Determination of endotoxin in vitamin-lipid syringes by the chromogenic kinetic method

Introduction

The hospital pharmacy needs to centralize the production for vitamin-lipid syringe. A sterility test and a physical stability test have been established.

The test for bacterial endotoxins (BET) is used to quantify endotoxin from gram-negative bacteria using amoebocyte lysate from the horseshoe crab. The purpose of this study is to validate the test in lipid emulsion.

Materials and methods

Endosafe®-MCS, Charles River® was used with Charles River®‘s cartridges (sensibility 0.005 EU/ml).

The cartridge contains 4 channels to which LAL (limulus amoebocyte lysate) reagent and a chromogenic substrate have been applied. Two of the four channels also contain an endotoxin spike and serve as the positive products controls.

According to the European pharmacopeia, the chosen method is the chromogenic kinetic method.

• The endotoxin limit and the maximum valid dilution (MVD) were calculated.
  Neonatology’s prescriptions (n=25) were analyzed to determinate the endotoxin limit which depends on both perfusion rate and body weight.
  MVD depends on cartridges sensibility, endotoxin limit and concentration of the sample.
• To validate the method, no interference should be detected. The test solution is considered free of interference factors if under the condition of the test, the measured concentration of the endotoxin added to the test solution, is within 50% -200% of the known added endotoxin concentration. A dilution (n=3) with NaCl or water BET is performed, not exceeding the MVD to remove any interference and the sample is put in the 4 channels.
• A last experiment has been carried out which consists in contaminating syringes with endotoxin at 5 UI/mL. The measured concentration is compared to the expected concentration.

Results and discussion

After analyzing the prescriptions, the worst case was weight 1.1 kg and infusion rate 1.1 ml/h. The endotoxin limit was established at 5 UI/mL.

With water for BET the endotoxin recovery is out of the specified range, with the apyrogenic NaCl a valid result was measured 3 times. So the DMS was fixed at 500 with NaCl 0.9 %.

NaCl increases the sensitivity of endotoxins.

Then, the surcharge of endotoxin 5 UI/ml was detected only 2 times out of 3. A negative and positive control was realized. The level of endotoxin quantified in the samples is 3UI/mL and 4UI/mL.

This test has shown that a concentration of 5 UI/ml is not necessarily detected despite the validation of the method and therefore we must investigate those cases.

Conclusion

The test is in conformity with the European pharmacopeia. The MVD has been fixed to 500. This test can be routinely performed.