Calibrator level
Spiking solution
Volume of spiking solution (μL)
Final volume (mL)
Final concentration (ng/dL)
Final concentration (pmol/L)
1
Low
35
50
1.6
45
2
Low
80
50
3.7
104
3
Low
160
50
7.5
207
4
High
32
50
15
415
5
High
62
50
29
804
6
High
125
50
58.5
1620
7
High
250
50
117
3241
8
High
500
50
234
6482
2.5 Analytical Equipment and Supplies
1.
Biotage ISOLUTE® SLE+ 400 μL plates (Biotage, Charlotte, NC).
2.
Vacuum manifold or positive pressure manifold, installed in robotic pipettor or manually controlled.
4.
MicroMat TFE/Silicone 96-well pre-slit square sealing mats (SUN-SRi, Rockwood, TN).
5.
API-5000 or API-5500 QTRAP® triple quadrupole mass spectrometer (AB SCIEX, Concord, ON) or other mass spectrometer capable of reaching the required limit of detection, equipped with appropriate software (e.g. Analyst®).
6.
Shimadzu 20AC LC System with pumps, column oven, degasser, autosampler, or equivalent system (Kyoto, Japan).
7.
Analytical column: Gemini NX-C18 3 μm 110 Å 100 mm × 2.0 mm (Phenomenex, Torrance) with a SecurityGuard™ C18 guard cartridge for columns with 2.0–3.0 mm internal diameters (Phenomenex, Torrance, CA).
3 Methods
3.1 Stepwise Procedure
1.
Thaw calibrators, patient pool, and patient samples. Allow an IS tube come to room temperature.
2.
Centrifuge the calibrators, patient pool, and patient samples for 5 min at 2100 × g.
3.
Add 10 mL of 1:1 methanol:water to the IS tube. Vortex mix for 45 s.
4.
Add 50 μL of 0.04 μg/mL d7-aldosterone IS prepared in step 3 to the appropriate number of wells in a Costar™ 96-well plate.
5.
Add 250 μL of calibrators, controls, and serum patient samples to the wells containing IS.
6.
Mix plate for 20 s at medium speed.
7.
9.
Add entire sample and IS (total 300 μL) to wells of SLE+ 400 μL plate.
10.
Apply a short (5–10 s) burst of vacuum to apply sample to SLE sorbent.
11.
Wait for 5 min.
12.
Add 900 μL of MtBE to all samples in appropriately ventilated conditions (fume hood or liquid handler under negative pressure).
13.
Apply short burst of vacuum.
14.
Wait for 5 min.
15.
Apply vacuum for 1–2 min to elute MtBE.
16.
Add an additional 900 μL of MtBE to all sample wells.
17.
Apply a short burst of vacuum.
18.
Wait for 5 min.
19.
Apply vacuum for 1–2 min to elute MtBE.
20.
Remove collection plate, and evaporate to dryness at room temperature in a fume hood.
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