SOP: Derivatized testosterone_01 Revision: 01 Date Effective: 05/17/15

Transcription

1 Chemicals needed: Precipitate solution, Methanol Derivatization Solution, Amplifex Keto Reagent Kit Mobile phases, H 2 0 with 0.1% Formic Acid LC/MS grade and Acetonitrile with 0.1% Formic Acid LC/MS grade Testosterone Standard Testosterone-D 3 Internal Standard. Materials needed: Labeled 0.3 ml microcentrifuge tubes Repeater Pipette Calibrated Micropipettes in various volumes (see table below) Appropriate Micropipette tips (see table below) Refrigerator Refrigerated Centrifuge SpeedVac Dryer with carrier for 96-well plates 96-well plate and pre-pierced silicon sealing mat LC- Accucore RP-MS (100 x 2.1 mm, 2.6μ) HPLC column Personal Protective Equipment Type Volumes (μl) Tip color P white P yellow P yellow P blue Precise Micropipette Volume and Transfer capabilities Instrumentation: Centrifuge, Eppendorf- 5417R: Samples are centrifuge below 10 C for 10 min at 20,000 rcf. SpeedVac, Savant AES1010: Supernatant is dried at low heat. HPLC, Thermo Scientific- Accela: Check the lines for air bubbles and purge line if required. pg. 1 of 5

2 Mass Spectrometer, Thermo Scientific- TSQ Quantum Ultra: The HESI II probe should be installed at position C. Extraction Procedure: 1- Add 50 µl of thawed vortexed sample to labeled, clean microcentrifuge tube using a P200 micropipette. 2- Add 20µL Testosterone-D 3 Internal Standard using a P20 micropipette.* 3- Allow equilibrate for at least 30 min at <10 C 4- Add 200 µl Methanol (100%) using a repeater pipette. Vortex sample to ensure mixing. 5- Cool sample at <10 C for 30 minutes to help precipitate proteins. 6- Spin at 20,000 rcf for 10 mins at <10 C to pellet proteins. 7- Using a P200 micropipette transfer 200µL of supernatant to 96-well plate, leaving behind protein pellet. 8- Dehydrate supernatant using SpeedVac. 9- Derivatize samples by adding 80 µl of derivatizing reagent (1:1 mixture of Amplifex Keto Reagent and Amplifex Keto Diluent). Vortex. 10- Incubate at room temperature for 60 min. 11- Quench reaction by adding 80 µl water. Vortex. 12- Load samples into auto sampler. *See APPENDIX A for preparation of Testosterone-D 3 Internal Standard. Data Collection: 1- Accucore RP-MS (100 x 2.1 mm, 2.6μ) HPLC column 2- Set up sequence starting with two methanol blanks, followed by Unknown Samples. After every 96 Unknown Samples, and following the Unknown Samples, run two methanol blanks, followed by a standard curve (low to high concentration). After the standard curve run two methanol blanks before running more Unknown Samples. 3- Injection volume is 10 ul. Gradient Information Flow rate is 600 ul/min. Duration of run is 2 min Initial conditions are 70% Solvent A (0.1% FA in Water) pg. 2 of 5

3 Solvent B (0.1% FA in Acetonitrile) linearly increases to 35% by 0.8 min, and to 80% by 1.0 min, where it is held until 1.9 min. The gradient is returned to initial conditions (70% Solvent A) by 2 min. Instrument Parameters HESI Probe Positive (+) Probe Temperature 300 C Spray Voltage 2500 V Capillary Temperature 300 C Sheath Gas 50 Auxillary Gas 20 Sweep gas 1 Nitrogen Collision Gas Pressure 1.5 mtorr Tune Lens 88 Collision Energy 60 ev SRM: Testosterone SRM: Testosterone-D Data Processing: 1- Integrate each of the two derivatized Testosterone peaks independently, and calculate the Testosterone: Testosterone-D3 peak ratio for each peak. 2- Add the peak area ratios to get the Total Peak Area Ratio 3- Plot the Standard Curve by graphing Testosterone concentrations against Total Peak Area Ratios.** 4- Calculate the Testosterone concentration for each Unknown Sample from the Standard Curve using reverse prediction. ** See APPENDIX B for preparation of Standard Curves. Created By: Eoin Quinlivan Date: 05/18/15 Reviewed By: Tim Garrett Date: 05/18/15 Approved By: Tim Garrett Date:05/18/15 Revision Number Name Reason for Revision Effective Date 01 Eoin Quinlivan Creation of SOP 05/18/15 pg. 3 of 5

4 APPENDIX A - Preparation of Testosterone-D3 Internal Standard Stock solution Prepare Internal Standard Stock solutions by diluting a vial (100 μg/ml in acetonitrile) of [16, 16, 17-d 3 ]testosterone (Product # T-046; Cerilliant) as follows: Stock A: Dilute 200 μl of neat Testosterone-D3 (100 μg/ml) to 200 μl with water = 50 μg/ml Stock B: Dilute 20 μl of Stock A (50 μg/ml) to 1000 μl with 1: 1:: water: acetonitrile = 1 μg/ml Testosterone-D3. Stock B was aliquoted and stored at -80 C until required. Prepare the Testosterone-D 3 Internal Standard by thawing Stock B, vortexing, and diluting 10 μl (1 μg/ml) to 1000 μl with water = 10 ng/ml (200 pg/ 20 μl) Testosterone. [The final concentration of Testosterone-D 3 per 50 μl sample = 200 pg/ 50 μl (4 ng/ ml)] pg. 4 of 5

5 APPENDIX B - Preparation of Testosterone Standard Curve stock solutions Prepare Standard Stock Solutions by diluting a vial (1 mg/ml in acetonitrile) of testosterone (Product # T-037; Cerilliant) as follows: Stock 1: Dilute 500 μl of neat Testosterone (1 mg/ml) to 1000 μl with water = 500 μg/ml Stock 2: Dilute 200 μl of Stock 1 (500 μg/ml) to 1000 μl with 1: 1:: water: acetonitrile = 100 μg /ml Testosterone. Stock 2 was aliquoted and stored at -80 C until required. Prepare the Testosterone Working Standard by thawing Stock 3, vortexing, and diluting as follows: Working Standard 1: Dilute 200 μl of Stock 3 (100 ng/ml) to 1000 μl with water = 20 ng/ml Testosterone. This Working Standard is used to prepare Standard Curves for Total Testosterone (Male), and for Total + Free Testosterone (Male) Working Standard 2: Dilute 50 μl of Working Standard 1 (20 ng/ml) to 1000 μl with water = 1 ng/ml Testosterone. This Working Standard is used to prepare Standard Curves for Total Testosterone (Female), and for Free Testosterone (Male). Prepared Testosterone Standard Curves by serially diluting the appropriate Testosterone Working Standard 1:1 with water. Standard Curve Ranges: Total Testosterone (Female): 1 ng/ml 62.5 pg/ml (100 pg/ dl 6.25 pg/dl) Free Testosterone (Male): 1 ng/ml 62.5 pg/ml (100 pg/ dl 6.25 pg/dl) Total Testosterone (Male): 20 ng/ml 625 pg/ml (2 ng/ dl 62.5 pg/dl) Total + Free Testosterone (Male): 20 ng/ml pg/ml (2 ng/ dl pg/dl) Process the Standard Curves for derivatization and injection onto LC- in the same manner as the Unknown Samples. pg. 5 of 5