Mass spectrometry method for measuring vitamin B6 in body fluid

A method for detecting the presence or amount of pyridoxal 5′-phosphate in a sample by tandem mass spectrometry, comprising:

• (i) adding an internal standard to a sample, wherein the internal standard is 2-hydroxy-6-methylpyridine-3-carboxylic acid;
• (ii) purifying the sample with an extraction column and an analytical column for chromatographic separation;
• (iii) ionizing pyridoxal 5′-phosphate and the internal standard in the sample to generate a parent ion of pyridoxal 5′-phosphate and a parent ion of the internal standard;
• (iv) generating one or more daughter ions of said parent ions, wherein said one or more daughter ions comprise a daughter ion of pyridoxal 5′-phosphate and a daughter ion of the internal standard;
• and
• (v) detecting the presence or amount of one or more said ions generated in step (iv), and determining the presence or amount of said pyridoxal 5′-phosphate in said sample from the presence or amount of one or more said ions.