Chemistry Reference
In-Depth Information
7.6.2 v
AlIdAtIon
of
P
ePtIde
m
APPIng
m
ethodS
There are several critical factors that must be considered to validate a method used
for peptide mapping, and each of the factors, along with the acceptance criteria,
should be designed into a protocol or SOP.
The critical factors include robustness, the limit of detection, specificity, linear-
ity, range, accuracy, and precision. Recovery and reagent stability are also important
to consider during method validation. Recovery can be addressed by performing
either quantitative amino acid analysis, spiking studies, or radiolabeling. Many of
the validation parameters must not only address the separation, but also the fragmen-
tation or digestion, particularly when considering robustness studies. The protocol
should also include written test procedures that give a detailed description of the
analytical method. Because there is a wealth of general validation guidance avail-
able, discussion will be restricted here to areas where peptide mapping validation
might differ from other types of methods (e.g., methods for synthetic drugs).
7.6.2.1 robustness
In order to evaluate a peptide map against a standard, the chromatographic separation
must be robust. While general chromatographic robustness is covered in Chapter 5 of
this volume and should be consulted for more details, the significance and role of pH
and mobile phase composition in peptide map robustness should not be overlooked.
However, there are additional issues to consider in a peptide map method, and these
include (enzyme) reagent quality or purity, and digest stability.
When determining the robustness of the reagents used for digestion, it is common
to evaluate a protein reference standard of known composition with cleavage agents
from different lots. The number of peaks obtained, their shape, and the peak areas
are all compared in the resulting chromatograms. Because in some cases, chromato-
graphic run times can by quite long, the length of time and the conditions under
which a digest can be stored before being analyzed must also be evaluated as part of
a robustness study. Digest stability is usually evaluated by looking for significant dif-
ferences in the map resulting from the analysis of several aliquots of a single digest
stored at different conditions. It may also be desirable to investigate stability through
to several freeze-thaw cycles.
Column considerations must also be made during any proper robustness study,
because it is a well-known fact that no two chromatographic columns are created
equal. Although manufacturers today have much better control of their processes
than in the past, minor column differences can have a significant effect on the sepa-
ration of these complex samples. It is a good idea to evaluate the reference standard
on several different column lots, and to evaluate column lifetime, because as a col-
umn ages, the separation can be affected. It is also a common precaution to examine
alternative columns and to make slight modifications to the gradient profile where
necessary to achieve equivalency.
7.6.2.2 limit of detection (lod)
The LOD in a peptide map is determined by the ability of the method to distinguish
changes in the map, for example, the presence or absence of a peak. Experiments
