2. The Analysis of 25-hydroxyvitamin D in Serum Using Semi-Automated Solid Phase Extraction and LC/MS/MS
Tue 4:30 PM - PosterSplash Track 1
Donald Mason
Lisa J Calton 1, Billy J Molloy 1, Brian Keevil 2, Donald S Mason 3 and Don P Cooper 1

1Waters Corporation, Atlas Park, Manchester, M22 5PP, UK
2University Hospital South Manchester (UHSM), Manchester, UK
3Waters Corporation, Beverly, MA 01915
In recent years the demand for serum 25-hydroxyvitamin D (25OHD) analysis has increased significantly. 25OHD concentration is accepted as the clinical indicator for determining vitamin D status and is important for monitoring supplementation therapy which is available in two forms; vitamin D2 and vitamin D3. Many clinical laboratories have now adopted LC/MS/MS based methods for measuring 25OHD to enable the simultaneous and reliable measurement of 25OHD2 and 25OHD3. The analysis of 25OHD by LC/MS/MS requires a number of sample pre-treatment steps to release 25OHD from Vitamin D binding protein and to minimise matrix effects. This method describes a semi-automated sample pre-treatment protocol in combination with UPLC/MS/MS for simultaneous analysis of 25OHD2 and 25OHD3.

Sixty five anonymised samples were analysed using an established manual liquid/liquid solvent extraction LC/MS/MS method at UHSM. Calibrators were prepared in horse serum (Sigma) over the concentration range 2.5-100ng/mL and QC samples were prepared independently. Commercially available calibrator and QC material from Chromsystems were also analysed. Primary serum samples and calibrators were placed on a robotic liquid-handling system and identified by bar code to be tracked throughout the extraction procedure. Internal standard was added to the dispensed samples prior to protein precipitation. Following centrifugation (off-line), the supernatant was transferred to a conditioned Oasis® µElution SPE plate and washed. The retained analytes were eluted by the liquid-handling system and directly separated using an ACQUITY UPLC™ and detected by multiple reaction monitoring mass spectrometry (Waters TQD).

The assay was linear over the range 2.5-100ng/mL with a coefficient of determination (R2) of > 0.997. The inter- and intra-assay imprecision at three levels was less than 10%CV (n=5, over 5 days). The calculated 25OHD concentrations of the twenty patient samples were in good agreement with the LC/MS/MS measurements from UHSM, using Passing-Bablok analysis Waters=1.01(UHSM)-1.45. Ion suppression studies were conducted and demonstrated minimal ion suppression for 25OHD3.

A routine semi-automated solid phase extraction UPLC/MS/MS method for the analysis of 25OHD2 and 25OHD3 in serum has been developed. The assay has good performance characteristics (linearity, sensitivity, precision and accuracy) and allows the analysis of at least two plates per work shift. The semi-automated procedure significantly reduces the manual operation steps, eliminates solvent evaporation, reconstitution and operator variability to ensure more consistent and reproducible results.