Mass spectrometry method for measuring vitamin b6 in body fluids

Abstract

Provided are methods of detecting the presence or amount of the active form of vitamin B6, pyridoxal 5′-phosphate, in a body fluid sample using tandem mass spectrometry coupled with liquid chromatography.

What is claimed is: 
     
         1 . A method for detecting the presence or amount of pyridoxal 5′-phosphate in a body fluid sample by tandem mass spectrometry, comprising:
 (i) purifying said sample with an extraction column and an analytical column for chromatographic separation; 
 (ii) generating a parent ion of said pyridoxal 5′-phosphate from said purified sample; 
 (iii) generating one or more daughter ions of said parent ion; and 
 (iv) detecting the presence or amount of one or more said ions generated in step (ii) or step (iii) or both, and relating the detected ions to the presence or amount of said pyridoxal 5′-phosphate in said sample. 
 
     
     
         2 . The method of  claim 1 , wherein the extraction column is a mixed mode anion exchange polymer column, and the analytical column is a C-8 analytical column. 
     
     
         3 . The method of  claim 2 , wherein the extraction column comprises particles of about 50 μm, and the analytical column comprises particles of about 3.5 μm. 
     
     
         4 . The method of  claim 1 , wherein steps (i)-(iv) are performed in an online automated fashion. 
     
     
         5 . The method of  claim 1 , wherein said parent ion has a mass/charge ratio of 248.03+/−1. 
     
     
         6 . The method of  claim 5 , wherein said one or more daughter ions comprise a daughter ion with a mass/charge ratio of 150.00+/−1. 
     
     
         7 . The method of  claim 1 , wherein said sample comprises plasma. 
     
     
         8 . The method of  claim 1 , wherein step (ii) is performed by electrospray ionization. 
     
     
         9 . The method of  claim 1 , wherein step (iii) is performed by Collision-Induced Dissociation using a neutral gas. 
     
     
         10 . The method of  claim 1 , wherein an internal standard is added to the body fluid sample prior to step (i). 
     
     
         11 . The method of  claim 1 , wherein said analytical column is a high performance liquid chromatography column. 
     
     
         12 . The method of  claim 1 , further comprising protein precipitation prior to the purification step.