The pKa values of the cancer drug methotrexate (MTX) were calculated, using experimental UV–Vis spectroscopic data and with the aid of the Stability Quotients from Absorbance Data (SQUAD) in addition to its deprotonation pathway from theoretical calculations using Gaussian 09 package (M062X functional and 6–311++g** basis set), since there is a discrepancy in the order of deprotonation. Also, the binding constants (K) of the complex formed between MTX and the nano clay called Laponite (Lap) were calculated at two pH values. From the log β values obtained with SQUAD, three pKa values for MTX were calculated: 2.68 ± 0.07, 4.11 ± 0.05 and 5.48 ± 0.01 associating the first pKa value to the carboxyl group attached to the alpha carbon and the second pKa value to the carboxyl group of the gamma carbon. The interaction between the chemical species of this drug (MTXH and MTX) and Lap was studied with UV–Vis spectrophotometry and SQUAD at pH values 2.0, and 7.5, resulting in a single equilibrium for the formation of the MTX-Lap complex with log K 4.36 ± 0.01 (1:1) and 1.99 ± 0.04 M (1:1) respectively. Lastly, the free and encapsulated MTX percentage was calculated from the chemical-species distribution diagram of the MTX-Lap system at pH 1.4, which agrees with those determined experimentally.

JCAE (CVU 508398) and EMA acknowledge and thank “Estancias Posdoctorales por México 2022 (CONACYT)” for the scholarship awarded and project CF-2019-263379, JCAE also thanks CONAHCYT for the SNI distinction awarded to him and to BYK Chemie de México for the donation of different types of Laponites.