Purpose Synthetic cathinones are encountered in a variety of antemortem and postmortem forensic toxicology investigations. Earlier experimental studies using fortified urine have evaluated analyte, temperature and pH-dependent variables associated with their stability. The purpose of this study was to compare experimental findings with those obtained using authentic urine from cathinone users. Methods In this report we compare cathinone concentrations in 180 authentic unpreserved urine specimens, following known periods of refrigerated storage. These findings are compared with previously published experimental data using fortified drug-free urine. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS) was used to target 22 cathinones. Quantitative results were compared in urine specimens (pH 4.5–10) following 5–17 months of storage. Results The 180 specimens resulted in 164 quantitative findings involving α-PVP, ethylone, methylone, MDPV and pentylone. Initial drug concentrations ranged from 25 ng/mL to over 100,000 ng/mL. Upon reanalysis, the percentage of drug remaining (0–119%) was correlated with storage time and specimen pH. The ability to reconfirm original results was not correlated with storage time. Instead, specimen pH was far more predictive. The relationship between initial and final drug concentration was highly pH-dependent, yielding significant correlations for α-PVP, ethylone and methylone, particularly under acidic conditions. Conclusions These results are in good agreement with experimental findings and highlight the critical importance of specimen pH, rather than conventional time dependent variables, when considering cathinone stability in biological samples. The potential for pre-analytical changes in cathinone concentrations must be carefully considered when interpreting their results.