Prominence 20 uflcxr system

Samples (5μl) were separated by reverse phase HPLC using a Prominence 20 UFLCXR system (Shimadzu, Columbia MD) with a Waters (Milford, MA) BEH Phenyl column (100mm × 2.1mm 1.7 μm particle size). Solvents used were HPLC grade water with 0.1% formic acid and HPLC grade acetonitrile with 0.1% formic acid. The initial conditions were 70% water and 30% acetonitrile, increasing to 50% acetonitrile at 10 min, 90% acetonitrile at 12 min where it was held at 90% acetonitrile until 13 min before returning to the initial conditions. The eluate was delivered into a 5600 (QTOF) TripleTOF using a Duospray™ ion source (AB Sciex, Framingham, MA). The capillary voltage was set at 5.5 kV in positive ion mode with a declustering potential of 80V. The mass spectrometer was operated with a 250 ms TOF scan from 50 to 950 m/z, and 7 100 ms MS/MS product ion scans (m/z 730.5, 733.5, 746.5, 749.5, 762.5, 765.5, 768.5) from 50 to 950 per duty cycle using a collision energy of 45V with a 30V spread. Chromatograms and ion spectra, and structure and fragmentation of DMEQ-TAD adducts of vitamin D, 25D, and 24,25D used for detection, are shown in Supplementary (S)Figure 1.

An equal volume of serum was mixed with acetonitrile containing 1 μM chlorpropamide (internal standard). The samples were vortexed and then spun at 10 min at 4 °C to precipitate particulates. The supernatant was transferred to an auto sampler vial and 20 μl was separated by reverse phase on a Prominence 20 UFLCXR system (Shimadzu, Columbia, MD) with a Waters BEH C18 column (100 mm × 2.1 mm, 1.7 um particle size) and the eluate delivered into a 5600 TripleTOF using a Duospray ion source (SCIEX, Framingham, MA) operating in enhanced mode and monitoring the transition m/z 401.3–177.1.