Table 1.
Conditions of the HPLC methods used.
| Conditions | Method 1 (USP34) |
Method 2 (in-house) |
|---|---|---|
| Apparatus | Chromatopac LC-10AS pump, ESA temperature-controlled autosampler and SPD-10 UV detector (Shimadzu, Kyoto, Japan) |
Agilent 1100 series with auto-sampler and UV–vis detector (Agilent, Santa Clara, CA, USA) |
| Stationary phase | C18 Synergi Hydro 4 μm column (150 × 3.9 mm)a | C18 Luna 3 μm column (150 × 4.6 mm)a |
| Mobile phase | Water: acetonitrile: H3PO4 85:15:0.1 (v/v) |
50 mM NaH2PO4: Acetonitrile 60:40 (v/v); pH 3 |
| Internal standard (IS) | Phenacetin | – |
| Flow rate (ml/min) | 1 | 0.6 |
| Detection wavelength (nm) | 237 | 220 |
| Temperature (°C) | 25 °C | Ambient |
| Injection volume (μl) | 50 | 40 |
| Thalidomide retention time (min) | 18 (24 for IS) | 4.8 |
a
Supplied by Phenomenex (Torrance, CA, USA).