Measurement of Interferents from Dosages in Metabolite Analyses

Abstract

Polar metabolites of and impurities from dosage solutions may elute similarly by HPLC and thereby yield inaccurate quantitative results. Therefore, impurities collected with eluate fractions containing metabolites from exposed animals may interfere in metabolite analyses. Marine organisms (shrimp) were exposed to ¹⁴C-labeled naphthalene and the resulting metabolites compared to dosage solution impurities using a ³H-labeled naphthalene internal standard obtained from exposed rats. HPLC purification is shown to remove impurities from dosage solutions prior to evaluation of metabolism. Procedures which test both for impurities and for effects of other compounds on metabolite studies are recommended, including criteria for evaluating when metabolism experiments should be conducted with purified solutions to avoid interferences due to impurities.

Determining metabolites of dosage substances is important in toxicity studies¹. For such bioanalytical measurements, trace impurities in exposure solutions may interfere with accurate analyses and lead to erroneous evaluations of metabolism'. If dosage materials are not pure, it is incumbent upon the investigator to devise purification methods appropriate to the task at hand. These needs seem to be well understood in mammalian toxicology¹. However, the special pharmacokinetics shown by aquatic organisms may require special tests for purity and extensive dosage purifications for metabolism studies using aquatic species. Reviewing the aquatic toxicology literature shows that such extensive evaluations and purifications are done only occasionally.

In aquatic organisms moderately polar aromatic metabolites often tend to accumulate while their parent aromatic compounds are eliminated rapidly^2–4. Measurements of metabolic analytes in systems displaying these toxicokinetics are therefore vulnerable to interferences by polar impurities froin dosage compounds. These impurities may mimic analytes, especially if the polar impurities and the metabolites of interest exhibit similar physiochemical properties.

As an example consider a case where 0.5% of a radiolabeled intraperitoneal dosage is polar impurities which are eliminated from an experimental animal with a half-life of 1 week, while the parent compound is eliminated with a half-life of 12 hours. Residual radioactive compounds isolated from an exposed organism after a short depuration, e.g. 60 hours, may have polar physiochemical properties and therefore be measured as metabolites of the dosage parent compound. However, these compounds may include impurities introduced by the dosage solution which were selectively retained because of their polarity. Impurities in this example may comprise only 0.5% of the original radioactivity, but are bioconcentrated to be 10% of the residual radioactivity. Thus, an apparently minor impurity in the dosage may prove to be a significant interferent, and thereby cause errors in analyses.

For toxicologic experiments, reagent evaluation or purification may use a variety of procedures which separate impurities from the radiolabeled compounds to be employed in biological experiments. Often the supplier performs purifications and typically guarantees that reagent purity exceeds 98% or 99%. However, we have found that radiolabeled compounds purchased for aromatic hydrocarbon metabolism studies, even those which contain low concentrations of polar impurities, may not be sufficiently pure for aquatic organism studies⁵. Thus, dosage evaluations must precede toxicologic experiments which are sensitive to small amounts of impurities, e.g., metabolite determinations.

We have observed and measured interferences in chromato-graphic analyses for metabolites⁵ due to small amounts of impurities in experiments, and herein illustrate effective procedures using HPLC which may be used to avoid errors in analyses. Small amounts of dosage solutions may be used for purity assessment. Up to 75 mg of exposure substance may be purified using commercially available analytical HPLC columns. Other advantages of these purifications include: (a) excellent separation of similar compounds due to the high resolution of HPLC, (b) similarity between the purification method used to test and prepare the dosage solution and the HPLC methods used to separate and detect metabolites, and (c) lower cost than performing purifications by other techniques.