Separation analysis of macrolide antibiotics with good performance on a positively charged C18HCE column

The separation of basic macrolide antibiotics suffers from peak tailing and poor efficiency on traditional silica‐based reversed‐phase liquid chromatography columns. In this work, a C18HCE column with positively charged surface was applied to the separation of macrolides. Compared with an Acquity BEH C18 column, the C18HCE column exhibited superior performance in the aspect of peak shape and separation efficiency. The screening of mobile phase additives including formic acid, acetic acid and ammonium formate indicated that formic acid was preferable for providing symmetrical peak shapes. Moreover, the influence of formic acid content was investigated. Analysis speed and mass spectrometry compatibility were also taken into account when optimizing the separation conditions for liquid chromatography coupled with tandem mass spectrometry. The developed method was successfully utilized for the determination of macrolide residues in a honey sample. Azithromycin was chosen as the internal standard for the quantitation of spiramycin and tilmicosin, while roxithromycin was used for erythromycin, tylosin, clarithromycin, josamycin and acetylisovaleryltylosin. Good correlation coefficients (r² > 0.9938) for all macrolides were obtained. The intra‐day and inter‐day recoveries were 73.7–134.7% and 80.7–119.7% with relative standard deviations of 2.5–8.0% and 3.9–16.1%, respectively. Outstanding sensitivity with limits of quantitation (S/N ≥ 10) of 0.02–1 μg/kg and limits of detection (S/N ≥ 3) of 0.01–0.5 μg/kg were achieved.