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Background and Importance Vitamin-lipid emulsions (ELIV) are parenteral nutrition preparations used for neonatal patients. Injectable preparations have to pass the European Pharmacopoeia (EP) guidelines for bacterial endotoxin testing (BET). The assay described in monograph 2.6.14, based on the reaction of limulus amebocyte lysate (LAL), raises ecological, ethical, and economic concerns and may produce false positives. The test in monograph 2.6.32 of the EP, based on the cleavage of a fluorogenic peptide by recombinant factor C (rFC), avoids these problems. However, the presence of lipids, in addition to opacifying the preparation, may interfere with BET quantification by forming a lipid-polysaccharide complex.Aim and Objectives The objective was to develop a sample preparation method for quantifying BET in a lipid matrix using the rFC method.Material and Methods Fluorescence was measured using a plate reader (Agilent BioTek) with the Endozyme II Go kit (Endonext-Biomérieux). Each sample was analysed with two sample wells and two control wells at 0.5 EU/mL associated with a calibration curve. According to the PE, the spike recovery (SR) must be between 50 and 200%. Sensitivity was set at 0.005 EU/mL in line with the BET limit concentration (3.3 EU/mL) and the maximum significant dilution calculated from the analysis of 600 ELIV prescriptions. Samples were prepared with or without heating and sonication, varying dilution factor (undiluted, 1/20, 1/40, 1/100 and 1/200) and diluent (NaCl and EPPI with or without addition of polysorbate 20 (PS) at 0.025%, 0.05% or 0.1%). After selecting the optimal parameters, the ELIVs were doped at 0.01, 0.02, 0.03 and 0.05 EU/mL to validate the absence of interference.Results Sonication, heating, and dilutions at 1/20 and 1/40 were not enough to remove the interference, unlike dilutions at 1/100 and 1/200 with PS concentrations of 0.05% and 0.025%. Dilutions at 1/100 and 1/200 with EPPI-PS at 0.025% gave an average SR ± 95% confidence interval (CI95) of 97% ± 4 and 103% ± 6, respectively, with an average error percentage ± CI95 of 31% ± 6.Conclusion and Relevance This work validates the BET using the rFC method in ELIVs. This approach opens the way to more environmentally friendly and ethical microbiological monitoring than the LAL method, applicable to other parenteral preparations.Conflict of Interest Corporate sponsored research or other substantive relationships: bioMérieux kindly supplied analytic equipment and consumables free of charge.