HyperSep Column Applications

Acetaminophen in Solution

Using 60mg 3mL HyperSep Retain PEP Extraction Column

(Part Number: 60107-203)

Sample Preparation

Prepare a 5ppm sample in 0.2% ammonium acetate, pH 5

Condition Retain PEP Extraction Column

2mL of methanol

2mL of DI H

Apply Sample

Load 4mL samples at 1-2mL/minute

Wash Column

1mL of DI H

Elute Acetaminophen

5mL of methanol

Dry Eluate and Reconstitute

Evaporate to full dryness under a stream of nitrogen

Reconstitute the sample to 1mL with water

Analysis

Mobile phase: ac

etonitrile:1mM

(30:70, v/v)

Flow: 1mL/minute

Temperature: 30°C

Detection: UV 254nm

Recommended HPLC Column

Part Number

Hypersil GOLD 5µm, 250 x 4.6mm

25005-254630

Pharmaceutical/Biotech

Applications

Benzodiazepines in Serum or Plasma for HPLC Analysis

Using 200mg 6mL HyperSep Verify-CX Extraction Column

(Part Number: 60108-722)

Prepare Sample

To 1mL 1.0mL of 100mM phosphate buffer (pH 6.0) add

internal standard(s)*

Add 1mL of serum or plasma

Mix/vortex

Sample pH should be 6.0±0.5

Adjust pH accordingly with 100mM monobasic or

dibasic sodium phosphate

Centrifuge as appropriate

Condition HyperSep Verify-CX Extraction Column

1 x 3mL CH

1 x 3mL DI H

1 x 1mL 100mM phosphate buffer (pH= 6.0)

NOTE:

Aspirate at <3"Hg to prevent sorbent drying

Apply Sample

Load at 1mL/minute

Wash Column

1 x 2mL DI H

1 x 2mL 20% acetonitrile in 100mM phosphate

buffer (pH 6.0)

Dry column (10 minutes at >10"Hg)

1 x 2mL hexane

Elute Benzodiazepines

1 x 5mL ethyl acetate containing 2% ammonium hydroxide

Collect eluate at 1 to 2mL/minute

Dry Eluate

Evaporate to dryness at <40°C

Reconstitute

Reconstitute in mobile phase

Quantitate

Inject sample onto HPLC

* Suggested internal standards: Alprazolam-D5, Alphahydroxyalprazolam-D5, Diazepam,

Lorazepam-D4, Oxazepam-D5, Temazepam-D5

Recommended HPLC Column

Part Number

BETASIL Phenyl/Hexyl 5µm, 150 x 4.6mm

73005-154630