US2011240840A1PendingUtilityA1

Mass spectrometric determination of cookson-derivatized, non-metabolized vitamin d

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Assignee: QUEST DIAGNOSTICS INVEST INCPriority: Dec 11, 2009Filed: Dec 9, 2010Published: Oct 6, 2011
Est. expiryDec 11, 2029(~3.4 yrs left)· nominal 20-yr term from priority
Inventors:Brett HolmquistNigel J. Clarke
G01N 30/88G01N 30/72H01J 49/004H01J 49/145G01N 2030/8813G01N 33/82G01N 2030/009G01N 30/7233G01N 2030/027
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Claims

Abstract

The invention relates to the detection of vitamin D. In a particular aspect, the invention relates to methods for detecting derivatized vitamin D by mass spectrometry.

Claims

exact text as granted — not AI-modified
1 . A method for determining the amount of vitamin D in a sample by tandem mass spectrometry, the method comprising the steps of:
 (i) subjecting Cookson-type-derivatized vitamin D from the sample to an ionization source to generate one or more precursor ions detectable by mass spectrometry;   (ii) fragmenting at least one of said precursor ions to generate one or more fragment ions detectable by mass spectrometry;   (iii) determining the amount of one or more precursor and fragment ions by mass spectrometry; and   (iv) relating the amount of ions determined in step (iii) to the amount of a vitamin D in the sample,   wherein the sample is subjected to a Cookson-type derivatization reagent under conditions sufficient to generate Cookson-type-derivatized vitamin D prior to step (i).   
     
     
         2 . The method of  claim 1 , wherein the Cookson-type-derivatized-vitamin D is subjected to an extraction column and an analytical column prior to ionization. 
     
     
         3 . The method of  claim 2 , wherein the extraction column is a solid-phase extraction (SPE) column. 
     
     
         4 . The method of  claim 2 , wherein the extraction column is a turbulent flow liquid chromatography (TFLC) column. 
     
     
         5 . The method of  claim 2 , wherein the analytical column is a high performance liquid chromatography (HPLC) column. 
     
     
         6 . The method of  claim 2 , wherein the extraction and analytical columns and the ionization source of step (i) are connected in an on-line fashion. 
     
     
         7 . The method of  claim 1 , wherein said ionization source is an atmospheric pressure chemical ionization (APCI) source. 
     
     
         8 . The method of  claim 1 , wherein said tandem mass spectrometry is conducted as multiple reaction monitoring, precursor ion scanning, or product ion scanning. 
     
     
         9 . The method of  claim 1 , wherein the Cookson-type derivatizing reagent is selected from the group consisting of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), 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-1,2,4-triazoline-3,5-dione (FMTAD), and isotopically labeled variants thereof. 
     
     
         10 . The method of  claim 1 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or an isotopically labeled variant thereof. 
     
     
         11 . The method of  claim 1 , wherein said vitamin D comprises one or more of vitamin D 2  and vitamin D 3 . 
     
     
         12 . The method of  claim 11 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); and said one or more precursor ions comprise one or more ions selected from the group consisting of ions with a mass/charge ratio (m/z) of 572.4±0.5 and 560.4±0.5. 
     
     
         13 . The method of  claim 11 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); and said one or more fragment ions comprise an ion with a mass/charge ratio (m/z) of 298.1±0.5. 
     
     
         14 . The method of  claim 1 , wherein said vitamin D comprises vitamin D 2 . 
     
     
         15 . The method of  claim 14 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); said one or more precursor ions comprise a vitamin D 2  precursor ion with a mass/charge ratio (m/z) of 572.4±0.5; and said one or more fragment ions comprise a vitamin D 2  fragment ion with a m/z of 298.1±0.5. 
     
     
         16 . The method of  claim 1 , wherein said vitamin D comprises vitamin D 3 . 
     
     
         17 . The method of  claim 16 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); said one or more precursor ions comprise a vitamin D 3  precursor ion with a mass/charge ratio (m/z) of 560.4±0.5; and said one or more fragment ions comprise a vitamin D 3  fragment ion with a m/z of 298.1±0.5. 
     
     
         18 . The method of  claim 1 , wherein two or more vitamin D derivatives are ionized simultaneously. 
     
     
         19 . The method of  claim 18 , wherein said vitamin D comprises vitamin D 2  and vitamin D 3 . 
     
     
         20 . The method of  claim 19 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); said one or more precursor ions comprise a vitamin D 2  precursor ion with a mass/charge ratio (m/z) of 572.4±0.5 and a vitamin D 3  precursor ion with a m/z of 560.4±0.5; and said one or more fragment ions comprise a vitamin D 2  fragment ion with a m/z of 298.1±0.5 and a vitamin D 3  fragment ion with a m/z of 298.1±0.5. 
     
     
         21 . The method of  claim 1 , wherein said sample comprises a biological sample. 
     
     
         22 . The method of  claim 21 , wherein said biological sample is from a human, and the amount of vitamin D determined in the sample is the amount present in the sample when taken from the human. 
     
     
         23 . The method of  claim 21 , wherein said sample comprises plasma or serum. 
     
     
         24 . A method for determining the amount of vitamin D in a sample by mass spectrometry, the method comprising the steps of:
 (i) subjecting the sample to turbulent flow liquid chromatography (TFLC);   (ii) subjecting Cookson-type-derivatized vitamin D from the sample to an ionization source under conditions suitable to generate one or more ions detectable by mass spectrometry;   (iii) determining the amount of one or more of the Cookson-type-derivatized vitamin Dions by mass spectrometry; and   (iv) relating the amount of Cookson-type-derivatized vitamin Dions determined in step (iii) to the amount of a vitamin D in the sample,   wherein the sample is subjected to a Cookson-type derivatizing reagent under conditions sufficient to generate Cookson-type-derivatized vitamin D in the sample prior to ionization.   
     
     
         25 . The method of  claim 24 , wherein the sample is subjected to high performance liquid chromatography (HPLC) after step (i) but prior to step (ii). 
     
     
         26 . The method of  claim 25 , wherein the TFLC of step (i), the HPLC, and the ionization of step (ii) are conducted in an on-line fashion. 
     
     
         27 . The method of  claim 24 , wherein the ionization source is an atmospheric pressure chemical ionization (APCI) source. 
     
     
         28 . The method of  claim 24 , wherein said mass spectrometry is tandem mass spectrometry. 
     
     
         29 . The method of  claim 28 , wherein said tandem mass spectrometry is conducted as multiple reaction monitoring, precursor ion scanning, or product ion scanning. 
     
     
         30 . The method of  claim 24 , wherein the Cookson-type derivatizing reagent is selected from the group consisting of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), 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-1,2,4-triazoline-3,5-dione (FMTAD), and isotopically labeled variants thereof. 
     
     
         31 . The method of  claim 24 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or an isotopically labeled variant thereof. 
     
     
         32 . The method of  claim 24 , wherein said vitamin D comprises one or more of vitamin D 2  and vitamin D 3 . 
     
     
         33 . The method of  claim 32 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); and said one or more precursor ions comprise one or more ions selected from the group consisting of ions with a mass/charge ratio (m/z) of 572.4±0.5 and 560.4±0.5. 
     
     
         34 . The method of  claim 32 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); and said one or more fragment ions comprise an ion with a mass/charge ratio (m/z) of 298.1±0.5. 
     
     
         35 . The method of  claim 24 , wherein said vitamin D comprises vitamin D 2 . 
     
     
         36 . The method of  claim 35 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); said one or more precursor ions comprise a vitamin D 2  precursor ion with a mass/charge ratio (m/z) of 572.4±0.5; and said one or more fragment ions comprise a vitamin D 2  fragment ion with a m/z of 298.1±0.5. 
     
     
         37 . The method of  claim 24 , wherein said vitamin D comprises vitamin D 3 . 
     
     
         38 . The method of  claim 37 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); said one or more precursor ions comprise a vitamin D 3  precursor ion with a mass/charge ratio (m/z) of 560.4±0.5; and said one or more fragment ions comprise a vitamin D 3  fragment ion with a m/z of 298.1±0.5. 
     
     
         39 . The method of  claim 24 , wherein two or more vitamin D derivatives are ionized simultaneously. 
     
     
         40 . The method of  claim 39 , wherein said vitamin D comprises vitamin D 2  and vitamin D 3 . 
     
     
         41 . The method of  claim 40 , wherein the Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); said one or more precursor ions comprise a vitamin D 2  precursor ion with a mass/charge ratio (m/z) of 572.4±0.5 and a vitamin D 3  precursor ion with a m/z of 560.4±0.5; and said one or more fragment ions comprise a vitamin D 2  fragment ion with a m/z of 298.1±0.5 and a vitamin D 3  fragment ion with a m/z of 298.1±0.5. 
     
     
         42 . The method of  claim 24 , wherein said sample comprises a biological sample. 
     
     
         43 . The method of  claim 42 , wherein said biological sample is from a human, and the amount of vitamin D determined in the sample is the amount present in the sample when taken from the human. 
     
     
         44 . The method of  claim 42 , wherein said sample comprises plasma or serum.

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