Automated Solid Phase Extraction of Allantoin from Cosmetics and Topical Pharmaceuticals Prior to Analysis by HPLC

February 11, 2011

The Application Notebook

The Application Notebook, The Application Notebook-02-11-2011, Volume 0, Issue 0

Allantoin is a heterocyclic organic compound derived from purine.

Michael Halvorson1, Torsten Kretschmer2, and Martin Roedel21Gilson, Inc., and 2Macherey-Nagel GmbH

Allantoin is a heterocyclic organic compound derived from purine. Allantoin has a long history of use in a variety of topical pharmaceuticals and cosmetics for skin care due to its keratolytic, moisturizing, soothing, and anti-irritant properties. Allantoin is typically used in these products at a level of 0.1 to 2.0%. Solid phase extraction (SPE) is often used as a purification step prior to analysis of allantoin in these products due to the complex nature of the matrices.

This study describes an automated SPE protocol using a Gilson GX-271 ASPEC™ System for the extraction of allantoin from cosmetics prior to analysis by HPLC using a NUCLEODUR® 100-3 HILIC column.

Experimental Conditions

Mix 1 g of sample containing allantoin with 100 mL of ultra-pure water. Allantoin-free cosmetics/topicals were spiked with 5 mg allantoin.

Solid Phase Extraction (SPE) Protocol

The SPE procedure used 3 mL Macherey-Nagel CHROMA-BOND®HR-XA (60 mg) cartridges.The SPE protocol is entirely automated using the Gilson GX-271 ASPEC system. The SPE steps are summarized with the schematic provided in the GX-271 ASPEC control software, Trilution LH™ (Figure 1).

  • Initialization Step: Gilson Mobile SPE Racks are moved above the waste rack

  • Condition the cartridge with 1 mL of methanol at 0.5 mL/min

  • Condition the cartridge with 1 mL of ammonia, w(NH3) = 5% at 0.5 mL/min

  • Dispense 4 mL of sample( 1g in 100 mL water) into a tube at 5 mL/min

  • Dispense 400 µL ammonia, w(NH3) = 26% at 0.5 mL/min into the same tube as step above

  • Load 1.1 mL of the sample mix created above onto the SPE cartridge at 0.5 mL/min

  • Wash cartridge with 1 mL of ammonia, w(NH3) = 5% at 0.5 mL/min

  • Wash cartridge with 1 mL of methanol at 0.5 mL/min

  • Dry with 5 mL air, 3 mL/min

  • Move the Gilson Mobile SPE Rack over the collection tubes

  • Elute with 2× 600 µL Hydrochloric acid, HCl, 0.1 mol/L at 0.5 mL/min

  • Eluent can be injected directly into the HPLC system

Figure 1: Trilution LH SPE and liquid handling tasks for extraction of Allantoin.

HPLC Analysis

Column: Macherey-Nagel EC 125/3 NUCLEODUR® 100-3 HILIC (Part no. 760 531.30)

Conditions:

Eluent A: 10 mmol/L Ammonium chloride, pH 3.0 20

Eluent B: Acetonitrile 80

Flow Rate: 0.3 mL/min

Temperature: Ambient

Injection Volume: 20 µL

Concentration: β(Allantoin) = 5 µg/mL eluent

Detection: UV, 214 nm

Figure 2: Chromatogram of Allantoin from cosmetic product following SPE extraction. The retention time for Allantoin is 3.66 min.

Results

The recovery of allantoin from the cosmetic product was 85.5% (n = 3).

CHROMABOND and NUCLEODUR are registered trademarks of Macherey-Nagel GmbH. ASPEC and Trilution are trademarks of Gilson, Inc.

Gilson, Inc.

3000 Paramenter St., P.O. Box 620027, Middleton, WI 53562

tel. (608) 836-1551, fax (608) 831-4451

Website: www.gilson.com