To make
Use source solution
Volume of source solution
Volume of 10 mM ammonium phosphate pH 6.5 (mL)
Metanephrine concentration (pg/mL)
Normetanephrine concentration (pg/mL)
Std7
Working solution
100 μL
QS to 100 mL
4000.00
4000.00
Std6
Std 7
50 mL
QS to 100 mL
2000.00
2000.00
Std5
Std 6
25 mL
QS to 100 mL
500.00
500.00
Std4
Std 5
25 mL
QS to 100 mL
125.00
125.00
Std3
Std 4
25 mL
QS to 100 mL
31.25
31.25
Std2
Std 3
50 mL
QS to 100 mL
15.63
Exclude
Std1
Std 2
25 mL
QS to 50 mL
7.81
Exclude
2.
Controls
(a)
Analyte spiking solution: Measure 10 μL of Cerilliant QC Stock (1.0 mg/mL) into 25 mL volumetric flask. Bring to volume with 10 mM Ammonium Phosphate, pH 6.5.
(b)
Level 1: Measure 3 μL of Analyte Spiking Solution and add to a 50 mL volumetric flask. Bring to volume with pooled plasma . Based on past pools, the concentrations may be approximately 60 pg/mL M and ~200 pg/mL NM.
(c)
Level 2: Measure 62.5 μL of Analyte Spiking Solution and add to a 50 mL volumetric flask. Bring to volume with pooled plasma . Based on past pools the concentrations may be approximately 535 pg/mL M and ~675 pg/mL NM.
2.5 Analytical Equipment and Supplies
1.
Nexera Shimadzu HPLC .
2.
AB Sciex 5500 QTRAP.
3.
Vacuum manifold.
4.
Biotage TurboVap.
5.
UltraShield UHPLC pre-column filter.
6.
Ultra PFP Propyl analytical column (3 μm, 2.1 × 100 mm).
7.
Oasis WCX SPE Cartridges.
8.
Injection Vials (12 × 32 mm, 350 μL).
9.
Injection Vial Caps (11 mm snap PTFE/SIL).
2.6 HPLC Parameters
They are given in Table 2.
Table 2
HPLC parametersa
Column temp. | 40 °C | ||
Flow rate | 0.500 mL/min | ||
Gradient | Time (min) | Event | Parameter |
0 | Pump B conc. | 1 | |
0.25 | Pump B conc. | 1 | |
0.26 | Pump B conc. | 40 | |
2.25 | Pump B conc. | 60 | |
2.26 | Total flow | 0.7 | |
2.26 | Pump B conc. | 60 | |
2.86 | Pump B conc. | 60 | |
2.87 | Pump B conc. | 1 | |
2.88 | Total flow | 0.5 | |
aOptimized for Nexera Shimadzu HPLC with Restek Ultra PFP Propyl analytical column (3 μm, 2.1 × 100 mm). Mobile phase A: 1 mM ammonium formate + 0.1 % formic acid; mobile phase B: 1 mM ammonium formate in methanol + 0.1 % formic acid
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