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The indirect chiral method, based on the o-phthaldialdehyde reaction using the chiral N-acetyl-L-cysteine reagent, has been optimized to determine D-amino acids in milk and oyster samples. Both the derivatization reaction and the HPLC separation have been improved, and simple sample treatments were proposed. The milk sample preparation involved centrifugation and filtration through polytetrafluoroethylene filters for determination of free D-AA. Two methods, nonenzymatic and enzymatic, were applied to determination of free and total D-AA, respectively, in oyster samples. The D-AA contents were in the range of 0.14 to 4.32 mg/L for milk samples and 0.80 to 15.9 mg/g for oyster samples, with relative standard deviations lower than 10% (n = 4), except for D-Tyr. In general, mean recoveries were greater than 90% with relative standard deviations lower than 9% (n = 4) at concentration levels from 0.055 to 0.426 mg/L for milk samples and 0.348 to 1.10 mg/ g for oyster samples.

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