2.5. Quantification of Tryptophan (Trp), Kynurenine (Kyn), Tyrosine (Tyr), and Phenylalanine (Phe)

Trp and Kyn quantities in basolateral cell culture samples were measured using HPLC connected to a photodiode array (PDA) detector. Trp and Kyn quantification in both basolateral and topical samples and Tyr and Phe quantification in topical samples were performed using HPLC-MS. A description of the preparation procedure of the stock solutions and calibration standards can be found in Section 2 (S2) of the Supplementary Materials.

To remove the proteins in basolateral cell culture samples, 10 kDa molecular weight cut-off filters (MWCO, PES modified; VWR International; Radnor, PA, USA) were used before Trp and Kyn analysis. A maximum of 500 µL of the sample was pipetted into the filter and centrifuged at 10,000–14,000× g at 10 °C for 15 min or until all of the sample solution went through the filter. The quantification of Trp and Kyn in cleaned-up samples was performed using an Agilent HPLC system equipped with a G1315 PDA detector, a G1313A autosampler, a G1316A column oven, a G1322-A in-line degasser, and a G1312A binary pump (Agilent 1100 Series; Agilent Technologies; Waldbronn, Germany). Chromatographic separation was performed on a reversed-phase Kromasil C18 column (250 × 4.6 mm, 5 μm particle size, 100 Å; ES Industries; West Berlin, NJ, USA). Solvents A (10 mM of NaH₂PO₄ adjusted to pH 2.8) and B (methanol) were used to create a linear 17 min gradient profile to elute analytes. The flow rate was set to 0.9 mL/min and the column compartment was heated to 40 °C. The separation profile was as follows: 25% of solvent B was held for 7 min, increased to 95% B in 4 min, and held for 4 min; then solvent B was decreased to 25% in 0.1 min and kept at 25% for an additional 1.9 min. Trp and Kyn were monitored at 280 nm and 360 nm, respectively.

The concentrations of Trp and Kyn in the samples were determined using the standard calibration curve approach. The calibration curves were prepared in the range of 0.78 to 100 μM (R² = 0.99). Automatic peak area integration was performed by OpenLAB software (Lab Advisor Basic Software; Agilent Technologies) followed by manual peak inspection. The limit of detection (LOD) was determined to be 0.2–0.4 µM for Kyn and 0.4–0.7 µM for Trp for mc and mm model samples, respectively. The limit of quantification (LOQ) was determined to be 0.5–0.7 µM for Kyn and 1.1–2.1 µM for Trp for mc and mm model samples, respectively. The LODs and LOQs for the mc and mm models differed slightly due to the difference in the type of cell culture medium used for growing these models. The LODs and LOQs were calculated from standard calibration curves as presented in Equations (2) and (3).

σ is the standard error of the y-intercept from the regression analysis of the calibration standards. All the samples, including the standards, were run in triplicate. The precision and accuracy of the HPLC-PDA method can be found in Table S1 (Supplementary Materials, S2).

HPLC-MS analysis of Tyr, Phe, Trp, and Kyn was carried out on an HPLC system (Alliance 2695; Waters; Milford, MA, USA) connected to a Waters micromass ZQ mass spectrometer equipped with an electrospray ion source. The separation of analytes was performed on a reversed-phase Kromasil C18 column (250 × 4.6 mm, 5 µm particle size, 100 Å; ES Industries). Solvents A (0.1% formic acid in water) and B (0.1% formic acid in methanol) were used to create the gradient to elute the analytes. The elution profile at a flow rate of 0.8 mL/min was performed as follows: 10% B was kept for 7 min and then increased to 95% B in 8 min and held at 95% B for 10 min. After that, solvent B was decreased to 10% over 0.1 min and held at 10% for 4.9 min. The quantification of analytes was performed in selected ion reaction (SIR) mode while operating at positive polarity (Table S2; Supplementary Materials, S2). The capillary voltage was set to 3.5 kV. The cone voltage was set to 20 eV, the extractor at 3 V, and the RF lens at 0.2 V. The source temperature was set to 120 °C. The flow of desolvation gas was 800 L/h, the cone gas flow was set to 25 L/h, and the desolvation temperature was 400 °C. The inter-channel and inter-scan delays were 0.02 and 0.1 s, respectively. Dwell times were 0.25 s for all selected ions. The span window was set to 1 Da.

Data analysis was performed by using MassLynx V4.1 software (Waters). The concentrations of analytes were calculated based on the calibration standards in the range of 0.15 to 5 µM for Phe, Trp, and Kyn, and from 0.45 to 15 µM for Tyr (R² > 0.99). The LODs for topical samples were as follows: 0.3 µM for Tyr, 0.01 µM for Phe, 0.1 µM for Trp, and 0.04 µM for Kyn. The LOQs were 0.9 µM for Tyr, 0.03 µM for Phe, 0.3 µM for Trp, and 0.1 µM for Kyn. The LODs and LOQs were calculated as described in Section 2.5.1. Precision, accuracy, LODs, and LOQs for topical samples measured with HPLC-MS can be found in Table S3 (Supplementary Materials, S2).