Calibrator
Tertiary combo standards (μL)
Secondary combo standards (μL)
Serum volume
Final conc. (ng/dL)
Blank
10 mL
1
10 μL
qs to 10 mL
10
2
20 μL
qs to 10 mL
20
3
50 μL
qs to 10 mL
50
4
20
qs to 10 mL
200
5
100
qs to 10 mL
1000
6
200
qs to 10 mL
2000
7 (Testosterone only)
5
2.
Quality Controls: Prepare controls 1–3 according to Table 2 (see Note 2 ).
Table 2
Preparation of quality controls using ultra-low steroids serum
QC | Tertiary combo standards (μL) | Secondary combo standards (μL) | Serum volume | Final conc. (ng/dL) |
|---|---|---|---|---|
1 | 50 μL | qs to 10 mL | 50 | |
2 | 100 μL | qs to 10 mL | 1000 | |
3 | 160 μL | qs to 10 mL | 1600 |
3.
Add appropriate amount of standards to 10 mL volumetric flask and qs to 10 mL with ultra-low steroids serum.
2.5 Analytical Equipment and Supplies
1.
AB Sciex LC/MS/MS 4000Q TRAP (Foster City, CA).
2.
Shimadzu Prominence HPLC system with autosampler, two pumps, and degasser (Lenexa, KS).
3.
Autosampler vials with caps.
4.
Disposable culture tubes 13 × 100 mm.
5.
Disposable culture tubes 10 × 75 mm.
6.
Analytical column: Kinetex C18, 100 × 3 mm, 2.6 μm, 100 A (Phenomenex).
7.
Guard column: Pinnacle, C18, 10 mm × 4 mm × 5 μm (Restek, Bellefonte, PA).
3 Methods
3.1 Stepwise Procedure
1.
Pipette 300 μL of each calibrator, control, and sample to the appropriately labeled 10 × 75 mm glass tubes.
2.
Add 50 μL working internal standard to each tube.
3.
Add 1.5 mL methyl tert-Butyl Ether (MTBE). Vortex and rock the tubes for 15 min.
4.
Centrifuge the tubes for 2000 × g for 10 min.
5.
Transfer the upper organic layer to a 13 × 100 mm.
6.
Evaporate the extract to dryness under stream of nitrogen at 45 °C.
7.
Reconstitute with 100 μL of methanol and vortex briefly.
8.
Transfer reconstituted samples to the autosampler vials and inject 20 μL into LC/MS/MS for analysis.
3.2 Instrument Operating Conditions
Instrument’s operating conditions are given on Table 3.
Table 3
Instruments operating conditions
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
