Extraction of Bases from Oral Fluid Using Orasure (OFC Device) on Kinetex Phenyl Hexyl column

LC Conditions

Column

Kinetex 2.6 µm Phenyl-Hexyl 100 Å, LC Column 50 x 4.6 mm, Ea

Brand

Kinetex

Part No

00B-4495-E0

Phase Name

Phenyl-Hexyl

Elution Type

Gradient

Mobile Phase

A: 10 mM Ammonium Formate

B: 0.1% Formic Acid in Methanol

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

95

5

2

4

0

100

3

6

0

100

Flow Rate

0.60 mL/min

Temperature

22°C

Detection

Tandem Mass Spec (MS-MS) @ (22°C)

Detection Info

Sample Preparation Details

Description

Strata-X-Drug B 33 µm Polymeric Strong Cation, 30 mg / 3 mL, Tubes, 50/Pk

Part No

8B-S128-TBJ

Pretreatment Note

SPE Conditions for Bases Cartridge: 30mg/3 CC of Strata-X Drug B Part No.: 8B-S128-TBJ Condition: 1 mL MeOH Equilibration: 1 mL 100 mM sodium acetate buffer, pH 5.0 Load: Pre-treated samples as below -Place the Swab into the 500 uL oral fluid spiked with analyte mix; wait 1 min -Transfer the Swab in cartridge containing the buffer (supplied by device) -Add 1 mL 50:50 (1%FA in H20:MeOH ) -Vortex 30 sec -Break up the cartridge -Centrifuge at 3000rpm for 20 min -Add 1.5 mL 100 mM sodium acetate buffer, pH 5.0 -Transfer contents Wash1: 1 mL 100 mM sodium acetate buffer, pH 5.0 Wash 2: 30% methanol/water Dry: 5 mins under full vacuum Elute: 2x0.5 mL methanol:ACN(1:1) + 5% NH4OH (28-30%) Add: 30 uL 0.1% HCl/methanol Dry down : To dryness under N2 (at 40-450C ) Reconstitute: In 500uL of mobile phase (A/B=85/15) spiked with internal standard Conc. of Spiked samples = 40ng/mL for bases

Condition 1

1 mL Methanol

Condition 2

1 mL 100 mM sodium acetate buffer, pH 5.0

Load

Load 0.50 mL Sample diluted in 100 mM sodium acetate buffer, pH 5.0

Wash 1

1 mL 100 mM sodium acetate buffer, pH 5.0

Wash 2

1 mL Acetonitrile: 100 mM sodium acetate buffer, pH 5.0 (30/70)

Dry

5 min. under vacuum at 10 inches Hg

Elution 1

0.5 mL Methanol:ACN(1:1) + 5% NH4OH (28-30%)

Elution 2

0.5 mL Methanol:ACN(1:1) + 5% NH4OH (28-30%)

Evaporation

Evaporate to dryness

Reconstitute

500 µL mobile phase (A/B=85/15)