25-OH Vitamin D2 and D3 Standards from Serum on Fully Porous 5µm C18 after PPT

LC Conditions

Column

Luna 5 µm C18(2) 100 Å, LC Column 50 x 2 mm, Ea

Brand

Luna

Part No

00B-4252-B0

Phase Name

C18(2)

Elution Type

Gradient

Mobile Phase

A: 0.05 % Formic acid

B: 5 mM Ammonium acetate + 0.1 % Formic acid in methanol

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

92

8

2

5

92

8

3

205

0

100

4

290

0

100

5

360

92

8

Flow Rate

0.90 mL/min

Temperature

35°C

Detection

Mass Spectrometer (MS) @ (22°C)

Detection Info

Sample Notes

SecurityGuard™ Guard Cartridge System extends column lifetime. - SecurityGuard Cartridges, C18 4 x 2.0mm ID, 10/Pk Part No.: AJ0-4286 - Holder Part No.: KJ0-4282

Sample Preparation Details

Description

Strata-X 33 µm Polymeric Reversed Phase, 30 mg / 3 mL, Tubes, 50/Pk

Part No

8B-S100-TBJ

Pretreatment Note

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

Condition 1

1 mL Acetonitrile

Condition 2

1 mL Acetonitrile/Water (65:35)

Load

Load 1.60 mL Sample diluted in Acetonitrile/Water (50:50)

Wash 1

0.5 mL Acetonitrile/Water (65:35) at 1 mL/min

Wash 2

0.5 mL Acetonitrile/Water (65:35) at 1 mL/min

Elution 1

0.5 mL Acetonitrile at 1 mL/min

Elution 2

0.5 mL Acetonitrile at 1 mL/min

Evaporation

Evaporate to dryness

Reconstitute

100 µL ethyl acetate/acetonitrile (5:95)