Under the auspices of the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a laboratory comparison, CCQM-P20.f, was coordinated by the Bureau International des Poids et Mesures (BIPM) in 2007/2008. Nine national measurement institutes, four expert laboratories and the BIPM participated in the comparison. Participants were required to assign the mass fraction of digoxin present as the main component in the comparison sample (CCQM-P20.f) which consisted of digoxin material obtained from a commercial supplier stated to comply with USP requirements.

In addition to assigning the mass fraction content of digoxin for the material, participants were requested, but not obliged, to provide mass fraction estimates for the minor components they identified in each sample.

In contrast with the previous round of the CCQM-P20 series, in which the mass fraction content of theophylline in two comparison samples (CCQM-P20.e.1 and CCQM-P20.e.2) was determined, a wider range of results were reported for the mass fraction content of digoxin in the CCQM-P20.f comparison.

A minority of participants did not appear to use conditions capable of fully resolving and/or quantifying the major related structure impurities present in the comparison sample. Among those that did achieve suitable separations, there was further variation in their reported quantifications of the individual and total related substance content which reflected in part the limited availability of reference standards for these materials and the resulting assumptions that had to be made regarding the structure and response factors relative to digoxin for each individual impurity. This was particularly relevant because of the span of molecular masses of the impurities present in the sample, which ranged from aglycones to glycones with tetrameric carbohydrate chains, relative to that of digoxin.

A significant additional factor also contributed to the observed variation of results. Unlike the CCQM-P20.e samples, in which the major impurities were solely related structure organic compounds, the CCQM-P20.f study material contained significant levels of residual organic solvents (ethanol, dichloromethane and to a lesser extent toluene). The majority of participants failed to detect and allow for the presence of this class of impurity, introducing a bias towards overestimation of digoxin content in most of the individual results.

However, the uncertainty budgets produced by several participants were sufficiently conservative such that their reported results were nevertheless consistent with the reference value for digoxin content assigned using a consensus mass balance approach. The results of the comparison reinforce the conclusion from previous rounds of the CCQM-P20 study that care in developing and validating the suitability of the chromatographic separation method used to resolve the main component from the related structure impurities present is essential to obtaining reliable, comparable results when using the mass balance approach to estimate purity.

This specific comparison has demonstrated that, in addition to developing an appropriate chromatographic separation, it is also important to use complementary techniques capable of detecting all potential orthogonal classes of impurities if it is desired to demonstrate a general capability to assign purity with a small (<0.2% relative) standard uncertainty.

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The final report has been peer-reviewed and approved for publication by the CCQM OAWG.