Diploma Work 30 hp - Planned for spring 2014

Development of bioanalytical methods for peptides using on-line SPE LC-MS/MS



Background:

The main route for analysis of therapeutic peptides in plasma samples utilizes protein precipitation (PP), or partial protein precipitation, as sample preparation followed by LC-MS/MS. Although this type of method is common in many applications it has its draw-backs:

. If the demand on lower limit of quantification (LLOQ) does not allow for sample dilution there is a need for an extra concentration step by evaporation or by other means.
. For larger peptides, or peptides strongly bound to proteins, the precipitation recovery can be low and variable.
. For low LLOQ methods, or as a trade of for point 2, the extract may not be pure enough giving rice to interfering matrix effects.


Solid Phase Extraction (SPE) has been established as an alternative sample preparation technique, using reversed phase or mixed mode ion exchange sorbents. For example Waters have commercialized a general peptide sample preparation approach based on Oasis WCX (weak cation exchange) or Oasis MAX (strong anion exchange). A few bioanalytical methods utilizing on-line SPE-LC or 2-dimentional LC, coupled to MS/MS have been reported, however this type of setup is widely used in the field of proteomics. Potential benefits from on-line SPE as compared to normal SPE, are:

. Faster method development.
. Only one manual step (dilution of sample incl addition of IS).
. Possibility to detect analyte from whole sample, gives lower LLOQ.
. Less lab work per analytical run


Goal:

. Establish a general method development protocol for on-line SPE-LC-MS/MS for peptides based on current knowledge of off-line SPE and proteomic methods. The protocol should be:
. Suitable for a wide range of peptides (differences in mw, pI, lipophilicity)
. Less time consuming then development of off-line SPE methods
. The developed methods should be proven to overcome the draw-backs for PP listed above.


Work will include:

. Optimization of reversed phase LC and MS/MS parameters for a range of different peptides.
. Establishment of a work flow for optimization of on-line SPE parameters for peptides.
. Comparison of different on-line SPE materials suitability for peptide analysis.
. Comparison between developed on-line SPE and PP methods for peptide quantification.


Relevant education for this work is believed to be:

Master in analytical chemistry or biochemistry.



Business: AstraZeneca



City: Mölndal



Deadline for applications: 2013-12-06



More information: Sara Ambertnsson, 031-7761000, Sara.Ambertnsson@astrazeneca.com.