Table 1.
Experimental conditions for LC-MS/MS analyses
| Method 1: AQC-RPLC-MS/MS | Method 2: HILIC-MS/MS | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Derivatization |
AQC |
none |
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| Chromatography |
RPLC |
HILIC |
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| Stationary phase |
Thermo Hypersil Gold C18 |
TosohBioscience TSK-gel Amide-80 |
||||||||||
| Column length × I.D. (mm) |
50 × 2 |
250 × 2 |
||||||||||
| Column packing size (μm) |
1.9 |
5.0 |
||||||||||
| Column temp (°C) |
20 |
40 |
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| Mobile phase A |
Water with 20 mM NH4COOH (pH 5 adjusted) |
Water with 50 mM HCOOH |
||||||||||
| Mobile phase B |
Methanol |
Acetonitrile: Water (95:5) with 50 mM HCOOH |
||||||||||
| Flow rate (mL/min) |
0.40 |
0.20 |
||||||||||
| Injection volume (uL) |
6.0 |
5.0 |
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| Elution conditions |
10-40% B in 6 min, to 85% B at 6.1 min, hold at 85% B until 8.5 min. |
90 to 60% B in 15 min, hold to 20 min, decrease to 55% B at 21 min, hold to 30 min. |
||||||||||
| MS Ionization |
Positive electrospray (Turbospray®) |
Positive electrospray (Turbospray®) |
||||||||||
| MS source temp (°C) |
450 |
450 |
||||||||||
| MS ionization voltage (V) |
5500 |
5500 |
||||||||||
| MS orifice potential (V) |
70 |
40 |
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| |
|
Relative intensities |
|
Relative intensities |
||||||||
|
SRM transitions for: |
Precursor m/z |
Product m/z |
CE (V) |
BMAA |
DAB |
AEG |
Precursor m/z |
Product m/z |
CE (V) |
BMAA |
DAB |
AEG |
| 1 BMAA, DAB, AEG |
459.2 |
171.1 |
35 |
1.00 |
1.00 |
1.00 |
119.1 |
102.1 |
15 |
1.00 |
0.40 |
1.00 |
| 2 BMAA, DAB, AEG |
459.2 |
119.1 |
30 |
0.28 |
0.07 |
0.28 |
119.1 |
101.1 |
20 |
0.55 |
1.00 |
0.65 |
| 3 BMAA, DAB, AEG |
459.2 |
289.1 |
20 |
0.39 |
0.13 |
0.27 |
- |
- |
- |
- |
- |
- |
| 4 BMAA specific |
459.2 |
258.1 |
30 |
0.04 |
na |
na |
119.1 |
88.1 |
20 |
0.29 |
na |
na |
| 5 DAB specific |
459.2 |
188.1 |
35 |
na |
0.02 |
na |
119.1 |
74.1 |
25 |
na |
0.11 |
na |
| 6 AEG specific |
459.2 |
214.1 |
35 |
na |
na |
0.02 |
- |
- |
- |
- |
- |
- |
| 7 d3-BMAA |
462.2 |
292.1 |
20 |
1.0 |
na |
na |
122.1 |
105.1 |
15 |
1.00 |
na |
na |
| 8 d3-BMAA |
462.2 |
122.1 |
30 |
0.7 |
na |
na |
122.1 |
88.1 |
20 |
0.24 |
na |
na |
| 8 mono-AQC BMAA |
230.1 |
171.1 |
30 |
na |
na |
na |
na |
na |
na |
na |
na |
na |
| 9 di-AQC lysine |
487.2 |
171.1 |
25 |
na |
na |
na |
na |
na |
na |
na |
na |
na |
| 10 mono-AQC lysine | 317.1 | 171.1 | 30 | na | na | na | na | na | na | na | na | na |