Development of a two-dimensional LC-MS/MS system for the determination of proline and 4-hydroxyproline enantiomers in biological and food samples

Chiharu Ishii; Yosuke Tojo; Komei Iwasaki; Akira Fujii; Takeyuki Akita; Masanobu Nagano; Masashi Mita; Tomomi Ide; Kenji Hamase

doi:10.1007/s44211-024-00530-w

Development of a two-dimensional LC-MS/MS system for the determination of proline and 4-hydroxyproline enantiomers in biological and food samples

Anal Sci. 2024 May;40(5):881-889. doi: 10.1007/s44211-024-00530-w.

Authors

Chiharu Ishii¹, Yosuke Tojo², Komei Iwasaki¹, Akira Fujii³, Takeyuki Akita¹, Masanobu Nagano³, Masashi Mita⁴, Tomomi Ide⁵, Kenji Hamase⁶

Affiliations

¹ Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
² MIRAI Technology Institute, Shiseido Co., Ltd., 1-2-11 Takashima, Nishi-ku, Yokohama, 220-0011, Japan.
³ Sakamoto Kurozu, Inc., 21-15 Uenosono-cho, Kagoshima, 890-0052, Japan.
⁴ KAGAMI, Inc., 7-7-15, Saito-asagi, Ibaraki, Osaka, 567-0085, Japan.
⁵ Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
⁶ Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. hamase@phar.kyushu-u.ac.jp.

PMID: 38598049
DOI: 10.1007/s44211-024-00530-w

Abstract

A two-dimensional LC-MS/MS system has been developed for the enantioselective determination of proline (Pro), cis-4-hydroxyproline (cis-4-Hyp) and trans-4-hydroxyproline (trans-4-Hyp) in a variety of biological samples. The amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the NBD-derivatives were separated by a reversed-phase column (Singularity RP18) as their D plus L mixtures in the first dimension. The collected target fractions were then introduced into the second dimension where the enantiomers were separated by a Pirkle-type enantioselective column (Singularity CSP-001S) and determined by a tandem mass spectrometer (Triple Quad™ 5500). The method was validated by the standard amino acids and also by human plasma, and sufficient results were obtained for the calibration, precision and accuracy. The method was applied to human plasma and urine, bivalve tissues and fermented food/beverages. D-Pro was widely found in the human physiological fluids, bivalves and several fermented products. Although trans-4-D-Hyp was not found in all the tested samples, cis-4-D-Hyp was present in human urine and tissues of the ark shell, and further studies focusing on the origin and physiological significance of these D-enantiomers are expected.

Keywords: 4-Hydroxyproline; Chiral separation; MS/MS; Proline; Two-dimensional LC.

Abstract

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