7 % BSA (mL)
1° Standard (μL)
PHE/TYR
2° Standard (μL)
PHE/TYR
Calibrator (mg/dL)
PHE/TYR
0.940
–/–
50/10
0.5/0.1
0.930
20/–
–/50
2.0/0.5
0.850
50/–
–/100
5.0/1.0
0.880
100/20
–/–
10.0/2.0
0.750
200/50
–/–
20.0/5.0
0.600
300/100
–/–
30.0/10.0
2.
Quality controls:
(a)
BIORAD Liquichek TDM Levels 1 and 2. Tyrosine is added to Biorad 1 for a target concentration of 0.5 mg/dL and to Biorad 2 for a target concentration of 2.5 mg/dL. Ranges for phenylalanine and tyrosine are established in-house.
(b)
In-house control spiked with phenylalanine (target: 25 mg/dL) and tyrosine (target: 7.5 mg/dL) in 7 % bovine serum albumin . Ranges established in-house.
Store calibrators and controls at −20 °C to −70 °C. Stable for 6 months.
2.5 Analytical Equipment and Supplies
1.
AB Sciex LC/MS/MS 4000Q TRAP with Shimadzu HPLC .
2.
Analytical column: Supelcosil, LC-18, 5 cm × 4.6 mm, 5 μm (Supelco).
3.
Turbovap LV (Biotage).
4.
Microcentrifuge tubes, 1.5 mL graduated tube with flat cap (Fisher Scientific).
5.
13 × 100 mm screw-cap test tubes.
6.
Autosampler vials with glass inserts and crimp caps (P.J. Cobert Associates).
3 Methods
3.1 Stepwise Procedure
1.
Pipette 20 μL sample, calibrator, blank, or control into a labeled microcentrifuge tube.
2.
Add 200 μL working internal standard reagent to all tubes.
3.
Vortex the tubes and let stand for 5 min.
4.
Centrifuge the tubes for 5 min at 10,000 × g.
5.
Remove 150 μL supernatant from each tube and transfer the extract to a Turbovap tube. Dry the extract for 2 min at 40 °C.
6.
Reconstitute the residue with 400 μL mobile phase mixture (80 % HPLC water:20 % ACN:0.1 % HBA).
7.
Transfer to labeled autosampler vials.
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