A NEW PROCEDURE FOR DETERMINING DL AMINO-ACID RATIOS IN FOSSILS USINGREVERSE-PHASE LIQUID-CHROMATOGRAPHY

Amino acid geochronology is based largely on the extent of racemizatio n in fossils, as measured by the ratio amounts of D- and L-isomers. He re we report a new, fully automated reverse phase HPLC procedure for s imple and precise stereoisomeric separations. At least nine pairs of a r-amino acids are separated with baseline resolution in 75 min using c ommercially available reagents and equipment. By optimizing precolumn derivatization, we attained compound detectability in the sub-picomole range, sufficient for milligram-size molluscan samples. Analytical re producibility for nine Dr ratios in four fossils spanning a broad rang e of ages averages 7% (n = 14-28). Asp and Glu Dr ratios are the most consistently well resolved and reproduced, with analytical variations of 2 and 3%, respectively. Ratios in three fossil mollusc samples anal yzed by the new method and measured previously by GC-based laboratorie s overlap in 17 out of 18 cases, when considering the fl sd analytical errors and +/-1 sd inter-laboratory variation. To determine the hydro lysis procedure that minimizes induced racemization while maximizing a mino acid recovery, nle hydrolyzed seven powdered molluscan fossils of different ages and genera for 0-48 h at 110 degrees C. Concentrations of most amino acids reached a stable plateau after 6-8 h. For young s amples, in which faster-racemizing amino acids are targeted (especiall y Asp), a hydrolysis time of 6 h minimizes induced racemization while attaining nearly complete amino acid recovery. For older samples, 22 h at 110 degrees C is preferred. (C) 1998 Elsevier Science Ltd. All rig hts reserved.