Rapid and Simple HPLC Method for the Simultaneous Determination of Urocanic Acid Isomers in Human Skin

Abstract

A high-performance liquid chromatography (HPLC) method for the determination of urocanic acid isomers extracted from human skin has been developed. Urocanic acid is a compound present in skin, accumulated in trans form with known photoprotective properties. This isomer can be photoisomerized to cis-Urocanic acid, a mediator of the cutaneous immunosuppression.

In this work, the HPLC separation was achieved on a Cyclobond I TM 2000 β-Cyclodextrin column. The mobile phase was a 15:85v/v solution of phosphate buffer-acetonitrile in an isocratic elution, at a flow rate of 3 mL min⁻¹. The diode array detector was operated at 276 nm, and column temperature was adjusted at 20°C.

The urocanic acid isomers peaks are well-resolved and free of interference from matrix components. The extraction and analytical method developed in this work allows the quantification of urocanic acid isomers with linearity and precision, establishing a simple and fast method of urocanic acid isomer quantification extracted from skin.

Keywords:

• Analytic methods
• HPLC
• Urocanic acid

Notes

^a Tailing is defined as W_0.05/2t_w, where W_0.05 is peak width at 5% of peak height (min) and t_w is distance between peak front and peak retention measured at 5% of the peak height (min).

^b Retention factor is defined as (t_R −t₀)/t₀, where t_R is retention time of peak (min) and t₀ is void time (min). Void time = 1.21 min for the method.