Analysis of the key intermediates for the regioselectivity control in the methylation of the manufacture of clarithromycin by reversed-phase high performance liquid chromatography

2′, 4″-O-Bis (trimethylsilyl) erythromycin A-9-O-(1- isopropoxycyclohexyl) oxime (2′, 4″-TMS-EMIPCH) and 2′, 4″-O-bis (trimethylsilyl)-6-O-methylerythromycin A-9-O-(1- isopropoxycyclohexyl) oxime (2′, 4″-TMS-IPCH) are the key intermediates for manufacturing clarithromycin. A qualitative and quantitative method for baseline separation of E- and Z-isomers and related process substances has been established. A DIKMA-Inertsil ODS-3 column (150 mm × 4. 6 mm i. d., 5 μm) was used. The column temperature was maintained at 40 °C. The mobile phase was CH₃CN-H₂O (95: 5, v/v). The flow rate was 1.5 mL/min and the detection wavelength was UV 205 nm. Good linearities for E-2′, 4″-TMS-EMIPCH and E-2′, 4″-TMS-IPCH were obtained in the ranges of 6-60 μg (r = 0.9994) and 6-72 μg (r = 0.9998), respectively. The method described has also been demonstrated to work equally well on other 2′, 4″-O-bis (trimethylsilyl) erythromycin 9-oxime hydroxyl derivatives, which provided the criterion for optimizing the protective groups at 9-oxime hydroxyl position and the study of regioselectivity of methylation at the 6-OH position.