Feasibility Study of Bacterial Endotoxin Testing in Hospital Preparations of 10% Glycerol / 5% Fructose

Introduction
In 2023, we initiated the production of injectable glycerol/fructose (GF) solutions upon the request of gastroenterologists for the excision of intestinal polyps through interventional endoscopy. The aim of this study is to explore and test the feasibility of implementing bacterial endotoxin testing (BET) to complement the release controls of these hospital preparations.

Materials and Methods
The reference method used is the "kinetic chromogenic" method of monograph 2.6.14 of the European Pharmacopoeia (EP). Measurements were performed with a portable spectrophotometer, Endosafe^® (Charles River). This method requires the determination of two factors: the endotoxin limit concentration (EL) in the produced solutions and the maximum valid dilution (MVD). To explore repeatability, four samples were tested: three samples tested 7 days post-production and one sample tested 50 days post-production, diluted in water for injectable preparations (WFI). The measurement cartridge (Charles River) contains four wells of 25 µl each analyzed simultaneously, with two wells used for measurement compliance controls and two for sample tests.

Results
According to the EP, the EL varies depending on the route of administration of the solutions. GF solutions are administered submucosally at the rectal or colonic level, considered a parenteral route. The EL is obtained by dividing the endotoxin threshold dose per body surface area (100 IU/m²) by the volume of the administered solution. The EL chosen for this study was set at 1.75 IU/ml, corresponding to a 70 kg patient with a body surface area of 1.8 m², for 100 ml of the administered solution. The choice of MVD involves accounting for the detection limit of the measuring device, which ranges from 0.05 to 5 IU/ml relative to this MVD. A 25-fold dilution of the samples was tested so that the smallest detectable value would be 1.25 IU/ml, well below the calculated EL of 1.75 IU/ml. Quality controls for the four samples are compliant with a recovery rate between 50 and 200. The results obtained are "less than 1.25 IU/ml" with an analysis time of 15 minutes and approximately 5 minutes of handling per sample.

Discussion / Conclusion
The samples are free of detectable endotoxins at the threshold of 1.25 IU/ml in accordance with EP recommendations. The implementation of these tests appears promising for routine application in our hospital center. The assay indeed requires a short acquisition time and minimal additional specific equipment. It would be pertinent to enhance the method by adding a positive control to ensure that the GF solutions do not inhibit the enzymatic reaction studied.

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