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Blank Plasma Samples Extracted from Phree Showing Endogenous Level of Vitamin D3 Using Kinetex C18

Sample Preparation Details

Part No:8E-S133-TGB
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.
Load:Dispense 300 uL of 85%ACN/15% MeOH in the Phree plate.
Add 100uL of Analyte & IS spiked (Vitamin D2 & D3 mix, to yield conc. = 75ng/mL) Human Plasma sample in each well with manual aspiration 2-3x (to ensure complete PPT).

Wait for 30 secs. Apply vacuum for 1-2 mins at high vacuum (15" Hg) and collect filtrate.
Dispense 200 uL more of 85%ACN/15% MeOH. Wait for 30 secs followed by vacuum application for 1-2 mins at 15" Hg and collect filtrate.
Inject the extract directly on to the column (no need to dry down the sample).
Final Prep and Analysis: Inject: 50 µL on HPLC / Mass Spectrometer (MS) @ amu (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)

Application Detail (App ID: 21870)

Part No:00A-4462-Y0
Dimensions:30 x 3 mm ID
Elution Type:Gradient
Elution A:0.1% formic acid/water
Elution B:0.1% Formic acid/methanol
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 40 60
2 0.5 5 95
3 2 5 95
4 2.01 40 60
5 3.5 40 60

Flow Rate:0.6 mL/min
Col. Temp: ambient
Detection: Mass Spectrometer (MS) @ amu (22°C)
Detector Info: AB Sciex API 5000™
Note:Instrument: API 5000 (AB SCIEX)
Ionization Source: APCI, Positive Polarity
Ionization Source Parameters
Curtain Gas: 25.0 psi
Gas 1 (Neb Gas): 40.00 psi
ihe: ON
CAD (Collision Gas): 10.0
NC: 5.00 µA
TEM: 360 °C
Note: Gas 2 is not available in APCI source
MRM Transitions and Mass Dependent Parameters:
Compound Q1, Da Q3, Da Dwell, msec DP, V EP, V CE, V CXP, V
Vit D2 (1) 395.3 209.3 45 69 10 25 10
Vit D2 (2) 395.3 269.2 45 69 10 25 10
IS (D3-3H2) 386.2 257.2 45 69 10 25 10
Vit D3 (1) 383.2 257.2 45 69 10 23 10
Vit D3 (2) 383.2 211.1 45 69 10 34 10


Analyte Details

Analyte Detail not available