Method validation for a greener approach to the quantification of 25-hydroxy vitamin D3 in patient serum using supported liquid extraction and liquid chromatography-tandem mass spectrometry

Appendix

For LC-MS/MS analyses, the Agilent (California, United States) 1,290 infinity LC and Agilent 6,490 triple quadrupole mass spectrometer were used. The LC column used was the Pursuit Pentafluorophenyl (150 mm × 2 mm × 3 µm, Cat no. A3051150) from Phenomenex (California, United States). SLE Cartridges (ISOLUTE^® SLE+ 200 µL Supported Liquid Extraction Plate) were purchased from Biotage (Uppsala, Sweden). 96-well sample preparation plates, SLE collection plates (2 mL 96 Well Square U 5,043–9,300), and collection plate sealing mats (96 Well Square Sealing Mat 5,043–9,319) were purchased from Agilent.

The method described by Albahrani et al. was used to prepare the samples using the LLE technique. Briefly, 100 µL of serum and 100 µL of Milli-Q water were added to a glass tube, which was then vortexed for 10 s. 200 µL of methanol containing the hexa-deuterated 25[OH]D3 (235 nmol/L) was then added to the tube and vortexed again for 10 s. This mixture was then left at RT for 10 min. Following this, 1.5 mL of n-Hexane was added and vortexed in 10 bursts of 2 s each to ensure adequate mixing. The mixture was then centrifuged for 5 min at 3,000 rpm. The organic layer was then transferred into a new tube and evaporated under a stream of nitrogen gas at RT. After the solvent was completely evaporated, the sample was reconstituted in 200 µL of methanol, vortexed for 20^sec and transferred to an amber glass vial, with an insert adapter for LC-MS/MS analysis.

For SLE, 100 µL of serum and 100 µL of Milli-Q filtered water were added to a U-bottom 96-well sample preparation plate and shaken for 2 min at RT. The hexa-deuterated ISTD (235 nmol/L) was then half diluted by adding ISTD to Milli-Q water and methanol (50:25:25 – ISTD:Milli-Q: Methanol). 200 µL of diluted ISTD was then added to the liquid matrix samples and the plate was shaken for 2 min at RT. The plate was then incubated at RT for 8 min.

Spiked samples were then transferred to the SLE plate using a multichannel pipette and absorbed for 1 min. Positive pressure was then applied to the SLE plate at 10 bar for <1 s and the plate was left for 5 min at RT. 1 mL of n-Hexane was then added to the SLE wells. The plate was then left to elute via gravity for 5 min, and then positive pressure of 10 bar was applied for 2–3 s.

n-Hexane eluate was then evaporated in a rotational speed vacuum (RCV 2–33 CD Plus, Martin Christ, Germany) at 500 rpm at 40 °C for 1 h. 120 µL of methanol was then added to reconstitute the samples and shaken for 30^sec. 80 µL of Milli-Q water was then added and the plate was shaken for 30^sec and resealed using a sealing mat (Agilent 96 Well Square Sealing Mat 5,043–9,319). Samples were then loaded for LC-MS/MS analysis.