TABLE 3

Analytic conditions and methods for HPLC glucuronide separation

The eluents were acetonitrile (B) and 50 mM phosphate buffer pH 3.0 (A) in all methods, except that in the R- and S-propranolol and bilirubin chromatographic methods 0.1% formic acid in water was used as eluent A. The column temperature and the flow rate were 40°C and 1 ml/min, respectively.

Analyte GlucuronideGradientInjection VolumeDetection Parameters (Wavelength)Retention Time of Glucuronide(s)
µlnmmin
4-Methylumbelliferone0–3 min, 20→50% B; 3–3.1 min, 50→20% B; 3.1–5 min, 20% B5–25Fluorescence, λex 316, λem 3821.7
17-α-Estradiol0–2 min, 20→40% B; 2–5 min, 40→50% B; 5–8 min, 50% B; 8–8.1 min, 50→20% B; 8.1–11 min, 20% B40–50UV, 281; fluorescence, λex 281, λem 3063.1, 3.8, and 4.2
Estradiol0–2 min, 20→40% B; 2–5 min, 40→50% B; 5–8 min, 50% B; 8–8.1 min, 50→20% B; 8.1–11 min, 20% B40–50Fluorescence, λex 281, λem 3062.8, 3.4, 3.6, and 3.9
Ethinylestradiol0–6 min, 25→50% B; 6–9 min, 50%B; 9–9.1 min, 50→25% B; 9.1–11 min, 25% B40UV, 281; fluorescence, λex 281, λem 3062.3, 4.0, and 4.6
Testosterone0–1.5 min, 27% B; 1.5–5 min, 27→50% B; 5–8 min, 50% B; 8–8.1 min, 50→27% B; 8.1–10 min, 27% B40UV, 2444.6
Epitestosterone0–2 min, 32% B; 2–5 min, 32→50% B; 5–9 min, 50% B; 9–9.1 min, 50→32% B; 9.1–11 min, 32% B40–60UV, 2444.7
Bilirubin0–6 min, 35→80% B; 6–6.3 min, 80→95% B; 6.3–12.5 min, 95% B; 12.5–12.7 min, 95→35% B; 12.7–15 min, 35% B15–25Visible, 4493.2 and 4.8
Entacapone0–1 min, 15→45% B; 1–3 min, 45→50% B; 3–4.5 min, 50% B; 4.5–4.6 min, 50→15% B; 4.6–6 min, 15% B20–25UV, 3092.9
Indomethacin0–4 min, 40→50% B; 4–9 min, 50% B; 9–9.1 min, 50→40% B; 9.1–11 min, 40% B50UV, 3193.3
Diclofenac0–4 min, 35→50% B; 4–9 min, 50% B; 9–9.1 min, 50→35% B; 9.1–11 min, 35% B40UV, 2763.8
Dexmedetomidine0–7 min, 10→45% B; 7–7.1 min, 45→10% B; 7.1–10 min, 10% B50UV, 2154.8 and 5.0
Levomedetomidine0–7 min, 10→45% B; 7–7.1 min, 45→10% B; 7.1–10 min, 10% B50UV, 2154.7 and 5.0
R-propranolol0–1.5 min, 23.5% B; 1.5–4 min, 23.5→28% B; 4–5.5 min, 28% B; 5.5→5.65 min, 28→50% B; 5.65–7 min, 50% B; 7–7.15 min, 50→23.5% B; 7.15–10 min, 23.5% B10UV, 227; fluorescence, λex 227, λem 3443.5
S-propranolol0–1.5 min, 23.5% B; 1.5–4 min, 23.5→28% B; 4–5.5 min, 28% B; 5.5→5.65 min, 28→50% B; 5.65–7 min, 50% B; 7–7.15 min, 50→23.5% B; 7.15–10 min, 23.5% B10UV, 227; fluorescence, λex 227, λem 3444.0