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 dayto-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.
Tojo
Yosuke
Y
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Hamase
Kenji
K
Nakata
Minako
M
Morikawa
Akiko
A
Mita
Masashi
M
Ashida
Yutaka
Y
Lindner
Wolfgang
W
Zaitsu
Kiyoshi
K
D000818:Animals; D001331:Automation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006909:Hydroxyproline / Q000032:analysis* / Q000097:blood; D051379:Mice; D011392:Proline / Q000032:analysis* / Q000097:blood; D012867:Skin / Q000737:chemistry; D013237:Stereoisomerism
D011392:Proline; D006909:Hydroxyproline