Leptin and leptin 1:1 conjugates were eluted from Jupiter C4 column (particle diameter 5 μm, pore diameter 300 Å, 4.6 × 100 mm) by gradient elution at 1 ml/min and 25 °C, and monitored by absorption at 220 nm. Mobile phase A: water + 0.1% TFA; mobile phase B: acetonitrile + 0.1% TFA: isopropanol 50:50 (v/v). The elution started from 5% B for 5 min, then linearly increased to 95% B at 1%/min, and stayed at 95% B till 100 min.