Robert Wardle (Presenter)
Waters Corporation
Authorship: Robert M Wardle, Lisa J Calton
Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, United Kingdom
| Short Abstract Many challenges are posed by matrix interferences when analyzing vitamin D metabolites by LC-MS/MS for clinical research. In particular, lysophosphatidylcholines (LysoPCs) cause ion suppression effects in mass spectrometry. Elution profile testing was carried out using Waters® Oasis® HLB and Oasis PRiME HLB µElution plates for the analysis of vitamin D metabolites by LC-MS/MS to optimize and compare SPE conditions to protein precipitation. Oasis PRiME HLB was shown to increase analytical sensitivity of vitamin D metabolites through the removal (>99%) of the targeted LysoPCs when compared to protein precipitation and Oasis HLB. Furthermore, the workflow created was minimal, simply load the protein precipitate, wash and elute. For Research Use Only, Not for Use in Diagnostic Procedures. |
Long Abstract
Background
Many challenges are posed by matrix interferences when analyzing vitamin D metabolites by LC-MS/MS for clinical research. In particular, lysophosphatidylcholines (LysoPCs 16:0, 18:1 and 18:0) which have similar hydrophobic properties to 25-hydroxyvitamin D (25OHD) despite being structurally very different have been shown to cause ion suppression effects in mass spectrometry. This is due to difficulties in removing the LysoPCs during sample preparation and challenges in obtaining chromatographic separation from the vitamin D metabolites.
Here we describe an approach using Waters® Oasis® HLB and Oasis PRiME HLB µElution solid phase extraction (SPE) plates to assess the reduction of such interferences. Chromatographic separation of the C3-epimer enabled peak area profiles of 25OHD2, 25OHD3, 24,25diOHD3 and C3-epi-25OHD3 along with targeted LysoPCs to be compared for sample extraction methods using SPE and protein precipitation.
Methods
Serum samples containing 25OHD3 were spiked with 25OHD2, C3-epi-25OHD3 and 24,25diOH2D3 for testing. Protein precipitation using methanol and zinc sulfate(aq) was performed on 100µL aliquots of serum (containing stable labeled internal standards for 25OHD3, 25OHD2, C3-epi-25OHD3). Following centrifugation, the supernatant was transferred onto either a Waters Oasis HLB or Oasis PRiME HLB µElution plate. Elution profile testing was performed by passing different concentrations (0 – 100%) of methanol(aq) and acetonitrile(aq) through each SPE plate and collecting the eluate. Using an ACQUITY UPLC® I-Class system, diluted eluate was injected onto a 2.1 x 100mm Waters ACQUITY UPLC HSS PFP column using a water/methanol/ammonium acetate/formic acid gradient and quantified with a Waters Xevo® TQ Detector. Chromatographic conditions enabled the quantification of 25OHD2, 25OHD3, 24,25diOH2D3 and C3-epi-25OHD3 with baseline resolution of the C3-epimer in under 8min. Multiple reaction monitoring (MRM) was used to obtain peak areas for the vitamin D metabolites and parent scan of m/z 184 to monitor LysoPCs. The LysoPCs with precursor ions of m/z496, m/z522 and m/z524 were targeted (16:0, 18:1 and 18:0 respectively) and peak areas were used to create elution profiles for the LysoPCs and vitamin D metabolites. The optimized method for each SPE sorbent was selected and compared against a protein precipitation extraction.
Results
The Oasis PRiME HLB µElution plate gave the best results when using high concentrations (90 – 100%) of acetonitrile, with >99% of the targeted LysoPCs being removed when compared to both Oasis HLB and protein precipitation. This resulted in >5x increase in peak areas for all vitamin D metabolites combined when compared to Oasis HLB and >10x increase when compared to protein precipitation. Oasis HLB using methanol removed approximately 50% of the targeted LysoPCs and >5x peak areas for all vitamin D metabolites combined when compared to protein precipitation. A simple 3-step SPE protocol; load, wash and elute using Oasis PRiME HLB µElution plates was developed from the elution profile data, with only one reagent being added to perform the protein precipitation step prior to SPE.
Conclusion
Elution profiling provides a useful and simple approach to SPE method development. An optimized SPE method using Oasis PRiME HLB has been developed for the LC-MS/MS analysis of vitamin D metabolites from serum for clinical research. Oasis PRiME HLB was shown to increase analytical sensitivity of vitamin D metabolites through the removal (>99%) of the targeted LysoPCs when compared to protein precipitation and Oasis HLB. Furthermore, the workflow created was minimal, simply load the protein precipitate, wash and elute.
For Research Use Only, Not for Use in Diagnostic Procedures.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Micromass UK Ltd. |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | Micromass UK Ltd. |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |