High Performance SPE-LC-MS/MS analysis of peptides in biological matrices

Peptides represent a growing number of drugs and drug candidates in the pharmaceutical portfolio. As per 2010, 60 peptide drugs have been FDA approved, 140 peptide drugs are in clinical trials and 500 – 600 are in pre-clinical development. Annual sales of peptide drugs amount 13 billion US$ which is 1.5% of total worldwide drug sales. Protein drugs such as therapeutic antibodies represent an even bigger share of the pharmaceutical market and the total market for biopharmaceuticals (peptides and proteins) is now valued over 70 billion US$. LC-MS/MS based platforms for peptide analysis are expected to become indispensable tools for quantitative bioanalysis in support of biopharma drug development in the very near future.

Suffice it to say that quantitative bioanalysis of peptides and proteins in support of biopharmaceutical drug development is a large and rapidly growing arena for analytical instrument providers. In this arena LC-MS/MS based bioanalysis is rapidly replacing traditional ligand binding assays (LBA’s), such as immunoassays. Especially for peptide analysis LC-MS/MS is already the method of choice for many bioanalytical assays. For proteins (or peptides larger than ~ 10 kD) LBA’s are still the predominant method of choice, but proteomics related LC-MS/MS workflows are now entering the biopharma labs. These workflows are usually based on digestion of the protein and subsequent LC-MS/MS analysis of unique, so called signature peptides for quantitation. LC-MS/MS based platforms for peptide analysis are therefore expected to become indispensable tools for quantitative bioanalysis  in support of biopharma drug development in the very near future.

For clinical diagnostic purposes, LC-MS/MS analysis of peptides and proteins is also rapidly gaining interest. In a recent review article Stefan Grebe and Ravinder Singh (Mayo clinic) anticipated that clinical protein/peptide analysis by LC-MS/MS will soon see similar growth as presently seen for low molecular compounds. 

Peptide analysis presents a number of additional challenges as compared to small molecule analysis. Because of charge state distribution and size, LC-MS/MS sensitivity is usually lower than for small molecules. Poor sample stability, high adsorption and solubility issues often complicate assay development and require the elimination of  as many sample transfer steps as possible.  Selective sample prep is critical to reduce matrix effects, especially important to reduce variability for incurred samples. Simple dilute-and-shoot type sample preparation, sometimes used for small molecules will prove inadequate in almost every attempt to develop a robust, sensitive peptide assay.

The Symbiosis system for Solid Phase Extraction coupled on-line to LC-MS/MS has a number of features that make it the perfect tool to meet the analytical challenges of peptide analysis. Simple protein precipitation including addition of internal standard is usually the only sample treatment required, before samples are placed in the autosampler for on-line SPE-LC-MS/MS analysis. Tedious and error prone extra handling such as liquid-liquid extraction, SPE eluate collection, evaporation,  reconstitution, dilution, etc. are thus avoided. A large volume of sample can be loaded and concentrated on the SPE cartridge to solve sensitivity problems because the cartridge is automatically exchanged for every new sample (if required). No need for regeneration and reduced risk of cross contamination. Controlled flow rates and volumes for the SPE process make recovery  less dependent of sample variability. The complex biological matrix and low abundance of most peptides require high resolution separations resulting in average LC run times of  > 3 minutes for even the fastest separations. Ample time to do on-line SPE in parallel within the LC run time, thus eliminating the contribution of SPE time to assay throughput!

For a few typical examples of successful peptide analysis with Symbiosis please see downloads below.

For additional information and references on Symbiosis for peptide bioanalysis,  please contact us, and let us help you streamline your peptide analysis!