Simultaneous determination of 18 amino acids in the rat plasma of diabetic nephropathy using pr-column derivation coupled with GC-MS

Objective To develop an pre-column derivation coupled with gas chromatography-mass spectrometry (GC-MS) method for the simultaneous determination of 18 amino acids in the rat plasma of diabetic nephropathy. Methods Rat plasma was determined by GC-MS after being derived using ethyl chloroformate. The conditions of GC-MS were as follows: The chromatographic column was Agilent HP-5MS UI (60 m × 0.25 mm × 0.25 μm); the injection mode was splitless mode and the injection port temperature was 240°C; He was used as carrier gas and the flow rate was 1 mL·min-1; The EI source interface was operated and the detector voltage was 70 eV; the solvent delay time was 5.45 min; all quantifications of analytes were performed in the selected ion monitoring mode. Results 18 amino acids in rat plasma were well separated and the compounds in the 1.6~2400 μmol·L-1 concentrations were detected with a good linear response correlation coefficient (r2＞0.99). The relative standard deviation (RSD) values of intra-batch and inter-batch precision and accuracy were all less than 10%. The average recoveries and matrix effects of samples were in the range of 72.17%~111.4% and 90.42%~114.6%, respectively. The plasma sample was stable for 8 h at room temperature and 28 d at -80℃. The derivatization sample was stable for 12 h at 4℃ and 24 h at -20℃. Except histidine and proline, the contents of 16 amino acids in the rat plasma of diabetic nephropathy were significantly different from that of normal rat plasma. Conclusion The developed method was simple and easy to use with good accuracy and specificity. The derivatization product was stable with wide linear range and low matrix effect, thus this method could be used for the determination of 18 amino acids in the normal and diabetic nephropathy rat plasma