Methods for detecting dihydroxyvitamin d metabolites by mass spectrometry

Abstract

Provided are methods of detecting the presence or amount of a dihydroxyvitamin D metabolite in a sample using mass spectrometry. The methods generally comprise ionizing a dihydorxyvitamin D metabolite in a sample and detecting the amount of the ion to determine the presence or amount of the vitamin D metabolite in the sample. In certain preferred embodiments the methods include immunopurifying the dihydroxyvitamin D metabolites prior to mass spectrometry. Also provided are methods to detect the presence or amount of two or more dihydroxyvitamin D metabolites in a single assay.

That which is claimed is: 
     
         1 . A method for determining an amount of one or more dihydroxyvitamin D metabolites in a biological sample by tandem mass spectrometry; the method comprising:
 (i) adding one or more internal standards to the sample;   (ii) purifying the one or more dihydroxyvitamin D metabolites and the one or more internal standards;   (iii) derivatizing the one or more dihydroxyvitamin D metabolites and the one or more internal standards with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD);   (iv) analyzing the amount of the derivatized one or more dihydroxyvitamin D metabolites and the derivatized one or more internal standards in the sample by tandem mass spectrometry.   
     
     
         2 . The method of  claim 1 , wherein the one or more internal standards comprise a deuterated dihydroxyvitamin D metabolite. 
     
     
         3 . The method of  claim 1 , wherein the one or more internal standards comprise at least one of d6-1α,25(OH) 2 D 2  and d3-1α,25(OH) 2 D 3 . 
     
     
         4 . The method of  claim 1 , wherein the one or more internal standards comprise at least one of 1α,25(OH) 2 D 2 -[26,26,26,27,27,27]- 2 H and 1α,25(OH) 2 D 3 -[6,19,19′]- 2 H. 
     
     
         5 . The method of  claim 1 , wherein the one or more dihydroxyvitamin D metabolites comprise at least one of 1α,25(OH) 2 D 2  and 1α,25(OH) 2 D 3 . 
     
     
         6 . The method of  claim 1 , wherein the one or more dihydroxyvitamin D metabolites comprise 1α,25(OH) 2 D 2  and 1α,25(OH) 2 D 3  and wherein the amount of the metabolites are determined in a single assay. 
     
     
         7 . The method of  claim 1 , wherein the one or more dihydroxyvitamin D metabolites comprise 1α,25(OH) 2 D 2  and wherein the analysis by tandem mass spectrometry comprises generating a precursor ion of the derivatized 1α,25(OH) 2 D 2  having a mass/charge ratio (m/z) of 411.35±0.5. 
     
     
         8 . The method of  claim 7 , wherein the analysis by tandem mass spectrometry comprises generating one or more fragment ions of the derivatized 1α,25(OH) 2 D 2  having a mass/charge ratio (m/z) selected from the group consisting of 151.12±0.5 and 135.12±0.5. 
     
     
         9 . The method of  claim 1 , wherein the one or more dihydroxyvitamin D metabolites comprise 1α,25(OH) 2 D 3  and wherein the analysis by tandem mass spectrometry comprises generating a precursor ion of the derivatized 1α,25(OH) 2 D 3  having a mass/charge ratio (m/z) of 399.35±0.5. 
     
     
         10 . The method of  claim 9 , wherein the analysis by tandem mass spectrometry comprises generating one or more fragment ions of the derivatized 1α,25(OH) 2 D 3  having a mass/charge ratio (m/z) selected from the group consisting of 151.12±0.5 and 135.12±0.5. 
     
     
         11 . The method of  claim 1 , wherein the one or more internal standards comprise 1α,25(OH) 2 D 2 -[26,26,26,27,27,27]- 2 H and wherein the analysis by tandem mass spectrometry comprises generating a precursor ion of the derivatized 1α,25(OH) 2 D 2 -[26,26,26,27,27,27]- 2 H having a mass/charge ratio (m/z) of 577.37. 
     
     
         12 . The method of  claim 11 , wherein the analysis by tandem mass spectrometry comprises generating one or more fragment ions of the derivatized 1α,25(OH) 2 D 2 -[26,26,26,27,27,27]- 2 H having a mass/charge ratio (m/z) of 317.12. 
     
     
         13 . The method of  claim 1 , wherein the one or more internal standards comprise 1α,25(OH) 2 D 3 -[6,19,19′]- 2 H and wherein the analysis by tandem mass spectrometry comprises generating a precursor ion of the derivatized 1α,25(OH) 2 D 3 -[6,19,19′]- 2 H having a mass/charge ratio (m/z) of 592.37. 
     
     
         14 . The method of  claim 13 , wherein the analysis by tandem mass spectrometry comprises generating one or more fragment ions of the derivatized 1α,25(OH) 2 D 3 -[6,19,19′]- 2 H having a mass/charge ratio (m/z) of 314.12. 
     
     
         15 . The method of  claim 1 , said purifying comprises purification by solid phase extraction, affinity separation, immunoaffinity separation, or a combination thereof. 
     
     
         16 . The method of  claim 1 , further comprising an extraction or a precipitation step. 
     
     
         17 . The method of  claim 1 , further comprising chromatography. 
     
     
         18 . The method of  claim 17 , wherein the chromatography is liquid chromatography. 
     
     
         19 . The method of  claim 17 , wherein the chromatography is high performance liquid chromatography. 
     
     
         20 . The method of  claim 1 , further comprising using a C18 column. 
     
     
         21 . The method of  claim 1 , wherein the ions generated in mass spectrometry are detected using multiple reaction monitoring (MRM). 
     
     
         22 . The method of  claim 1 , further comprising constructing a standard curve based on the precursor and fragment ions of the internal standard and determining the amount of the one or more dihydroxyvitamin D metabolites in the biological sample using the standard curve. 
     
     
         23 . The method of  claim 1 , wherein the biological sample comprises serum or plasma. 
     
     
         24 . A method for determining an amount of 1α,25(OH) 2 D 2  and 1α,25(OH) 2 D 3  in a biological sample by tandem mass spectrometry, the method comprising:
 (i) adding d6-1α,25(OH) 2 D 2  and d3-1α,25(OH) 2 D 3  to the sample; 
 (ii) extracting the 1α,25(OH) 2 D 2 , 1α,25(OH) 2 D 3 , d6-1α,25(OH) 2 D 2 , and d3-1α,25(OH) 2 D 3  from the sample; 
 (iii) purifying the 1α,25(OH) 2 D 2 , 1α,25(OH) 2 D 3 , d6-1α,25(OH) 2 D 2 , and d3-1α,25(OH) 2 D 3 ; 
 (iv) derivatizing the 1α,25(OH) 2 D 2 , 1α,25(OH) 2 D 3 , d6-1α,25(OH) 2 D 2 , and d3-1α,25(OH) 2 D 3  with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); 
 (v) analyzing the amount of the derivatized 1α,25(OH) 2 D 2 , 1α,25(OH) 2 D 3 , d6-1α,25(OH) 2 D 2 , and d3-1α,25(OH) 2 D 3  by tandem mass spectrometry; and 
 (vi) determining the amount of 1α,25(OH) 2 D 2  and 1α,25(OH) 2 D 3  in the biological sample from step (v). 
 
     
     
         25 . The method of  claim 24 , wherein the d6-1α,25(OH) 2 D 2  is 1α,25(OH) 2 D 2 -[26,26,26,27,27,27]- 2 H and wherein d3-1α,25(OH) 2 D 3  is 1α,25(OH) 2 D 3 -[6,19,19′]- 2 H. 
     
     
         26 . The method of  claim 24 , wherein the analysis by tandem mass spectrometry comprises generating a precursor ion of the derivatized 1α,25(OH) 2 D 2  having a mass/charge ratio (m/z) of 411.35±0.5 and a precursor ion of the derivatized 1α,25(OH) 2 D 3  having a m/z of 399.35±0.5. 
     
     
         27 . The method of  claim 24 , wherein the analysis by tandem mass spectrometry comprises generating one or more fragment ions of the derivatized 1α,25(OH) 2 D 2  having a mass/charge ratio (m/z) selected from the group consisting of 151.12±0.5 and 135.12±0.5 and one or more fragment ions of the derivatized 1α,25(OH) 2 D 3  having a m/z selected from the group consisting of 151.12±0.5 and 135.12±0.5. 
     
     
         28 . The method of  claim 24 , wherein the d6-1α,25(OH) 2 D 2  comprises 1α,25(OH) 2 D 2 -[26,26,26,27,27,27]- 2 H and d3-1α,25(OH) 2 D 3  comprises 1α,25(OH) 2 D 3 -[6,19,19′]- 2 H, and wherein the analysis by tandem mass spectrometry comprises generating a precursor ion of the derivatized 1α,25(OH) 2 D 2 -[26,26,26,27,27,27]- 2 H having a mass/charge ratio (m/z) of 577.37 and a precursor ion of the derivatized 1α,25(OH) 2 D 3 -[6,19,19′]- 2 H having a m/z of 592.37. 
     
     
         29 . The method of  claim 28 , wherein the analysis by tandem mass spectrometry comprises generating one or more fragment ions of the derivatized 1α,25(OH) 2 D 2 -[26,26,26,27,27,27]- 2 H having a mass/charge ratio (m/z) of 317.12 and one or more fragment ions of the derivatized 1α,25(OH) 2 D 3 -[6,19,19′]- 2 H having a mass/charge ratio (m/z) of 314.12. 
     
     
         30 . The method of  claim 24 , said purifying comprises purification by solid phase extraction, affinity separation, immunoaffinity separation, or a combination thereof. 
     
     
         31 . The method of  claim 24 , wherein said extraction comprises a precipitation step. 
     
     
         32 . The method of  claim 24 , further comprising chromatography. 
     
     
         33 . The method of  claim 32 , wherein the chromatography is liquid chromatography. 
     
     
         34 . The method of  claim 32 , wherein the chromatography is high performance liquid chromatography. 
     
     
         35 . The method of  claim 24 , further comprising using a C18 column. 
     
     
         36 . The method of  claim 24 , wherein the ions generated in mass spectrometry are detected using multiple reaction monitoring (MRM). 
     
     
         37 . The method of  claim 24 , further comprising constructing a standard curve based on the precursor and fragment ions of the internal standard and determining the amount of the one or more dihydroxyvitamin D metabolites in the biological sample using the standard curve. 
     
     
         38 . The method of  claim 23 , wherein the biological sample comprises serum or plasma.