This application note provides a method for enhancing the detection sensitivity of tobramycin using pre-column derivatization with orthophthalaldehyde (OPA). The method can be used to analyze poorly detectable polar compounds, such as tobramycin.
This document details a method for analyzing tobramycin, a polar pharmaceutical compound with low UV-absorptivity, using reversed phase HPLC and fluorescence detection. The method employs pre-column derivatization with orthophthalaldehyde (OPA) to improve sensitivity.
The procedure utilizes the Varian 9100 AutoSampler to automate the derivatization process. Key parameters include a reaction time of 30 minutes and a solvent composition of 60% acetonitrile. The resulting fluorescence detection offers a detection limit approximately 400 times better than UV absorbance.
| Detection Limit (Fluorescence) | 1 pmol |
|---|---|
| Detection Limit (UV) | 400 pmol |
| Optimum Reaction Time | 30 minutes |
| Linearity Range | up to 1.2 nmoles |
| Correlation Coefficient | 0.998 |
The optimum reaction time is 30 minutes.
The optimum reaction time is 30 minutes.
Fluorescence detection is about 400 times better than using UV absorbance.
Linearity was tested up to 1.2 nmoles.