Accuracy of the dimethylmethylene blue spectrophotometric assay in measuring the amount of encapsulated pentosan polysulfate into nanoparticles

Dimethylmethylene blue (DMMB) spectrophotometric assay was used to estimate the amount of pentosan polysulfate (PPS), a heparin-like glycosaminoglycan (GAG), encapsulated in chitosan-based nanoparticles (NPs) as an alternative method for capillary zone electrophoresis (CZE) that has been shown to lack sensitivity. The analytical performance of the DMMB method was evaluated, which revealed the response of PPS is linear in the investigated concentration range (R2 = 0.9996). The detection and quantification limits were < 0.22 µg/ml and < 0.75 µg/ml, respectively. The average recovery of the PPS was roughly 97.5%, with %DFA and %RSD < 5%. Based on indirect and direct estimation of the entrapment efficiency of the NPs, PPS encapsulated in NPs was 82.9 ± 0.084% and 84.1 ± 0.10%, respectively, with a %RSD of less than 1%. Comparison between the DMMB and CZE performances revealed that all the uncertainties related to results produced by the DMMB assay were at least 30-fold smaller than those produced by the CZE, revealing the higher precision and accuracy of the DMMB assay. The sensitivity of the DMMB was several times higher than the CZE. The DMMB also was found to have high stability and binding specificity for PPS. Overall, the DMMB was demonstrated to be more convenient than CZE in analytical terms and also in terms of practicality as a routine assay. Thus, the DMMB assay offers an alternative tool for the quantification of PPS, perhaps even in biological samples.