Analytical methods used for separation, detection, and quantification of metabolites formed in the human skin explants
For all methods, eluents A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. Analytes were separated on the following high-performance liquid chromatography (HPLC) columns: Agilent StableBond C18 (column A: 50 × 1.0 mm; column B: 150 × 1.0 mm; 3.5 μm particle size; Agilent), Phenomenex Luna pentafluorophenyl (column C: 150 × 1.0 mm, 3 μm particle size; Phenomenex, Torrance, CA), or Waters SunFire C18 column (column D: 150 × 2.1 mm, 3.5 μm particle size; Waters). Eluent flow rates were 0.1 and 0.3 ml/min for 1.0- and 2.1-mm diameter analytical columns, respectively. Column temperature was 40°C and injection volume was 10 μl.
| Analyte | HPLC Column | Gradient | Internal Standard | Ionization | Transitions | Cone/ Collision | LOD/LOQ |
|---|---|---|---|---|---|---|---|
| m/z | V | nM | |||||
| 4-Methylumbelliferone-β-D-glucuronide | C | 0–5 min, 3→95% B; 5–8 min, 95% B; 8–8.1 min, 95→3% B; 8.1–17 min, 3% B | p-Nitrophenol-β-D-glucuronide | ESI– | 351 > 175 | 50/14 | 2.28/7.63 |
| 17β-Estradiol-17-β-D-glucuronide | A | 0–1 min, 3% B; 1–9 min, 3→95% B; 9–17 min, 95% B; 17–17.1 min, 95→3% B; 17.1–25 min, 3% B | 4-Methylumbelliferone-β-D-glucuronide | ESI– | 447 > 271 | 50/30 | <1 |
| 17β-Estradiol-3-β-D-glucuronide | 447 > 271 | 50/32 | <1 | ||||
| 17β-Estradiol-3-sulfate | 352 > 272 | 50/30 | <1 | ||||
| Minoxidil sulfate | C | 0–5 min, 5→95% B; 5–11 min, 95% B; 11–11.1 min, 95→5% B; 11.1–20 min, 5% B | 4-Methylumbelliferone-β-D-glucuronide | ESI– | 288 > 96; 288 > 97; 288 > 105; 288 > 208 | 60/22 | <1 |
| 4′-Methylacetanilide | B | 0–5 min, 5→95% B; 5–14 min, 95% B; 14–14.1 min, 95→5% B; 14.1–22 min, 5% B | 4-Hydroxycarbazeran | ESI+ | 150 > 93; 150 > 108 | 30/20 | <1 |
| 3-Methoxy-2-naphthol | D | 0–12 min, 15→95% B; 12–16 min, 95% B; 16–16.1, 95→15% B; 16.1–26 min, 15% B | External quantification | Fluorescence | λex = 254 nm λem = 360 nm | n.a. | 1.85/5.48 |
| 4-Nitroguaiacol | A | 0–1 min, 5% B; 1–7 min, 5→95% B; 7–12 min, 95% B; 12–12.1 min, 95→5% B; 12.1–18 min, 9% B | 2,3-Dihydroxynaphthalene | ESI– | 168 > 123; 168 > 153 | 35/17; 35/12 | 4.01/13.36 |
| N-Acetylprocainamide | C | 0–1 min, 5% B; 1–6 min, 5→95% B; 6–12 min, 95% B; 12–12.1 min, 95→5% B; 12.1–22 min, 5% B | 4′-Methylacetanilide | ESI+ | 278 > 121; 278 > 162; 278 > 164; 278 > 205 | 30/20 | <1 |
| Indomethacin acyl-β-D-glucuronide | A | 0–1.5 min, 3% B; 1.5–3 min, 3→95% B; 3–13 min, 95% B; 13–13.1 min, 95→3% B; 13.1–20 min, 3% B | p-Nitrophenol-β-D-glucuronide | ESI– | 532 > 312; 532 > 356 | 50/11 | <1 |
| Diclofenac acyl-β-D-glucuronide | A | 0–1 min, 5% B; 1–5 min, 5→95% B; 5–15 min, 95% B; 15–15.1 min, 95→5% B; 15.1–22 min, 5% B | Triclosan O-β-D-glucuronide | ESI– | 470 > 193; 470 > 294; 472 > 193; 472 > 296 | 25/8 | <1 |
| Triclosan O-sulfate | A | 0–1 min, 10% B; 1–5 min, 10→95% B; 5–15 min, 95% B; 15–15.1 min, 95→10% B; 15.1–22 min, 10% B | 17β-Estradiol-3-β-D-glucuronide | ESI– | 367 > 287; 369 > 289 | 20/13 | <1 |
| Triclosan O-β-D-glucuronide | 463 > 287; 465 > 289 | 20/13 | <1 |
ESI+, electrospray ionisation in positive mode; ESI–, electrospray ionization in negative mode; LOD, lower limit of detection; LOQ, lower limit of quantification; n.a., not applicable.