Mass balance studies are also called “C-14 studies” or “Absorption, Metabolism, and Excretion (AME) studies”. It is important to understand what you are trying to learn from the experiment. The primary objectives of a mass balance study are generally:
- To determine the mass balance of drug-related material following dose administration
- To determine the ratio of parent drug to metabolite(s) in circulation
- To determine the primary route of excretion of drug-related material
Let’s discuss each of these in order … First, mass balance is a term that refers to balancing the amount of drug administered as a dose to the amount of drug-related material collected in human excreta (normally feces and urine, but also could include expired air and sweat). One would expect if we administered 100 drug molecules to a human subject, we should collect 100 drug-related molecules in the excreta to achieve mass balance. Because there are always errors in any measurement technique, we normally use recovery of >90% as reference for nearly complete recovery. The difference between the theoretical 100% and the actual 90% could be due to measurement errors, sample processing errors, or missing samples. Since the experiment requires accurate collection and measurement of all feces and urine for up to 14 days, there are many opportunities for errors.
To calculate mass balance, we need an accurate method for measuring drug-related material in the various human excreta. While LC/MS-MS methods are sensitive, they can only be used to quantify the amount of an analyte with a known structure. In this situation, we need to measure parent drug AND all metabolites (even ones that are previously unknown). Thus, a different tool is used. The most common tool is “labeling” the parent drug molecule with a Carbon-14 atom. Carbon-14 (C-14) only represents 0.1% of carbon in the world, so it is not commonly found in any molecule. But, we can make a drug product with extra C-14 to “label” it in a way that we can follow it with sensitive radiometric detection methods (liquid scintillation or accelerated mass spectroscopy). Thus we can compare the amount of “radioactivity” in the original dose to the amount of radioactivity in the excreta to calculate the mass balance. Radioactivity measurements are independent of chemical structure, thus total radioactivity measurements can be thought of as “parent + all metabolites”.
Second, we want to learn how much of the circulating drug is parent drug versus metabolites. This is important to evaluate the safety of each metabolite, and identify unique human metabolites. The blood or plasma can be analyzed for parent drug concentrations using standard techniques (e.g. LC/MS-MS) to allow for estimates of total exposure (AUCparent). Then the blood or plasma can be analyzed for total drug product (parent + metabolites) using radiometric detection methods to allow for estimates of total exposure (AUCparent+metabolites). The ratio of the two AUC measurements gives the proportion of total exposure represented by parent drug. Similarly, if specific assays are available for some metabolites, the proportion of each metabolite relative to total drug exposure can be calculated. These ratios are important for addressing development questions around safety metabolite testing and drug-drug interaction studies. Further, the presence of the “label” allows for identification of metabolites using LC/MS-MS methods combined with radiometric detection.
Third, the primary route of excretion (feces or urine) can be determined in a mass balance study. Normally only feces and urine are collected as human excreta, but in certain situations expired air and sweat might be obtained if excretion by those routes is expected. Depending on the specific excretion profile of the drug, the majority of radioactivity will normally end up in the urine or the feces. Radioactivity can only appear in the urine after systemic absorption, suggesting that the bioavailability is at least equal to the fraction of drug appearing in the urine. The amount of drug in the feces is a mixture of unabsorbed drug (assuming oral administration), drug excreted in the GI tract, and drug excreted in the bile.
A properly designed human mass balance study will allow you to address these three main objectives with a small number of healthy volunteers.
To learn more about how to systematically leverage the many benefits of M&S across a drug development program, read this white paper.