Robert Wardle (Presenter)
Waters Corporation
Authorship: Robert M Wardle, Dominic Foley and Lisa J Calton
Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, United Kingdom
| Short Abstract Matrix interferences are challenging when analyzing vitamin D metabolites by LC-MS/MS for clinical research. An extraction method using Waters® Oasis® PRiME HLB was developed demonstrating an increase in analytical sensitivity of vitamin D metabolites through the removal of >99% of the targeted lysophosphatidylcholines when compared to protein precipitation. A simple workflow was created; loading the protein precipitate, wash and elute. The extraction method coupled with chromatographic conditions to separate 25OHD2, 25OHD3, 24,25diOHD3 and C3-epi-25OHD3 enabled good precision, analytical sensitivity and accuracy for these vitamin D metabolites using an ACQUITY UPLC Xevo TQ-S micro system. For Research Use Only, Not for use in diagnostic procedures. |
Long Abstract
Background
Matrix interferences are challenging 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 compared to protein precipitation. Chromatographic separation of 25OHD2, 25OHD3, 24,25diOHD3 and C3-epi-25OHD3 enabled peak area profiles to be compared with targeted LysoPCs for the sample extraction methods. Precision, accuracy and analytical sensitivity testing were performed using an ACQUITY UPLC I-Class Xevo TQ-S micro system to assess feasibility of the optimized method.
Methods
To assess matrix interference from LysoPCs, serum samples containing 25OHD3 were spiked with 25OHD2, C3-epi-25OHD3 and 24,25diOH2D3. Prior to extraction, stable labeled internal standards for 25OHD3, 25OHD2, 24,25diOHD3 and C3-epi-25OHD3 were added. Protein precipitation using methanol and zinc sulphate(aq) was performed on 100µL of serum sample. 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 methanol(aq) (0 – 100%) and acetonitrile(aq) (0 – 100%) through each SPE plate and collecting the eluate. 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. Multiple reaction monitoring (MRM) was used to obtain peak areas for the vitamin D metabolites and parent ion scan mode to look for LysoPCs.
The optimized developed method was assessed for precision, accuracy and analytical sensitivity using an ACQUITY I-Class UPLC Xevo TQ-S micro system. Precision was assessed using serum samples spiked low, mid and high concentrations of vitamin D metabolites over five days. Analytical sensitivity testing was performed over three days and accuracy evaluated using DEQAS and NIST SRM972a material.
Results
Matrix interference from LysoPCs studies showed that Oasis PRiME HLB µElution plate provided the highest LysoPC depletion when using high concentrations (90 – 100%) of acetonitrile, >99% of the targeted LysoPCs were removed when compared to Oasis HLB and protein precipitation. This resulted in >5x increase in peak area for all vitamin D metabolites combined when compared to Oasis HLB and >10x increase when compared to protein precipitation. Oasis HLB using a methanol elution removed approximately 50% of the targeted LysoPCs and resulted in >5x peak areas for all vitamin D metabolites combined when compared to protein precipitation.
The optimized method utilized a simple 3-step SPE protocol; load, wash and elute using Oasis PRiME HLB µElution plates, with only one reagent being added to perform the protein precipitation and release from the VDBP step, prior to SPE. Chromatographic conditions provided baseline resolution of the C3-epimers of 25OHD and also an isobaric peak in the 24,25diOHD3 transition which was later confirmed to be 25,26diOHD3.
The performance of the optimized method demonstrated a within-run and total precision of <10% CV for all vitamin D metabolites at low, mid and high concentration levels. Analytical sensitivity was determined to have a %CV of <20% and a signal to noise ratio of >10:1 at 0.5nmol/L for 24,25diOHD3 and C3-epi-25OHD3 and 1nmol/L for 25OHD2 and 25OHD3. Accuracy assessments showed that the mean bias was within ±10% bias when compared to the assigned concentrations for 25OHD3, 25OHD2 and C3-epi-25OHD3. The 25OHD3 to 24,25diOHD3 ratio was also calculated in 24 DEQAS samples, the determined ratio range was 9.9-19.0 with a mean of 13.2.
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. Good precision, accuracy and analytical sensitivity were achieved from the method, displaying the potential for a clinical research method for the analysis of 25OHD2, 25OHD3, 24,25diOHD3 and C3-epi-25OHD3 in human serum by LC-MS/MS.
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 |