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

LC Conditions

Column

Kinetex 5 µm C18 100 Å, LC Column 100 x 4.6 mm, Ea

Brand

Kinetex

Part No

00D-4601-E0

Phase Name

C18

Elution Type

Gradient

Mobile Phase

A: 25mM Na2HPO4, 10% Methanol, pH 7.0

B: 25mM Na2HPO4, 70% Methanol, pH 7.0

Gradient Profile

Step No.

Time(min)

%A

%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

1.00 mL/min

Temperature

25°C

Detection

Fluorescence (FLUOR) @ (22°C)

Detection Info

Sample Notes

Using Impact Protein Precipitation Plate (Cat Log #CE0-7565) for sample preparation

Sample Preparation Details

Description

Impact Protein Precipitation, 2mL Square Well Filter Plate, 2/Pk

Part No

CE0-7565

Pretreatment Note

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.

Load

Load Sample

Evaporation

Evaporate to dryness