Quantifying Penicillin G in Culture Supernatant

Quantification of penicillin G in culture supernatant was performed by LC-MS as developed previously, using an Agilent 1290 LC and 6550 quadropole time-of-flight mass spectrometer with electrospray ionisation and an Agilent Zorbax Extend C-18, 2.1 × 50 mm, 1.8 μm LC column^{19 (link)}. penicillin G (Sigma Aldrich) standards of concentration 0 ng ml⁻¹, 10 ng ml⁻¹, 100 ng ml⁻¹, 1 μg ml⁻¹ and 10 μg ml⁻¹ were used to construct a curve of best fit that was then used to quantify penicillin G in culture supernatant samples. Samples were prepared by growing cells to an OD₆₀₀ of 0.6, in 96-well format, centrifuging and removing the supernatant, which was used as the sample.