Table 1 Experimental conditions for LC-MS/MS analyses
Method 1: AQC-RPLC-MS/MS | Method 2: HILIC-MS/MS | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
Derivatization | AQC | none | ||||||||||
Chromatography | RPLC | HILIC | ||||||||||
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 | ||||||||||
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 | ||||||||||
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 | ||||||||||
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 |