50 February / March 2020


lower molecular mass phospholipids elute in a very small number of chromatographic cycles, however the heavier molecular mass phospholipids require a substantial number of cycles to elute from the column, indeed even after 10th injection cycles some of the phospholipids are still eluting from the C18 column. The consequence of this is that the amount of suppression will vary from one injection to the next and that the amount of suppression can depend on the nature of the previous sample. Selective removal of the phospholipids will alleviate this issue, which can occur with the appropriate choice of SPE.


Conclusion


The use of sample preparation to remove matrix components is something that separation scientists need to be aware of, however it is also important to be aware of the consequences that not performing adequate sample preparation can have on the overall performance characteristics of the assay. This has greater significance within the regulated environment with the introduction of ISR, which was introduced to ensure the robustness of an assay. It has been demonstrated that the use of simple, cost effective approaches such as protein precipitation can result in greater matrix components being present in the final sample which can have a detrimental effect on the assay performance, due to the common use of mass spectrometry within a bioanalytical laboratory. Where variability is seen in a bioanalytical assay then time should be spent investigating the effects that the matrix has on the system, and then looking to address these issues through improved chromatography or the application of more selective sample preparation techniques.


In order to reduce the deleterious effects of matrix components co-eluting, it is important to be aware of the effects that the matrix components can have, and one approach is to monitor the TIC to identify when co-eluting components are coming off the column. It will also aid in determining potential types of matrix components which will allow for more selective choice of sample preparation.


Phospholipids are present at high concentrations within a range of biological fluids and are renowned for causing ion suppression with a range of compounds. Monitoring these common transitions will allow the extraction process to be optimised to remove a large proportion of these components.


References


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2 T. Sangster, M. Spence, P. Sinclair, R. Payne, C. Smith, Rapid Comms. Mass Spectrom. 8 (2004) 1361-1364


3 B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Anal. Chem. 75 (2003) 3019-3030


4 G. Liumbruno, A. A’Alessandro, G. Grazzini, L. Zolla, J., Proteomics 73 (2010) 883-507


5 G.R. Wilkinson, Advanced Drug Delivery Reviews, 27 (1997) 129- 159


6 P.J. Larger, M. Breda, D. Fraier, H. Hughes, C.A. James, J. Pharm. Biomed. Anal., 39 (2005) 206-216


7 O.A. Ismaiel, M.S. Halquist, M.Y. Elmamly, A. Shalaby, H.T. Karnes, J. Chrom. B, 875 (2) (2008) 333-343


8 R.B. Cole, A.K. Harrata, J. Amer. Soc. Mass Spectrom., 4 (7) (1993) 546-556


9 L.L. Jessome, D.A. Volmer, LCGC North America, 24 (5), 498- 510 (2006)


10 Workshop Report: Fast, D., AAPS Journal: 2009; 11: 238-241


11. F. Michopoulos, A.M. Edge, Y-T. Hui, T. Liddicoat, G. Theodoridis, I.D. Wilson, Bioanalysis 3 (24) (2011) 2747-2755


PREP-2020 Preparative & Process Chromatography Conference Announced


PREP-2020, the 33rd International Symposium on Preparative & Process Chromatography Conference, will be held at the Hyatt Regency Hotel in Baltimore, USA, from 31 May - 3 June, will be showcasing the latest scientific advances. The programme is packed with papers on biochromatography, downstream processing, QbD, monoclonal antibodies, plasmids, enzymes, vaccines, viral vectors for gene delivery, VLPs and other biopharmaceuticals, clearance of high-risk impurities such as host cell proteins, chiral molecules, SFC, fine chemicals, peptides, proteins, oligonucleotides, APIs, natural products, batch, multi-column and continuous SMB processes, column technology and equipment, monoliths, new and improved stationary phases, membrane chromatography, product quality, stability, safety and/or immunogenicity and related process control strategies, regulatory aspects and more.


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