MSACL Conference Schedule

High Sensitivity Quantitation of 1,25-Dihydroxy Vitamin D3 (Calcitriol) in Human Plasma Using Online Sample Preparation and Triple Quadrupole Mass Spectrometry
Mon 10:00 AM - Session: Small Molecule Analytes

Calcitriol (1,25-dihydroxy vitamin D3) is the active form of vitamin D3. Endogenous to humans, vitamin D3 is involved in the regulation of calcium levels by increasing the absorption of calcium from the GI tract and is also used as a therapeutic agent in hypocalcaemia and osteoporosis. Measurement of Calcitriol is analytically challenging due to the high sensitivity and selectivity requirements. Unlike the mono-hydroxylated metabolites which are present at ng/mL levels, the dihydroxy metabolite levels can be less than 10 pg/mL.

Mass spectrometry based methods have been recently proposed which require derivatization and/or extensive off-line SPE sample cleanup to achieve the required LOQ. Here we describe an analytical strategy that incorporates two-dimensional liquid chromatography coupled to tandem mass spectrometry, minimizing upfront sample preparation and significantly improving the chromatographic resolution required for reaching low limits of detection and selectivity against endogenous interferences.


Sample preparation consisted of a simple protein precipitation step. The supernatant was directly injected into a two dimensional chromatographic system consisting of a perfusion column for the first dimension. The plug carrying the analyte was trapped and introduced onto the second chromatographic dimension coupled to electrospray ionization tandem mass spectrometry for the quantitative measurement. Results were generated through calibration against standard solutions of calcitriol using d6-calcitriol as the internal standard.


With the proposed configuration, total analysis time per sample was 16 minutes with a sample consumption of 30