The first step in the determination of porphyrins includes a straightforward sample preparation. The pH value of the sample and the calibrator is adjusted below 2.5 by adding 20 μl of hydrochloric acid. After centrifugation, the supernatant is injected into the HPLC system.
The separation via HPLC follows a gradient method, using a reversed-phase column at 30 °C; one run lasts 25 minutes. The quantification is performed with the delivered calibrator. The concentration is calculated by integrating the peak areas or heights.