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Licensed Unlicensed Requires Authentication Published by De Gruyter June 9, 2023

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

  • Gavin Turrell , Thilini Thrimawithana , Catherine Itsiopoulos , Ronda F. Greaves ORCID logo and Rosita Zakaria EMAIL logo

Corresponding author: Rosita Zakaria, PhD, Royal Melbourne Institute of Technology, Melbourne, VIC, 3083, Australia; and Murdoch Children’s Research Institute, Melbourne, VIC, 3052, Australia, E-mail:

  1. Research funding: The research described in our article was funded by RMIT University, School of Health and Biomedical Sciences, supporting the student’s Honours project.

  2. Author contributions: Mr Gavin Turrell performed the studies as part of his Honours project and wrote the first draft of the manuscript. RZ, TT, CI and RG supervised and consulted for this project as part of GT’s Honours project. GT and RZ together developed and validated the method. All authors contributed to the writing of the subsequent drafts, reviewed, edited, and approved the final manuscript.

  3. Competing interests: The author(s) state(s) no conflict of interest.

  4. Informed consent: Not applicable.

  5. Research ethics: Not applicable.


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 20sec 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 30sec. 80 µL of Milli-Q water was then added and the plate was shaken for 30sec 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.


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Received: 2023-05-03
Accepted: 2023-05-31
Published Online: 2023-06-09
Published in Print: 2023-11-27

© 2023 Walter de Gruyter GmbH, Berlin/Boston

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