LC-MS separation and detection of vitamin D metabolites

摘要

Systems, kits, and methods for quantitation of metabolites of vitamin D by liquid chromatography-mass spectrometry (LC-MS). The systems, kits, and methods described herein stabilize and/or promote the formation of the protonated molecular ion ([M+H]+) for the vitamin D metabolites in the ionization source (e.g., electrospray ionization (“ESI”)). Formation of the molecular ion does not involve loss of a water molecule from the parent molecule. Subsequent fragmentation of the [M+H]+ ion yields product ions that are specific to each molecular ion. The systems, kits, and methods described herein provide for no compromise in specificity and provide for a significant increase in sensitivity relative to previously described methods.

主权项

1. A method for detecting and/or quantifying at least one vitamin D metabolite including 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in a sample by liquid chromatography-mass spectrometry, the method comprising:
purifying 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2, if present in the sample, by liquid chromatography using a mobile phase buffer containing a source of ammonium ions to stabilize and/or promote formation of a protonated molecular ion specific to each of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2; ionizing the 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in a mass spectrometer by electrospray ionization to produce the protonated molecular ion specific to each of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2, wherein the protonated molecular ion of 25-hydroxyvitamin D3 has a mass/charge ratio (Da/e) of about 401.2 and the protonated molecular ion of 25-hydroxyvitamin D2 precursor ion has a Da/e of about 413.2; fragmenting the protonated molecular ions to produce prevalent product ions specific to each of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2, wherein the prevalent product ions specific to 25-hydroxyvitamin D3 have a Da/e of about 159.1, 131.1, 105.1, and 91.1 and the prevalent product ions specific to 25 hydroxyvitamin D2 have a Da/e of about 131.1, 107.1, 105.1, and 91.1, wherein the product ions specific to each of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 are formed by fragmentation of the protonated molecular ion without water loss; and detecting and/or quantifying a presence or an amount of at least one protonated molecular ion or product ion specific to each of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2, wherein the presence or quantity of the detected ions is related to the presence or quantity of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in the sample.