Absciex 4000 triple quadrupole mass spectrometer

25-Hydroxyvitamin D (OH-D) levels were assessed in plasma via liquid chromatography-tandem mass spectrometry on a Dionex Ultimate 3000RS HPLC (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an ABSciex 4000 triple quadrupole mass spectrometer (ABSciex, Foster City, CA, USA)^{16 (link)}. The cutoff values used for the classification of vitamin D status were as follows: < 25 nmol/l, severe deficiency; 25–49 nmol/l, deficiency; 50–74 nmol/l, insufficiency; and ≥ 75 nmol/l, normal vitamin D status.
Calcium and phosphate levels were measured via a colorimetric assay (Vista – Siemens). Calcium levels < 2.12 mmol/l and > 2.60 mmol/l indicated hypocalcemia and hypercalcemia, respectively. Phosphate levels < 0.80 mmol/l and > 1.45 mmol/l indicated hypophosphatemia and hyperphosphatemia, respectively. Calcium, phosphate and 25-OHD levels were collected from the patients’ medical records by one of the investigators (XR).

The quantification of the lesions was carried out by liquid chromatography isotope dilution tandem mass spectrometry technique. The calibration of the spectrometer with the natural lesions was based on the parameters reported previously (Belmadoui et al., 2010 (link)). The analytes were resolved on a 2 mm × 150 mm Luna C18 (2) 100 Å column (3 μ min particle size, Phenomenex) loaded with a pre-column C18 (2) cartridge using an LC system (Perkin Elmer Inc., USA) coupled with an AB Sciex 4000 triple quadrupole mass spectrometer (AB Sciex Inc., USA) working on multiple reaction monitoring mode. The chromatographic method used for the separation of the analytes started with 99% of 2 mM ammonium formate (solvent A) and 1% acetonitrile (solvent B). A gradient program using 2 mM ammonium formate (A) and acetonitrile (B) was involved, 0 → 20 min solvent B 1 → 9.8%. The system was washed for extra 5 min with isocratic solution B 15% and additional 10 min were given for re-equilibration after each analysis. The flow rate remained constant at 0.2 mL/min, the column was thermostated at 30°C and the injection volume was 15 μL. Linear responses were found for injection volumes up to 30 μL and upon dilution (see Figures S8, S10, respectively, in Supplementary Material).