Testosterone Assay Protocol 

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Chemicals:

• Precipitating solution (Methanol)

• Derivatization solution (Amplifex Keto Reagent Kit)

• Mobile phases: LC/MS-grade water with 0.1% formic acid (Solvent A) and LC/MS-grade acetonitrile with 0.1% formic acid (Solvent B)

• Testosterone standard

• Testosterone-D3 internal standard

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Materials:

• 0.3 mL microcentrifuge tubes (labeled)

• Repeater pipette

• Calibrated micropipettes (various volumes)

• Micropipette tips (appropriate sizes and colors)

• Refrigerator

• Refrigerated centrifuge

• SpeedVac dryer with 96-well plate carrier

• 96-well plate

• Pre-pierced silicon sealing mat

• LC-MS/MS system

• Accucore RP-MS HPLC column (100 x 2.1 mm, 2.6 μm)

• Personal protective equipment (PPE)

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Instrumentation:

• Centrifuge: Eppendorf 5417R (samples centrifuged below 10°C, 10 min, 20,000 rcf)

• SpeedVac: Savant AES1010 (supernatant dried at low heat)

• HPLC: Thermo Scientific Accela (lines checked for air bubbles and purged if necessary)

• Mass Spectrometer: Thermo Scientific TSQ Quantum Ultra (HESI II probe installed at position C)

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Extraction Procedure:

1. Add 50 µL of thawed, vortexed sample to a labeled microcentrifuge tube using a P200 pipette.

2. Add 20 µL of Testosterone-D3 internal standard using a P20 pipette.

3. Equilibrate for at least 30 min at <10°C.

4. Add 200 µL of 100% methanol using a repeater pipette. Vortex to mix.

5. Cool at <10°C for 30 minutes to precipitate proteins.

6. Centrifuge at 20,000 rcf for 10 min at <10°C.

7. Transfer 200 µL of supernatant to a 96-well plate using a P200 pipette, leaving the protein pellet behind.

8. Dry the supernatant using a SpeedVac.

9. Derivatize by adding 80 µL of derivatizing reagent (1:1 Amplifex Keto Reagent:Amplifex Keto Diluent). Vortex.

10. Incubate at room temperature for 60 min.

11. Quench the reaction by adding 80 µL of water. Vortex.

12. Load samples into the autosampler.

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Data Collection:

1. Use an Accucore RP-MS (100 x 2.1 mm, 2.6 μm) HPLC column.

2. Run a sequence: two methanol blanks, unknown samples, two methanol blanks, standard curve (low to high), two methanol blanks, unknown samples, etc. (96 unknown samples between blanks and curves).

3. Injection volume: 10 µL.

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Gradient Information:

• Flow rate: 600 µL/min

• Run duration: 2 min

• Initial conditions: 70% Solvent A (0.1% FA in water)

• Solvent B (0.1% FA in acetonitrile): Linearly increase to 35% by 0.8 min, then to 80% by 1.0 min (held until 1.9 min). Return to 70% Solvent A by 2 min.

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Instrument Parameters:

• HESI Probe: Positive (+)

• Probe Temperature: 300°C

• Spray Voltage: 2500 V

• Capillary Temperature: 300°C

• Sheath Gas: 50

• Auxiliary Gas: 20

• Sweep Gas: 1

• Nitrogen Collision Gas Pressure: 1.5 mTorr

• Tune Lens: 88

• Collision Energy: 60 eV

• SRM: Testosterone 403.303→164.200; Testosterone-D3 406.303→164.200

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Data Processing:

1. Integrate each of the two derivatized testosterone peaks independently. Calculate the testosterone:testosterone-D3 peak ratio for each peak.

2. Sum the peak area ratios (Total Peak Area Ratio).

3. Plot the standard curve (testosterone concentration vs. Total Peak Area Ratio).

4. Calculate unknown sample testosterone concentrations from the standard curve using reverse prediction.

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