Calibrator number
Volume of previous calibration material (ml)
Volume of deionized water (ml)
Final concentration (pg/ml)
6
3.75 (working stock)
2.25
625
5
2.0 (Cal #6)
4.0
208
4
2.0 (Cal #5)
4.0
69
3
2.0 (Cal #4)
4.0
23
2
2.0 (Cal #3)
4.0
7.7
1
2.0 (Cal #2)
4.0
2.6
2.
Controls: Bio-Rad Lyphochek Immunoassay Plus Control Level 1–3: To prepare controls 2–4 add appropriate volume of working estrone control material and deionized water to each vial of lyphochek for a total volume of 5.0 ml (see below, Table 2.) and allow to stand for 15 min prior to use. Mix by swirling. Control 1 will be prepared by diluting control 2 1:10 with deionized water during each run. Aliquot into 300 μl aliquots and store at <−20 °C. Stable for up to 30 days (see Note 3 ).
Table 2
Preparation of controls
Control | Volume of control estrone material (μl) | Volume of deionized water (ml) | Final concentration of estrone (pg/ml) |
|---|---|---|---|
2 | 500 | 4.5 | 100 |
3 | 1000 | 4.0 | 200 |
4 | 2000 | 3.0 | 400 |
3.
Operating parameters: Set instrument parameters according to Tables 3 and 4. Parameters are optimized specifically for an API 5500 with Shimadzu 20A Prominence HPLC System; therefore, tune setting may vary slightly between instruments.
Table 3
HPLC operating conditions
Column temp | 45 °C | ||
Flow rate | 0.45 ml/min | ||
Equilibration time | 0.5 min | ||
Gradient | Step | Time (min) | Parameter (%) |
1 | 0.3 | 45 | |
2 | 0.5 | 45 | |
3 | 2.00 | 80 | |
4 | 2.20 | 80 | |
5 | 2.50 | 90 | |
6 | 2.80 | 90 | |
7 | 3.00 | 45 | |
8 | 3.50 | Stop | |
Table 4
Mass spectrometry operating conditions
Mode | MRM |
Polarity | Negative |
Curtain gas | 15.0 psi |
Ion source gas 1 | 70.0 psi |
Ion source gas 2 | 75.0 psi |
Nebulizing current | −5.00 |
Collision cell exit potential | −15.00 |
Entrance potential | −13.0 |
TEMP | 700.0 |
2.5 Equipment and Supplies
1.
2.0 ml 9 mm Short-cap Screw Thread Vial (Restek, Bellefonte, PA).
2.
2.0 ml 9 mm Short-cap Screw Thread Vial Closure (Restek, Bellefonte, PA).
3.
Vial Inserts, 100 μl, tri-spring (Phenomenex, Foster City CA).
4.
13 × 100 ml Aliquot Tube.
5.
Eppendorf Pipette Tips EP 2–200 μl.
6.
Eppendorf Tips, 2.5 ml.
7.
Repeater pipet tips 50 ml.
8.
Centrifuge: Capable of Speeds of 3267 × g (RCF).
9.
TurboVap® IV Evaporator (Zymark Corporation, Hopkinton, MA).
10.
Instrumentation: API 5500 with Shimadzu 20A Prominence HPLC System (AB Sciex, Foster City CA).
11.
Software: Analyst 1.6 quantitative software and Multiquant Software Version 2.1 (AB Sciex, Foster City CA).
12.
Analytical Column: Kinetex XB-C18, 2.6 μl, 50 × 2.10 mm 100 Å (Phenomenex, Torrance, California).
3 Methods
3.1 Stepwise Procedure
1.
Label an appropriate number of 13 × 100 ml glass tubes: Cal 1–6, Blank (Negative/Carry-Over), QC1, QC2, QC3, & QC4.
2.
For QC1 Add 25 μl of Bio-Rad control 1 (QC2) to QC1 tube plus 225 μl of deionized water. For all other controls and calibrators, add 250 μl of control or calibration material to the appropriate tubes, to the Blank and Negative/Carry-Over control add 250 μl of deionized water.
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