Enantioselective micro‐2D‐HPLC determination of aspartic acid in the pineal glands of rodents with various melatonin contents

Enantioselective determination of aspartic acid (Asp) in the pineal gland of rodents with various melatonin contents was performed using a highly sensitive and selective two‐dimensional HPLC system. After derivatization of the amino group with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), NBD‐Asp was separated using a capillary monolithic ODS column in the first dimension. The fraction of NBD‐Asp was automatically collected and transferred to the second dimension, and the D ‐ and L ‐Asp were separated and determined using a narrowbore enantioselective column. Large amounts of D ‐Asp were observed in the pineal gland of the rats and specific strains of mice (C3H and CBA) possessing a high concentration of melatonin in their pineal gland. On the other hand, the amounts of D ‐Asp were small in the pineal gland of mice possessing a trace or no melatonin in their pineal gland (ddY, ICR, C57BL and BALB/c). In other tissues and physiological fluids, no significant strain‐dependent changes of the D ‐Asp amounts were observed. These results indicate that large amounts of D ‐Asp are present only in the pineal gland containing large amounts of melatonin, and special care should be taken when selecting mouse strains for the investigation of D ‐Asp.