Simple method for the simultaneous analysis of pipecolic acid and lysine by high-performance liquid chromatography and its application to rumen liquor and plasma of ruminants

摘要

A high-performance liquid chromatography method for the simultaneous determination of pipecolic acid (Pip) and lysine (Lys),a precursor of Pip,in the unmen liquor and plasma of ruminant animals was established. Samples of urmen liquor and plasma were deproteinized with 50% acetonitrile and derivatized with a fluorescent agent 9-fluorenylmethyloxy carbonyl chloride (Fmoc-Cl). Chromatographic separation was achieved on a TSK gel ODS-80TM column using a reversed-phase gradient elution system. For the gradient elution,two mobile phases,A and B,were needed,both commonly consisted of:5 mM L-proline,2.5 mM cupric sulfate and 6.5 mM ammonium acetate. Mobile phase B additionally contains 50% (v/v) acetonitrile. The pH of both mobile phases was adjusted to 7.0. Derivatized Pip and Lys were detected on a fluorescent detector at excitation and emission wavelengths of 260 and 313 nm,respectively. The calibration curves were linear within the range 0 to 1 mM (r>0.999). The average recoveries for Pip and lys were 95.9+-1.8 and 93.2+-2.5% in rumen liquor and 98.3+-1.4 and 97.5+-1.3% in plasma,respectively. The limits of detection for Pip and Lys were 0.6 and 0.7 #mu#M in rumen liquor and 0.01 and 0.05 #mu#M in plasma. The assay has acceptable precision,relative standard deviation (RSD) for reproducibility (within-day and day-to-day variation) were less than 5.2% for aqueous (5.0 #mu#M Pip and Lys),MB9 (5.0 #mu#M Pip and Lys),plasma (7.1 #mu#M Pip and 85.6 #mu#M Lys) and rumen liquor (28.4 #mu#M Pip and 10.2 #mu#MLys) samples. The levels of Pip and Lys in faunated goats,determined from three animals over a period of two days sampling,were found to be 36.8+-18.1 and 14.6+-2.8 #mu#M in rumen liquor,and 7.3+-2.5 and 137.3+-38.0 #mu#M in plasma at 1 h after feeding. This is the first report on the normal levels of Pip in the rumen liquor and plasma of faunated goat.