LC-MS analysis of vitamin D2/D3 in human plasma and serum

03/25/2015

Analysis and quantification of vitamin D2/D3 metabolites 25-hydroxyvitamin D2/D3 in two human patient plasma and serum samples via reversed phase LC-MS/MS.
Samples were prepared via simple protein precipitation and two internal standards 2H3-25-OH vitamin D2/D3 were utilized.
As complete elimination of endogenous levels of 25-OH Vitamin D2/D3 in human serum/plasma is impossible, deuterated 2H6-25-OH Vitamin D2/D3 were used.
Standard curves were created from standard samples where ratio of deuterated 25-OH vitamin D2/D3 and two internal standards 2H3-25-OH vitamin D2/D3 were plotted against ratio of concentrations of the same.

Chromatographic Conditions
Column	Purospher® STAR RP-18 endcapped (2μm) Hibar® HR 100-2.1
Injection volume	10 μL
Detection	APCI-MS/MS, MRM transitions: m/z 419.3/355.1, 416.3/358.1 (vitamin D2) and m/z 407.3/159.0, 404.3/162.0 (vitamin D3)
Flow Rate	0.4 - 0.5 mL/min
Mobile Phase	A: Water, B: Methanol
Gradient	see table
Temperature	50 °C
Sample diluent (v/v)	Water
Sample/sample preparation	Patient plasma and serum prepared with commercially available vitamin D kit.

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