Chemical Purity D-Luciferin

QUALITY CONTROL PROCEDURES - HPLC analysis of chemical purity

The biochemical performance of each lot of D-luciferin is assayed at 25°C by an automatic procedure using a special quality control kit containing standardised preparations of all components (luciferase, ATP, buffer etc.) required for the measurement.
Light emission is measured at the following final concentrations of D-luciferin: 0.1 mg/mL (below optimum), 0.2 mg/mL (optimum) and 0.3 mg/mL (above optimum). Each run begins and ends with 0.2 mg/mL of a reference lot of D-luciferin. Assays are performed in triplicates for each sample.

The analysis is performed with Shimadzu SPD-10A UV Spectrometric Detector in Shimadzu Liquid Chromatograph. The procedure uses a buffered-reverse phase technique with a gradient elution. An example of a chromatogram obtained with Lot 1035 at 330 nm is shown in Fig. 1. Results from 5 samples performed in duplicates are summarized in Table 1. Results obtained at 265 nm were similar. In the interval 220-540 nm no additional chromatographic peaks were detected. The average retention time of D-Luciferin was 7.126±0.013 min.

Peak no.2 at 9.302±0.021 min corresponds to deoxyluciferin. The average percent purity for Lot 1035 is 99.65±0.03% D-Luciferin. According to specifications, the percent purity of D-Luciferin (free acid) must be greater than 99.5%.

HPLC analysis of chemical purity

Fig. 1: Chromatogram of D-Luciferin (Lot 1035) at 330 nm. Insert shows absorbance using an enlarged scale.

Table 1: Chemical purity analysed by HPLC. UV detection at 330 nm