The analysis is performed by a method using the same HPLC equipment as described under 'Chemical Purity'. An example of a chromatogram obtained with Lot 1035 is shown in Fig. 2. Results from 5 samples performed in duplicates are summarized in Table 2. According to experiments with pure D- and L-Luciferin assayed alone and in mixtures the average retention time for L-Luciferin is 2.622±0.062 min and for D-Luciferin 5.011±0.2019 min. For Lot 2019 the first two peaks correspond to D- and L-Luciferin, respectively. Additional three peaks were found in all 10 chromatograms. These peaks (no. 3, 4 and 5) had the retention times: 7.286±0.041 min, 7.768±0.241 min and 9.325±0.089 min.

In Table 2 the D- and L-Luciferin levels would be estimated as 0.17±0.02% and 99.51±0.05%. The chemical purity was 99.68% (cf. Table 2) from which 0.17% L-Luciferin should be subtracted to give a D-Luciferin purity (chemical and optical) of 99.5%. According to specifications the L-Luciferin level must be lower than 0.7%.

HPLC analysis of optical purity

Fig 2: Chromatogram of D-Luciferin (Lot No.2019) at 220 nm. Insert shows absorbance using an enlarged scale.

Chromatogram of D-Luciferin (Lot No.2019) at 220 nm.

Table 2: Optical purity analysed by HPLC. UV detection at 220 nm

Retention time	Area (%)
2.622	0.17
5.011	99.51
7.286	0.16
7.768	0.10
9.325	0.06

The pure form of D-Luciferin is stable for at least four years when stored dry at -18°C under an inert gas like argon or nitrogen. Alkaline pH and contact with oxygen deteriorates the product rapidly. The corresponding L- Luciferin, suitable as signal prolonger for "glow form" bioluminescence, is also available.

The recommended use of D-Luciferin in ATP assay vary in details dependent if it is used in cell proliferation or in bioluminescent assays. It is important a suitable concentration of magnesium is available for the subsequent light production.

Chemical and optical purity are analyzed by reverse phase and chiral chromatography, respectively. The UV detection at 330 nm is performed with Shimadzu SPD-10A UV Spectrometric Detector.