Validation of an assay method for lidocaine, prilocaine and tetracaine using UV detection high- performance liquid chromatography

Introduction
Currently, there are limited options for immediate pain relief in cases of traumatic skin injuries, with peri-lesional lidocaine injection being the primary method. This approach is often challenging for medical personnel to administer and can cause discomfort for patients. To address these issues, researchers have formulated a novel topical anesthetic combining lidocaine (L), prilocaine (P), and tetracaine (T). The objective of this work was to develop and validate a High-Performance Liquid Chromatography (HPLC) method to initiate stability studies and biopharmaceutical assessments of this new formulation.

Materials & methods
Analytical validation was conducted in accordance with the recommendations of the International Conference on Harmonisation (ICH). Separation by HPLC (Shimadzu^®) was carried out using: Kinetex^® column (C18, 2.6 µm, 150 mm x 4.6 mm) thermostated at 25°C; mobile phase composed of acetonitrile and 40 mM phosphate buffer solvent at pH 7 (40/60, v/v); flow rate set at 1 mL/min for an injection volume of 20 µL. UV detection was used (λ = 220 nm and λ = 330 nm). The concentration range was defined as 12 to 28 µg/mL, with a target of 20 µg/mL. To validate specificity, three standard solutions were prepared and compared with a blank, one for each active compound. Linearity, precision and accuracy were determined. Forced degradation tests were carried out using 1 mL of 80 µg/mL solutions for each compound, placed in contact (100°C) in a sealed glass bottle with: 1 mL of 1 N HCl for 6 h, or 1 mL of 1 N NaOH for 1 h, or 1 mL of 10% H2O2 for 3 h. On leaving the oven, the solutions were neutralised with HCl or NaOH and/or completed with the mobile phase.

Results
The retention times (Rt) remained consistent for each compound: 7.1 ± 0.4 min for L, 3.7 ± 0.2 min for P, and 5.7 ± 0.3 min for T. The assay demonstrated: Specificity: good chromatographic peak separation was achieved (R > 1.5); The 330 nm wavelength was found to be specific for T; Linearity: the method was linear in the range of 12 to 28 µg/mL (n = 15), with a correlation coefficient (r) of 0.999 (p < 0.05) for all three compounds; Intermediate precision: the coefficient of variation was less than 1.52% (n = 27); Accuracy: Recovery rates ranged between 99% and 101%. In forced degradation studies, L and P remained stable under acidic and basic conditions. However, in oxidative conditions, 78% of L and 32% of P were degraded. T was completely degraded under all stress conditions. Importantly, no degradation products were detected at the retention times of the parent compounds

Discussion & conclusion
The analytical method has been successfully validated according to the criteria outlined in the ICH guideline Q2 (R1). This validated method allows for the accurate and precise quantification of L, P and T. With this robust analytical tool, researchers can now proceed with stability studies and biopharmaceutical assessments of the novel topical formulation.