The term “pharmaceutical impurity reference standard” is used to denote a substance whose properties are accepted as representative of the various impurities that may appear in pharmaceutical preparations. Such reference standards are typically provided by manufacturers of chemicals or excipients used in the manufacture of pharmaceutical products. The assignment of purity specifications for these materials is often based on results obtained from assays performed on samples taken from production batches manufactured by the supplier. As with all manufacturing processes, small quantities of unintended molecules can be generated during production, which can have an adverse impact on product quality and safety if present at levels above specified thresholds. Typical examples include environmental pollutants such as pesticides or heavy metals, where potential contamination should be below specified limits according to local regulatory requirements (e.g., the “maximum residue limit” in the European Union).
The application of pharmaceutical impurity reference standards is important to obtain meaningful assay results, which are representative of each production batch and meet the quality specifications of the manufacturer. However, there is no international standard concerning impurity reference standards that manufacturers provide or use during their manufacturing processes. There are also no established guidelines defining how impurities should be identified, isolated, and analyzed during impurity profiling assays; this process requires significant technical expertise. Such expertise can be acquired through education and training at specialized workshops (see below), but experienced scientists usually develop their own strategies based on experience gained by trial & error procedures (sometimes referred to as “torturing tools”). The latter approach represents a potential source of errors in the experimental design and, therefore, a significant challenge for scientists who want to apply reference standards in their Quality Control analytical laboratories.
Pharmaceutical impurity reference standards are therefore typically employed in either quantitative or qualitative assay systems, but not both. A typical example is the “Reference Standard for Impurities E (R1)”, which has been introduced by ICH as a non-GLP analytical method to estimate the content of anti-epileptic drug degradation products. This pharmaceutical reference standard provides relative response factors that are used to calibrate an HPLC assay targeting these impurities, although its precise identity remains undisclosed by manufacturer GlaxoSmithKline. Another example of a pharmaceutical impurity standard applied in several laboratories is amoxicillin and its impurities: clavulanic acid, cis- and trans-aconitic, and thujone (a ketone also found in absinthe). These reference materials have been purchased from Sigma-Aldrich and are available as aqueous solutions of 200, 500, and 1000 ppm for each analyte.
The application of pharmaceutical impurity reference standards is particularly important when the assays are applied to samples derived from studies carried out on patients (e.g., drug interaction studies). The compositions, volumes, and durations between consecutive study periods may not always be constant due to variability in patient compliance or other body fluid sampling issues. Furthermore, different types of biological matrices lead to different matrix effects that need to be considered during assay development (see article on “Sample Preparation”).