| Chirality is an important feature of endogenous metabolites,However,the studied the optimal of biomarkers in metabonomics has not been solved.Therefore,the identification of chiral biomarkers is an important subject in the field of metabolomics research.However,D-amino acid neurotransmitters are widely found in the brain tissue of mammals,E.g D-Ser,D-Asp.D-amino acids as a new physiologically active substance,has gradually become a new target compound in metabonomics research.Now the method of pre-column derivatization in the method of analyzing chiral metabolites.The detection sensitivity can be improved and good chiral separation can be achieved at the cheap of the reversed phase column,has been widely used.The modern metabolomics uses analysis with high resolution and high throughput LC-MS ability conduct a metabolomics analysis.But the chiral compound m/z is the same,cannot be separated and analysis in the mass spectrometry,and some metabolites have low content and low ionization efficiency,it is difficult to obtain high sensitivity detection.In view of this,this study is to develop amino functional chiral metabolites labeled with positively charged new mass spectrometric chiral reagents.First,triphenylphosphine structure(TPPP)for the mother structure,with L-thiopr oline(TZD),N-hydroxysuccinimide(NHS)was reacted.Synthesized having targeted amino functional groups and chiral metabolites positively charged mass spectrometry chiral reagents N-[1-Oxo-5-(triphenylphosphonium)pentyl]-(R)-1,3-thiazolidinyl-4-N-hydroxysuccinimide ester bromide salt(OTPTHE).gradient analysis at AQ-C18(150 mm×4.6 mm,3 μm i.d.)column using 0.1%of formic acid including water and acetonitrile mobile phase.The chiral separation efficiency was evaluated by HPLC-UV using 11 D,L-amino acids(Ala,Thr,Lys,Ile,Ser,His,Pro,Met,Trp,Val,Leu).And the structure of the chiral derivatives under different bombardment energy was investingated on LC-MS.And D and L-Ser as an example,Conducted a methodological study.Within 30 min of 11 D and L-Ser the separation of amino acids(Rs)1.62-2.41,up to the complete separation.D and L-Ser in the range of 15-1000μM,20-2000 μM a good linearity was achieved from the calibration curves(r2≥0.9997);the limit of detection(S/N=3)was 5 pmol;the limit of quantitation(S/N=10)was 12.5 pmol;D and L-Ser the inter-day and intra-day assay precisions were 1.66%-4.56%,1.18%-3.64%;D and L-Ser plasma addition average recovery rate of 99.48%-104.06%.All meet the requirements of biological sample determination.In addition,the quantitative analysis of D and L-Ser in plasma of ten healthy subjects was performed.The average content of D and L-Ser in plasma was 2.65 ± 1.31 μg/mL and 20.36 ± 4.51 μg/mL.The average L-Ser content is about 10 times that of D-Ser.This study,synthesized having targeted amino functional groups and chiral metabolites positively charged mass spectrometry chiral reagents OTPTHE.And can be separated from 11 different amino acids.Can also be quantified in human plasma D,L-Ser.Therefore,this reagent can be used for pharmacokinetic study of chiral drugs in biological samples and quantitative analysis of chiral amino metabolites.Which provides a new analytical method for the separate ion and quantitative analysis of amino chiral metabolites. |
