Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals

A fully automated 2D-HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column has been developed for the simultaneous enantiomer determination of proline, trans-4-hydroxyproline and cis-4-hydroxyproline in mammals. As a first dimension, a monolithic ODS column of 0.53 mm i.d. showed 3-6 times larger theoretical plate numbers than those of particle-packed ODS columns, and the best enantioseparations were obtained by a Chiralpak QN-2-AX column of 1.5 mm i.d. in the second dimension (separation factors: 1.44-1.83). The R.S.D. values for within-day and day-to-day precisions were less than 5.8%, and the lower limits of quantitation for the d-enantiomers were 1 fmol. The present method was successfully applied to the determination of proline and hydroxyproline enantiomers in the serum and collagen-rich skin tissue. Small amounts of d-proline were found both in the serum (1.57 ± 0.19 nmol/mL) and in the skin (0.093 ± 0.015 nmol/mg protein), while the amounts of d-hydroxyproline were smaller than the lower limit of quantitation.