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Protease degradation of polyglutamated molecules in plasma is very fast.Protease degradation of polyglutamated molecules is variable between individuals.Polyglutamates of pemetrexed and of methotrexate are both affected.This metabolism has serious consequences for quantification studies.Most drugs are metabolized in the human body. Therefore, it is essential for therapeutic drug monitoring studies to also take into account the concentrations of drug metabolites. One of the possible metabolic activities on drugs such as pemetrexed or methotrexate is (poly)glutamation. Here, we report on a series of experiments that we performed to investigate the stability of polyglutamate metabolites in plasma.Removal of glutamate residues from pemetrexed polyglutamate by most likely proteases in human plasma is influenced by temperature as it is observed at 25 °C and even more strongly at 37 °C, but not at 4 °C. The observed protease activity is highly variable among patients; in approximately 15–20% of the patients tested it is not observed, whereas in other individuals the activity is so extensive that after 10 min, more than 50% of spiked polyglutamated pemetrexed is degraded at room temperature (5–10% of the tested individuals). Similar observations also pertain to methotrexate polyglutamates. These observations do not extend to pemetrexed and methotrexate themselves which are unaffected by this activity.Due to the considerable and, among individuals, variable protease activities on polyglutamated drug metabolites in plasma, these metabolites are virtually impossible to quantify if no precautions are taken.