2.4 Internal Standards
1.
1α,25-Dihydroxyvitamin D3 (26,26,26,27,27,27-d6), Medical Isotopes, Pelham, NH.
2.
1α,25-Dihydroxyvitamin D3 (26,26,26,27,27,27-d6), Medical Isotopes, Pelham, NH.
3.
Preparation of individual standard stocks (20 μg/mL): Check the vial label to ensure the content is approximately 1 mg. Add 0.5 mL 2-propanol to each vial then quantitatively transfer the contents of the individual d6-1,25(OH)2D2 and d6-1,25(OH)2D3 standard vials to separate 50 mL Class A volumetric flasks. Repeat 2-propanol transfer step 3 times to ensure complete transfer of material. Bring to volume with 2-propanol (stable for 2 years when stored at <−60 °C).
4.
Preparation of individual internal standard substock (200 ng/mL): Using a Class A volumetric pipette, add 1 mL of each 20 μg/mL stock internal standard into separate 100 mL Class A volumetric flasks. Bring to volume with 2-propanol (stable for 2 years when stored at < −60 °C).
5.
Preparation of combined working internal standard (d6-1,25(OH)2D3 and d6-1,25(OH)2D2): Using a Class A volumetric pipette add 1.1 mL of the d6-1,25(OH)2D3 200 ng/mL substock internal standard and 2 mL of the d6-1,25(OH)2D2 200 ng/mL substock internal standard into the same 100 mL Class A volumetric flask. Bring to volume with 70:30 methanol:water (stable for 1 year when stored at < −20 °C). This recipe will change based on the concentration of the original internal stock vial.
6.
Preparation of injection standard (test mix): In a 100 mL Class A volumetric flask, add the following using a Hamilton glass syringe:
(a)
d6-1,25(OH)2D3 internal standard substock (50 μL; 200 ng/mL d6-1,25(OH)2D3).
(b)
d6-1,25(OH)2D2 internal standard substock (25 μL; 200 ng/mL d6-1,25(OH)2D2).
(c)
Combined substock calibration standard (500 μL; 10 ng/mL).
(d)
Bring to volume with 70:30 methanol:water (stable for 3 months when stored at < −20 °C).
2.5 Calibrators and Controls
1.
Calibrators: Standard 5 Solution (160 pg/mL) [Note: Defrost and pool at least 300 mL of aliquoted SeraCon II for this step.]: Using a Hamilton syringe, add 1600 μL of the 10 ng/mL Combined substock to a 100 mL class A volumetric flask. Bring to volume with SeraCon II. (Note: Allow this solution to rest >1 min before further dilution.)
2.
Cal 5 is serially diluted with volumetric pipettes into 125 mL bottles to prepare the rest of the working standards as shown in Table 1.
Table 1
Preparation of calibrators
To make calibrator | Use source solution | Volume of source solution (mL) | Volume of SeraCon II | Final concentration (pg/mL) |
|---|---|---|---|---|
Cal 4 | Cal 5 | 50 | 50 | 80 |
Cal 3 | Cal 4 | 25 | 75 | 20 |
Cal 2 | Cal 3 | 50 | 50 | 10 |
Cal 1 | Cal 2 | 50 | 50 | 5 |
3.
All working standards are aliquoted (0.6 mL) into labeled clear glass screw top 2 mL vials (stable for 6 months when stored at < −60 °C).
2.6 Analytical Equipment and Supplies
1.
Onyx monolithic C18 columns (100 × 3.0 mm).
2.
UV/visible spectrophotometer.
3.
Savant SpeedVac.
4.
Vortex genie mixer.
5.
TLX2 Autosampler and HPLC (Thermo Fisher Scientific).
6.
Liquid chromatography parameters include:
(a)
Mobile phase A: 0.5 mM lithium acetate in 70:30 methanol:water.
(b)
Mobile phase B: 0.5 mM lithium acetate in methanol.
(c)
Mobile phase C: 0.1 % glacial acetic acid in 80:20 water:methanol.
(d)
Injection volume: 40 μL.
(e)
Elution gradients, see Table 2.
Table 2
Elution gradients
Method time (min) | Duration (s) | Flow rate (mL/min) | Step/ramp | Mobile phase A% | Mobile phase B% |
|---|---|---|---|---|---|
0 | 30 | 0.5 | Step | 100 | NA |
0.5 | 300 | 0.5 | Ramp | NA | 100 |
5.5 | 90 | 0.5 | Step | NA | 100 |
7 | 180 | 0.5 | Step | 100 | NA |
7.
Get Clinical Tree app for offline access
Thermo TSQ Vantage Tandem Mass Spectrometer (Thermo Fisher Scientific) parameters include H-ESI Probe settings as follows:
(a)
Spray voltage: 500 V.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree