Figure 1.
Ionization (MS 1) and fragmentation (MS 2) of each amino acid in tandem MS analysis.
The data was collected as a 40 scan MCA (2 s/scan), i.e. each spectra is an accumulative result of 40 scans. The most abundant precursor and product ions for the amino acids were (M+H)+ and (M−COOH)+. The isobaric amino acids (e.g., Leu and Ile) have the similar and principal transition of m/z 132→m/z 86. The transitions of m/z 132→m/z 43 and m/z 132→m/z 69 were observed to be characteristic of Leu and Ile, respectively. It was also observed that the transitions were not specific, and two minor signals of m/z 132→m/z 69 and m/z 132→m/z 43 occurred for Leu and Ile, respectively. Val, valine; Ile, isoleucine; Leu, leucine; Tyr, tyrosine; Phe, phenylalanine; M, molecular weight; m/z, mass-to-charge ratio; MS, mass spectrometry.
Figure 2.
Multiple reaction monitoring (MRM) chromatograms of the five amino acids from a serum pool.
The prepared sample was eluted with a mobile phase of A) 0.01% formic acid in water: acetonitrile (90∶10) or B) 0.01% formic acid in water: acetonitrile (98∶2). Val, valine; Ile, isoleucine; Leu, leucine; Tyr, tyrosine; Phe, phenylalanine.
Figure 3.
Correlation of the two sets of results from 51 serum samples under different mobile phase conditions.
Mobile phase A: 0.01% formic acid in water. Mobile phase B: acetonitrile. Val, valine; Ile, isoleucine; Leu, leucine; Tyr, tyrosine; Phe, phenylalanine.
Table 1.
Ion transitions for the unlabeled and labeled amino acids (internal standards) and instrumental parameters for their MS/MS detection in MRM mode.
Table 2.
Comparison of the two sets of results from 51 serum samples under different mobile phase conditions.
Figure 4.
Representative calibration curves for each amino acid.
Val, valine; Ile, isoleucine; Leu, leucine; Tyr, tyrosine; Phe, phenylalanine.
Table 3.
Calibration parameters of serum BCAAs and AAAs by the LC/MS/MS method.
Table 4.
Analytical recoveries of the LC/MS/MS method.
Table 5.
Precision of LC/MS/MS analysis of serum BCAAs and AAAs.
Table 6.
The concentrations of BCAAs and AAAs and percentiles (µmol/l) obtained by the LC/MS/MS method in 192 healthy subjects.
Table 7.
Partial correlations (r) of BCAAs and AAAs with other parameters in 192 healthy subjects, adjusted for age and gender.
Table 8.
Intra-subject (CVI) and inter subject (CVG) biological variations of BCAAs and AAAs.