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

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.

That which is claimed is: 
     
         1 . A method for determining the amount of non-metabolized vitamin D in a sample by tandem mass spectrometry, the method comprising the steps of:
 (i) purifying the sample by liquid-liquid extraction;   (ii) subjecting the sample to turbulent flow liquid chromatography (TFLC);   (iii) subjecting Cookson-type-derivatized non-metabolized vitamin D from the sample to an ionization source to generate one or more precursor ions detectable by mass spectrometry;   (iv) fragmenting at least one of said precursor ions to generate one or more fragment ions detectable by mass spectrometry;   (v) determining the amount of one or more precursor and fragment ions by mass spectrometry; and   (vi) relating the amount of ions determined in step (iii) to the amount of a non-metabolized vitamin D in the sample.   
     
     
         2 . The method of  claim 1 , wherein the Cookson-type-derivatized non-metabolized vitamin D is subjected to an extraction column and an analytical column. 
     
     
         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 analytical column is a high performance liquid chromatography (HPLC) column. 
     
     
         5 . 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. 
     
     
         6 . The method of  claim 1 , wherein said ionization source is an atmospheric pressure chemical ionization (APCI) source. 
     
     
         7 . 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. 
     
     
         8 . 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. 
     
     
         9 . The method of  claim 1 , wherein said vitamin D comprises one or more of vitamin D 2  and vitamin D 3 . 
     
     
         10 . The method of  claim 9 , 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. 
     
     
         11 . The method of  claim 9 , 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. 
     
     
         12 . The method of  claim 1 , wherein two or more non-metabolized vitamin D derivatives are ionized simultaneously. 
     
     
         13 . The method of  claim 1 , wherein said sample comprises plasma or serum. 
     
     
         14 . A method for determining the amount of non-metabolized vitamin D in a sample by tandem mass spectrometry, the method comprising the steps of:
 (i) purifying the sample by liquid-liquid extraction;   (ii) subjecting the sample to turbulent flow liquid chromatography (TFLC);   (iii) subjecting Cookson-type-derivatized non-metabolized vitamin D from the sample to an ionization source to generate one or more precursor ions detectable by mass spectrometry;   (iv) fragmenting at least one of said precursor ions to generate one or more fragment ions detectable by mass spectrometry, wherein said one or more fragment ions comprise an ion with a mass/charge ratio (m/z) of 298.1±0.5;   (v) determining the amount of one or more precursor and fragment ions by mass spectrometry; and   (vi) relating the amount of ions determined in step (iii) to the amount of a non-metabolized vitamin D in the sample.   
     
     
         15 . The method of  claim 14 , wherein the Cookson-type-derivatized non-metabolized vitamin D is subjected to an extraction column and an analytical column. 
     
     
         16 . The method of  claim 15 , wherein the extraction column is a solid-phase extraction (SPE) column. 
     
     
         17 . The method of  claim 15 , wherein the analytical column is a high performance liquid chromatography (HPLC) column. 
     
     
         18 . The method of  claim 15 , wherein the extraction and analytical columns and the ionization source of step (i) are connected in an on-line fashion. 
     
     
         19 . The method of  claim 14 , wherein said ionization source is an atmospheric pressure chemical ionization (APCI) source. 
     
     
         20 . The method of  claim 14 , 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.