TABLE 1

Analytical conditions in the kinetic analysis of eight UGT1A isoenzymes




Column

Eluent

Flow

Injection Vol.

Detection

RT, gluc.

RT, aglycon

Quantitation
ml/min μl nm min
EED Chromolith SpeedRod 50 mM PB, pH 3/MeOH, 60:40 1 (40°C) 100 Fluorescence; λex 280, λem 305 4.1 19.1 Radiolabeled [14C]UDPGA
Scopoletin Chromolith SpeedRod 50 mM PB, pH 3/MeOH, 90:10 0.8 (5-11 min, 2.5) (40°C) 80 Fluorescence; λex 335, λem 455 4.2 8.5 External standard
4-ABP Waters Symmetry C18, 5 μm, 3.9 × 50 mm 20 mM PB, pH 6.8/MeOH, gradient 0-8 min (35-40%), 8-20 min (40-80%), 20-23 min (80-35%), 23-35 min (35% MeOH) 1 (40°C) 100 UV 275 5.8 15.6 Radiolabeled [14C]UDPGA
1-Hydroxpyrene Hypersil BDS C18, 5 μm, 4 × 150 mm 0.5% aquous acetic acid/ACN, 40:60 0.9 (20°C) 20 Fluorescence; λex 237, λem 388 2.3 6.8 External standard
1-Naphthol Waters Symmetry C18, 5 μm, 3.9 × 150 mm 50 mM PB, pH 3/MeOH, 58:42 1 (40°C) 40 Fluorescence; λex 285, λem 335 4.2 13.2 External standard
Entacapone Hypersil BDS C18, 5 μm, 4 × 150 mm 0.5% aqueous acetic acid/ACN, 65:35 1 (40°C) 50 UV 310 2.6 6.5 External standard
4-Hydroxyestrone Chromolith SpeedRod 50 mM PB, pH 3/MeOH, gradient 0-8 min (15-40%), 8-11 min (40%), 11-12 min (40-15%), 12-15 min (15% MeOH) 2 (40°C) 100 UV 220 6.7 8.2 Radiolabeled [14C]UDPGA
4-Nitrophenol Chromolith SpeedRod 50 mM PB, pH 3/MeOH, 85:15 1 50 UV 300 1.5 6.0 External standard
Umbelliferone
Chromolith SpeedRod
50 mM PB, pH 3/MeOH, 85:15
1.5
25
Fluorescence; λex 316, λem 382
1.3
5.3
External standard
  • EED, ethinylestradiol; 4-ABP, 4-aminobiphenyl; PB, phosphate buffer; MeOH, methanol; ACN, acetonitrile.