Table 1:Abbreviations, molecular weights, and mass transitions (MRM) for the 26 amino acids measured by FIA-MS/MS after butylation
IS- isotope labelled internal standard, used for quantification (see Table 3, supplement)
Notation / Common Name / MW [Da] / Q1 m/z / Q3 m/z / Experiment / ISGln / Glutamine / 146.1 / 203.1 / 84 / MRM / 1
Lys / Lysine / 146.2 / 203.2 / 84.1 / MRM / 12
OH-Prol / Hydroxyproline / 131.1 / 188.1 / 86.1 / NL / 2
PiPA / Pipecolic Acid / 129.2 / 186.2 / 84.1 / NL / 2
Aba / Abscisic Acid / 263.3 / 320.3 / 218.2 / NL / 2
Ala / Alanine / 89.1 / 146.1 / 44 / NL / 2
Arg / Arginine / 174.2 / 231.2 / 70.1 / MRM / 12
Asp / Aspartic Acid / 133.0 / 246.2 / 144.1 / NL / 8
Carn / Carnosin / 226.2 / 283.2 / 110 / MRM / 1
Cit / Citrulline / 175.2 / 232.2 / 113.1 / MRM / 11
Glu / Glutamic acid / 147.1 / 260.2 / 158.1 / NL / 9
Gly / Glycine / 75.1 / 132.1 / 76 / MRM / 1
His / Histidine / 155.1 / 212.1 / 110.1 / NL / 5
Leu/Ile / Leucine/Isoleucine / 131.2 / 188.2 / 86.1 / NL / 4
MeHis / Methyl-Histidine / 169.2 / 226.2 / 124.1 / NL / 6
Met / Methionine / 149.1 / 206.1 / 104.1 / NL / 5
Orn / Ornithine / 132.2 / 189.2 / 70.1 / MRM / 10
Phe / Phenylalanine / 165.2 / 222.2 / 120.1 / NL / 6
Pro / Proline / 115.1 / 172.1 / 70.1 / NL / 4
Sarc / Sarcosine / 89.1 / 146.1 / 90 / MRM / 1
Ser / Serine / 105.1 / 162.1 / 60 / NL / 2
Tau / Taurine / 126.0 / 126.0 / 108 / MRM / 1
Thr / Threonine / 119.1 / 176.1 / 74.1 / NL / 3
Trp / Tryptophan / 204.2 / 261.2 / 159.1 / NL / 7
Tyr / Tyrosine / 181.1 / 238.1 / 136.1 / NL / 7
Val / Valine / 117.2 / 174.2 / 72.1 / NL / 3
Table 2:Abbreviations, molecular weights, and mass transitions (MRM) of carnitine and 34 acylcarnitines measured by FIA-MS/MS after butylation[1]
IS- isotope labelled internal standard, used for quantification (see Table 3, supplement)
Notation / Common Name / MW [Da] / Q1 m/z / Q3 m/z / Experiment / ISC0 / Carnitine free / 161.2 / 218.2 / 85 / PS / 13
C2 / Acetylcarnitine / 203.2 / 260.2 / 85 / PS / 14
C3 / Propionylcarnitine / 217.2 / 274.2 / 85 / PS / 15
C3DC / Malonylcarnitine / 248.1 / 360.2 / 85 / PS / 15
C4 / Butyrylcarnitine / 231.2 / 288.2 / 85 / PS / 16
C4-OH / 3-Hydroxy-Butyryl-Carnitine / 247.2 / 304.2 / 85 / PS / 16
C5 / Isovalerylcarnitine / 245.2 / 302.2 / 85 / PS / 17
C5:1 / Tiglylcarnitine / 243.2 / 300.2 / 85 / PS / 17
C5OH/HMG / 2-Hydroxyisovalerylcarnitine, 3S-Hydroxy-3-methyl-glutarylcarrnitine / 261.4 / 318.4 / 85 / PS / 17
C6 / Hexanoylcarnitine / 259.3 / 316.3 / 85 / PS / 17
C6DC / Adipylcarnitine / 290.2 / 402.3 / 85 / PS / 20
C8 / Octanoylcarnitine / 287.3 / 344.3 / 85 / PS / 18
C8:1 / Octenoylcarnitine / 285.3 / 342.3 / 85 / PS / 18
C10 / Decanoylcarnitine / 315.3 / 372.3 / 85 / PS / 18
C10:1 / Decenoylcarnitine / 313.3 / 370.3 / 85 / PS / 18
MMA / Methylmalonycarnitine / 262.1 / 374.3 / 85 / PS / 18
Glut / Glutarylcarntine / 276.1 / 388.3 / 85 / PS / 18
C12 / Dodecanoylcarnitine / 343.3 / 400.3 / 85 / PS / 19
MeGlut / 3-Methylglutarylcarnitine / 345.6 / 402.6 / 85 / PS / 19
C14 / Tetradecanoylcarnitine / 371.4 / 428.4 / 85 / PS / 19
C14:1 / Tetradecenoylcarnitine / 369.4 / 426.4 / 85 / PS / 19
C14OH / 3-Hydroxy-tetradecanoylcarnitine / 387.4 / 444.4 / 85 / PS / 19
C16 / Hexadecanoylcarnitine / 399.4 / 456.4 / 85 / PS / 20
C16:1 / Hexadecenoylcarnitine / 397.4 / 454.4 / 85 / PS / 20
C16:1OH / 3-Hydroxy-hexadecenoylcarnitine / 413.4 / 470.4 / 85 / PS / 19
C16OH / 3-Hydroxy-hexadecanoylcarnitine / 415.4 / 472.4 / 85 / PS / 19
C18 / Octadecanoylcarnitine / 427.4 / 484.4 / 85 / PS / 20
C18:1 / Octadecenoylcarnitine / 425.4 / 482.4 / 85 / PS / 20
C18:1OH / Hydroxy-octadec-1-enoylcarnitine / 441.4 / 498.4 / 85 / PS / 20
C18:2OH / Hydroxy-octadec-2-enoylcarnitine / 441.4 / 498.4 / 85 / PS / 20
C18OH / 3-Hydroxy-octadecanoylcarnitine / 443.4 / 500.4 / 85 / PS / 20
C18:2 / trans,trans-9,12-octadecadienoic acid / 423.4 / 480.4 / 85 / PS / 20
Table 3:Abbreviations, molecular weights, and mass transitions (MRM) for 20 isotopic labelled internal standards measured by FIA-MS/MS after butylation
Notation / Internal standards / MW [Da] / Q1 m/z / Q3 m/z / Experiment / IS1 / 15N; 2-13C-Glycine / 78.0 / 135.0 / 79 / MRM
2 / 2H4-Alanine / 93.2 / 150.2 / 48.1 / NL
3 / 2H8-Valine / 125.3 / 182.3 / 80.2 / NL
4 / 2H3-Leucine / 134.3 / 191.3 / 89.2 / NL
5 / 2H3-Methionine / 152.3 / 209.3 / 107.2 / NL
6 / 13C6-Phenylalanine / 171.0 / 228.0 / 126 / NL
7 / 13C6-Tyrosine / 187.3 / 244.3 / 142.2 / NL
8 / 2H3-Aspartate / 136.3 / 193.3 / 91.2 / NL
9 / 2H3-Glutamate / 206.3 / 263.3 / 161.2 / NL
10 / 2H2-Ornithine / 134.1 / 191.1 / 72 / MRM
11 / 2H2-Citrulline / 177.0 / 234.0 / 115 / MRM
12 / 2H4; 5-13C-Arginine / 179.2 / 236.2 / 75.1 / MRM
13 / 2H9-Carnitine (free carnitine) / 170.2 / 227.2 / 85 / PS
14 / 2H3-Acetylcarnitine (C2) / 206.2 / 263.2 / 85 / PS
15 / 2H3-Propionylcarnitine (C3) / 220.2 / 277.2 / 85 / PS
16 / 2H3-Butyrylcarnitine (C4) / 234.2 / 291.2 / 85 / PS
17 / 2H9-Isovalerylcarnitine (C5) / 254.3 / 311.3 / 85 / PS
18 / 2H3-Octanoylcarnitine (C8) / 290.3 / 347.3 / 85 / PS
19 / 2H9-Myristoylcarnitine (C14) / 380.4 / 437.4 / 85 / PS
20 / 2H3-Palmitoylcarnitine (C16) / 402.4 / 459.4 / 85 / PS
Table 4:Recovery (R, %),coefficient of variation (CV, %) andregression coefficients (R2)for amino acids c=2.5- 250µmol/L(n=5)
Level 1c=2.5 µmol/L / Level 2
c=25 µmol/L / Level 3
c=250 µmol/L
Mean [µmol/L] / CV [%] / R
[%] / Mean [µmol/L] / CV [%] / R
[%] / Mean [µmol/L] / CV [%] / R
[%] / R2
OH-Prol / 18.6 / 10.6 / 744 / 179 / 27.0 / 718 / 1015 / 8.7 / 406 / 0.9953
Aba / 0.6 / 19.9 / 26 / 4 / 12.2 / 18 / 61 / 11.1 / 24 / 0.9993
Ala / 3.2 / 9.6 / 129 / 22 / 11.8 / 86 / 242 / 10.9 / 97 / 0.9998
Asp / 3.0 / 10.7 / 120 / 25 / 15.9 / 100 / 661 / 8.8 / 265 / 0.9972
Cit / 2.3 / 11.6 / 93 / 19 / 5.1 / 76 / 214 / 5.4 / 86 / 0.9999
Glu / 3.6 / 19.1 / 144 / 26 / 6.5 / 103 / 280 / 6.4 / 112 / 0.9999
Gly / 3.2 / 8.1 / 128 / 20 / 3.9 / 81 / 208 / 6.8 / 83 / 1.0000
Leu/Ile / 9.1 / 12.6 / 182 / 79 / 24.5 / 158 / 400 / 7.1 / 80 / 0.9925
Met / 2.9 / 12.8 / 115 / 19 / 8.7 / 75 / 194 / 8.7 / 78 / 0.9999
Phe / 2.6 / 10.9 / 105 / 21 / 11.3 / 83 / 196 / 8.5 / 79 / 1.0000
Pro / 3.1 / 9.3 / 123 / 23 / 8.3 / 90 / 168 / 7.9 / 67 / 0.9993
Sarc / 7.5 / 4.0 / 298 / 55 / 12.9 / 218 / 483 / 4.8 / 193 / 0.9999
Ser / 3.6 / 22.5 / 145 / 30 / 30.1 / 120 / 101 / 7.2 / 40 / 0.9672
Tau / 0.09 / 7.6 / 3.74 / 0.28 / 21.0 / 1.13 / 1.17 / 8.6 / 0.47 / 0.9932
Thr / 0.5 / 8.2 / 18 / 3 / 12.3 / 12 / 20 / 6.2 / 8 / 0.9987
Tyr / 2.3 / 5.9 / 93 / 20 / 9.2 / 82 / 218 / 9.4 / 87 / 1.0000
Val / 24.9 / 14.8 / 995 / 43 / 15.5 / 172 / 219 / 10.5 / 88 / 1.0000
Lys / 2.5 / 6.4 / 100 / 23 / 4.7 / 91 / 231 / 3.2 / 92 / 1.0000
Arg / 2.0 / 4.6 / 80 / 20 / 3.9 / 80 / 217 / 5.4 / 87 / 0.9999
Carnosin / 1.2 / 7.4 / 47 / 8 / 11.9 / 34 / 152 / 22.1 / 61 / 0.9984
His / 8.5 / 8.1 / 338 / 90 / 4.3 / 360 / 821 / 3.0 / 338 / 1.0000
MeHis / 6.8 / 10.4 / 273 / 67 / 7.1 / 267 / 693 / 4.1 / 282 / 1.0000
Orn / 2.2 / 5.0 / 88 / 20 / 3.1 / 81 / 213 / 3.7 / 85 / 1.0000
Trp / 0.4 / 16.3 / 16 / 6.0 / 13.0 / 24 / 91 / 11.1 / 37 / 0.9992
Table 5:Medians, within- and between-day variability (n=20)of amino acids and acylcarnitines at physiological concentration (Level 1) and elevated concentrations (Level 2)
Analyte / Variability / Level 1 / Level 2Median [µmol/L] / CV
[%] / Median [µmol/L] / CV
[%]
Lysin / Within-day / 110.6 / 18.7 / 129.7 / 10.9
Between-day / 118.5 / 19.7 / 156.6 / 16.7
OH-Prolin / Within-day / 559.6 / 9.3 / 1441.3 / 12.3
Between-day / 746.2 / 11.1 / 1418.6 / 20.1
Aba / Within-day / 9.0 / 14.0 / 7.2 / 15.2
Between-day / 22.0 / 15.0 / 21.4 / 20.4
Ala / Within-day / 434.4 / 7.1 / 849.0 / 7.8
Between-day / 478.6 / 9.0 / 776.3 / 11.7
Arg / Within-day / 7.0 / 12.7 / 43.5 / 7.2
Between-day / 8.0 / 17.9 / 40.7 / 12.1
Cit / Within-day / 58.2 / 10.7 / 252.5 / 7.3
Between-day / 46.7 / 11.8 / 209.1 / 7.8
Glu / Within-day / 323.5 / 9.5 / 603.1 / 11.9
Between-day / 356.9 / 11.1 / 628.6 / 15.0
Gly / Within-day / 296.5 / 6.2 / 936.5 / 6.8
Between-day / 326.0 / 6.5 / 791.8 / 7.1
Leu/Ile / Within-day / 207.8 / 10.7 / 474.9 / 6.9
Between-day / 250.1 / 6.9 / 454.4 / 15.7
Met / Within-day / 104.2 / 13.1 / 203.6 / 10.25
Between-day / 65.7 / 9.3 / 230.2 / 14.1
Orn / Within-day / 98.4 / 12.7 / 274.1 / 9.5
Between-day / 122.8 / 19.8 / 293.9 / 19.7
Phe / Within-day / 112.6 / 8.7 / 478.3 / 7.7
Between-day / 134.8 / 9.7 / 465.7 / 22.8
Pro / Within-day / 157.0 / 14.5 / 146.6 / 14.6
Between-day / 259.9 / 17.8 / 242.0 / 26.6
Sarc / Within-day / 180.4 / 13.9 / 318.7 / 7.7
Between-day / 127.1 / 14.4 / 188.3 / 18.3
Ser / Within-day / 103.2 / 8.3 / 112.2 / 11.2
Between-day / 63.0 / 20.6 / 60.8 / 26.6
Tau / Within-day / 0.7 / 32.7 / 0.8 / 26.0
Between-day / 0.7 / 20.3 / 0.9 / 15.4
Thr / Within-day / 16.2 / 12.3 / 14.0 / 12.2
Between-day / 61.4 / 16.4 / 58.4 / 18.7
Trp / Within-day / 26.2 / 22.5 / 49.1 / 17.2
Between-day / 24.5 / 15.7 / 38.9 / 17.7
Tyr / Within-day / 152.7 / 8.0 / 483.2 / 7.6
Between-day / 162.8 / 22.0 / 414.6 / 17.1
Analyte / Variability / Level 1 / Level 2
Median [µmol/L] / CV
[%] / Median [µmol/L] / CV
[%]
Val / Within-day / 222.6 / 7.9 / 464.2 / 9.8
Between-day / 320.3 / 9.9 / 556.9 / 15.4
C0 / Within-day / 52.7 / 12.0 / 123.5 / 11.1
Between-day / 37.8 / 14.3 / 88.9 / 18.3
C2 / Within-day / 38.3 / 11.5 / 76.1 / 10.4
Between-day / 29.7 / 12.9 / 74.7 / 12.3
C3 / Within-day / 6.3 / 11.8 / 12.5 / 19.7
Between-day / 4.6 / 19.2 / 12.3 / 22.4
C4 / Within-day / 1.11 / 22.2 / 4.16 / 14.7
Between-day / 0.86 / 21.6 / 3.48 / 18.6
C5 / Within-day / 0.52 / 21.2 / 2.09 / 14.6
Between-day / 0.47 / 20.5 / 2.29 / 11.3
C6 / Within-day / 0.44 / 23.9 / 1.92 / 13.8
Between-day / 0.39 / 21.4 / 1.79 / 18.2
C8 / Within-day / 0.73 / 19.4 / 2.90 / 13.5
Between-day / 0.48 / 27.7 / 2.25 / 18.3
C10 / Within-day / 0.78 / 21.4 / 2.75 / 12.4
Between-day / 0.56 / 22.8 / 2.23 / 18.3
C14 / Within-day / 0.60 / 21.8 / 2.35 / 13.5
Between-day / 0.67 / 29.2 / 2.38 / 16.0
C16 / Within-day / 4.80 / 13.9 / 11.42 / 10.5
Between-day / 3.85 / 17.9 / 9.84 / 14.5
MMA / Within-day / 0.50 / 22.7 / 0.58 / 19.2
Between-day / 0.33 / 24.2 / 0.34 / 36.7
C18 / Within-day / 2.76 / 12.7 / 8.45 / 9.2
Between-day / 2.10 / 14.0 / 6.88 / 18.4
C18:1 / Within-day / 1.02 / 20.1 / 0.87 / 15.3
Between-day / 0.53 / 13.6 / 0.52 / 25.0