AMINO ACIDS continued
Table 3 – RTs and SIR windows/values for the 17 analytes RT (min) 5.18 5.80 6.57 6.90 7.19 8.87
Amino acid Phe Tyr
Leu Met Ile
10.40 10.70 11.22 11.68 11.97 12.90 13.40 16.40 21.40 22.20 23.80
Val Glu Pro Thr Asp Ala Ser Gly Cys His Lys Arg
SIR–RT windows 0.00–6.40 4.60–6.50 6.00–8.00 6.00–8.00 6.00–8.00 8.00–10.00 9.30–11.50 9.30–11.50 10.40–12.20 10.60–12.90 11.40–12.90 12.30–14.20 12.30–14.20 15.30–17.50 20.50–22.50 20.50–22.50 22.50–25.0
SIR (m/z) 166.0 182.0 132.0 150.0 132.0 118.2 148.0 116.0 120.1 134.0 90.0
106.1 76.0
241.0 156.0 147.0 175.1
acid was 2.50 μmol/mL in 0.1N HCl, except for cysteine, which was present at 1.25 μmol/ mL. The standard was diluted to 1.0 μmol/ mL for the working standard 1 (WS1) and to 0.1 μmol/mL for the working standard 2 (WS2). WS1 was used for the calibration of alanine and cysteine, because these two amino acids have lower sensitivity. The lower level standards were prepared from each of the two working standards via serial dilution with the diluent. Prior to injection, all calibration standards and samples were fi ltered through 0.45-μm fi lters to remove any small particles.
Results and discussion Figure 1 shows the overlay of the selected ion
recordings (SIRs) for all 17 amino acids, using the optimized conditions described above. The analysis time was under 25 minutes. Table 3 shows the retention times (RTs) and SIR win- dows/values used for each amino acid.
An overlay of 10 replicates of 0.1 µmol/mL histidine standard is shown in Figure 2. A fi ve- level calibration suite was used, covering a concentration range of 0.006 to 0.10 μmol/mL, except for alanine and cysteine, which covered a concentration range of 0.06 to 1.00 μmol/mL.
Figure 3 shows the representative calibration data for aspartic acid, glycine, histidine and methionone. All amino acids followed a qua- dratic (2nd-order) fi t.
Figure 2 – Overlay of 10 replicates of 0.1 µmol/mL histidine standard. AMERICAN LABORATORY 24 APRIL 2016
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