An Automated Sequential Injection Analysis System for the Determination of Trace Endotoxin Levels in Water

Abstract

A new automated instrument based on the Limulus amebocyte lysate (LAL)-chromogenic substrate kinetic assay for the determination of bacterial endotoxins is reported. A computer controlled syringe pump–multiport valve combination was used to aspirate the sample and other reagents into a holding coil. The syringe was always filled with air; liquid did not enter the syringe. The valve could address up to four individual radial paths, fully referenced optical fiber LED-based absorbance detectors that were housed collectively in a single, small (20 × 20 × 30 mm) metal block with a heater and temperature sensor, and maintained at 37°C. Assay mixtures containing sample or standards were delivered to the individual detector cells for the simultaneous collection of the time based absorbance data. The automated system determined endotoxins with good accuracy and reproducibility in the range of 0.005 – 0.5 endotoxin units (EU)/mL (r² ≥ 0.99). Based on three times the standard deviation of the blank and the slope of the calibration curve, the lower limit of detection was ≤ 0.003 EU/mL. The variability of the assay method is less than 5% (n=10). Analysis time required for a 0.005 EU/mL standard was <100 min. Appropriately diluted tap water samples were simultaneously analyzed by the present system and a manually loaded commercial microplate reader based instrument; the data were statistically indistinguishable at the 95% confidence level.