Jonathan Danaceau (Presenter)
Waters Corporation
Bio: Dr. Jonathan Danaceau is a Sr. Applications Chemist within Waters Corporation's Consumable Business Unit. He received his B.S. in Biology from Allegheny College (Meadville, PA) and his Ph.D. in Neuroscience from the University of Utah (Salt Lake City, UT). He has an extensive background in bioanalysis including experience in the pharmaceutical industry, forensic toxicology, and anti-doping analysis. Since 2011 he has been in the Chemistry Applied Technology group and has been focusing on sample preparation and chromatography solutions for forensic toxicology and clinical research applications.
Authorship: Jonathan Danaceau, Xin Zhang, and Erin Chambers
Waters Corporation
| Short Abstract A novel reversed-phase SPE sorbent has been developed that is designed to additionally remove phospholipids from biological samples. Pretreated samples were loaded directly onto the SPE sorbent without conditioning and equilibration. The three-step protocol (load-wash-elute) eliminated >95% of phospholipids compared to protein precipitation. This reduction was shown to have direct impacts on ion suppression of co-eluting analytes. |
Long Abstract
Background: Bioanalysis of drugs and endogenous molecules from plasma samples often involves using solid phase extraction (SPE) to clean up the matrix and concentrate the analytes of interest. Despite the advances in SPE sorbents and formats, residual phospholipids often remain in extracts and can interfere with analyses by causing ion suppression and by prematurely fouling analytical columns and MS sources. A novel reversed-phase SPE sorbent has been developed that is designed to additionally remove phospholipids from biological samples. This sorbent is also water wettable, enabling extraction without the usual requisite preconditioning and equilibration steps. Application examples of plasma corticosteroids and synthetic cannabinoids in whole blood are highlighted. The resulting methods demonstrate consistent recovery, reduced matrix effects, and the elimination of >95% of phospholipids compared to protein precipitation or other reversed-phase sorbents.
Methods: Plasma or whole blood samples were pretreated to lyse blood cells and disrupt protein binding as necessary. After pretreatment, samples were directly loaded onto the wells of SPE plates. All wells were then washed with 25% MeOH and eluted with 90:10 ACN:MeOH. The sample eluates were evaporated and reconstituted or diluted with water and analyzed by UPLC/MS/MS.
Results: Recoveries were efficient and consistent for all compounds in both panels, with %CVs of less than 7%. Matrix effects were modest and highly consistent for both methods. For corticosteroids, matrix effects averaged -10% with mean %CVs of only 2.2%. The synthetic cannabinoid panel had a mean matrix effect magnitude of 15% with an average %CV of only 5.6%. Phospholipid removal was nearly complete. Compared to protein precipitation, more than 95% of phospholipids were removed from both plasma and whole blood samples. Comparison with traditional reversed-phase SPE showed similar results, with the new sorbent providing up to a 90% reduction in residual phospholipids versus traditional reversed-phase sorbents. This reduction had a direct impact on method performance. The synthetic cannabinoid JWH-203 normally coelutes with lysophosphatidyl choline 18:0. When phospholipids remained in the extracts, matrix effects were substantial with up to 94% ion suppression that appeared to be directly related to phospholipid abundance. When phospholipids were eliminated, ion suppression was reduced to a negligible -11%.
Conclusion: The combination of phospholipid removal and water wettability and has enabled the development of simple, fast and clean extraction methods that result in excellent reproducibility, minimal matrix effects and the elimination of residual phospholipids. The advances highlighted should enable improvements in other reversed-phase SPE methods, resulting in more robust analyses with improved performance over traditional reversed-phase SPE sorbents.
For Research Use Only, Not for Use in Diagnostic Procedures.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Waters Corporation |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |