.

FIELD OF THE INVENTION

[0002] The invention relates to the detection of vitamin D metabolites. In a particular aspect, the invention relates to mass spectrometry methods for detecting vitamin D metabolites ionized by a laser diode thermal desorption ionizer.

BACKGROUND OF THE INVENTION

[0003] Vitamin D is an essential nutrient with important physiological roles in the positive

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regulation of calcium (Ca ) 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 supplemented 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 the 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-hydroxycholecalciferol (calcifediol; 250HD₃). Calcifediol is the major form of Vitamin D₃ in the circulation. Circulating 250HD₃ is then converted by the kidney to form 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.

[0004] Vitamin D₂ is derived from fungal and plant sources. Many 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 250HD₂ and 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 (Armas et. al., (2004) J. Clin. Endocrinol. Metab. 89:5387-5391).

[0005] Serum levels of 25 -hydro xyvitamin D3, 25-hydroxyvitamin D₂, and total 25- hydroxyvitamin D are useful indexes of vitamin D nutritional status and the efficacy of certain vitamin D analogs. 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.

[0006] Measurement of l,25(OH)₂D is also used in clinical settings, however, this test has a more limited diagnostic usefulness than measurements of 250HD. Factors that contribute to limitations of the diagnostic values of l,25(OH)₂D as an index of Vitamin D status include the precision of the endogenous regulation of renal production of the metabolite and its short half- life in circulation. However, certain disease states can be reflected by circulating levels of l,25(OH)₂D, for example kidney disease and kidney failure often result in low levels of l,25(OH)₂D. Elevated levels of l,25(OH)₂D may be indicative of excess parathyroid hormone or can be indicative of certain diseases such as sarcoidosis or certain types of lymphomas.

[0007] Detection of vitamin D metabolites has been accomplished by radioimmunoassay with antibodies co-specific for 250HD₂ and 25OHD₃. Because the current immuno logically- based assays do not separately resolve 250HD₂ and 250Ηϋ₃, the source of any deficiency nutritional of vitamin D cannot be determined without resorting to other tests. More recently, reports have been published that disclose methods for detecting specific vitamin D metabolites using mass spectrometry. In some of the reports, the vitamin D metabolites are derivatized prior to mass spectrometry, but in others, they are not. 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; Higashi T, et al, J Pharm Biomed Anal. 2002, 29(5):947-55; Higashi T, et al., Anal. Biochanal Chem, 2008, 391 :229-38; and Aronov, et al., Anal Bioanal Chem, 2008, 391 : 1917-30 disclose methods for detecting various vitamin D metabolites by derivatizing the metabolites prior to 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; Clarke, et al, in U.S. Patent Application Serial Nos. 11/101,166, filed April 6, 2005, and 11/386,215, filed March 21, 2006, and Singh, et al, in U.S. Patent Application Serial No. 10/977,121, filed October 24, 2004.

SUMMARY OF THE INVENTION

[0008] In one aspect of the present invention, the amount of one or more vitamin D metabolites in a sample is determined by mass spectrometry methods which comprise volatizing a sample by heating the sample with a laser. In these methods, one or more vitamin D metabolites in the volatized sample are ionized to produce one or more ions detectable by mass spectrometry; and detecting the amount of one or more of the ions by mass spectrometry. The amount of the detected ion or ions is related to the amount of one or more vitamin D metabolites in the sample. In some embodiments, the sample is not subjected to chromatography, including liquid chromatography (such as HPLC or TFLC), prior to mass spectrometry. In alternative embodiments, the sample may be subjected to chromatography, including liquid

chromatography, prior to mass spectrometry. In some embodiments, the sample comprises a biological sample. In related embodiments, the biological sample comprises plasma or serum. In some embodiments, the sample is dried prior to heating with a laser. In some embodiments, the one or more ions detectable by mass spectrometry are ionized with atmospheric pressure chemical ionization (APCI). In some embodiments, the mass spectrometry is tandem mass spectrometry.

[0009] In some embodiments, the one or more vitamin D metabolites comprises 25- hydroxyvitamin D₃. In some related embodiments, the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass- to-charge ratio of 383.2 ± 0.5, 257.1 ± 0.5, and 211.1 ± 0.5. In further related embodiments, the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to- charge ratio of 383.2 ± 0.5 and one or more fragment ions selected from the group consisting of ions with a mass-to-charge ratio of 257.1 ± 0.5 and 211.1 ± 0.5.

[0010] In some embodiments, the one or more vitamin D metabolites comprises 25- hydroxyvitamin D₂. In some related embodiments, the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass- to-charge ratio of 395.3 ± 0.5, 251.1 ± 0.5, 209.1 ± 0.5, and 179.1 ± 0.5. In further related embodiments, the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 395.3 ± 0.5 and one or more fragment ions selected from the group consisting of ions with a mass-to-charge ratio of 251.1 ± 0.5, 209.1 ± 0.5, and 179.1 ± 0.5.

[0011] In some embodiments, the one or more vitamin D metabolites comprise 25- hydroxyvitamin D₃ and 25-hydroxyvitamin D₂. In related embodiments, 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ are volatilized simultaneously.

[0012] In some embodiments, the one or more vitamin D metabolites comprises 1,25- dihydroxyvitamin D₂. In some related embodiments, the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass- to-charge ratio of 375.1 ± 0.5, 393.1 ± 0.5, 411.1 ± 0.5, 105.3 ± 0.5, 156.9 ± 0.5, and 135.3 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 375.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 105.3 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 393.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 156.9 ± 0.5. In some related

embodiments, the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 411.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 135.3 ± 0.5.

[0013] In some embodiments, the one or more vitamin D metabolites comprises 1,25- dihydroxyvitamin D₃. In some related embodiments, the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass- to-charge ratio of 363.1 ± 0.5, 381.1 ± 0.5, 399.1 ± 0.5, 156.8 ± 0.5, 157.0 ± 0.5, and 158.8 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 363.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 156.8 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 381.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 157.0 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of comprise a precursor ion with a mass-to-charge ratio of 399.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 158.8 ± 0.5.

[0014] In some embodiments, the one or more vitamin D metabolites comprise 1,25- dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂. In related embodiments, 1,25- dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂ are volatilized simultaneously.

[0015] In some embodiments, the one or more vitamin D metabolites are derivatized with a Cookson-type reagent prior to volatilization of the sample to generate one or more derivatized vitamin D metabolites. In related embodiments, the one or more vitamin D metabolites derivatized with a Cookson-type reagent comprise one, two, three, or four vitamin D metabolites selected from the group consisting of 25-hydroxyvitamin D₂, 25-hydroxyvitamin D_3; 1,25- dihydroxyvitamin D₂, and 1,25-dihydroxyvitamin D₃. In some related embodiments, the one or more vitamin D metabolites derivatized with a Cookson-type reagent are 25-hydroxyvitamin D₂, 25-hydroxyvitamin D_3, 1,25-dihydroxyvitamin D₂, and 1,25-dihydroxyvitamin D₃.

[0016] In embodiments where the one or more vitamin D metabolites are derivatized with a Cookson-type reagent, the one or more derivatized vitamin D metabolites comprise one, two, three, or four derivatized vitamin D metabolites selected from the group consisting of 4-phenyl- l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₂, 4-phenyl-l,2,4-triazoline- 3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₃, 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₂, and 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₃. In some embodiments, the one or more derivatized vitamin D metabolites are 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25- hydroxyvitamin D₂, 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₃, 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₂, and 4- phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₃.

[0017] In any embodiment with more than one derivatized vitamin D metabolite, the one or more derivatized vitamin D metabolites may be volatized at the same time.

[0018] In embodiments where the one or more derivatized vitamin D metabolites comprises PTAD derivatized 25-hydroxyvitamin D₃, the one or more ions detectable by mass spectrometry include one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 558.3 ± 0.5 and 298.1 ± 0.5. In related embodiments, the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 558.3 ± 0.5 and a fragment ion with a mass-to-charge ratio of 298.1 ± 0.5.

[0019] In embodiments where the one or more derivatized vitamin D metabolites comprises PTAD derivatized 25-hydroxyvitamin D₃, the one or more ions detectable by mass spectrometry include one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 570.3 ± 0.5 and 298.1 ± 0.5. In related embodiments, the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 570.3 ± 0.5 and a fragment ion with a mass-to-charge ratio of 298.1 ± 0.5.

[0020] In some embodiments, the one or more vitamin D metabolites derivatized with a Cookson-type reagent are selected from the group consisting of 25-hydroxyvitamin D₂, 25- hydroxyvitamin D₃, 1,25-dihydroxyvitamin D₂, and 1,25-dihydroxyvitamin D₃. In some embodiments, the Cookson-type reagent is 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD). In some related embodiments, the one or more derivatized vitamin D metabolites are selected from the group consisting of PTAD derivatized 25-hydroxyvitamin D₂, PTAD derivatized 25- hydroxyvitamin D₃, PTAD derivatized 1,25-dihydroxyvitamin D₂, and PTAD derivatized 1,25- dihydroxyvitamin D₃.

[0021] In embodiments where the one or more derivatized vitamin D metabolites comprises PTAD derivatized 1,25-dihydroxyvitamin D₂, the one or more ions detectable by mass spectrometry include one or more ions selected from the group consisting of ions with a mass-to- charge ratio of 550.4 ± 0.5, 568.4 ± 0.5, 586.4 ± 0.5, 227.9 ± 0.5, 298.0 ± 0.5, and 314.2 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 550.4 ± 0.5 and a fragment ion with a mass-to- charge ratio of 227.9 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 568.4 ± 0.5 and a fragment ion with a mass-to-charge ratio of 298.0 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 586.4 ± 0.5 and a fragment ion with a mass-to-charge ratio of 314.2 ± 0.5.

[0022] In embodiments where the one or more derivatized vitamin D metabolites comprises PTAD derivatized 1,25-dihydroxyvitamin D₃, the one or more ions detectable by mass spectrometry include one or more ions selected from the group consisting of ions with a mass-to- charge ratio of 538.4 ± 0.5, 556.4 ± 0.5, 574.4 ± 0.5, 278.1 ± 0.5, 298.0 ± 0.5, and 313.0 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 538.4 ± 0.5 and a fragment ion with a mass-to- charge ratio of 278.1 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 556.4 ± 0.5 and a fragment ion with a mass-to-charge ratio of 298.0 ± 0.5. In some related embodiments, the one or more ions detectable by mass spectrometry include a precursor ion with a mass-to-charge ratio of 574.4 ± 0.5 and a fragment ion with a mass-to-charge ratio of 313.0 ± 0.5.

[0023] In some embodiments, the one or more vitamin D metabolites comprise 1,25- dihydroxyvitamin D₂ and 1,25-dihydroxyvitamin D3. In some embodiments, the one or more derivatized vitamin D metabolites comprise 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₂ and 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D3. In some related embodiments, the PTAD derivatized 1,25-dihydroxyvitamin D₂ and PTAD derivatized 1,25-dihydroxyvitamin D3 are volatilized simultaneously.

[0024] The features of the embodiments listed above may be combined without limitation for use in methods of the present invention.

[0025] In certain preferred embodiments of the methods disclosed herein, mass spectrometry is performed in positive ion mode. Alternatively, mass spectrometry is performed in negative ion mode. The preferred ionization technique used in methods described herein is a laser diode thermal desorption ionization technique.

[0026] In preferred embodiments, one or more separately detectable internal standards is provided in the sample, the amount of which is also determined in the sample. In these embodiments, all or a portion of both the analyte of interest and the one or more internal standards is ionized to produce a plurality of ions detectable in a mass spectrometer, and one or more ions produced from each are detected by mass spectrometry. Without intending to be limited to any particular internal standard or standards, exemplary compounds suitable for use as internal standards include 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- ¾ and 25- hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- ¾. In embodiments utilizing 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ as an internal standard, ions detectable in a mass spectrometer are selected from the group consisting of positive ions with a mass/charge ratio (m/z) of 401.3 ± 0.5, 251.1 ± 0.5, 209.1 ± 0.5, and 179.1 ± 0.5. In some preferred embodiments, a 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ precursor ion has m/z of 401.3 ± 0.5, and one or more fragment ions are selected from the group of ions with m/z of 251.1 ± 0.5, 209.1 ± 0.5, and 179.1 ± 0.5. In embodiments utilizing 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- ¾ as an internal standard, ions detectable in a mass spectrometer are selected from the group consisting of positive ions with a mass/charge ratio (m/z) of 389.3 ± 0.5, 257.1 ± 0.5, and 211.1 ± 0.5. In some preferred embodiments, a 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- ¾ precursor ion has m/z of 389.3 ± 0.5, and one or more fragment ions are selected from the group of ions with m/z of 257.1 ± 0.5 and 211.1 ± 0.5. Other exemplary compounds suitable for use as internal standards may include vitamin D analogues that are not isotopic variants of 25-hydroxyvitamin D₂ or 25-hydroxyvitamin D₃. In these embodiments, the presence or amount of ions generated from the analyte of interest may be related to the presence of amount of analyte of interest in the sample.

[0027] In other embodiments, the amount of the one or more vitamin D metabolite ion or ions may be determined by comparison to one or more external reference standards. Exemplary external reference standards include blank plasma or serum spiked with 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆, 25-hydroxyvitamin D₃, and 25- hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆-

[0028] As used herein, unless otherwise stated, the singular forms "a," "an," and "the" include plural reference. Thus, for example, a reference to "a protein" includes a plurality of protein molecules.

[0029] As used herein, the term "vitamin D metabolite" refers to any vitamin D analog or any chemical species related to vitamin D. Vitamin D metabolites may include analogs of, or a chemical species related to, vitamin D₂ or vitamin D₃. Vitamin D metabolites may be found in the circulation of an animal and/or may be generated by a biological organism, such as an animal, or by biotransformation of vitamin D₂ or vitamin D₃. Vitamin D metabolites may be metabolites of naturally occurring forms of vitamin D or may be metabolites of synthetic vitamin D analogs. In certain embodiments a vitamin D metabolite is one or more compounds selected from the group consisting of 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₂, 1,25- dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂.

[0030] As used herein, "derivatizing" means reacting two molecules to form a new molecule. Thus, a derivatizing agent is an agent that may be reacted with another substance to derivatize the substance. For example, 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) is a derivatizing reagent that may be reacted with a vitamin D metabolite to form a PTAD-derivatized vitamin D metabolite.

[0031] As used here, the names of derivatized forms of vitamin D metabolites include an indication as to the nature of derivatization. For example, the PTAD derivative of 25- hydroxyvitamin D₂ is indicated as PTAD derivatized 25- hydroxyvitamin D₂, PTAD-25- hydroxyvitamin D₂, or PTAD-250HD₂.

[0032] As used herein, a "Cookson-type derivatizing agent" is a 4-substituted 1,2,4-triazoline- 3,5-dione compound. Exemplary Cookson-type derivatizing agents include 4-phenyl- 1,2,4- triazoline-3,5-dione (PTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4- dihydroquinoxalyl)ethyl]- 1 ,2,4-triazoline-3,5-dione (DMEQTAD), 4-(4-nitrophenyl)- 1 ,2,4- triazoline-3,5- dione (NPTAD), and 4-ferrocenylmethyl-l,2,4-triazoline-3,5-dione (FMTAD). Additionally, isotopically labeled variants of Cookson-type derivatizing agents may be used in

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some embodiments. For example, the C₆-PTAD isotopic variant is 6 mass units heavier than normal PTAD and may be used in some embodiments. Derivatization of vitamin D metabolites by Cookson-type reagents can be conducted by any appropriate method. See, e.g., 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; Higashi T, et al, J Pharm Biomed Anal. 2002, 29(5):947-55; Higashi T, et al, Anal. Biochanal Chem, 2008, 391 :229-38; and Aronov, et al, Anal Bioanal Chem, 2008, 391 : 1917-30.

[0033] As used herein, the term "purification" or "purifying" does not refer to removing all materials from the sample other than the analyte(s) of interest. Instead, purification refers to a procedure that enriches the amount of one or more analytes of interest relative to other components in the sample that may interfere with detection of the analyte of interest.

Purification of the sample by various means may allow relative reduction of one or more interfering substances, e.g., one or more substances that may or may not interfere with the detection of selected parent or daughter ions by mass spectrometry. Relative reduction as this term is used does not require that any substance, present with the analyte of interest in the material to be purified, is entirely removed by purification.

[0034] As used herein, the term "sample" refers to any sample that may contain an analyte of interest. As used herein, the term "body fluid" means any fluid that can be isolated from the body of an individual. For example, "body fluid" may include blood, plasma, serum, bile, saliva, urine, tears, perspiration, and the like. In preferred embodiments, the sample comprises a body fluid sample; preferably plasma or serum.

[0035] As used herein, the term "solid phase extraction" or "SPE" refers to a process in which a chemical mixture is separated into components as a result of the affinity of components dissolved or suspended in a solution (i.e., mobile phase) for a solid through or around which the solution is passed (i.e., solid phase). In some instances, as the mobile phase passes through or around the solid phase, undesired components of the mobile phase may be retained by the solid phase resulting in a purification of the analyte in the mobile phase. In other instances, the analyte may be retained by the solid phase, allowing undesired components of the mobile phase to pass through or around the solid phase. In these instances, a second mobile phase is then used to elute the retained analyte off of the solid phase for further processing or analysis. SPE, including utilization of a turbulent flow liquid chromatography (TFLC) column as an extraction column, may operate via a unitary or mixed mode mechanism. Mixed mode mechanisms utilize ion exchange and hydrophobic retention in the same column; for example, the solid phase of a mixed-mode SPE column may exhibit strong anion exchange and hydrophobic retention; or may exhibit column exhibit strong cation exchange and hydrophobic retention.

[0036] As used herein, the term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

[0037] As used herein, the term "liquid chromatography" or "LC" means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). Examples of separation techniques which employ "liquid chromatography" include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (TFLC) (sometimes known as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography). In some embodiments, an SPE column may be used in combination with an LC column. For example, a sample may be purified with a TFLC extraction column, followed by additional purification with a HPLC analytical column.

[0038] As used herein, the term "high performance liquid chromatography" or "HPLC" (sometimes known as "high pressure liquid chromatography") refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column.

[0039] As used herein, the term "turbulent flow liquid chromatography" or "TFLC" (sometimes known as high turbulence liquid chromatography or high throughput liquid chromatography) refers to a form of chromatography that utilizes turbulent flow of the material being assayed through the column packing as the basis for performing the separation. TFLC has been applied in the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, e.g., Zimmer et al., J Chromatogr A 854: 23-35 (1999); see also, U.S. Patents No. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further explain TFLC. Persons of ordinary skill in the art understand "turbulent flow". When fluid flows slowly and smoothly, the flow is called "laminar flow". For example, fluid moving through an HPLC column at low flow rates is laminar. In laminar flow the motion of the particles of fluid is orderly with particles moving generally in straight lines. At faster velocities, the inertia of the water overcomes fluid frictional forces and turbulent flow results. Fluid not in contact with the irregular boundary "outruns" that which is slowed by friction or deflected by an uneven surface. When a fluid is flowing turbulently, it flows in eddies and whirls (or vortices), with more "drag" than when the flow is laminar. Many references are available for assisting in determining when fluid flow is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction, P.S. Bernard & J.M. Wallace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)). [0040] As used herein, the term "gas chromatography" or "GC" refers to chromatography in which the sample mixture is vaporized and injected into a stream of carrier gas (as nitrogen or helium) moving through a column containing a stationary phase composed of a liquid or a particulate solid and is separated into its component compounds according to the affinity of the compounds for the stationary phase.

[0041] As used herein, the terms "on-line" and "inline", for example as used in "on-line automated fashion" or "on-line extraction" refers to a procedure performed without the need for operator intervention. In contrast, the term "off-line" as used herein refers to a procedure requiring manual intervention of an operator. Thus, if samples are subjected to precipitation, and the supernatants are then manually loaded into an autosampler, the precipitation and loading steps are off-line from the subsequent steps. In various embodiments of the methods, one or more steps may be performed in an on-line automated fashion.

[0042] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique to identify compounds by their mass. MS refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m/z". MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometric instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z"). See, e.g., U.S. Patent Nos. 6,204,500, entitled "Mass Spectrometry From Surfaces;" 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry;" 6,268,144, entitled "DNA Diagnostics Based On Mass Spectrometry;"

6,124,137, entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes;" Wright et al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis 2000, 21 : 1164-67.

[0043] As used herein, the term "operating in negative ion mode" refers to those mass spectrometry methods where negative ions are generated and detected. The term "operating in positive ion mode" as used herein, refers to those mass spectrometry methods where positive ions are generated and detected.

[0044] As used herein, the term "ionization" or "ionizing" refers to the process of generating an ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units.

[0045] As used herein, the term "desorption" refers to the removal of an analyte from a surface and/or the entry of an analyte into a gaseous phase. Laser diode thermal desorption ("LDTD") is a technique wherein a sample containing the analyte is thermally desorbed into the gas phase by a laser pulse. In one example described herein, the laser is directed to the back of a specially made 96-well plate with a metal base, wherein the laser pulse heats the base and the heat causes the analyte to transfer into the gas phase. The analyte may be pre-dried onto the base of the plate prior to heating with the laser, or may be in solution at the beginning of the heating process.

[0046] As used herein, the term "LDTD ionization technique" refers to a method for ionizing an analyte (e.g., a vitamin D metabolite) in which LDTD is used to volatilize an analyte, which is then ionized by any suitable technique. In preferred embodiments, the volatilized sample is ionized via atmospheric pressure chemical ionization; that is, the volatilized analyte is drawn past a corona discharge and ionized before being drawn into the mass spectrometer. An exemplary ionization source comprising LDTD is described in detail in US Patent No. 7,321,116 (filed 5/20/05), incorporated herein by reference in its entirety.

[0047] As used herein, the term "selective ion monitoring" is a detection mode for a mass spectrometric instrument in which only ions within a relatively narrow mass range, typically about one mass unit, are detected.

[0048] As used herein, "multiple reaction mode," sometimes known as "selected reaction monitoring," is a detection mode for a tandem mass spectrometric instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0049] As used herein, the term "lower limit of quantification", "lower limit of quantitation" or "LLOQ" refers to the point where measurements become quantitatively meaningful. The analyte response at this LOQ is identifiable, discrete and reproducible with a relative standard deviation (RSD %) of less than 20% and an accuracy of 85% to 115%.

[0050] As used herein, the term "limit of detection" or "LOD" is the point at which the measured value is larger than the uncertainty associated with its measurement. The LOD is defined as three times the RSD of the mean at the zero concentration.

[0051] As used herein, an "amount" of an analyte in a body fluid sample refers generally to an absolute value reflecting the mass of the analyte detectable in volume of sample. However, an amount also contemplates a relative amount in comparison to another analyte amount. For example, an amount of an analyte in a sample can be an amount which is greater than a control or normal level of the analyte normally present in the sample.

[0052] The term "about" as used herein in reference to quantitative measurements not including the measurement of the mass of an ion, refers to the indicated value plus or minus 10%. Mass spectrometry instruments can vary slightly in determining the mass of a given analyte. The term "about" in the context of the mass of an ion or the mass/charge ratio of an ion refers to +/- 0.50 atomic mass unit.

[0053] The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figures 1 A-D show exemplary chromatograms for 25-hydroxyvitamin D₂, 25- hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ (internal standard), 25-hydroxyvitamin D₃, and 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ (internal standard), respectively. Details are discussed in Example 3.

[0055] Figure 2 shows a plot of analytical results for calibrator solutions of various

concentrations of 25-hydroxyvitamin D₂. Details are discussed in Example 4.

[0056] Figure 3 shows a plot of analytical results for calibrator solutions of various

concentrations of 25-hydroxyvitamin D₃. Details are discussed in Example 4.

[0057] Figure 4A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D₂ ions. Figure 4B shows an exemplary product ion spectra (covering the m/z range of about 100 to 396) for fragmentation of the 25-hydroxyvitamin D₂ precursor ion with m/z of about 395.2. Details are described in Example 5.

[0058] Figure 5 A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D₂-[6, 19, 19]-Ή₃ ions. Figure 5B shows an exemplary product ion spectra (covering the m/z range of about 100 to 402) for fragmentation of the 25- hydroxyvitamin D₂-[6, 19, 19]- H₃ precursor ion with m/z of about 398.2. Details are described in Example 5.

[0059] Figure 6A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ ions. Figure 6B shows an exemplary product ion spectra (covering the m/z range of about 100 to 402) for fragmentation of the 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 401.2. Details are described in Example 5.

[0060] Figure 7A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D₃ ions. Figure 7B shows an exemplary product ion spectra (covering the m/z range of about 100 to 396) for fragmentation of the 25-hydroxyvitamin D₃ precursor ion with m/z of about 383.2. Details are described in Example 5.

[0061] Figure 8A shows an exemplary Ql scan spectrum (covering the m/z range of about 350

2

to 450) for 25-hydroxyvitamin D₃-[6, 19, 19]-Ή₃ ions. Figure 8B shows an exemplary product ion spectra (covering the m/z range of about 100 to 402) for fragmentation of the 25- hydroxyvitamin D₃-[6, 19, 19]- H₃ precursor ion with m/z of about 386.2. Details are described in Example 5.

[0062] Figure 9A shows an exemplary Ql scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ ions. Figure 9B shows an exemplary product ion spectra (covering the m/z range of about 100 to 402) for fragmentation of the 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 389.2. Details are described in Example 5.

[0063] Figure 10A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamin D₂ ions. Figure 10B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25 - hydroxyvitamin D₂ precursor ion with m/z of about 570.3. Details are described in Example 6.

[0064] Figure 11A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamm D₂-[6, 19, 19]-Ή₃ ions. Figure 1 IB shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD- 25 -hydroxyvitamm D₂-[6, 19, 19]- H₃ precursor ion with m/z of about 573.3. Details are described in Example 6.

[0065] Figure 12A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamm D₂-[26, 26, 26, 27, 27, 27]-²H₆ ions. Figure 12B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25 -hydroxyvitamm D₂-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 576.3. Details are described in Example 6.

[0066] Figure 13A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamm D₃ ions. Figure 13B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25 - hydroxyvitamm D₃ precursor ion with m/z of about 558.3. Details are described in Example 6.

[0067] Figure 14A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamm D₃-[6, 19, 19]-Ή₃ ions. Figure 14B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD- 25 -hydroxyvitamm D₃-[6, 19, 19]- H₃ precursor ion with m/z of about 561.3. Details are described in Example 6.

[0068] Figure 15A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25 -hydroxyvitamm D₃-[26, 26, 26, 27, 27, 27]-²H₆ ions. Figure 15B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25 -hydroxyvitamm D₃-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 564.3. Details are described in Example 6.

[0069] Figure 16A shows an exemplary Ql scan spectrum (covering the m/z range of about 340 to 440) for 1,25-dihydroxyvitamin D₂ ions. Figure 16B shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₂ precursor ion with m/z of about 375.1. Figure 16C shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₂ precursor ion with m/z of about 393.1. Figure 16D shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₂ precursor ion with m/z of about 411.1. Details are described in Example 7.

[0070] Figure 17A shows an exemplary Ql scan spectrum (covering the m/z range of about 340 to 440) for 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ ions. Figure 17B shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 381.1. Figure 17C shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 399.1. Figure 17D shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ precursor ion with m/z of about 417.1. Details are described in Example 7.

[0071] Figure 18A shows an exemplary Ql scan spectrum (covering the m/z range of about 340 to 440) for 1,25-dihydroxyvitamin D₃ ions. Figure 18B shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₃ precursor ion with m/z of about 363.1. Figure 18C shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₃ precursor ion with m/z of about 381.1 Figure 18D shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₃ precursor ion with m/z of about 399.1. Details are described in Example 7.

[0072] Figure 19A shows an exemplary Ql scan spectrum (covering the m/z range of about 340 to 440) for 1,25-dihydroxyvitamin D₃-[6, 19, 19]-Ή₃ ions. Figure 19B shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25- dihydroxyvitamin D₃-[6, 19, 19]- H3 precursor ion with m/z of about 366.1. Figure 19C shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₃-[6, 19, 19]- H₃ precursor ion with m/z of about 384.1. Figure 19D shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for 2

fragmentation of the 1,25-dihydroxyvitamin D₃-[6, 19, 19]- H₃ precursor ion with m/z of about 402.1. Details are described in Example 7.

[0073] Figure 20A shows an exemplary Ql scan spectrum (covering the m/z range of about 340 to 440) for 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ ions. Figure 20B shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 369.1. Figure 20C shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 387.1. Figure 20D shows an exemplary product ion spectra (covering the m/z range of about 100 to 420) for fragmentation of the 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ precursor ion with m/z of about 405.1. Details are described in Example 7.

[0074] Figure 21 A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD- 1,25-dihydroxyvitamin D₂ ions. Figure 21B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-1,25- dihydroxyvitamin D₂ precursor ion with m/z of about 550.4. Figure 21C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D₂ precursor ion with m/z of about 568.4. Figure 21D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D₂ precursor ion with m/z of about 586.4. Details are described in Example 8.

[0075] Figure 22A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-l,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ ions. Figure 22B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 556.4. Figure 22C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 574.4 Figure 22D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 592.4. Details are described in Example 8. [0076] Figure 23 A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-l,25-hydroxyvitamin D₃ ions. Figure 23 B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-1,25- dihydroxyvitamin D₃ precursor ion with m/z of about 538.4. Figure 23C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D₃ precursor ion with m/z of about 556.4. Figure 23D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D₃ precursor ion with m/z of about 574.4. Details are described in Example 8.

[0077] Figure 24A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-l,25-dihydroxyvitamin D₃-[6, 19, 19]-Ή₃ ions. Figure 24B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D₃-[6, 19, 19]- H₃ precursor ion with m/z of about 541.4. Figure 24C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D₃-[6, 19, 19]- H₃ precursor ion with m/z of about 559.4. Figure 24D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D₃-[6, 19, 19]- H₃ precursor ion with m/z of about 577.4. Details are described in Example 8.

[0078] Figure 25 A shows an exemplary Ql scan spectrum (covering the m/z range of about 520 to 620) for PTAD-l,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ ions. Figure 25B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 544.4. Figure 25C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PT AD- 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 562.4. Figure 25D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD- 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ precursor ion with m/z of about 580.4. Details are described in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0079] Methods are described for measuring the amount of one or more vitamin D metabolites in a sample. More specifically, mass spectrometric methods are described for ionizing one or more vitamin D metabolites and detecting ion thereby produced by mass spectrometry. These methods may include purifying one or more vitamin D metabolites in the sample prior to ionization and mass spectrometry. However, the methods may be performed without purifying the sample with chromatography. In some embodiments, the methods of the invention may be used to detect two or more vitamin D metabolites in a single assay. Preferred embodiments are particularly well suited for application in large clinical laboratories for automated vitamin D metabolite quantification.

[0080] In some embodiments, the vitamin D metabolites may be derivatized prior to mass spectrometric analysis. Derivatization of vitamin D metabolites is known in the art and described, for example, by Aronov, et al, Anal Bioanal Chem, 2008, 391 : 1917-30; Higashi, et al, Anal. Bioanal Chem, 2008, 391 :229-238; and Yeung, et al, J. Chromatog, 1993, 645: 115- 23. Derivatization may occur at any point prior to thermal desorption by LDTD, and the ions monitored are selected depending on the derivatization agent utilized. For example, mass spectrometric detection of 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-OH vitamin D₂ and 25-OH vitamin D₃ may be conducted by monitoring mass transitions (in a MS/MS technique) from 570.3 ± 0.5 to 298.1 ± 0.5 and 558.3 ± 0.5 to 298.1 ± 0.5, respectively.

[0081] Suitable test samples for use in methods of the present invention include any test sample that may contain the analyte of interest. In some preferred embodiments, a sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, a cell culture, an organ culture, etc. In certain preferred embodiments, samples are obtained from a

mammalian animal, such as a dog, cat, horse, etc. Particularly preferred mammalian animals are primates, most preferably male or female humans. Preferred samples comprise bodily fluids such as blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples; preferably plasma and serum. Such samples may be obtained, for example, from a patient; that is, a living person, male or female, presenting oneself in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. [0082] The present invention also contemplates kits for a vitamin D metabolite quantitation assay. A kit for a vitamin D metabolite quantitation assay may include a kit comprising the compositions provided herein. For example, a kit may include packaging material and measured amounts of an isotopically labeled internal standard, in amounts sufficient for at least one assay. In embodiments where vitamin D metabolite quantitation is conducted with derivatized vitamin D, the kit may include a derivatizing agent, preferably PTAD, in a quantity sufficient for at least one assay. Typically, the kits will also include instructions recorded in a tangible form (e.g., contained on paper or an electronic medium) for using the packaged reagents for use in a vitamin D metabolite quantitation assay.

[0083] Quality control (QC) pools having known concentrations, for use in embodiments of the present invention, are preferably prepared using a matrix similar to the intended sample matrix. Calibration pools for use in embodiments of the present invention may be prepared using a matrix similar to the intended sample matrix, or may be prepared from a vitamin D free matrix containing albumin and phosphate buffered saline solution.

Sample Preparation for Mass Spectrometric Analysis

[0084] In preparation for mass spectrometric analysis, vitamin D metabolites may be enriched relative to one or more other components in the sample (e.g. protein) by various methods known in the art, including for example, solid phase extraction (SPE), LC, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis including capillary electrophoresis, affinity separations including immunoaffinity separations, extraction methods including ethyl acetate or methanol extraction, and the use of chaotropic agents or any combination of the above or the like. In preferred embodiments, chromatography, including LC, is not used.

[0085] Protein precipitation is one method of preparing a test sample, especially a biological sample, such as serum or plasma. Protein purification methods are well known in the art, for example, Poison et ah, Journal of Chromatography B 2003, 785:263-275, describes protein precipitation techniques suitable for use in methods of the present invention. Protein

precipitation may be used to remove most of the protein from the sample leaving vitamin D metabolites in the supernatant. The samples may be centrifuged to separate the liquid supernatant from the precipitated proteins; alternatively the samples may be filtered to remove precipitated proteins. The resultant supernatant or filtrate may then be applied directly to mass spectrometry analysis; or alternatively to liquid chromatography and subsequent mass spectrometry analysis.

[0086] In preferred embodiments, liquid-liquid extraction methods including ethyl acetate or methanol extraction are used to extract vitamin D metabolites from the a sample. In these embodiments, between 10 and 500 μΐ of sample, such as between 10 and 250 μΐ, such as between 10 and 100 μΐ, such as about 50 μΐ, is added to a portion of extraction solvent. The quantity of extraction solvent is commensurate with sample volume and may vary depending on the extraction solvent used, but is preferably between about 50 and 1000 μΐ, such as about 200 μΐ. The sample/solvent mixtures are mixed and centrifuged, and a portion of the supernatant or organic phase (depending on solvent used) is drawn off for further analysis. Solvent may be removed from the drawn off portion, for example under a nitrogen flow, and the residue reconstituted in a different solvent such as methanol. A portion of the resulting solution is then spotted in a well of a LDTD plate. A LDTD well plate is similar to a standard 96-well plate, but has a metal coating at least on the bottom of the plate at each well.

Detection and Quantitation by Mass Spectrometry

[0087] Vitamin D metabolites present in the LDTD well plate are then ionized by an ionization technique comprising LDTD. LDTD is described in great detail in U.S. Patent No. 7,321,116 (filed May 20, 2005). In LDTD, thermal desorption is induced indirectly by a laser beam. A laser is directed to the metal coating on the bottom of the plate beneath a well containing a spotted sample. Vitamin D metabolites in the spotted sample desorb from the well and are directed past a corona discharge, creating vitamin D metabolite ions. Ions of multiple vitamin D metabolites (e.g., 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃) may be formed from a single sample volatilization.

[0088] Samples prepared for LDTD according to this method can be thermally desorbed without a support matrix, and ionization achieved without introduction of a liquid mobile phase into the system. Thus, the LDTD method described herein differs from MALDI and SELDI, even though all three methods employ a laser.

[0089] In the LDTD method described herein, the laser intensity used may be varied but must be sufficient to create a strong enough source of ions for mass spectrometric detection. Vitamin D metabolites may be ionized in positive or negative mode. In preferred embodiments, vitamin D metabolites are ionized in positive mode.

[0090] In mass spectrometry techniques generally, after the sample has been ionized, the positively or negatively charged ions thereby created may be analyzed to determine a mass-to- charge ratio. Suitable analyzers for determining mass-to-charge ratios include quadrupole analyzers, ion traps analyzers, and time-of-flight analyzers. Exemplary ion trap methods are described in Bartolucci, et αί, Rapid Commun. Mass Spectrom. 2000, 14:967-73.

[0091] The ions may be detected using several detection modes. For example, selected ions may be detected, i.e. using a selective ion monitoring mode (SIM), or alternatively, mass transitions resulting from collision induced dissociation or neutral loss may be monitored, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). Preferably, the mass-to-charge ratio is determined using a quadrupole analyzer. For example, in a "quadrupole" or "quadrupole ion trap" instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and the mass/charge ratio. The voltage and amplitude may be selected so that only ions having a particular mass/charge ratio travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments may act as both a "mass filter" and as a "mass detector" for the ions injected into the instrument.

[0092] One may enhance the resolution of the MS technique by employing "tandem mass spectrometry," or "MS/MS". In this technique, a precursor ion (also called a parent ion) generated from a molecule of interest can be filtered in an MS instrument, and the precursor ion subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure. By careful selection of precursor ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collisions with atoms of an inert gas produce the fragment ions. Because both the precursor and fragment ions are produced in a reproducible fashion under a given set of

ionization/fragmentation conditions, the MS/MS technique may provide an extremely powerful analytical tool. For example, the combination of filtration/fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples. [0093] Alternate modes of operating a tandem mass spectrometric instrument include product ion scanning and precursor ion scanning. For a description of these modes of operation, see, e.g., E. Michael Thurman, et al., Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, Chapter 8 (Amadeo R. Fernandez -Alba, ed., Elsevier 2005) (387).

[0094] The results of an analyte assay, that is, a mass spectrum, may be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion may be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, external standards may be run with the samples, and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion may be converted into an absolute amount of the original molecule. In certain preferred embodiments, one or more internal standards are used to generate standard curves for calculating the quantity of a vitamin D metabolite. Methods of generating and using such standard curves are well known in the art and one of ordinary skill is capable of selecting an appropriate internal standard. For example, isotopically labeled vitamin D₂ (e.g., 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆, or 25-hydroxyvitamin D₂-[6, 19, 19]- H₃) may be used as an internal standard. Numerous other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.

[0095] As used herein, an "isotopic label" produces a mass shift in the labeled molecule relative to the unlabeled molecule when analyzed by mass spectrometric techniques. Examples of

2 13 15

suitable labels include deuterium ( H), C, and N. For example, 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ has a mass about six units higher than 25-hydroxyvitamin D₂. The isotopic label can be incorporated at one or more positions in the molecule and one or more kinds of isotopic labels can be used on the same isotopically labeled molecule.

[0096] One or more steps of the methods may be performed using automated machines. In certain embodiments, one or more purification steps are performed on-line, and more preferably all of the purification and mass spectrometry steps may be performed in an on-line fashion. [0097] In certain embodiments, such as MS/MS, where precursor ions are isolated for further fragmentation, collision activated dissociation (CAD) is often used to generate fragment ions for further detection. In CAD, precursor ions gain energy through collisions with an inert gas, and subsequently fragment by a process referred to as "unimolecular decomposition." Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy. In preferred embodiments, vitamin D metabolite ions are in a gaseous state and an inert collision gas is argon or nitrogen; preferably argon.

[0098] In particularly preferred embodiments, vitamin D metabolites in a sample are detected and/or quantified using MS/MS as follows. The vitamin D metabolites are purified in a sample by liquid-liquid extraction, with the a portion of the resulting solution spotted in a LDTD well. The spotted sample is heated indirectly with a laser which results in thermal desorption of vitamin D metabolites, and the desorbed vitamin D metabolites then pass a corona discharge and are ionized before being introduced into a triple quadrupole MS instrument. Quadrupoles 1 and 3 (Ql and Q3) are mass filters, allowing selection of ions (i.e., selection of "precursor" and "fragment" ions in Ql and Q3, respectively) based on their mass-to-charge ratio (m/z).

Quadrupole 2 (Q2) is a collision cell, where precursor ions are fragmented. The vitamin D metabolite ions, e.g. precursor ions, pass through the orifice of the instrument and enter a first quadrupole (Ql). The first quadrupole of the mass spectrometer (Ql) selects for molecules with the mass-to-charge ratios of a vitamin D metabolite. Precursor ions with the correct mass/charge ratios are allowed to pass into the collision chamber (Q2), while unwanted ions with any other mass/charge ratio collide with the sides of the quadrupole and are eliminated. Precursor ions entering Q2 collide with neutral gas molecules, preferably argon, and fragment. The fragment ions generated are passed into quadrupole 3 (Q3), where the fragment ions of a vitamin D metabolite are selected while other ions are eliminated. By switching the m/z ratio selected in Ql, multiple precursor ions detected from a single sample.

[0099] The methods may involve MS/MS performed in either positive or negative ion mode; preferably positive ion mode. Using standard methods well known in the art, one of ordinary skill is capable of identifying one or more fragment ions of a particular precursor ion of a vitamin D metabolite that may be used for selection in quadrupole 3 (Q3). [00100] As ions collide with the detector they produce a pulse of electrons that are converted to a digital signal. The acquired data is relayed to a computer, which plots counts of the ions collected versus time. The resulting mass chromatograms are similar to chromatograms generated in traditional HPLC-MS methods. The areas under the peaks corresponding to particular ions, or the amplitude of such peaks, may be measured and correlated to the amount of the analyte of interest. In certain embodiments, the area under the curves, or amplitude of the peaks, for fragment ion(s) and/or precursor ions are measured to determine the amount of vitamin D metabolite. As described above, the relative abundance of a given ion may be converted into an absolute amount of the original analyte using calibration standard curves based on peaks of one or more ions of an internal molecular standard.

[00101] In some embodiments, multiple vitamin D metabolites may be determined

simultaneously by mass spectrometry (i.e., determined from a single sample volatilization). For example, the amounts of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ may be determined by mass spectrometric analysis of the ions formed from the volatilization of a single sample.

[00102] The following Examples serve to illustrate the invention. These Examples are in no way intended to limit the scope of the methods. In particular, the following Examples demonstrate quantitation of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ by mass spectrometry with the use of 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- ¾ and 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ as internal standards. The use of these compounds as internal standards is not meant to be limiting in any way. Any appropriate chemical species, easily determined by one in the art, may be used as an internal standard for vitamin D metabolite quantitation.

EXAMPLES

Example 1: Reagent and Sample Preparation

[00103] Extraction/internal standard solutions were prepared with 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ and 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ at a

concentration of 40 ng/mL in methanol, and 40 ng/dL in ethyl acetate.

[00104] Calibrant solutions were prepared with 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ in a vitamin D metabolite free matrix containing albumin and phosphate buffered saline. Concentration of the calibrant solutions ranged from 4 to 128 ng/mL for each analyte. [00105] Serum samples were obtained from Golden West Biologicals (Temecula, CA) and were certified free of common human pathogens (SP1060-VD (defibrinated, delipidized, charcoal stripped and solvent extracted serum) and SP1010 (defibrinated and delipidized, Lots

GO 11005020 (Lot 20) and GO 11005032 (Lot 32))).

Example 2: Enrichment of Vitamin D Metabolites

[00106] All extractions were performed in a 96-well plate. 50 of serum or calibrant solution was added to each well, along with 200 of either methanol or ethyl acetate extraction/internal standard solution. Plates were vigorously mixed for about 2 minutes at 1600 RPM, and subsequently centrifuged for about 30 minutes at 6000 g.

[00107] An automated pipettor was used to transfer 150 μΐ_^ οΐ supernatant (from methanol extractions) or organic layer (from ethyl acetate extractions) to a new 96 well plate. Liquids were evaporated under a stream of nitrogen. The residue was reconstituted in 100 of methanol, and about 2 to 5 of the methanol solution was spotted onto a well of a LazWell plate.

Example 3: Detection and Quantitation of Vitamin D Metabolites by MS/MS

[00108] MS/MS was performed using a Thermo Finnigan TSQ Vantage MS/MS system (Thermo Electron Corporation). The ionization source was a Laser Diode Thermal Desorption (LDTD) source from Phytronix Technologies (Quebec, Qc, Canada). Laser intensity varied from 10% to 60% of maximum output during development experiments, and was set to about 25% maximum output for quantitative analysis.

[00109] Ions passed to the first quadrupole (Ql), which selected 25-hydroxyvitamin D₂ and 25- hydroxyvitamin D₃ precursor ions with a mass-to-charge ratio of 395.3 ± 0.50 m/z and 383.3 ± 0.50 m/z, respectively. Ions entering quadrupole 2 (Q2) collided with argon gas to generate ion fragments, which were passed to quadrupole 3 (Q3) for further selection. Simultaneously, the same process using isotope dilution mass spectrometry was carried out with internal standards, 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆ and 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- ¾. The mass transitions used for detection and quantitation during validation on positive polarity are shown in Table 1.

Table 1. Mass Transitions for 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 2

27]- H (internal standard), 25-hydroxyvitamin D₃, and 25-hydroxyvitamin D₃-[26, 26, 26, 27,

27, 27]- ¾ (internal standard) (Positive Polarity)

[00110] Exemplary chromatograms for the four analytes shown in Table 1 are found in Figures 1 A-D. During the three minutes of data collection shown in the chromatograms, five different wells were lased/ionized under different laser intensity settings to find the optimal laser intensity setting for the desired analytes.

Example 4: Analytical Results for Vitamin D Metabolites Extracted with Ethyl Acetate

[00111] Calibration curves were developed for both 25-hydroxyvitamin D₂ and 25- hydroxyvitamin D₃ using the samples extracted with ethyl acetate. These curves are shown in Figures 2 and 3.

[00112] Goodness of fit (R ) values for the 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ calibration curves were 0.9924 and 0.9917, respectively.

Example 5: Analytical Sensitivity for Vitamin D Metabolites

[00113] Several samples of the serum matrices described in Example 1 were analyzed as unknowns. The results of these analyses are presented in Tables 2 and 3. Table 2. Analytical Results of Analysis of SP1060-VD Serum Samples

Table 3. Analytical Results of Analysis of SPlOlO Lots 20 and 32 Serum Samples

[00114] For the SP1060-VD and SPlOlO Lot 20 materials, both analytes were at or near the LOQ (not yet defined, but presumed to be in the 4 ng/mL range).

[00115] For the SPlOlO Lot 32 material, 25-hydroxyvitamin D3 was present at about 38 ng/mL (normal range is about 10 to 55 ng/mL) with only 22% coefficient of variation. These results suggest that this measurement method is quantitative in nature with minimal sample preparation.

Example 5: Exemplary spectra from LDTD-MS/MS analysis of 25-hydroxyvitamin D? and 25- hydroxyvitamin D₃

[00116] Exemplary Ql scan spectra from the analysis of samples containing 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₂-[6, 19, 19]-²H₃, and 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ are shown in Figures 4A, 5A, and 6A, respectively. These spectra were collected by scanning Ql across a m/z range of about 350 to 450.

[00117] Exemplary product ion scans from each of these species are presented in Figures 4B, 5B, and 6B, respectively. The precursor ions selected in Ql and the collision energies used to generate these product ion spectra are indicated in Table 4.

[00118] An exemplary MRM transition for the quantitation of 25-hydroxyvitamin D₂ is fragmenting a precursor ion with a m/z of about 395.2 to a product ion with a m/z of about 208.8. An exemplary MRM transition for the quantitation of 25-hydroxyvitamin D₂-[6, 19, 19]- H₃ is fragmenting a precursor ion with a m/z of about 398.2 to a product ion with a m/z of about 211.9. A preferred MRM transition for the quantitation of 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ is fragmenting a precursor ion with a m z of about 401.2 to a product ion with a m/z of about 269.2. However, as can be seen in the product ion scans in Figures 4B, 5B, and 6B, several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 4B, 5B, and 6B to replace or augment the exemplary fragment ions. Table 4. Precursor Ions and Collision Cell Energies for Fragmentation of 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₂-[6, 19, 19]-²H₃, and 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆

[00119] Exemplary Ql scan spectra from the analysis of 25-hydroxyvitamin D₃, 25-

2 2 hydroxyvitamin D₃-[6, 19, 19]- H₃, and 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ are shown in Figures 7A, 8 A, and 9A, respectively. These spectra were collected by scanning Ql across a m/z range of about 350 to 450.

[00120] Exemplary product ion scans from each of these species are presented in Figures 7B, 8B, and 9B, respectively. The precursor ions selected in Ql and the collision energies used to generate these product ion spectra are indicated in Table 5.

[00121] An exemplary MRM transition for the quantitation of 25-hydroxyvitamin D₃ is fragmenting a precursor ion with a m/z of about 383.2 to a product ion with a m/z of about 186.9. An exemplary MRM transition for the quantitation of 25-hydroxyvitamin D₃-[6, 19, 19]- H₃ is fragmenting a precursor ion with a m/z of about 386.2 to a product ion with a m/z of about 256.8. An exemplary MRM transition for the quantitation of 25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- ¾-PTAD is fragmenting a precursor ion with a m/z of about 389.2 to a product ion with a m/z of about 210.0. However, as can be seen in the product ion scans in Figures 7B, 8B, and 9B, several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 7B, 8B, and 9B to replace or augment the exemplary fragment ions. Table 5. Precursor Ions and Collision Cell Energies for Fragmentation of 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₃-[6, 19, 19]-²H₃, and 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆

Example 6: Exemplary spectra from LDTD-MS/MS analysis of PTAD derivatized 25- hydroxyvitamin D? and 25-hydroxyvitamin D₃

[00122] PTAD derivatives of 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₂-[6, 19, 19]-²H₃, 25- hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₃-

2 2

[6, 19, 19]- H₃, and 25-hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ were prepared by treating aliquots of stock solutions of each analyte with PTAD in acetonitrile. The derivatization reactions was allowed to proceed for approximately one hour, and were quenched by adding water to the reaction mixture. The derivatized analytes were then analyzed according to the procedure outlined above in Examples 2 and 3.

[00123] Exemplary Ql scan spectra from the analysis of samples containing PTAD-25- hydroxyvitamin D₂, PTAD-25 -hydroxyvitamin D₂-[6, 19, 19]-²H₃, and PTAD-25- hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ are shown in Figures 10A, 11A, and 12A, respectively. These spectra were collected by scanning Ql across a m/z range of about 520 to 620.

[00124] Exemplary product ion scans from each of these species are presented in Figures 10B, 1 IB, and 12B, respectively. The precursor ions selected in Ql, and collision energies used in fragmenting the precursors are indicated in Table 6.

[00125] An exemplary MRM transition for the quantitation of PTAD-25 -hydroxyvitamin D₂ is fragmenting a precursor ion with a m/z of about 570.3 to a product ion with a m/z of about 298.0. An exemplary MRM transition for the quantitation of PTAD-25 -hydroxyvitamin D₂-[6, 19, 19]- H₃ is fragmenting a precursor ion with a m/z of about 573.3 to a product ion with a m/z of about 301.0. An exemplary MRM transition for the quantitation of PTAD-25-hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- ¾ is fragmenting a precursor ion with a m/z of about 576.3 to a product ion with a m/z of about 298.0. However, as can be seen in the product ion scans in Figures 10B, 1 IB, and 12B, several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 10B, 1 IB, and 12B to replace or augment the exemplary fragment ions.

Table 6. Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-25- hydroxyvitamin D₂, PTAD-25-hydroxyvitamin D₂-[6, 19, 19]-²H₃, and PTAD-25- hydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆

[00126] Exemplary Ql scan spectra from the analysis of PTAD-25 -hydroxyvitamin D₃, PT AD- 25 -hydroxyvitamin D₃-[6, 19, 19]-²H₃, and PTAD-25 -hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- ¾ are shown in Figures 13 A, 14 A, and 15 A, respectively. These spectra were collected by scanning Ql across a m/z range of about 520 to 620.

[00127] Exemplary product ion scans from each of these species are presented in Figures 13B, 14B, and 15B, respectively. The precursor ions selected in Ql and the collision energies used to generate these product ion spectra are indicated in Table 7.

[00128] An exemplary MRM transition for the quantitation of PTAD-25 -hydroxyvitamin D₃ is fragmenting a precursor ion with a m/z of about 558.3 to a product ion with a m/z of about 298.1. An exemplary MRM transition for the quantitation of PTAD-25 -hydroxyvitamin D₃-[6, 19, 19]- H₃ is fragmenting a precursor ion with a m/z of about 561.3 to a product ion with a m/z of about 300.9. An exemplary MRM transition for the quantitation of PTAD-25 -hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ is fragmenting a precursor ion with a m/z of about 564.3 to a product ion with a m/z of about 298.0. However, as can be seen in the product ion scans in Figures 13B, 14B, and 15B, several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 13B, 14B, and 15B to replace or augment the preferred fragment ions.

Table 7. Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-25- hydroxyvitamin D₃, PTAD-25-hydroxyvitamin D₃-[6, 19, 19]-²H₃, and PTAD-25- hydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆

Example 7: Exemplary spectra from LDTD-MS/MS analysis of 1,25-dihydroxyvitamin D? and 1,25-dihydroxyvitamin D₃

[00129] Exemplary Ql scan spectra from the analysis of samples containing 1,25- dihydroxyvitamin D₂ and 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ are shown in Figures 16A, and 17A, respectively. These spectra were collected by scanning Ql across a m/z range of about 350 to 450.

[00130] Exemplary product ion scans generated from three different precursor ions for each of 1,25-dihydroxyvitamin D₂ and 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ are presented in Figures 16B-D, and 17B-D, respectively. The precursor ions selected in Ql and the collision energies used to generate these product ion spectra are indicated in Table 8.

[00131] Exemplary MRM transitions for the quantitation of 1,25-dihydroxyvitamin D₂ include fragmenting a precursor ion with a m/z of about 375.1 to a product ion with a m/z of about 105.3; fragmenting a precursor ion with a m/z of about 393.1 to a product ion with a m/z of about 156.9; and fragmenting a precursor ion with a m/z of about 411.1 to a product ion with a m/z of about 135.3. Exemplary MRM transitions for the quantitation of 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ include fragmenting a precursor ion with a m/z of about 381.1 to a product ion with a m/z of about 104.9; fragmenting a precursor ion with a m/z of about 399.1 to a product ion with a m/z of about 156.6; and fragmenting a precursor ion with a m/z of about 417.1 to a product ion with a m/z of about 134.9. However, as can be seen in the product ion scans in Figures 16B-D and 17B-D, several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 16B-D and 17B-D to replace or augment the exemplary fragment ions.

Table 8. Precursor Ions and Collision Cell Energies for Fragmentation of 1,25-dihydroxyvitamin

D₂ and 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆

[00132] Exemplary Ql scan spectra from the analysis of 1,25-dihydroxyvitamin D3, 1,25- dihydroxyvitamin D₃-[6, 19, 19]-²H₃, and 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ are shown in Figures 18 A, 19A, and 20A, respectively. These spectra were collected by scanning Ql across a m/z range of about 340 to 440.

[00133] Exemplary product ion scans generated from three different precursor ions for each of 1,25-dihydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃-[6, 19, 19]- H₃, and 1,25- dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ are presented in Figures 18B-D, 19B-D, and 20B-D, respectively. The precursor ions selected in Ql and the collision energies used to generate these product ion spectra are indicated in Table 9.

[00134] Exemplary MRM transitions for the quantitation of 1,25-dihydroxyvitamin D₃ include fragmenting a precursor ion with a m/z of about 363.1 to a product ion with a m/z of about 156.8; fragmenting a precursor ion with a m/z of about 381.1 to a product ion with a m/z of about 157.0; and fragmenting a precursor ion with a m/z of about 399.1 to a product ion with a m/z of about 158.8. Exemplary MRM transitions for the quantitation of 1,25-dihydroxyvitamin D₃-[6, 19, 19]- H₃ include fragmenting a precursor ion with a m/z of about 366.1 to a product ion with a m/z of about 158.8; fragmenting a precursor ion with a m/z of about 384.1 to a product ion with a m/z of about 160.2; and fragmenting a precursor ion with a m/z of about 402.1 to a product ion with a m/z of about 162.0. Exemplary MRM transitions for the quantitation of 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- ¾ include fragmenting a precursor ion with a m/z of about 369.1 to a product ion with a m/z of about 156.9.9; fragmenting a precursor ion with a m/z of about 387.1 to a product ion with a m/z of about 156.8; and fragmenting a precursor ion with a m/z of about 405.1 to a product ion with a m/z of about 158.8. However, as can be seen in the product ion scans in Figures 18B-D, 19B-D, and 20B-D, several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 18B-D, 19B-D, and 20B-D to replace or augment the exemplary fragment ions.

Table 9. Precursor Ions and Collision Cell Energies for Fragmentation of 1,25-dihydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃-[6, 19, 19]- H₃, and 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27,

27, 27]-²H₆

Example 8: Exemplary spectra from LDTD-MS/MS analysis of PTAD derivatized 1,25- dihydroxyvitamin D? and 1,25-dihydroxyvitamin D₃

[00135] PTAD derivatives of 1,25-dihydroxyvitamin D₂, 1,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆, 1,25-dihydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃-[6, 19, 19]-²H₃, and 1,25- dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-Ή₆ were prepared by treating aliquots of stock solutions of each analyte with PTAD in acetonitrile. The derivatization reactions was allowed to proceed for approximately one hour, and were quenched by adding water to the reaction mixture. The derivatized analytes were then analyzed according to the procedure outlined above in Examples 2 and 3.

[00136] Exemplary Ql scan spectra from the analysis of samples containing PTAD- 1,25- dihydroxyvitamin D₂ and PTAD- 1,25 -dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ are shown in Figures 21 A, and 22A, respectively. These spectra were collected by scanning Ql across a m/z range of about 520 to 620.

[00137] Exemplary product ion scans generated from three different precursor ions for each of PTAD-l,25-dihydroxyvitamin D₂ and PTAD-l,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- ¾ are presented in Figures 21B-D, and 22B-D, respectively. The precursor ions selected in Ql and the collision energies used to generate these product ion spectra are indicated in Table 10.

[00138] Exemplary MRM transitions for the quantitation of PTAD-l,25-dihydroxyvitamin D₂ include fragmenting a precursor ion with a m/z of about 550.4 to a product ion with a m/z of about 277.9; fragmenting a precursor ion with a m/z of about 568.4 to a product ion with a m/z of about 298.0; and fragmenting a precursor ion with a m/z of about 586.4 to a product ion with a m/z of about 314.2. Exemplary MRM transitions for the quantitation of PTAD-1,25- dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ include fragmenting a precursor ion with a m/z of about 556.4 to a product ion with a m/z of about 278.1; fragmenting a precursor ion with a m/z of about 574.4 to a product ion with a m/z of about 298.1; and fragmenting a precursor ion with a m/z of about 592.4 to a product ion with a m/z of about 313.9. However, as can be seen in the product ion scans in Figures 21B-D and 22B-D, several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 21B-D and 22B-D to replace or augment the exemplary fragment ions.

Table 10. Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-1,25- dihydroxyvitamin D₂ and PTAD-1,25 -dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-²H₆

[00139] Exemplary Ql scan spectra from the analysis of PTAD-1,25 -hydroxyvitamin D3, PTAD- 1,25 -dihydroxyvitamin D₃-[6, 19, 19]-²H₃, and PTAD-1,25 -dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆ are shown in Figures 23 A, 24A, and 25 A, respectively. These spectra were collected by scanning Ql across a m/z range of about 520 to 620. [00140] Exemplary product ion scans generated from three different precursor ions for each of PTAD-l,25-hydroxyvitamin D₃, PTAD-l,25-dihydroxyvitamin D₃-[6, 19, 19]-²H₃, and PTAD- 1,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]- H₆ are presented in Figures 23B-D, 24A-D, and 25B-D, respectively. The precursor ions selected in Ql and the collision energies used to generate these product ion spectra are indicated in Table 11.

[00141] Exemplary MRM transitions for the quantitation of PTAD- 1,25 -hydroxyvitamin D₃ include fragmenting a precursor ion with a m/z of about 538.4 to a product ion with a m/z of about 278.1 ; fragmenting a precursor ion with a m/z of about 556.4 to a product ion with a m/z of about 298.0; and fragmenting a precursor ion with a m/z of about 574.4 to a product ion with a m/z of about 313.0. Exemplary MRM transitions for the quantitation of PTAD- 1,25- dihydroxyvitamin D₃-[6, 19, 19]- H₃ include fragmenting a precursor ion with a m/z of about 541.4 to a product ion with a m/z of about 280.9; fragmenting a precursor ion with a m/z of about 559.4 to a product ion with a m/z of about 301.1; and fragmenting a precursor ion with a m/z of about 577.4 to a product ion with a m/z of about 317.3. Exemplary MRM transitions for the quantitation of PTAD- 1,25 -dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]- H₆ include fragmenting a precursor ion with a m/z of about 544.4 to a product ion with a m/z of about 278.0; fragmenting a precursor ion with a m/z of about 562.4 to a product ion with a m/z of about 298.2; and fragmenting a precursor ion with a m/z of about 580.4 to a product ion with a m/z of about 314.0. However, as can be seen in the product ion scans in Figures 23B-D, 24B-D, and 25B-D, several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in Figures 23B-D, 24B-D, and 25B-D to replace or augment the exemplary fragment ions.

Table 11. Precursor Ions and Collision Cell Energies for Fragmentation of PT AD- 1,25- dihydroxyvitamin D₃, PTAD-l,25-dihydroxyvitamin D₃-[6, 19, 19]-²H₃, and PT AD- 1,25- dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-²H₆

[00142] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[00143] The methods illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the invention embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. [00144] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the methods. This includes the generic description of the methods with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00145] Other embodiments are within the following claims. In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

THAT WHICH IS CLAIMED IS:

1. A method for determining the amount of one or more vitamin D metabolites in a sample, said method comprising: a. volatilizing the sample by heating the sample with a laser; b. ionizing one or more vitamin D metabolites in the volatilized sample to produce one or more ions detectable by mass spectrometry; and c. detecting the amount of one or more of the ions produced in step b) by mass spectrometry; wherein the amount of the one or more ions detected in step c) is related to the amount of the one or more vitamin D metabolites in the sample.

2. The method of claim 1, wherein the sample is not subjected to liquid chromatography prior to mass spectrometry.

3. The method of claim 1, wherein the sample is not subjected to chromatography prior to mass spectrometry.

4. The method of claim 1, wherein the sample is subjected to liquid chromatography prior to mass spectrometry.

5. The method of claim 1, wherein the sample is subjected to chromatography prior to mass spectrometry.

6. The method of any one of claims 1-5, wherein the one or more vitamin D metabolites in the volatilized sample are ionized with atmospheric pressure chemical ionization (APCI).

7. The method of any one of claims 1-6, wherein the sample comprises a biological sample.

8. The method of claim 7, wherein the sample comprises plasma or serum.

9. The method of claim any one of claims 1-8, wherein the sample is dried prior to heating with the laser.

10. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites comprises 25-hydroxyvitamin D₂.

11. The method of claim 10, wherein said one or more ions comprise one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 395.3 ± 0.5, 251.1 ± 0.5, 209.1 ± 0.5, and 179.1 ± 0.5.

12. The method of claim 10, wherein the mass spectrometry is tandem mass spectrometry.

13. The method of claim 12, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 395.3 ± 0.5 and one or more fragment ions selected from the group consisting of ions with a mass-to-charge ratio of 251.1 ± 0.5, 209.1 ± 0.5, and 179.1 ± 0.5.

14. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites comprises 25-hydroxyvitamin D₃.

15. The method of claim 14, wherein the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 383.2 ± 0.5, 257.1 ± 0.5, and 211.1 ± 0.5.

16. The method of claim 14, wherein the mass spectrometry is tandem mass spectrometry.

17. The method of claim 16, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 383.2 ± 0.5 and one or more fragment ions selected from the group consisting of ions with a mass-to-charge ratio of 257.1 ± 0.5 and 211.1 ± 0.5.

18. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites comprise 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂.

19. The method of claim 18, wherein the 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ are volatilized simultaneously.

20. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites are derivatized with a Cookson-type reagent prior to step a to generate one or more derivatized vitamin D metabolites.

21. The method of claim 20, wherein the Cookson-type derivatizing agent is 4-phenyl- 1,2,4- triazoline-3,5-dione (PTAD).

22. The method of claim 20, wherein the one or more vitamin D metabolites derivatized with a Cookson-type reagent comprise 25-hydroxyvitamin D₂.

23. The method of claim 20, wherein the one or more derivatized vitamin D metabolites comprise 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₂.

24. The method of claim 23, wherein said one or more ions comprise one or more ions selected from the group consisting of ions with a mass-to-charge ratio 570.3 ± 0.5 and 298.1 ± 0.5.

25. The method of claim 23, wherein the mass spectrometry is tandem mass spectrometry.

26. The method of claim 25, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 570.3 ± 0.5 and a fragment ion with a mass-to-charge ratio of 298.1 ± 0.5.

27. The method of claim 20, wherein the one or more vitamin D metabolites derivatized with a Cookson-type reagent comprise 25-hydroxyvitamin D₃.

28. The method of claim 20, wherein the one or more derivatized vitamin D metabolites comprise 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₃.

29. The method of claim 28, wherein the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 558.3 ± 0.5 and 298.1 ± 0.5.

30. The method of claim 28, wherein the mass spectrometry is tandem mass spectrometry.

31. The method of claim 30, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 558.3 ± 0.5 and a fragment ion with a mass-to-charge ratio of 298.1 ± 0.5.

32. The method of claim 20, wherein the one or more vitamin D metabolites derivatized with a Cookson-type reagent comprise 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D3.

33. The method of claim 20, wherein the one or more derivatized vitamin D comprise 4- phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D3 and 4-phenyl- 1,2,4- triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₂.

34. The method of claim 33, wherein the PTAD derivatized 25-hydroxyvitamin D3 and PTAD derivatized 25-hydroxyvitamin D₂ are volatilized simultaneously.

35. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites comprises 1,25-dihydroxyvitamin D₂.

36. The method of claim 35, wherein the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 375.1 ± 0.5, 393.1 ± 0.5, 411.1 ± 0.5, 105.3 ± 0.5, 156.9 ± 0.5, and 135.3 ± 0.5.

37. The method of claim 35, wherein the mass spectrometry is tandem mass spectrometry.

38. The method of claim 37, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 375.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 105.3 ± 0.5.

39. The method of claim 37, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 393.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 156.9 ± 0.5.

40. The method of claim 37, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 411.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 135.3 ± 0.5.

41. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites comprises 1,25-dihydroxyvitamin D₃.

42. The method of claim 41, wherein said one or more ions comprise one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 363.1 ± 0.5, 381.1 ± 0.5, 399.1 ± 0.5, 156.8 ± 0.5, 157.0 ± 0.5, and 158.8 ± 0.5.

43. The method of claim 41, wherein the mass spectrometry is tandem mass spectrometry.

44. The method of claim 43, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 363.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 156.8 ± 0.5.

45. The method of claim 43, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 381.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 157.0 ± 0.5.

46. The method of claim 43, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 399.1 ± 0.5 and a fragment ion with a mass-to-charge ratio of 158.8 ± 0.5.

47. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites comprise 1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂.

48. The method of claim 47, wherein the 1,25-dihydroxyvitamin D₃ and 1,25- dihydroxyvitamin D₂ are volatilized simultaneously.

49. The method of claim 20, wherein the one or more vitamin D metabolites derivatized with a Cookson-type reagent comprise 1,25-dihydroxyvitamin D₂.

50. The method of claim 20, wherein the one or more derivatized vitamin D metabolites comprise 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₂.

51. The method of claim 50, wherein the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 550.4 ± 0.5, 568.4 ± 0.5, 586.4 ± 0.5, 227.9 ± 0.5, 298.0 ± 0.5, and 314.2 ± 0.5.

52. The method of claim 50, wherein the mass spectrometry is tandem mass spectrometry.

53. The method of claim 52, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 550.4 ± 0.5 and a fragment ion with a mass-to-charge ratio of 227.9 ± 0.5.

54. The method of claim 52, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 568.4 ± 0.5 and a fragment ion with a mass-to-charge ratio of 298.0 ± 0.5.

55. The method of claim 52, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 586.4 ± 0.5 and a fragment ion with a mass-to-charge ratio of 314.2 ± 0.5.

56. The method of claim 20, wherein the one or more vitamin D metabolites derivatized with a Cookson-type reagent comprise 1,25-dihydroxyvitamin D3.

57. The method of claim 20, wherein the one or more derivatized vitamin D metabolites comprise 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D3.

58. The method of claim 57, wherein the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass-to-charge ratio of 538.4 ± 0.5, 556.4 ± 0.5, 574.4 ± 0.5, 278.1 ± 0.5, 298.0 ± 0.5, and 313.0 ± 0.5.

59. The method of claim 57, wherein the mass spectrometry is tandem mass spectrometry.

60. The method of claim 59, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 538.4 ± 0.5 and a fragment ion with a mass-to-charge ratio of 278.1 ± 0.5.

61. The method of claim 59, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 556.4 ± 0.5 and a fragment ion with a mass-to-charge ratio of 298.0 ± 0.5.

62. The method of claim 59, wherein the one or more ions detectable by mass spectrometry comprise a precursor ion with a mass-to-charge ratio of 574.4 ± 0.5 and a fragment ion with a mass-to-charge ratio of 313.0 ± 0.5.

63. The method of claim 20, wherein the one or more vitamin D metabolites comprise 1,25- dihydroxyvitamin D₂ and 1,25-dihydroxyvitamin D3.

64. The method of claim 20, wherein the one or more derivatized vitamin D metabolites comprise 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₂ and 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₃.

65. The method of claim 64, wherein the PTAD derivatized 1,25-dihydroxyvitamin D₂ and PTAD derivatized 1,25-dihydroxyvitamin D3 are volatilized simultaneously.

66. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites comprise two or more selected from the group consisting of 25-hydroxyvitamin D₂, 25- hydroxyvitamin D_3; 1,25-dihydroxyvitamin D₂, and 1,25-dihydroxyvitamin D₃.

67. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites comprise three or more selected from the group consisting of 25-hydroxyvitamin D₂, 25- hydroxyvitamin D_3; 1,25-dihydroxyvitamin D₂, and 1,25-dihydroxyvitamin D₃.

68. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites comprise 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₃, 1,25-dihydroxyvitamin D₂, and 1,25- dihydroxyvitamin D₃.

69. The method of any one of claims 1-9, wherein the one or more vitamin D metabolites are 25-hydroxyvitamin D₂, 25-hydroxyvitamin D_3; 1,25-dihydroxyvitamin D₂, and 1,25- dihydroxyvitamin D₃.

70. The method of claim 20, wherein the one or more vitamin D metabolites derivatized by a Cookson-type reagent comprise two or more selected from the group consisting of 25- hydroxyvitamin D₂, 25-hydroxyvitamin D_3; 1,25-dihydroxyvitamin D₂, and 1,25- dihydroxyvitamin D₃.

The method of claim 20, wherein the one or more vitamin D metabolites derivatized by Cookson-type reagent comprise three or more selected from the group consisting of 25- hydroxyvitamin D₂, 25-hydroxyvitamin D_3, 1,25-dihydroxyvitamin D₂, and 1,25- dihydroxyvitamin D₃.

72. The method of claim 20, wherein the one or more vitamin D metabolites derivatized by a Cookson-type reagent comprise 25-hydroxyvitamin D₂, 25-hydroxyvitamin D_3, 1,25- dihydroxyvitamin D₂, and 1,25-dihydroxyvitamin D₃.

73. The method of claim 20, wherein the one or more vitamin D metabolites derivatized by a Cookson-type reagent are 25-hydroxyvitamin D₂, 25-hydroxyvitamin D_3; 1,25-dihydroxyvitamin D₂, and 1,25-dihydroxyvitamin D₃.

74. The method of claim 20, wherein the one or more derivatized vitamin D metabolites comprise two or more selected from the group consisting of 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₂, 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₃, 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25- dihydroxyvitamin D₂, and 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25- dihydroxyvitamin D₃.

75. The method of claim 20, wherein the one or more derivatized vitamin D metabolites comprise three or more selected from the group consisting of 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₂, 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₃, 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25- dihydroxyvitamin D₂, and 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25- dihydroxyvitamin D₃.

76. The method of claim 20, wherein the one or more derivatized vitamin D metabolites comprise 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₂, 4- phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₃, 4-phenyl- 1,2,4- triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₂, and 4-phenyl- 1,2,4- triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₃.

77. The method of claim 20, wherein the one or more derivatized vitamin D metabolites are 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₂, 4-phenyl- 1,2,4- triazoline-3,5-dione (PTAD) derivatized 25-hydroxyvitamin D₃, 4-phenyl-l,2,4-triazoline-3,5- dione (PTAD) derivatized 1,25-dihydroxyvitamin D₂, and 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) derivatized 1,25-dihydroxyvitamin D₃.

78. A method for determining the amount of one or more vitamin D metabolites in a biological sample, said method comprising: a. ionizing vitamin D metabolites in the sample to produce one or more ions detectable by mass spectrometry; and b. detecting the amount of one or more of the ions produced in step a) by mass spectrometry; wherein the amount of the one or more ions detected in step b) is related to the amount of one or more vitamin D metabolites in the sample and wherein the sample is not subjected to chromatography prior to mass spectrometry.

79. The method of claim 78, wherein the sample comprises plasma or serum.

80. The method of any one of claims 78-79, further comprising volatilizing the biological sample by heating with a laser prior to ionizing.

81. The method of any one of claims 78-80, wherein said ionizing comprises ionizing with atmospheric pressure chemical ionization (APCI).

82. The method of any one of claims 78-81 , wherein the biological sample is dried prior to heating with the laser.

83. The method of any one of claims 78-82, wherein the one or more vitamin D metabolites are derivatized with a Cookson-type derivatizing reagent prior to step a.

84. The method of any one of claims 78-83, wherein the one or more vitamin D metabolites are derivatized with 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD) prior to step a.