Allicin is an unstable and reactive compound under gas chromatography settings65 , therefore, high-performance liquid chromatography (HPLC) was used to determine allicin concentrations65 .
Various preparations of Bald’s eyesalve were analysed by reversed-phase HPLC on an Agilent 1,200 series system fitted with an Agilent ZORBAX Eclipse XDB-C18, 150 × 4.6 mm, 5 μm particle size column and diode array detection at 210 nm. A gradient of methanol (5–95%) in water was used at a flow rate of 1 ml·min−1 over 30 min. Injection volume was 10 μl, and the column temperature was 25 °C.
To identify the concentration of allicin in eyesalve preparations, a calibration curve was prepared by serially diluting an external allicin standard (Abcam, Cambridge) in water (twofold dilutions, from 750 μg·ml−1 to 0.5 μg·ml−1). External standards were run through HPLC as described above (n = 2) and allicin peak area, in the 210 nm reading, was plotted against concentration (μg·ml−1) (Fig.S2 and Data Supplement). Allicin external standards had a retention time of approximately 15 min (Fig. S2 ). This peak was confirmed in Bald’s eyesalve by comparing fresh eyesalve to the same batch spiked with additional allicin standard. The suspected peak increased as expected (Fig. S2 ).
Various preparations of Bald’s eyesalve were analysed by reversed-phase HPLC on an Agilent 1,200 series system fitted with an Agilent ZORBAX Eclipse XDB-C18, 150 × 4.6 mm, 5 μm particle size column and diode array detection at 210 nm. A gradient of methanol (5–95%) in water was used at a flow rate of 1 ml·min−1 over 30 min. Injection volume was 10 μl, and the column temperature was 25 °C.
To identify the concentration of allicin in eyesalve preparations, a calibration curve was prepared by serially diluting an external allicin standard (Abcam, Cambridge) in water (twofold dilutions, from 750 μg·ml−1 to 0.5 μg·ml−1). External standards were run through HPLC as described above (n = 2) and allicin peak area, in the 210 nm reading, was plotted against concentration (μg·ml−1) (Fig.