Development of a method for the quantification of bacterial endotoxins in parenteral vitamin-lipid emulsion preparations using the recombinant C factor

Introduction
Vitamin-lipid emulsions (ELIV) are parenteral nutrition preparations used for neonatal patients. These injectable preparations have to pass the European Pharmacopoeia (EP) guidelines for bacterial endotoxin testing (BET). The assay described in monograph 2.6.14 is based on the reaction of horse-shoe crab amoebocyte lysate (LAL). However, it raises ecological, ethical and economic concerns, and may produce false positives, whereas the test described in monograph 2.6.32 of the EP, based on the cleavage of a fluorogenic peptide by recombinant factor C (rFC), avoids these problems. Nevertheless, the presence of lipids, in addition to opacifying the preparation, can interfere with the quantification of BET by forming a lipid-polysaccharide complex. The objective was to develop a sample preparation method that would allow the quantification of BET in a lipid matrix using the rFC method.

Materials and methods
Fluorescence was measured using a plate reader (Agilent - BioTek Synergy HTX - Endonext^® - Biomérieux) with the Endozyme II Go kit. Each sample was analysed with two sample wells and two control wells at 0.5 EU (endotoxin unit)/ml associated with a calibration curve (0.005 to 5 EU/ml). According to the PE, the spike recovery (SR), calculated from the difference in BET concentration between a spiked and unspiked sample, must be between 50 and 200% and the coefficients of variation < 25%. 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. The samples were prepared with or without heating and sonication, varying the dilution factor (undiluted, 1/20, 1/40, 1/100 and 1/200) and the diluent (NaCl and EPPI with or without the 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 IU/ml to validate the absence of interference (n=5).

Results and discussion
Sonication, heating, and dilutions at 1/20 and 1/40 weren’t 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. Despite the inherent underestimation due to the sensitivity of the method, these dilutions allow quantification adapted to the concentration limit calculated in BET.

Conclusion
This work validates the testing of 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 drug preparations.