Simultaneous chiral analysis of DL-asp and DL-α-HB with derivatization in human fingernail by UHPLC-MS/MS: an application in colorectal cancer

With the development of untargeted metabolomics, studies have shown that alterations in the serum levels of specific amino acids are strongly correlated with the development of early-stage colorectal cancer (CRC). However, the relationship between optically active free amino acids in human nails and the risk of CRC remains unclear. In this study, a highly sensitive UHPLC-MS/MS chiral separation method based on ( S )-(−)-DBD-Pro-COCl derivatization was established for the simultaneous detection of DL-Asp, DL-Kyn, DL-α-hydroxybutyric acid (DL-α-HB), and Cystamine in the fingernails of patients with CRC. The derivatization reagents were incubated with DL-amino acids, DL-α-HB and Cyst at 60 °C for 15 min to form diastereomers. Subsequently, an Imtakt cadenza CD-C 18 column (3.0 × 150 mm, 3.0 μm) was used for the separation of diastereomers under gradient elution of 10 mM HCOONH 4 in H 2 O as mobile phase A and 0.1 % HCOOH in CH 3 CN as mobile phase B. The degrees of separation (Rs) ranged from 1.76 to 3.21. R 2 was >0.9995, indicating good linearity; the limit of detection (LOD) was 25–100 fmol; the interday and intraday precision of DL-Asp and DL-α-HB detection were 0.85 %–4.91 %; and the average recovery ranged from 81.12 % to 110.2 %. However, during the determination process of fingernail samples, only DL-Asp and DL-α-HB were successfully detected, while DL-Kyn and Cyst were not detected. Therefore, this novel method was used to quantify DL-Asp and DL-α-HB in fingernail samples from 32 healthy volunteers (HVs) and 16 patients with CRC (CPs). The results revealed significant differences in D-Asp ( p  < 0.05), L-Asp ( p  < 0.01), and L-α-HB ( p  < 0.05) levels between male HVs and CPs and in D-α-HB ( p  < 0.05) levels between female HVs and CPs. In addition, the D/L-α-HB ratio ( p  < 0.01) was significantly different between male HVs and CPs. These findings validate the use of DL-amino acids, DL-α-HB and Cyst from human fingernails as potential diagnostic biomarkers for CRC, providing a basis for the clinical application of human fingernail samples for the noninvasive diagnosis of CRC.