Detection of lipopolysaccharide in hemoglobin‐vesicles by Limulus amebocyte lysate test with kinetic–turbidimetric gel clotting analysis and pretreatment of surfactant

Abstract

A method to quantitatively measure the bacterial endotoxin content (lipopolysaccharide, LPS) in phospholipid vesicles or liposomes is necessary because the conventional Limulus amebocyte lysate (LAL) test does not provide an accurate measurement due to the hydrophobic interaction of LPS and vesicles that shields the activity of LPS to clot the LAL coagulant. This interference was evident from isothermal titration calorimetry results in our study that clearly demonstrated the insertion of the LPS molecule into the phospholipid bilayer membrane. Hemoglobin‐vesicles (HbVs; particle diameter = 251 ± 80 nm; [Hb] = 10 g/dL) are artificial oxygen carriers encapsulating a conc. Hb solution in phospholipid vesicles, and their oxygen transporting ability has been extensively studied. To accurately measure the LPS content in the HbV suspension, we tested the solubilization of HbV with deca(oxyethylene) dodecyl ether (C12E10), used to release the LPS entrapped in the vesicles, as a pretreatment for the succeeding LAL assay of the kinetic–turbidimetric gel clotting (detecting wavelength, 660 nm). The C12E10 surfactant interferes with the gel clotting in a concentration‐dependent manner, and the optimal condition was determined in terms of minimizing the dilution factor and C12E10 concentration. We clarified the condition that allowed the measurement of LPS at >0.1 endotoxin units (EU)/mL in the HbV suspension. Moreover, the utilization of histidine‐immobilized agarose gel effectively concentrated the trace amount of LPS from the C12E10‐solubilized HbV solution and washed out C12E10 as an inhibitory element. The LAL assay with the LPS‐adsorbed gel resulted in the detection limit of 0.0025 EU/mL. Pretreatment with C12E10 would be applicable not only to HbVs but also to other drug delivery systems using phospholipid vesicles encapsulating or incorporating functional molecules. © 2004 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:310–321, 2004