B-004 Method Validation of Lactate in the Plasma of Rats

Abstract

Abstract Background The objective of this study is to validate Lactate (LAC) measurement in rat plasma using the ADVIA 1800 analyzer. Methods LAC was validated in rat plasma using the ADVIA 1800 analyzer. Validation testing included intra-assay and inter-assay precision, accuracy, linearity of dilution, limit of quantitation (LOQ), limit of detection (LOD), reference interval, carry-over, correlation, recovery, and stability. Samples were collected into tubes containing sodium fluoride. Intra-assay precision was determined by analyzing two biological samples and the two quality control levels of Bio-Rad Lyphochek Assayed Chemistry Control 10 consecutive times. Inter-assay precision was determined by analyzing the two quality control levels in duplicate for six days over a 10-day period. Accuracy was calculated using the data obtained from the inter-assay precision testing. For linearity of dilution, Audit MicroControl General Chemistry Linearity Kit and biological samples were diluted and analyzed in duplicate. The LOQ was determined by diluting the calibrator material to produce a value at the low end of the reportable range. Diluted material was tested six times. The LOD was determined by assaying the appropriate blank 10 times. The reference interval was determined by analyzing 21 plasma samples. Correlation between the two ADVIA 1800 analyzers was determined by assaying 10 samples on both analyzers once. The carry-over of the assay was determined by analyzing the high-level quality control material followed by the low-level quality control material. Recovery was determined by analyzing a pair of test samples in duplicate that have been spiked and diluted. The proportional error between the two test samples was calculated. Stability of plasma samples was tested on wet ice, refrigerated, and frozen (at -70°C) for various durations of storage. Results Measurement of LAC in rat plasma met the acceptance criteria for precision, accuracy, linearity of dilution, limit of quantitation, carry-over, recovery, and stability. For intra-assay precision in specimen and control material, the %CV was within ±20%. The inter-assay precision %CV was within ±20%. The total error observed (TEobs) was within ±20% for accuracy and the obtained mean of the control material was within the manufacturer’s acceptable range. For linearity of dilution, the coefficient of determination was ≥ 0.9000, the slope was within 1.00 ± 0.25, and TEobs for each plasma and linearity kit dilution was ≤20%. The LOQ was set at the lowest concentration for which the TEobs was within ±20%. The LOD was calculated by adding the mean concentration obtained and three times the standard deviation. The reference interval was set to the 2.5th to 97.5th percentile interval using Excel software. The carry-over was acceptable at ≤2%. Correlation between the two ADVIA 1800 analyzers was determined to describe any bias in result interpretation. Recovery was acceptable as the proportional error was within ±20%. Stability at all storage conditions was found to be acceptable with a mean %difference within ±20%. Conclusions LAC in rat plasma met the acceptance criteria for all required validation parameters. The ADVIA 1800 analyzer can be used for preclinical studies to test LAC in rat plasma.