Nitrocefin stock solution (20 mM) was prepared in acetonitrile and then diluted with Hepes buffer (50 mM, pH 7.4) to a concentration of 2 mM (further dilutions were made from this stock). All nitrocefin samples were kept on ice, and subsequent dilutions or mixing were performed under cold conditions to prevent background self-hydrolysis. Pre-cooled nitrocefin solutions (50 µL) were mixed with pre-incubated PFK solutions (200 µM PFK, incubated in Hepes 50 mM, KCl 0.2 M, pH 7.4) in pre-cooled clear 96-well plates (Greiner flat-bottom; 360 µL). The plate was then transferred to a pre-cooled plate reader (Multiskan GO, Thermo Scientific, Waltham, MA) at 18°C and shaken slowly for 5 seconds. The absorbance of degraded nitrocefin was measured every 1 minute at λ=480 nm for two hours. Initial rates (V0) were calculated based on the slope of the initial linear range of degraded nitrocefin production within the first 15 minutes. Reported results represent the average of at least five separate measurements (N, the exact number of repeats, is provided). Each experiment included blank samples of nitrocefin substrate diluted with buffer instead of PFK. All presented results were adjusted for buffer control subtraction. Initial rate calculations also accounted for substrate self-degradation, ensuring that product absorbance resulted from the presence of PFK. 10-12 min 10% of A, 12-13 min 90% of A, 13-15 min 90% of A. The flow rate was 0.4 mL/min, and the column temperature was 40°C. The parameters of the MS were set according to the manufacture recommendations. The samples were analyzed in a full-scan mode (70-1000 m/z) with 140,000 FWHM resolution. Relative abundance of the intact/degraded compounds was calculated by the peak area of the extracted ion chromatogram: for amoxicillin 366.1-366.12 m/z, for degraded-amoxicillin 384.1-384.13 m/z, for penicillin 335.1-335.12 m/z, and for degradedpenicillin 353.1-353.12 m/z. The data was analyzed using Xcalibur software, v4.2 (Thermo Fisher Scientific, California, USA).