There are two ways in which 'difficult' samples are usually categorized: either by the problems posed by the physical nature of the post-vivo sample matrix containing the chemical entity to be analysed, or by taking into account the known reactivity, interaction and stability of the chemical entity or analyte contained within the matrix and sample tube. An assessment of all physicochemical contributions will be made before an opinion is given regarding the difficulty of analysis.

On many occasions, biologically derived samples are submitted for analysis with insufficient thought given to the post-sample integrity of the chemical entities of interest. The samples are often frozen and stored at either –20 or –80 °C eventually, with the assumption that both the matrix and chemical entities of interest are of the same composition as when sampled. This view can be seriously flawed and changes to the chemical content of the sample can occur during the sampling and storage processes. Contributions from the storage containers also complicate future determination of the analytes of interest: either through adsorption onto the containers or addition of manufactured products from the container to the solution of sample matrix.

In light of such complexities and analytical challenges, the tasks of developing and validating robust analytical methods are often outsourced to provide more cost-effective and strategic solutions.

Usually, the solution is the quantitative or qualitative determination of chemical substances found in various matrices of biological origin. This article will refer to these as 'analytes' and will propose solutions to overcoming some of the challenges to their accurate determination.

Sample preparation

The overall analytical process from sample collection to analysis can be summarized into four stages:

• Sample collection, transportation and storage.
• Homogeneous sampling and measurement.
• Isolation of the analytes from the matrix.
• Preparation of the extracts for analysis.

It is useful if the analyst(s) is/are involved in toxicological and clinical studies from the conception and planning stages so that he or she understands the physicochemical nature and fate of the drug(s) after administration. Any measures proposed to safeguard the integrity of the parent drug and metabolites of interest must also be considered and put into place. At each stage, the physicochemical contributions of endogenous and exogenous entities in the sample should be considered, along with the controlled environmental conditions.

Assessing physical problems

Analytical methods are often required to determine concentrations or identify 'parent' compounds and metabolites from samples derived from toxicological or clinical studies.

On the go...

Treatment for some of these matrices, such as plasma, serum and saliva, after initial workup, can be generalized and grouped as being similar for extraction purposes. Tears and nasal fluid require minor modifications to the group analytical procedure.

Most matrices will contain different proportions of lipids, carbohydrates, proteins, lipoproteins, nucleic acids, salts and cellular debris, and various strategies have been employed to reduce the impact of these constituents in the determination of the targeted analytes. A possible solution for the removal of particulates is to induce precipitates using filtration or centrifugation, but this may also remove the analytes from the sample. It is important to carefully consider the interaction of the analytes during the simple pretreatment clean-up strategy that is proposed.