Vitamin D metabolite determination utilizing mass spectrometry following derivatization

A method for determining an amount of one or more vitamin D metabolites in a sample by mass spectrometry, the method comprising:

• (i) adding one or more internal standards to a sample;
• (ii) precipitating one or more vitamin D metabolites and one or more internal standards with acetonitrile;
• (iii) subjecting the one or more vitamin D metabolites and one or more internal standards to 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatization,
• (iv) subjecting the one or more PTAD derivatized vitamin D metabolites and one or more PTAD derivatized internal standards to solid phase extraction (SPE),
• (v) purifying the one or more PTAD derivatized vitamin D metabolites and one or more internal standards by turbulent flow liquid chromatography (TFLC);
• (vi) ionizing the one or more PTAD-derivatized vitamin D metabolites derivatives and the one or more PTAD-derivatized internal standards to produce one or more ions comprising a mass/charge (m/z) ratio selected from the group consisting of 550.4, and 568.4, and which are detectable by mass spectrometry;
• and
• (vii) determining an amount of the one or more PTAD-derivatized vitamin D metabolites. ions and the one or more PTAD-derivatized internal standards ions by mass spectrometry, wherein the amount of the one or more PTAD-derivatized vitamin D metabolites is determined from the amount of ions.