Effect of micropipette inaccuracy on performance of spectrophotometric assay for bacteria endotoxin dosage (chromogenic LAL) employed in products subjected to sanitary surveillance

Abstract

The ISO 17025 Guidelines states that all instruments used in performing experiments must be calibrated. For micropipettes no maximum error limit has established, which makes difficult to determine as far as a micropipette can be used without interfering on the final results. The aim of this study was to determine the percentage of errors which could be accepted in the micropipettes volume without interfering on results from different steps of LAL toxicological assay. The error percentages were determined based on the information from 44 certificates of micropipettes survey. A monochannel variable volume calibrated micropipette (Eppendorf) and QCL Chromogenic LAL kit (Lonza) were employed. Endotoxin curve was plotted using 0.1, 0.25, 0.5 and 1 EU/mL concentrations. The error percentages of ±2%, ±4% and ±10% were compared with the reference concentration (0.5 EU/mL). Results were analyzed by Student’s t-Test in relation to the reference concentration (p<0.05). Statistical analysis showed that the error of 2% did not interfere on standard endotoxin and LAL substrate results, but errors above this percentage caused alterations on results. Micropipette errors did not interfere on the reaction of substrate color. Errors higher than ±2% should be corrected by adjusting the micropipette volume.