Quantitative analysis of vitamin D3, vitamin D2, and metabolites thereof

A method for quantitating a vitamin D analyte in a sample, the vitamin D analyte comprising one or more of vitamin D2, vitamin D3, a metabolite of vitamin D2, and a metabolite of vitamin D3, the method comprising:

• treating a vitamin D analyte with a dienophile-containing one-step tagging reagent to form a labeled Diels-Alder adduct;
• providing a standard comprising a known concentration of a known vitamin D analyte;
• treating the known vitamin D analyte in the standard with a one-step dienophile-containing labeled Diels-Alder reagent to form a labeled standard adduct;
• mixing the labeled standard adduct and the labeled Diels-Alder adduct to form a mixture;
• separating the mixture to form separated labeled analytes;
• and
• analyzing the labeled Diels-Alder adduct using mass spectrometry;
• wherein analyzing the labeled Diels-Alder adduct comprises analyzing the separated labeled analytes and wherein the one-step dienophile-containing labeled Diels-Alder reagent used to label the standard comprises a first isobaric tag from a set of isobaric tags and the dienophile-containing one-step tagging reagent used to form the labeled Diels-Alder adduct comprises a second isobaric tag from the set of isobaric tags, that differs from the first isobaric tag.