Simultaneous quantification of 2-pyrrolidone-5-carboxylic acid (PCA), urocanic acid (UCA), and histidine (His) in the stratum corneum by HPLC-PDA

[Display omitted] •HPLC-PDA method for PCA, UCA, and His quantification in stratum corneum was proposed.•The proposed method was selective, linear, accurate, precise, with proper LOD/LOQ.•Measurement uncertainty was assessed using a top-down approach.•Impact of gender, phototype and age in PCA, UCA and His concentrations was studied. Located in the epidermis, the stratum corneum is the most superficial layer of the skin, acting as a barrier against aggression from the external environment, preventing dehydration, and maintaining the water balance of the skin. The stratum corneum contains the Natural Hydration Factor (NMF), a mixture of hygroscopic molecules derived from filaggrin. The NMF includes 2-pyrrolidone-5-carboxylic acid (PCA), urocanic acid (UCA), and histidine (His), target biomarkers that were extracted by tape-stripping from the stratum corneum of participants for quantification in HPLC-PDA. By extracting the stratum corneum, we developed a protocol to optimize, through audience definition, the quantification of NMF biomarkers. Chromatographic analysis was performed using a YMC-Triart C18 chromatographic column, with a gradient elution of mobile phase composed of triethylammonium phosphate and acetonitrile mixture, and a photodiode array detector. HPLC-PDA procedure was selective, linear (in the range from 0.2 to 5.0 µg/mL), accurate (recovery from 92.7 to 115.1 %), precise (RSD from 0.3 to 12.1 %), and with proper detection and quantification limits. The measurement uncertainty was evaluated from validation data, with combined standard uncertainty values of 0.025–0.12 µg/mL (2.1–5.6 %), 0.004–0.28 µg/mL (2.4–12.6 %), and 0.016–0.16 µg/mL (3.2–7.9 %) for His, PCA, and UCA, respectively. Statistical analyses were performed using Monte Carlo simulation and the Mann-Whitney test, as our results were not homoscedastic and deviated from normality. The results indicate that the best audience for quantifying biomarkers were participants up to 35 years old, with all phototypes, and, preferably, female.