Help Prevent Intravascular Devices-Related Bloodstream Infections With Minocycline

 By R. L. Steffen, A. K. Wilson, D.G. Cuttell, M.S. Medrano, L.R. Brewster, and A. Hoffa

This method uses a 50:50 THF:water extraction solvent for the preparation of standards and samples. Preservative at a level of 0.1 weight percent in solution was added to stabilize the preparations. Analysis was accomplished by HPLC carried out on a Phenomenex Luna Phenyl Hexyl (150 x 4.6 mm) column at a flow rate of 1.0 mL/min. The separation was completed with a mobile phase of 35 mM KH2PO4:Triethylamine:CH3CN: (74.5:0.5:25 v:v:v), pH 7.5. The detector wavelength was set at 280 nm and the column temperature adjusted to 50 ºC. A run time of 12 minutes was sufficient to elute the peaks of interest for isocratic separation and 23 minutes for gradient separation.

Standard solutions made with sodium sulfite preservative were found to be stable, showing only nominal amounts of epiminocycline. However, atypical degradation patterns were sometimes observed with this solvent combination. Chromatograms of degraded solution showed the main band splitting into two peaks. Detergent carryover from labware cleaning was found to facilitate the degradation. The chromatographic profile of degraded solution was very similar to that observed with forced thermal degradation. Conversely, using sodium citrate as a preservative in place of sulfite did not show this atypical degradation pattern, even after aging for more than seven days in the presence of detergent. Chromatographic interference prevented the use of ascorbic acid as an antioxidant for this assay.