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TMP and Thiamine Standard by Impact PPT on Kinetex 5u C18 100x4.6mm

Sample Preparation Details

SPE Product:Impact™ Protein Precipitation, 2mL Square Well Filter Plate, 2/Pk
Part No:CE0-7565
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a Proprietary bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Final Prep and Analysis: Add 400µL of methanol to a well of a Protein Precipitation Plate; Add 100µL plasma to the well. Mix 3 times with pipette tip (or vortex the whole plate briefly); Wait for 5 minutes; Filter through the Protein Precipitation Plate under vacuum (5 Hg) for 5 minutes. Transfer 50 µL to an autosampler vial (or a well of 96-well plate). Add 100µL water and 25 µL of derivatizing reagent*. Cover the vial with a lid. Vortex for 15 seconds. Put the vial into an autosampler. All conditions are at room temperature (RT). It is ready to inject.

*Derivatizing reagent: Potassium ferricyanide [K3Fe(CN)6] was prepared as the following: dissolve 6mg of K3Fe(CN)6 in 15mL 15% sodium hydroxide solution. Prepare fresh daily.
Inject: 20 µL on HPLC / Fluorescence (FLUOR) @ Ex. 375 nm (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)
1 Thiamine monophosphate 200 
2 Thiamine 200 

Application Detail (App ID: 20875)

Part No:00D-4601-E0
Dimensions:100 x 4.6 mm ID
Elution Type:Gradient
Elution A:25mM Na2HPO4, 10% Methanol, pH 7.0
Elution B:25mM Na2HPO4, 70% Methanol, pH 7.0
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 97 3
2 0.25 75 25
3 0.75 75 25
4 3 65 35
5 4 40 60
6 4.5 97 3
7 5 97 3

Flow Rate:1 mL/min
Col. Temp: 25 °C
Detection: Fluorescence (FLUOR) @ Ex. 375 nm (22°C)
Note:Using Impact Protein Precipitation Plate (Cat Log #CE0-7565) for sample preparation

Analyte Details

Analyte Detail not available

1
Thiamine monophosphate
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2
Thiamine
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