Calibrator
Cys (μM)
Phe, Tyr, Gly, PEA (μM)
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0
0
Cal 1
15.6
31.2
Cal 2
31.2
62.5
Cal 3
62.5
125
Cal 4
125
250
Cal 5
250
500
Cal 6
500
1000
Cal 7
1250
2500
6.
Quality controls: Mix 6.5 mL of amino acid standards (500 μM, Sigma) and 500 μL of 10 mM in 0.1 N HCl phosphoethanolamine (Sigma). This provides concentrations of 464 μM for Phe, Tyr, Gly and Cys, and 357 μM for PEA (QC 3). Dilute QC3 to make QC 1 and QC 2 (Table 2) (see Note 1 ).
Table 2
Preparation of quality controls
Quality control | PEA (μM) | Phe, Tyr, Gly, Cys (μM) |
|---|---|---|
QC 1 | 89.3 | 58 |
QC 2 | 178.5 | 116 |
QC 3 | 357 | 464 |
2.4 Analytical Equipment and Supplies
1.
Triple TOF™5600 (AB Sciex).
2.
Acuity UPLC (Waters).
3.
Analytical Column: Kinetex C18, 100 × 3 mm, 2.6 μm (Phenomenex).
3 Methods
3.1 Stepwise Procedure
1.
Pipette 50 μL calibrators, sample or control to 1.5 mL microcentrifuge tubes.
2.
Pipette 450 μL of working internal standard mixture and 20 μL cystine -D4 internal standard.
3.
Vortex tubes for 1 min and let stand for 5 min.
4.
Centrifuge the tubes for 10 min at 12,000 × g.
5.
Transfer 200 μL of supernatant to two separate disposable glass tubes (Tubes A and B).
7.
Add 100 μL of 3 N HCl in butanol to one disposable tube for butylation (Tube A).
8.
Add 50 μL of 0.1 M NaHCO3 (Tube B).
9.
Add 50 μL dansyl chloride solution (Tube B).
10.
Incubate both tubes A and B in dry block for 20 min at 60 °C.
