Endotoxin assay optimization and validation in binary parenteral nutrition mixtures

Binary Parenteral Nutrition Bags (BP-NBs) must be bacterial endotoxins free. Bacterial Endotoxin Test (BET) knonw LAL test as described in the European Pharmacopoeia (EP) is a sensitive assay highly influenced by operating conditions, environmental and qualitative and quantitative interferences of mixtures contents. The aim of this study was the development, optimization and validation of an automated LAL test taking in consideration local formulation of BP-NBs compounded in our pharmacy department.

Endotoxins assay was performed by spectrophotometric assessment of the absorbance of a reactive gel. This matrix is produced when a Limulus amoebocyte lysate (LAL) reacts with endotoxins present in media (Endosafe MCS multi-cartridge spectrophotometer, Charles River). The sensitivity of used preloaded reagent cartridges was 0.005 IU/mL.

The study assessed the following elements:
1- Lower limit of Endotoxin Concentration (LEC) in BP-NBs determination. LEC calculation depends on patient weight and administration rate. It was assessed by retrospective calculation using data of formerly BP-NBs (n=157 bags, 30 patients) and by selecting the lowest found values.
2- Qualitative and quantitative determination of ionic components: ions included in compounded BP-NBs are major interfering factors. Worst case conditions when realistic highest ions concentrations could be met in BP-NBs were assessed using local prescription guidelines and published data, leading to the assessment of the Worst Case Bag (WCB) formulation that’ll be used in the validation study.
3- Dilution factor (DF) that determines the optimal dilution conditions regarding the sensitivity assay and practical considerations.
4- Finally, BET assay validation was assessed using upper prefixed conditions (WCB, DF), evaluating repeatability (n=9), intermediate precision (n=9) and reproducibility (n=9), according to EP acceptance criteria.

LEC was assessed within these conditions: 9.3 mL/h administration rate and 1.43 kg weight. LEC was assessed at 0.7 IU/mL. The interfering factors identified were calcium, magnesium, and zinc; their maximum acceptable concentrations were respectively 1.54 mg/mL, 15.38 µmol/mL, and 5.77 µmol/mL. The calculated DF was 41 (25 µL in 1 mL), allowing to maintain a sensitivity <0.205 IU/mL when using accurate fixed-volume 25 µL and 1000 µL pipettes. Repeatability, intermediate fidelity, and reproducibility were within EP specifications.

BET in BN-PBs compounded by our compounding unit has been therefore validated by defining optimal operating conditions allowing to avoid chemical interferences and to respect the sensitivity of the technique.
Once validated, the next step will be pharmacy staff training and qualification for an efficient and accurate the use of this technique that requires a particular mastering and dexterity.

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