The first step in determining vitamin B6 includes sample preparation with additional derivatization. During the precipitation, higher molecular substances are removed. After centrifugation, the supernatant is used for derivatization (10 min at 60 °C), transforming the vitamin B1 into a fluorescent product. The sample is cooled, centrifuged, and injected into the HPLC system.
The separation via HPLC follows an isocratic method at 30 °C using a reversed-phase column; one run lasts about 12 minutes. The quantification is performed with the delivered EDTA-whole blood calibrator; the concentration is calculated by integrating the peak area/heights.