Extraction of Opiates in Human Serum Using Strata-X PRO and a Kinetex 2.6 µm Biphenyl LC Column

LC Conditions

Column

Kinetex 2.6 µm Biphenyl 100 Å, LC Column 50 x 3.0 mm, Ea

Brand

Kinetex

Part No

00B-4622-Y0

Phase Name

Biphenyl

Elution Type

Gradient

Mobile Phase

A: 0.1%Formic acid in Water

B: 0.1%Formic acid in Methanol

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

90

5

2

4

5

95

3

5.5

5

95

4

5.51

95

5

5

7

95

5

Flow Rate

0.50 mL/min

Temperature

45°C

System

Agilent Technologies 1260 Infinity SCIEX 4500

Detection

LC/MS/MS

Detection Info

Sample Notes

Human Serum

Sample Preparation Details

Sample Pretreatment

200 µL Human serum diluted 1:1 with 1% Formic acid in Water

Load

400 µL Pre-treated Sample

Wash 1

600 µL of 5% Methanol in Water

Dry

2-3 minutes @ 5 Hg

Elution 1

600 µL Acetonitrile/0.1% Formic acid in Methanol (90:10)

Evaporation

Evaporate to dryness

Reconstitute

200 µL 0.1% Formic acid in water/0.1% Formic acid in Methanol

Steps

0

Load

400 µL Pre-treated Sample

1

Wash

600 µL of 5% Methanol in Water

2

Dry

2-3 minutes @ 5 Hg

3

Elute

600 µL Acetonitrile/0.1% Formic acid in Methanol (90:10)

4

Dry Down

Under a gentle stream of Nitrogen at 40°C to dryness

5

Reconstitute

200 µL 0.1% Formic acid in water/0.1% Formic acid in Methanol