Mass spectrometry method for measuring vitamin b6 in body fluids

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, wherein purifying comprises:
• (a) applying a body fluid sample and a first solvent to an extraction column under conditions suitable to reversibly retain pyridoxal 5′-phosphate on said extraction column;
• (b) applying a second solvent to said extraction column under conditions suitable to elute retained pyridoxal 5′-phosphate from the extraction column;
• (c) applying eluted pyridoxal 5′-phosphate from the extraction column to an analytical column for chromatographic separation;
• and
• (d) applying a third solvent to said analytical column to elute pyridoxal 5′-phosphate from the analytical column;
• (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.