Development of an RP-HPLC Method for Quantifying Diclofenac Diethylamine, Methyl Salicylate, and Capsaicin in Pharmaceutical Formulation and Skin Samples

An RP-HPLC method with a UV detector was developed for the simultaneous quantification
of diclofenac diethylamine, methyl salicylate, and capsaicin in a pharmaceutical formulation and
rabbit skin samples. The separation was achieved using a Thermo Scientific ACCLAIMTM 120 C18
column (Waltham, MA, USA, 4.6 mm × 150 mm, 5 µm). The optimized elution phase consisted of
deionized water adjusted to pH = 3 using phosphoric acid mixed with acetonitrile in a 35:65% (v/v)
ratio with isocratic elution. The flow rate was set at 0.7 mL/min, and the detection was performed
at 205 nm and 25 ◦C. The method exhibits good linearity for capsaicin (0.05–70.0 µg/mL), methyl
salicylate (0.05–100.0 µg/mL), and diclofenac diethylamine (0.05–100.0 µg/mL), with low LOD values
(0.0249, 0.0271, and 0.0038 for capsaicin, methyl salicylate, and diclofenac diethylamine, respectively).
The RSD% values were below 3.0%, indicating good precision. The overall greenness score of the
method was 0.61, reflecting its environmentally friendly nature. The developed RP-HPLC method
was successfully applied to analyze Omni Hot Gel® pharmaceutical formulation and rabbit skin
permeation samples.