High resolution separation of related compounds of Erythromycin

Synthetic impurities as well as degradation products are becoming increasingly important in today’s analytical laboratories. Over the past few years there have been steps made by The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) to produce a consensus guideline on the assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk. In this current M7 step 2 draft document lower thresholds of impurities are mentioned.  It therefore appears that there will be a need in the near future for HPLC with improved sensitivity and resolution.

It is proposed that the ChromasterUltra Rs together with the LaChrom Ultra II high resolution column is able to fulfill some of these future needs. The ChromasterUltra Rs is equipped with Liquid Beat Technology (LBT), which is the automatic continual adjustment of the piston stroke using four independent pressure sensors in order to compensate for the change in compressibility of the solvent as the mobile phase composition and the back pressure changes over time.  LBT, along with the new double cork mixing technology, allows exceptional baseline stability.  The ChromasterUltra Rs DAD has an optional 65 mm capillary flow cell.  These key features help to deliver the excellent sensitivity and resolution required for more stringent requirements of impurity and related compound testing. Erythromycin is a widely prescribed antibiotic manufactured using either fungal fermentation or a highly complex chemical synthesis, owing to the presence of ten asymmetric carbon atoms.   Therefore, there is an increased probability of structurally similar impurities being present in the final product. There are a number of publications of methods involving high end mass spectroscopy for the analysis of related substances,  however, these methods are not suitable for the tight budgets of small and medium sized pharmaceutical companies.Column temperature:  50 °C
Eluent: 20 mmol/L Phosphate Buffer/CH3CN/CH3OH = 45/40/15 (pre-mixed)
Wavelength: 210 nm
Column Type:  LaChromUltra II C18
Max Pressure:  1350 bar (Coupled columns) Sample:  Erythromaycin A:  Wako pure chemicals 
056-07361  DCH57893

Figure 1: Zoom of main peak (Poor separation between main peak and suspect impurity peak) Flow rate: 1.000 mL/min. Inj. Vol.: 20 µL Column: 4.6 x 150 x 5µm

Figure 2: Flow rate: 0.50 mL/min. Inj. Vol.: 10 µL. Two columns coupled (3.0 mm x 250+250, 1.9 µm). Showing excellent separation of the suspected impurities.

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