It is important to consider the influence of the sample matrix when setting up an analytical method.
When we set up an analytical liquid chromatography (LC) method, our focus is usually on the analyte of interest. However,
other things that potentially are present in the sample can have a profound influence on the results we obtain. As a generic
term, we use "sample matrix" to describe everything that is present in the typical sample except for the analytes of interest.
If we are analysing an environmental water sample, the matrix would be water without the analyte. For a bioanalytical method
intended to measure a drug in plasma, the matrix would be untreated plasma. For a pharmaceutical formulation, it would be
a placebo with all the excipients, but no active ingredient. Only if our samples comprise a pure compound, such as with the
analysis of a raw material for purity, can we ignore the sample matrix. And in some cases, even a supposedly pure compound
may contain other things, such as reaction impurities or by-products. As a result, whenever we are developing or transferring
an LC method, it is important to consider what effect the sample matrix may have on the results. If we don't, we may find
ourselves in a troubleshooting situation when the results don't make sense.
What Do the Authorities Say?
In the pharmaceutical industry, the regulatory agencies are aware of the importance of the sample matrix. Let's consider a
few of these. The International Conference on Harmonization (ICH) publishes guidelines on validation of analytical methods
(1). One of the important factors to consider is specificity. The ICH definition states:
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.
Typically these might include impurities, degradants, matrix, etc.
You can see that the implication of this definition is that things, other than the analyte, that are present in the sample
can confuse the identification or ability to quantify the analyte of interest.
The United States Pharmacopoeia (USP) includes Chapter 1226, "Verification of Compendial Procedures" (2). Although these guidelines technically apply only to USP methods, many workers apply them to non-USP methods, as well. Included in the discussion are suggestions of tests to consider when evaluating methods:
. . . an assessment of specificity is a key parameter in verifying that a compendial procedure is suitable for use in assaying
drug substances and drug products . . . drug substances from different suppliers may have different impurity profiles that
are not addressed by the compendial test procedure. Similarly, the excipients in a drug product can vary widely among manufacturers
and may have the potential to directly interfere with the procedure or cause the formation of impurities that are not addressed
by the compendial procedure. In addition, drug products containing different excipients, antioxidants, buffers, or container
extractives may affect the recovery of the drug substance from the matrix.
Here again we see that specificity is an important consideration. The USP highlights the possibility that different drug sources may contain different impurities and that different matrices may affect
the recovery of the analytes of interest.
A third source of regulatory opinion is seen in advice about the validation of analytical methods to measure drugs present
in biological matrices (3) from the United States Food and Drug Administration (FDA). What is referred to as specificity by
the ICH and USP is called selectivity by the FDA:
Selectivity is the ability of an analytical method to differentiate and quantify the analyte in the presence of other components
in the sample. For selectivity, analyses of blank samples of the appropriate biological matrix (plasma, urine, or other matrix)
should be obtained from at least six sources. Each blank sample should be tested for interference, and selectivity should
be ensured at the lower limit of quantification (LLOQ).
You can see that the influence of the matrix is important in bioanalytical methods, as well. And, whereas drug product or
drug substance matrices are very consistent for a certain product over time, plasma can vary widely from individual to individual,
so at least six different sources of blank plasma matrix should be tested for interferences.