Removal of Phospholipids from Plasma Samples with an On-line Zirconium Particle Packed Guard Cartridge Followed by LC/MS/MS Analysis

Paul Rodwell, Merck/Sigma-Aldrich Company Ltd, Cambridge , United Kingdom
Xiaoning Lu, Milliporesigma/supelco, Bellefonte, Pa, U.s.a.
Hillel Brandes, Milliporesigma/supelco, Bellefonte, Pa, U.s.a.
David S. Bell, Milliporesigma/supelco, Bellefonte, Pa, U.s.a.
Klaus Buckenahl, Merck/sigma-aldrich Chemie Gmbh, Taufkirchen, Germany

Protein precipitation is widely used for sample preparation of biological fluid samples such as plasma, serum and whole blood, prior to an LC/MS/MS analysis. While the method is simple and efficient removing proteins, other highly abundant interference's in the biological fluids are not removed. In particular, phospholipids have been reported giving rise to severe effects on LC/MS/MS analysis including ion suppression and fouling on chromatographic separation. This poster describes the development and application of an on-line guard cartridge with the zirconia coated silica particles to further simplify the sample cleanup workflow.

The plasma samples spiked with analytes were prepared by standard protein precipitation followed by centrifugation. The resulting supernatants were directly injected and loaded onto the on-line guard cartridge (4 mm i.d. x 2 cm length) with zirconia coated particles. The phospholipids were retained by the cartridge while the analytes passed onto an analytical reversed-phase column (Acentis^® Express C18 5 cm x 2.1 mm).

The phospholipid removal from the protein precipitation rat plasma by the on-line guard was monitored by LC/MS/MS. The results show >95% phospholipids were removed by the guard even after 120 times of 1 µL injection of the samples.

The applicability of the on-line guard was validated with three sets of analytes including vitamin D2 and D3 metabolites, digoxin and digitoxin, and three basic pharmaceuticals. To improve sample loading mobile phase compatibility and resulting peak shapes, an LC system including a 6-port switching valve and an additional sample loading pump was set up & configured resulting in sharp peak shapes and recoveries over 95%, with a RSD <10% for over 20 injections.

The on-line cartridge for removal of phospholipids from protein precipitation Biological Fluid Samples is a viable alternative to off line sample preparation to increase throughput in LC/MS/MS with good recovery and acceptable RSDs.