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An706 b agonists:an603

This Application Note contains important information about this product
AFFINILUTE MIP – β-agonists
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Molecularly imprinted polymers (MIPs) are a class of highly cross-linked polymers- engineered to bind one target compound Application Note
or a class of structurally related target compounds with high selectivity. Selectivity is introduced during MIP synthesis inwhich a template molecule, designed to mimic the analyte, guides the formation of specific cavities or imprints that aresterically and chemically complementary to the target analyte(s). It is therefore critical for analysts to use the methodologydescribed below when using this phase. Conventional generic methodologies employed with conventional SPE chemistries(e.g., reversed-phase C18) will yield sub-optimal results when employed with this phase.
Extraction of β-agonists from biological matrices
The following methods have been developed for the selective extraction of beta-agonists from biological matrices. Methodshave been developed for both biological tissues (e.g. bovine muscle) and fluids (e.g. urine). The methods are highlyreproducible and offer β-agonist recoveries in the range of 35-90%. Since the methods are amenable to the extraction of awide range of β-agonists, recoveries may vary for each specific molecule. It is recommended to use the prescribed methodas a screening tool to identify which β-agonists are present. Once specific β-agonists are identified, conditioning, wash, andelution steps can be further optimized to offer higher recoveries if required. Extraction Procedure:
Recommended flow rate is ~0.5 mL/min. For analyte elution, a flow rate at ~0.2 mL/min. is recommended.

Extraction of β-agonists from bovine
Extraction of β-agonists from urine
Application Name:
muscle and other tissues1
and other biological fluids2
Analyte:
Validated for bovine muscle but is amenable with other Sample Matrix:
tissues such as rabbit, duck, turkey, liver, and fish. The compounds cimaterol, cimbuterol, ractopamine, Typical recoveries are over 70% for ritrodrine, clenbuterol, clenproperol, clenbuterol, brombuterol, mabuterol, formoterol, salmeterol, ractopamine, tulobuterol, mapenterol and isoxsuprine meet the requirements for brombuterol, and mapenterol; between 40-70% recovery is General Comments:
quantitative determination. Screening is reliable to below observed for terbutaline, metaproterenol and cimbuterol.
Isoproterenol, salbutamol, fenoterol, and isoxsuprine cannotbe determined via HPLC-UV due to interfering peaks. • Combine 5 g thawed minced muscle; 50 μL of 0.1 ng/μL Urine (centrifuged at 3000 x g for 10 min.) diluted 1:1 (v/v) of internal standard (deuterated analog in methanol); 5 mL Tris buffer, pH 9.5; and ~ 5 mg protease. 50 μL β-glucoronidase – arylsulfatase 4h at room • After cooling to room temperature, hydrolyze conjugates by adding: 15 μL concentrated acetic acid; 1 mL 2 M acetic buffer, pH 5.2; and 40 μL suc d’Helix Pomatia For β-glucoronidase treatment, please refer to Widstrand, • Incubate for 2 hours at 37 °C. Adjust to pH > 12 with • Liquid-liquid extract the mixture with 10 mL ethyl Sample Pre-treatment:
acetate. Isolate upper organic layer, and extract mixture again with 5 mL ethyl acetate. Combine the organic layer from both extractions, and evaporate under N2 at 55 °C. • Reconstitute with 4 mL 20% methanol in water. Adjust to • Remove fats from the sample by adding 1 mL heptane, shake vigorously, centrifuge at 4000 g, and remove/discard the upper and intermediate layers3. Repeat the fat removal procedure with an additional 1 mL heptane. Neutralize the sample (lower aqueous layer) with 50 μL 10 M NaOH and 2 mL 0.1 M phosphate buffer, pH 6. 1. Condition/equili-
brate cartridge with: • 1 mL 25 mM ammonium or sodium acetate, pH 6.7
2. Load sample:
• Apply 2 min. of full vacuum to remove residual moisture • 1 mL DI water (selective elution/removal of salts and (interference
elution):
• Apply full vacuum through cartridge for 2 min to remove • 1 mL 0.5% acetic acid in acetonitrile (selective removal • 1 mL 1% acetic acid in acetonitrile (selective removal of • 1 mL 50 mM ammonium acetate, pH 6.7 (selective removal of electrostatically bonded interferences)4 • 1 mL 50 mM ammonium acetate, pH 6.7 (selective removal • 1 mL 60% acetonitrile/40% DI Water (selective removal of electrostatically bonded interferences)4 • 1 mL 60% acetonitrile/40% DI Water (selective removal of • Apply full vacuum through cartridge for 2 min. to remove • Apply full vacuum through cartridge for 2 min. to remove 4 Analyte elution:
Elute β-agonists with 2 x 5 mL 10% acetic acid in Elute β-agonists with 2 x 1 mL 10% acetic acid in methanol.
methanol. Apply a gentle vacuum between each fraction.
Apply a gentle vacuum between each fraction. Evaporate and flow rate ~0.2 mL/min. Evaporate and reconstitute with LC mobile phase prior to reconstitute with LC mobile phase prior to analysis. Recommended Analytical Technique:
HPLC-UV or LC-MS
Column: Ascentis® Express C18, 5 cm x 2.1 mm I.D., 2.7 μm particle size (53822-U) 10 mM ammonium acetate (pH unadjusted) in methanol (A) and MS-grade water (B) MRM transitions: 1. Metaproterenol 212.19/152.10 1. Procedure based on:
The analysis of beta-agonists in bovine muscle using molecular imprinted polymers with ion trap LCMS screening, Kootstra PR, CJPF Kuijpers, KL Wubs, D van Doorn, SS Sterk, LA van Ginkel and RW Stephany, 2005, Anal. Chim. Acta, 529:75-81 2. Procedure based on:
Multi-residue liquid chromatography/tandem mass spectrometric analysis of beta-agonists in urine using molecularly imprinted polymers. Van Hoof et al., Rapid Commun. Mass Spectrom. 2005; 19: 2801-2808 Evaluation of MISPE for the multi-residue extraction of beta-agonist from calves urine. Withstrand et al., J Chromatogr B Analyt Technol Biomed Life Sci. 2004, May 5; 804(1):85-91 Evaluation of two different clean-up steps, to minimize ion suppression phenomena in ion trap liquid chromatography-tandem mass spectrometry for the multi-residue analysis of beta agonists in calves urine. Fiori M. et al., Analytica Chimica Acta 529 (2005) 207-210 3. If the intermediate layer is very viscous, only the top uppermost layer is removed, and an additional 4 mL 20% methanol is added prior to further extraction with 1 mL hexane.
4. The prescribed wash procedure has been optimized to maximize sample clean-up prior to analysis. To increase recovery, reduce the acetic acid content of the second 1 mL 1% acetic acid in acetonitrile wash step. Recovery can be further improved by eliminating the last two 50 mM ammonium acetate and 60% acetonitrile wash step. Ordering Information
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AFFINILUTE MIP - β-agonists (class selective)
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