Jet stream ion source

Serum samples were analyzed using a method described in detail elsewhere [29 (link)]. In short, 100 µL of serum was added internal standards (80 ng ¹³C-vitamin D₃ and ¹³C-25(OH) D₃) followed by precipitation of the protein by use of 300 µL acetonitrile. Then the solution was cleaned up by HybridSPE and acetonitrile as eluent. After evaporation, the extract was derivatized by 188 µg 4-phenyl-1,2,4-triazoline-3,5-dione for five minutes in the dark. Separation was performed on an Agilent 1200 Series HPLC mounted with a C18-column, combined with a gradient of methanol:water added ammonium formate as additive, and quantification of 25(OH)D₃, 25(OH)D₂, vitamin D₃ and vitamin D₂ were performed on an Agilent 6470 Triple Quadrupole MS equipped with a Jet Stream ion source (Agilent Technologies, Santa Clara, CA, USA). All samples were analyzed in duplicate, and a house reference sample was included in each series to ensure validity. The house reference sample was measured against a certified reference sample.

For liquid chromatography and high-resolution accurate mass spectrometry, we coupled an Agilent Technologies 1290 Infinity II UHPLC system to an Agilent Technologies 6545 Quadrupole Time-of-Flight Mass Spectrometer (i.e., QTOF-MS). Using the LC gradient profile described above, electrospray ionization (ESI) was achieved with an Agilent Jet Stream ion source. ESI was conducted in the negative ion mode (for measurement of [M – H]^- ions) with capillary and nozzle voltages of 3.5 kV and 2 kV, respectively. Drying and nebulizing gases were set to 10 L/min and 25 lb/in² (172 kPa), respectively, and a drying-gas temperature of 300 °C was used throughout. The Jet Stream ion source employed a heated nitrogen sheath gas (at 350 °C with a gas flow of 10 L/min) to improve droplet desolvation for signal enhancement. The fragmentor, skimmer, and OCT 1 RF Vpp voltages were set to 140 V, 50 V, and 300 V, respectively. The data acquisition range was from 100-2500 m/z, and the acquisition rate was 1 spectra/s. Data acquisition (Workstation B.08.00) and processing (Qualitative Analysis B.06.00) were performed via Agilent Technologies MassHunter software.