|*Yun Wei, *Diane McIntosh, #Stéphane Montpas, *Connie Prosser.|
*Department of Laboratory Medicine and Pathology, University of Alberta Hospital, Edmonton, Alberta Canada
#Waters Corp. Montreal, Quebec Canada
Various liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been established to investigate Vitamin D status. The increasing demand for vitamin D testing has created pressure for diagnostic laboratories to increase analytical capacity. Developing a method with higher sample throughput and better assay sensitivity was highly desirable. Ultra Performance Liquid Chromatography (UPLC) has gained more attention recently as an attractive technique to reduce runtime and improve sensitivity. The purpose of this study was to developed an UPLC-MS/MS method to measure the serum level of 25-hydroxyvitamin D2 and D3 (25-OH D2 and D3) and compared it to the HPLC-MS/MS method in current use.
Serum samples spiked with deuterated 25-OH D3 internal standard were incubated with buffer to release the analytes and the internal standard from Vitamin D binding protein. After adding 2-propanol to precipitate the protein, the samples were vortex mixed, extracted with n-heptane, and centrifuged. The hexane layer was then removed, dried under nitrogen, and reconstituted in MeOH. The reconstituted extract was analyzed by a Waters Acquity UPLC coupled to an Applied Biosystems Sciex API 4000 Qtrap. The chromatographic separation was performed with an Acquity UPLC BEH C18 column 1.7 £gm (2.1„e30 mm) and the quantification was conducted using positive electrospray ionization in selected reaction monitoring mode.
The elution of 25-OH D2 and D3, and the internal standard was achieved between 0.62 and 0.68 minutes with a total run time of 1.6 minutes. Intra- and inter-assay CVs for 25-OH D2 were 3.3% and 4.1% at 18 nmol/L, and for 25-OH D3 were 2.7% and 3.1% at 43 nmol/L, 1.6% and 4.0% at 195 nmol/L, respectively. The linear range was 2.4 nmol/L to 1200 nmol/L for both analytes with r=0.999. In a method comparison with the HPLC-MS/MS method in use, good correlations were achieved for 25-OH D2 with y=1.0076x+0.0665 (r2=0.999, n=50) and for 25-OH D3 with y=1.0015x+0.867 (r2=0.996, n=98). However, the UPLC method has a lower detection limit of 1 nmol/L for 25-OH D2 and D3, while that of the HPLC method is 7.6 nmol/L and 4.8 nmol/L for 25-OH D2 and D3, respectively. With the reduction of runtime by 3.75-fold, the UPLC method dramatically increases throughput.
This UPLC-MS/MS method provides a rapid, sensitive and cost-effective alternative for detection of 25-OH D2 and D3 in serum. It is suitable for routine use for the investigation of Vitamin D status.