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

Abstract

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

1 . A method for determining the amount of vitamin D 2  in a sample by tandem mass spectrometry, the method comprising the steps of:
 (i) subjecting vitamin D 2  from a sample to an ionization source under conditions suitable to generate one or more precursor ions detectable by mass spectrometry selected from the group consisting of ions with a mass to charge ratio (m/z) of 397.2±0.5 or 379.2±0.5;   (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 of the ions generated in steps (i) and (ii) by mass spectrometry; and   (iv) relating the presence of vitamin D 2  ions determined in step (iii) to the presence of vitamin D 2  in the sample,   wherein if the fragmented precursor ions comprise an ion with m/z of 397.2±0.5, the fragment ions comprise one or more ions selected from the group consisting of ions with m/z of 159.0±0.5, 146.9±0.5, 133.1±0.5, and 121.0±0.5, and if the fragmented precursor ions comprise an ion with m/z of 379.2±0.5, the fragment ions comprise one or more ions selected from the group consisting of ions with m/z of 283.2±0.5, 187.3±0.5, 175.2±0.5, and 159.0±0.5.   
     
     
         2 . The method of  claim 1 , wherein the fragmented precursor ion is an ion with a m/z of 397.2±0.5. 
     
     
         3 . The method of  claim 1 , wherein the fragmented precursor ion is an ion with a m/z of 379.2±0.5. 
     
     
         4 . The method of  claim 1 , wherein the sample is subjected to an extraction column prior to ionization. 
     
     
         5 . The method of  claim 4 , wherein the extraction column is a solid phase extraction (SPE) column. 
     
     
         6 . The method of  claim 4 , wherein the extraction column is a turbulent flow liquid chromatography (TFLC) column. 
     
     
         7 . The method of  claim 4 , wherein the sample is further subjected to an analytical column prior to ionization. 
     
     
         8 . The method of  claim 7 , wherein the analytical column is a high performance liquid chromatography (HPLC) column. 
     
     
         9 . The method of  claim 7 , wherein the extraction and analytical columns and the ionization source of step (i) are connected in an on-line fashion. 
     
     
         10 . The method of  claim 1 , wherein said ionization source is an atmospheric pressure chemical ionization (APCI) source. 
     
     
         11 . The method of  claim 1 , wherein said tandem mass spectrometry is conducted as multiple reaction monitoring, precursor ion scanning, or product ion scanning. 
     
     
         12 . The method of  claim 1 , further comprising detecting vitamin D 3  in the sample. 
     
     
         13 . The method of  claim 12 , wherein the vitamin D 2  and vitamin D 3  are ionized simultaneously. 
     
     
         14 . The method of  claim 1 , wherein the sample comprises a biological sample. 
     
     
         15 . The method of  claim 14 , wherein said biological sample is from a human, and the amount of vitamin D 2  determined in the sample is the amount present in the sample when taken from the human. 
     
     
         16 . The method of  claim 14 , wherein the sample comprises serum or plasma. 
     
     
         17 . A method for determining the amount of vitamin D 3  in a sample by tandem mass spectrometry, the method comprising the steps of:
 (i) subjecting vitamin D 3  from a sample to an ionization source under conditions suitable to generate one or more precursor ions detectable by mass spectrometry selected from the group consisting of ions with a mass to charge ratio (m/z) of 385.2±0.5 or 367.2±0.5;   (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 of the ions generated in steps (i) and (ii) by mass spectrometry; and   (iv) relating the presence of vitamin D 3  ions determined in step (iii) to the presence of vitamin D 3  in the sample,   wherein if the fragmented precursor ions comprise an ion with m/z of 385.2±0.5, the fragment ions comprise one or more ions selected from the group consisting of ions with m/z of 159.0±0.5, 147.0±0.5, 133.1±0.5, and 107.1±0.5, and if the fragmented precursor ions comprise an ion with m/z of 367.2±0.5, the fragment ions comprise one or more ions selected from the group consisting of ions with m/z of 172.2±0.5, 145.0±0.5, and 119.1±0.5.   
     
     
         18 . The method of  claim 17 , wherein the fragmented precursor ion is an ion with a m/z of 385.2±0.5. 
     
     
         19 . The method of  claim 17 , wherein the fragmented precursor ion is an ion with a m/z of 367.2±0.5. 
     
     
         20 . The method of  claim 17 , wherein the sample is subjected to an extraction column prior to ionization. 
     
     
         21 . The method of  claim 20 , wherein the extraction column is a solid phase extraction (SPE) column. 
     
     
         22 . The method of  claim 20 , wherein the extraction column is a turbulent flow liquid chromatography (TFLC) column. 
     
     
         23 . The method of  claim 20 , wherein the sample is further subjected to an analytical column prior to ionization. 
     
     
         24 . The method of  claim 23 , wherein the analytical column is a high performance liquid chromatography (HPLC) column. 
     
     
         25 . The method of  claim 23 , wherein the extraction and analytical columns and the ionization source of step (i) are connected in an on-line fashion. 
     
     
         26 . The method of  claim 17 , wherein said ionization source is an atmospheric pressure chemical ionization (APCI) source. 
     
     
         27 . The method of  claim 17 , wherein said tandem mass spectrometry is conducted as multiple reaction monitoring, precursor ion scanning, or product ion scanning. 
     
     
         28 . The method of  claim 17 , further comprising detecting vitamin D 2  in the sample. 
     
     
         29 . The method of  claim 28 , wherein the vitamin D 2  and vitamin D 3  are ionized simultaneously. 
     
     
         30 . The method of  claim 17 , wherein the sample comprises a biological sample. 
     
     
         31 . The method of  claim 30 , wherein said biological sample is from a human, and the amount of vitamin D 3  determined in the sample is the amount present in the sample when taken from the human. 
     
     
         32 . The method of  claim 30 , wherein the sample comprises serum or plasma. 
     
     
         33 . A method for determining the amounts of vitamin D 2  and vitamin D 3  in a sample by tandem mass spectrometry, the method comprising the steps of:
 (i) subjecting vitamin D 2  and vitamin D 3  in the sample to an ionization source under conditions suitable to generate one or more vitamin D 2  precursor ions detectable by mass spectrometry selected from the group of ions with a mass to charge ratio (m/z) of 397.2±0.5 and 379.2±0.5, and one or more vitamin D 3  precursor ions detectable by mass spectrometry selected from the group of ions with a mass to charge ratio (m/z) of 385.2±0.5 and 367.2±0.5;   (ii) fragmenting at least one of said vitamin D 2  precursor ions to generate one or more vitamin D 2  fragment ions detectable by mass spectrometry,
 wherein if the fragmented vitamin D 2  precursor ions comprise an ion with m/z of 397.2±0.5, the vitamin D 2  fragment ions comprise one or more ions selected from the group consisting of ions with m/z of 159.0±0.5, 146.9±0.5, 133.1±0.5, and 121.0±0.5, and if the fragmented vitamin D 2  precursor ions comprise an ion with m/z of 379.2±0.5, the vitamin D 2  fragment ions comprise one or more ions selected from the group consisting of ions with m/z of 283.2±0.5, 187.3±0.5, 175.2±0.5, and 159.0±0.5; 
   (iii) fragmenting at least one of said vitamin D 3  precursor ions to generate one or more vitamin D 3  fragment ions detectable by mass spectrometry,
 wherein if the fragmented vitamin D 3  precursor ions comprise an ion with m/z of 385.2±0.5, the vitamin D 3  fragment ions comprise one or more ions selected from the group consisting of ions with m/z of 159.0±0.5, 147.0±0.5, 133.1±0.5, and 107.1±0.5, and if the fragmented vitamin D 3  precursor ions comprise an ion with m/z of 367.2±0.5, the vitamin D 3  fragment ions comprise one or more ions selected from the group consisting of ions with m/z of 172.2±0.5, 145.0±0.5, and 119.1±0.5; 
   (iv) determining the amount of one or more of the vitamin D 2  and vitamin D 3  ions generated in steps (i), (ii) and (iii) by mass spectrometry; and   (v) relating the amounts of vitamin D 2  and vitamin D 3  ions determined in step (vi) to the amounts of vitamin D 2  and vitamin D 3  in the sample.   
     
     
         34 . The method of  claim 33 , wherein the fragmented vitamin D 2  precursor ion is an ion with m/z of 397.2±0.5. 
     
     
         35 . The method of  claim 33 , wherein the fragmented vitamin D 2  precursor ion is an ion with m/z of 379.2±0.5. 
     
     
         36 . The method of  claim 33 , wherein the fragmented vitamin D 3  precursor ion is an ion with m/z of 385.2±0.5. 
     
     
         37 . The method of  claim 33 , wherein the fragmented vitamin D 3  precursor ion is an ion with m/z of 367.2±0.5. 
     
     
         38 . The method of  claim 33 , wherein the sample is subjected to an extraction column prior to ionization. 
     
     
         39 . The method of  claim 38 , wherein the extraction column is a solid phase extraction (SPE) column. 
     
     
         40 . The method of  claim 38 , wherein the extraction column is a turbulent flow liquid chromatography (TFLC) column. 
     
     
         41 . The method of  claim 38 , wherein the sample is further subjected to an analytical column prior to ionization. 
     
     
         42 . The method of  claim 41 , wherein the analytical column is a high performance liquid chromatography (HPLC) column. 
     
     
         43 . The method of  claim 41 , wherein the extraction and analytical columns and the ionization source of step (i) are connected in an on-line fashion. 
     
     
         44 . The method of  claim 33 , wherein said ionization source is an atmospheric pressure chemical ionization (APCI) source. 
     
     
         45 . The method of  claim 33 , wherein said tandem mass spectrometry is conducted as multiple reaction monitoring, precursor ion scanning, or product ion scanning. 
     
     
         46 . The method of  claim 33 , wherein the sample comprises a biological sample. 
     
     
         47 . The method of  claim 46 , wherein said biological sample is from a human, and the amounts of vitamin D 2  and vitamin D 3  determined in the sample are the amounts present in the sample when taken from the human. 
     
     
         48 . The method of  claim 46 , wherein the sample comprises serum or plasma.