Development, validation, and application of a liquid chromatography-tandem mass spectrometry method for quantitative determination of trans-4-hydroxy-l-proline concentration in the serum of dogs with chronic hepatitis.

Abstract

To develop and analytically validate a liquid chromatography-tandem mass spectrometry method for measurement of endogenous trans-4-hydroxy-l-proline concentrations in canine serum and to assess serum trans-4-hydroxy-l-proline concentrations in dogs with chronic hepatitis. Serum samples obtained from 20 dogs with histopathologically confirmed chronic hepatitis and 20 healthy control dogs. A liquid chromatography-tandem mass spectrometry method for quantification of trans-4-hydroxy-l-proline concentration was developed and assessed for analytic sensitivity, linearity, accuracy, precision, and reproducibility. Serum concentration of trans-4-hydroxy-l-proline in dogs with chronic hepatitis and healthy control dogs was measured. Observed-to-expected ratios for dilutional parallelism ranged from 72.7% to 111.5% (mean ± SD, 91.3 ± 19.6%). Intra-assay and interassay coefficients of variation ranged from 2.1% to 3.0% and 3.2% to 5.3%, respectively. Relative error ranged from -2.3% to 7.8%. Trans-4-hydroxy-l-proline concentrations were significantly lower in serum obtained from dogs with chronic hepatitis (median, 0.24 ng/mL; range, 0.06 to 1.84 ng/mL) than in serum obtained from healthy control dogs (median, 0.78 ng/mL; range, 0.14 to 4.90 ng/mL). The method described here for the quantification of trans-4-hydroxy-l-proline concentration in canine serum was found to be sensitive, specific, precise, accurate, and reproducible. Dogs with chronic hepatitis had significantly lower serum trans-4-hydroxy-l-proline concentrations than did healthy control dogs, possibly as a result of altered hepatic metabolism of amino acids.