|
Erythromycin |
|
|
Introduction Commercial erythromycin preparations contain three, structurally similar analogs with the difference in mass between the three species being less than 2%. The erythromycin structure (figure below) does not contain a chromophore assessable above 220 nm making derivatization necessary for sensitive detection. However, because of their hydrophobic similarity, these derivatives are not easily separated by reverse phase HPLC.
Detection without Derivatization The figure below shows a reverse phase HPLC separation of a commercial preparation of erythromycin using the PDR-Chiral Advanced Laser Polarimeter. The total amount injected was 2.9 mg. The eluent was a mixture of acetonitrile/methanol/ 0.2M ammonium acetate/water (45:30:10:15) at pH 6.2 and a flow rate of 0.7 mL/min. The baseline offset beginning at 3 minutes is the signal from a rotational standard corresponding to 29 mdeg.
Without detection enhancing derivatization, the three erythromycins were well separated with an analysis time of less than 13 min. Our sensitivity is excellent with detection limits below the 50-ng level. Erythromycin Ethylsuccinate For special purposes, erythromycin can be derivatized with an ethylsuccinate moiety to enhance its ability to resist acid hydrolysis in the stomach. In a specific quality control application, a pharmaceutical preparation of erythromycin ethylsuccinate (EES) is ground, weighed and subsequently dissolved in the chromatographic mobile phase. The sample is then injected directly into the HPLC.
The above chromatogram shows a reverse phase HPLC separation of the erythromycin ethylsuccinate sample. The total amount injected was 3.0 m g. The mobile phase was methanol / water (97:3) at pH 6.5 and a flow rate of 0.7 mL/min. The rotational standard in this analysis is centered at approximately 7 minutes. EES is much more hydrophobic than underivatized erythromycin and therefore the mobile phase must contain substantially more methanol (97%). As before, the three structural analogs are separated although the EES-C peak is present only as a small shoulder on the major EES-A peak. The average amount of EES for a ten-tablet sample was found to be within the FDA specifications. The analysis time was less than 6 minutes.
Specific Rotation The large difference in specific rotations for the three, erythromycin analogs is surprising given the small structural differences between the analogs. These differences are much larger than the experimental uncertainty inherent in such measurements and from these results, it appears that specific rotation may be used as a qualitative attribute in identifying a specific erythromycin analog. Obviously a useful tool in studies of the antibiotic's fate or location in a physiological pathway. | ||||||||||||||||||||||
