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

A high-performance liquid chromatography method for the simultaneous determ ination of pipecolic acid (Pip) and lysine (Lys), a precursor of Pip, in th e rumen liquor and plasma of ruminant animals was established. Samples of r umen liquor and plasma were deproteinized with 50% acetonitrile and derivat ized with a fluorescent agent 9-fluorenylmethyloxy carbonyl chloride (Fmoc- Cl). Chromatographic separation was achieved on a TSK gel ODS-80TM column u sing a reversed-phase gradient elution system. For the gradient elution, tw o mobile phases, A and B, were needed, both commonly consisted of: 5 mM L-p roline, 2.5 mM cupric sulfate and 6.5 mM ammonium acetate. Mobile phase B a dditionally contains 50% (v/v) acetonitrile. The pH of both mobile phases w as adjusted to 7.0. Derivatized Pip and Lys were detected on a fluorescent detector at excitation and emission wavelengths of 260 and 313 nm, respecti vely. The calibration curves were linear within the range 0 to 1 mM (r>0.99 9). 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, relat ive 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 liqu or (28.4 mu M Pip and 10.2 mu M Lys) samples. The levels of Pip and Lys in faunated goats, determined from three animals over a period of two days sam pling, were found to be 36.8+/-18.1 and 14.6+/-2.8 mu M in rumen liquor, an d 7.3+/-2.5 and 137.3+/-38.0 mu M in plasma at 1 h after feeding. This is t he first report on the normal levels of Pip in the rumen liquor and plasma of faunated goat. (C) 1999 Elsevier Science BN. All rights reserved.