Quantification of L- and D-enantiomers of proline, trans -4-hydroxyproline, and cis -4-hydroxyproline in natural samples using differential labeling with 12C- and 13C6-dabsyl chloride, chiral separation, and liquid chromatography-mass spectrometry

L-Proline (L-Pro) and trans-4-hydroxy-L-proline (t-4-HPL) are major components of collagen and plant cell wall proteins. During their breakdown, free L-Pro and t-4-HPL are produced abundantly and play unique biological roles through complex metabolisms including isomerization. Therefore, it is essential to quantify all the 6 stereoisomers of proline and 4-hydroxyproline. Here, analytes were first labelled with 12C-dabsyl group, which has strong red color and stability under light. Second, each of the 13C6-dabsyl stereoisomers were added to 12C-dabsylated samples as internal standards before cleanup. Finally, the molar ratio of 12C- and 13C6-dabsyl analytes was determined as the intensity ratio of doublet molecular ions with m/z difference of 6 in mass spectra, which were obtained by subjecting the cleaned sample to capillary chromatography-mass spectrometry. The total amounts of proline, t-4-HP, and cis-4-hydroxyproline were determined using the standard addition method. Linear calibration curves (R2 > 0.99) were obtained in the range of 10-1000 pmol. To determine the molar ratio of D- and L-enantiomers, enantiopure dabsyl analyte fraction was obtained using reverse-phase and chiral chromatography. Mouse serum contained 0.20 μmol/L trans-4-hydroxy-D-proline. Achatina fulica hemolymph contained 0.070 μmol/L D-proline. The Santalum album wood powder contained all the 6 stereoisomers and unexpectedly the D-enantiomer was dominant for trans-4-hydroxyproline.