Dionex ultimate 3000 rs hplc

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).

Liquid chromatography was performed on a Dionex UltiMate 3000 RS HPLC from Thermo Scientific (Waltham, MA, USA) equipped with a Nucleosil 100-3 C18 HD (125 × 2 mm; particle size: 3 μm) from Macherey–Nagel (Düren, Germany). The injection volume was 2 μL and the column temperature was set to 40 °C. The mobile phase consisted of solvent A: 3% ACN and 0.1% formic acid in water and solvent B: 90% ACN and 0.1% formic acid in water. The LC run (flow rate: 0.25 mL/min, total time: 52 min) started with 2% B for 3 min, followed by a linear gradient to 60% B in 30 min and another linear gradient to 100% B in 1 min, followed by an isocratic step for 10 min. After switching to 2% B in 1 min, the column was allowed to equilibrate at 2% B for 7 min.
Data for the peptide identification (peak lists of precursor and fragment ions) were obtained by data-dependent high-resolution MS/MS on a maXis UHR-ToF system (Bruker Daltonik, Bremen, Germany) in the positive ESI mode (capillary voltage: 3500 V). The ESI interface setting parameters were 180 °C drying gas temperature and 4 bar ESI nebulizer gas (N₂) pressure. The mass range of the LC-MS/MS measurements was m/z 100–1600 with a spectra scan rate of 2 Hz. Selected precursors analyzed more than twice were actively excluded from analysis for 60 s. The collision energy of the quadrupole ranged between 25 and 50 V [28 (link)].

Separation of peptides was performed with a Dionex UltiMate 3000 RS HPLC from Thermo Scientific (Waltham, MA, USA) equipped with a Nucleodur C18 Gravity-SB column (50 × 2 mm; particle size 1.8 μm) from Macherey–Nagel (Düren, Germany). The injection volume was 5 μL and the column temperature was set to 50 °C. The mobile phase consisted of solvent A and solvent B (see Section 2.2.2). The LC run (flow rate: 0.5 mL/min) started with 2% B and a gradient to 25% B in 7.9 min. After switching to 100% B in 0.1 min, an isocratic step followed for 4.5 min at 100% B (flow rate: 0.7 mL/min). At the end of the run, the column was allowed to equilibrate at 2% B for 2.5 min at a flow rate of 0.5 mL/min (total time: 15 min).
Peptide detection was carried out on an AB Sciex QTrap 5500 (Darmstadt, Germany) in positive ESI mode using the following parameters: Source temperature: 430 °C, ion spray voltage: 5.5 kV, curtain gas flow: 35 µL/min, and an entrance potential of 10 V. Details of the scheduled MRM method are shown in Table 2.