Spectrophotometric recognition of tramadol using toluidine blue as acidophilic metachromatic optical probe

Tramadol (TRD), a centrally acting analgesic of significant clinical importance due to its opioid nature, necessitates precise monitoring of its levels in the body to ensure therapeutic efficacy and prevent potential toxicity. This research, aimed at overcoming the challenges of conventional methods, developed a rapid and sensitive strategy based on UV-Vis spectrophotometry method utilizing the toluidine blue (TB) as excellent optical probe. The core principle of this method relies on the selective interaction of TRD with the TB probe, leading to a measurable physical-chemical change (a stable notable shift in the absorption spectrum of UV-Vis) where the peak wavelength shifts from the TB baseline of 289 to 278.5 nm (∆λ = 11 nm). Quantitative validation demonstrated that the method exhibits an efficient linear range for TRD measurements from 0.4 µM to 2 mM (in human BP) and 4–30 mM (in human EBC), with the low limit of quantification of 0.4 µM successfully determined within this range. Also, the zeta potential results showed that a broad change from 13.4 to -0.918 mV which confirmed successful interaction of TRA with TB. Intra-day repeatability was confirmed at higher concentrations (1 mM) and it was obtained with a standard deviation (SD) of 0.038 and Inter-day repeatability demonstrated exceptional analytical robustness by recording SD = 0.0603 at a lower concentration (0.4 mM) across three working days. Furthermore, the method’s capability was validated by successfully measuring TRD in real samples such as human blood plasma (BP) and, notably, in the challenging matrix of human exhaled breath condensate samples, confirming its potential for non-invasive monitoring. Finally, selectivity analysis revealed that while the probe is inherently selective, the existence of caffeine and Phenobarbital can cause significant spectral interference that requires careful management in clinical applications; overall, this research introduces a rapid, robust, and sensitive analytical tool with high potential for use in non-invasive and rapid biomedical analysis.