Chiral Separation of Dextromethorphan/Levomethorphan and Dextrorphan/Levorphanol on Lux 3u AMP, 150x4.6mm

LC Conditions

Column

Lux 3 µm AMP, LC Column 150 x 4.6 mm, EA

Brand

Lux

Part No

00F-4751-E0

Phase Name

AMP

Elution Type

Gradient

Mobile Phase

A: 5mM ammonium bicarbonate, adjusted to pH 11 with ammonium hydroxide

B: Acetonitrile

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

55

45

2

4.5

55

45

3

4.6

20

80

4

8

20

80

5

8.1

55

45

6

10

55

45

Flow Rate

1.00 mL/min

Temperature

40°C

System

Agilent Technologies 1200 Series Binary LC System SCIEX 4500

Detection

LC/MS/MS

Detection Info

Sample Notes

Urine hydrolyzed with beta-glucuronidase and extracted with Strata-X-C

Sample Preparation Details

Description

Strata X-C 33 µm Polymeric Strong Cation, 30 mg / well, 96-Well Plates, 2/Pk

Part No

8E-S029-TGB

Condition 1

1 mL methanol

Sample Pretreatment

Urine sample pretreatment: Combine 200µL urine sample, 200µL 100mM ammonium acetate buffer (pH 4.0), 20µL internal standard solution (250ng/mL), and 20µL of beta-glucuronidase (>100,000 units/mL) and incubate for 1 hour at 55°C. Centrifuge for 10 minutes at 13,000rcf. Dilute 400µL supernatant 1:1 with 0.1% aqueous formic acid. Serum sample pretreatment: Combine 250µL serum, 250µL 0.1% formic acid, and 20µL internal standard solution (250 ng/mL).

Load

Full pretreated sample (from above)

Wash 1

1.0% formic acid in water

Wash 2

1.0% formic acid in methanol

Dry

3-4 minutes at high vacuum

Elution 1

2 x 0.5 mL 5% NH4OH in 50:50 methanol/acetonitrile

Evaporation

Evaporate to dryness

Reconstitute

100 µL 40:60 acetonitrile/water

Steps

0

Condition

1 mL methanol

1

Equilibrate

1 mL 0.1% formic acid in water

2

Load

Full pretreated sample (from above)

3

Wash

1.0% formic acid in water

4

Wash

1.0% formic acid in methanol

5

Dry

3-4 minutes at high vacuum

6

Elute

2 x 0.5 mL 5% NH4OH in 50:50 methanol/acetonitrile

7

Dry Down

at 40°C under a gentle stream of nitrogen

8

Reconstitute

100 µL 40:60 acetonitrile/water