INTERLABORATORY COMPARISON OF AMINO ACID D/L VALUES

It has been over 25 years since the last multi-laboratory comparison of amino acid racemization (AAR) values (Wehmiller, 1984). During this time, new methods and new laboratories have been established, while older methods have improved. Although most labs use internal controls to monitor consistency of results, and occasional informal comparisons between labs occur related to specific projects, only two systematic and comprehensive inter-laboratory comparisons have occurred during the history of AAR research.

In this phase of an on-going effort, six laboratories analyzed three samples of Pleistocene mollusk (the original ILC-A, -B, and –C samples, prepared at Delaware) and two samples of Pleistocene eggshell from the extinct bird Genyornis (prepared at Colorado). Dried residues of sample hydrolyzates were distributed, eliminating all inter-laboratory variables except those inherent to instrumentation. D/L values in these five samples span most of the D/L range from ~0.15 to racemic equilibrium. Results were obtained by reverse phase liquid chromatography (RP, 4 labs), ion exchange liquid chromatography (IEX, 2 labs), and gas chromatography (GC, 1 lab). D/L values for Ala, Asx, Glx, A/I, Leu, Phe, Ser, and Val were reported from most labs. One lab obtained results by both RP and IEX.

Coefficients of variation (CV) vary with amino acid, sample and sample type, but individual lab CV’s are usually less than 1% and almost always less than 5%. CV’s for the two IEX A/I values are <0.2% for the eggshells but range from 0.6 % to 13% for the mollusk powders, the largest CV seen in ILC-A, the least racemized of the samples (hence with the smallest D- amino acid peaks). CV’s for multi-lab results for other amino acids in ILC-A (by either RP or GC) are also larger than those seen in either the eggshells (which have larger concentrations of amino acids) or the more extensively racemized mollusks ILC-B and ILC-C. GC values for Asx, , Leu and Val are offset from those obtained by RP, Asx being ~4-8% higher, Leu and Val being ~6-10% lower. For the most extensively racemized sample, RP D/L Leu and Val values significantly exceed the racemic limit of 1.0. Overall, instrumental differences are smaller than those encountered in the original 1984 study. Long term comparability of results will require future periodic interlab comparisons such as these.