This document describes a method for sample cleanup of tobramycin in serum using pre-SPE derivatization with orthophthalaldehyde (OPA) to enhance selectivity. The method improves the cleanup of tobramycin from a biological matrix by pre-SPE derivatization with OPA and enhances detection sensitivity through fluorescence detection of the derivative formed.
This document details a method for improving the selectivity and recovery of tobramycin, an aminoglycoside antibiotic, from biological samples like serum. The technique involves pre-column derivatization with orthophthalaldehyde (OPA) followed by solid phase extraction (SPE) using Bond Elut Cys sorbent. This approach overcomes the low selectivity and poor recovery typically associated with SPE of polar compounds without derivatization.
The note provides a step-by-step procedure for preparing reagents, processing the sample, and performing the SPE. It includes results demonstrating the linearity of peak heights versus concentration and high recovery rates for spiked serum samples. The document also presents chromatograms comparing the performance of the derivatization method against a standard SPE-only method.
| Sorbent | Bond Elut Cys |
|---|---|
| 2-ME Concentration | 1% v/v |
| Sulfosalicylic Acid Concentration | 10% (w/v) |
| Mobile Phase Composition | 0.02M Phosphate pH 6.5:Acetonitrile 50:50 |
| Flow Rate | 2 mL/min |
| Excitation Wavelength | 340 nm |
| Emission Wavelength | 450 nm |