Troubleshooting carry-over of LC–MS/MS method for rifampicin, clarithromycin and metabolites in human plasma

Clarithromycin and rifampicin are used for the treatment of Mycobacteria. Pharmacokinetic drug interaction is possibly due to the influence of the two drugs on the liver enzymes. Using a Hypurity Aquastar C18 column (50 mm × 2.1 mm × 5 μm) for liquid chromatography including a polar end-capped phase for the determination of clarithromycin, rifampicin and their metabolites together in plasma using LC–MS/MS resulted in a substantial carry-over. As a consequence, the throughput of the method is not assured. Using a step-by-step troubleshooting procedure, such carry-over was found originating from column memory effect. With the use of another type of C18 column, the carry-over is eliminated. Due to the absence of carry-over, the analytical concentration ranges are extended and are therefore more appropriate for the analysis of patient samples. The method was re-validated for linearity, reproducibility and dilution integrity.

► “Carry-over” of rifampicin and 25 desacetyl rifampicin was originated in the column.
► Replacing the polar end-capped phase column excluded the “carry-over”.
► The new method is more convenience for clinical practice than previous method.