25-OH Vitamin D2 and D3 Standards from Serum on Kinetex 2.6µm C18 after PPT

Sample Preparation Details

Part No:8B-S100-TBJ
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a 30 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Final Prep and Analysis: Note: Avoid over drying eluates to avoid loss of analytes and/or internal standard, which will impact both precision and accuracy of data
Reconstitute with 100 µL ethyl acetate/acetonitrile (5:95), and vortex mix for 5 seconds
Centrifuge for 10 min at 2000g to settle any precipitate
Transfer clear liquid to amber-colored autosampler vial and cap Inject: 50 µL on HPLC / Mass Spectrometer (MS) @ amu (450 °C)


Name Spiked
Log P pKa % Rec %RSC (n=0)
1 25-Hydroxy vitamin D3 (25-OH D3)
2 25-Hydroxy vitamin D3-d6 (25-OH D3-d6)
3 25-Hydroxy vitamin D2 (25-OH D2)

Application Detail (App ID: 19313)

Part No:00B-4462-E0
Dimensions:50 x 4.6 mm ID
Elution Type:Gradient
Elution A:0.05 % Formic acid
Elution B:5 mM Ammonium acetate + 0.1 % Formic acid in methanol
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 92 8
2 5 92 8
3 205 0 100
4 290 0 100
5 360 92 8

Flow Rate:1 mL/min
Col. Temp: 35 °C
Detection: Mass Spectrometer (MS) @ amu (450°C)
Detector Info: AB Sciex API 4000™
Note:SecurityGuard™ ULTRA Guard Cartridge System extends column lifetime.
- SecurityGuard ULTRA Cartridges, UHPLC C18 for 4.6mm ID Columns, 3/Pk Part No.: AJ0-8768
- Holder Part No.: AJ0-9000

Analyte Details

Analyte Detail not available

25-Hydroxy vitamin D3 (25-OH D3)

25-Hydroxy vitamin D3-d6 (25-OH D3-d6)

25-Hydroxy vitamin D2 (25-OH D2)