Validation of a Graphite Furnace Atomic Absorption Spectrometry (GFAAS) Method for Determining Total Antimony in Organs of Mice

For more than 70 years, antimony (Sb)-based drugs have been the mainstay for leishmaniasis treatment, despite their high cardiac, hepatic, and renal toxicity. In this context, the development of alternative Sb-based formulations remains essential, and liposomal drug delivery systems represent a promising strategy to reduce toxicity and improve therapeutic outcomes. This study addresses the evaluation of Sb quantification by validating an analytical method in various biological matricesheart, liver, spleen, kidneys, and plasmausing graphite furnace atomic absorption spectrometry (GFAAS). Method validation was performed according to the INMETRO DOQ-CGCRE-008 guideline, assessing the matrix effect, linearity, limit of detection (LoD), limit of quantification (LoQ), accuracy, and precision. Significant matrix effects were observed in all of the evaluated tissues, reinforcing the need for matrix-matched calibration curves. Linearity was demonstrated over the ranges 20-100 μg/L (organs) and 20-120 μg/L (plasma), with correlation coefficients (r) exceeding 0.99. LoD values ranged from 0.10 to 3.1 mg/kg, while LoQ values were confirmed between 0.3 and 10.4 mg/kg, with recoveries from 93% to 97%. Accuracy assessments yielded recoveries from 90% to 109%, and precisionevaluated through intra- and interday analysesresulted in relative standard deviation values below 7% and Horwitz ratio values below 1.0. The method proved to be reliable, reproducible, and sensitive, meeting the validation criteria of the adopted guidelines. As pharmacokinetic and biodistribution profiles of Sb have already been established in our previous work, the validated method presented here reinforces the analytical basis required for ongoing and future preclinical evaluations of antimonial formulations.