A new procedure for determining DL amino acid ratios in fossils using reverse phase liquid chromatography
Amino acid geochronology is based largely on the extent of racemization in fossils, as measured by the ratio amounts of D- and L-isomers. Here we report a new, fully automated reverse phase HPLC procedure for simple and precise stereoisomeric separations. At least nine pairs of DL-amino acids are separated with baseline resolution in 75 min using commercially available reagents and equipment. By optimizing precolumn derivatization, we attained compound detectability in the sub-picomole range, sufficient for milligram-size molluscan samples. Analytical reproducibility for nine DL ratios in four fossils spanning a broad range of ages averages 7% ( n=14-28). Asp and Glu DL ratios are the most consistently well resolved and reproduced, with analytical variations of 2 and 3%, respectively. Ratios in three fossil mollusc samples analyzed by the new method and measured previously by GC-based laboratories overlap in 17 out of 18 cases, when considering the ±1 sd analytical errors and ±1 sd inter-laboratory variation. To determine the hydrolysis procedure that minimizes induced racemization while maximizing amino acid recovery, we hydrolyzed seven powdered molluscan fossils of different ages and genera for 0-48 h at 110°C. Concentrations of most amino acids reached a stable plateau after 6-8 h. For young samples, in which faster-racemizing amino acids are targeted (especially Asp), a hydrolysis time of 6 h minimizes induced racemization while attaining nearly complete amino acid recovery. For older samples, 22 h at 110°C is preferred.