Extraction Methods for Quantitation of Gentamicin Residues from Tissues Using Fluorescence Polarization Immunoassay

Abstract

Sodium hydroxide digestion of unhomogenized kidney and skeletal muscle for 20 min at 70 degrees C was a superior method for extracting gentamicin from tissues, compared with simple homogenization, trichloroacetic acid precipitation of homogenized tissue, and sodium hydroxide digestion of homogenized tissue. Fluorescence polarization immunoassay was used to quantitate gentamicin. Sodium hydroxide digestion of unhomogenized tissue allowed for the recovery of 90.0 +/- 5.9% (means +/- SD) from renal cortex and 79.9 +/- 3.5% from skeletal muscle. The limit of sensitivity was 17.4 ng/g kidney tissue, 15.8 ng/g digested muscle, and 39.0 ng/g digested heart. The within-assay coefficient of variation (CV) at 100 ng/g kidney was 9.2%; at 500 ng/g kidney, the CV was 2.5%; and at 2000 ng/g kidney, the CV was 1.5%. The between-assay coefficient of variation was less than 7.5% for all concentrations from kidney, and the 99% confidence interval at 100 ng/g kidney was 71.7-112.4 ng gentamicin/g kidney. The within-assay coefficient of variation (CV) at 100 ng/g muscle was 15%; at 500 ng/g muscle, the CV was 2.6%; and at 2000 ng/g muscle, the CV was 2.3%. The between-assay coefficient of variation was less than 15% for all concentrations from muscle, and the 99% confidence interval at 100 ng/g muscle was 72.5-136.8 ng gentamicin/g muscle. Gentamicin-free milk could be distinguished from milk containing gentamicin concentrations of 10 ng/mL milk with 95% confidence, and from milk containing concentrations of 30 ng gentamicin/mL milk with 99% confidence. Quantitative results at or below the tolerance level can be obtained within 90 min of sample acquisition using these extraction and assay methods.