Development of Eco-friendly Microextraction-TLC Approach for Diphenhydramine Detection in Different Samples

Diphenhydramine (DPH) is an antihistamine drug commonly incorporated into pharmaceutical formulations such as cough syrups, antipyretics and anti-allergy medications. It has been a widely abused drug in over-the-counter medicines for day-to-day use without any prescription. Therefore, a new, greener, rapid and cost-effective methodology was developed by integrating vortex-assisted dispersive liquid-liquid microextraction (VA-DLLME) with TLC coupled to image colorimetry for the detection of diphenhydramine in biological and pharmaceutical samples. Optimisation of DPH was performed by evaluating extraction solvent and its volume (trichloroethylene, 100 µL), disperser solvent and its volume (methanol, 1000 µL), pH (11) and vortex speed and time (2000 rpm, 1 min). The sediment phase obtained from the VA-DLLME method was subjected to TLC-image colorimetry, where ethyl acetate: ammonia solution: methanol (42.5:2.5:5 v/v/v) was used as the mobile phase. The developed TLC plate was photographed under 254 nm UV light and the image was analysed using ImageJ software. Further, validation was performed according to ICH guidelines assessing parameters including the limit of detection (2.08, 1.56, 1.56 µg/spot), the limit of quantification (6.25, 3.12, 5.61 µg/spot), linearity, accuracy, precision (inter-day and intra-day), recovery (119.70%, 141.99%, 172.53%) and enrichment factor (94.74%, 90.64%, 80.47%) for blood, urine and pharmaceutical formulation, respectively.