An706 b agonists:an603
This Application Note contains important information about this product
AFFINILUTE MIP –
AFFINILUTE MIP β-agonists 25 mg/10 mL
AFFINILUTE MIP β-agonists 25 mg/3 mL
Molecularly imprinted polymers (MIPs) are a class of highly cross-linked polymers- engineered to bind one target compound
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.
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
muscle and other tissues
and other biological fluids
Validated for bovine muscle but is amenable with other
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
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.
• Digest samples overnight at 60 °C.
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,
2004; and Fiori., 2005.
• Incubate for 2 hours at 37 °C. Adjust to pH > 12 with
• Liquid-liquid extract the mixture with 10 mL ethyl
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
pH 1 with concentrated HCl.
• 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 mL methanol
• 1 mL DI water
brate cartridge with:
• 1 mL 25 mM ammonium or sodium acetate, pH 6.7
2. Load sample:
Apply sample to the cartridge.
flow rate 0.5 mL/min.
• Apply 2 min. of full vacuum to remove residual moisture
• 1 mL DI water (selective elution/removal of salts and
from the cartridge.
hydrophilic matrix components)
• 1 mL acetonitrile
• Apply full vacuum through cartridge for 2 min to remove
Note: Apply gentle
• 1 mL 0.5% acetic acid in acetonitrile (selective removal
residual moisture from cartridge.
vacuum between each
of hydrophobic interferences)4
• 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
of hydrogen bonded interferences)4
• 1 mL 60% acetonitrile/40% DI Water (selective removal of
• Apply full vacuum through cartridge for 2 min. to remove
hydrogen bonded interferences)4
• 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)
Applied Biosystems 3200 Q-TRAP
10 mM ammonium acetate (pH unadjusted) in methanol (A) and MS-grade water (B)
MRM transitions: 1. Metaproterenol 212.19/152.10
Ion spray voltage: 2700 V
Injection volume: 30 µL
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.
AFFINILUTE MIP -
β-agonists (class selective)
AFFINILUTE MIP -
β-Blocker (class selective)
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TENDER DOCUMENT Livestock and Dairy Development Board Main Park Road, NARC Gate No.2, Islamabad Tell:051-9255701-2 Fax:051-9255703 www.lddb.org.pk Contents Terms and Conditions Other Conditions List and Specification of Items Government of Pakistan Ministry of National Food Security and Research Livestock and Dairy Development Board
Rambabu.Kuchi et al, Research Desk, 2012, Oct-Dec, 1(2).66-73 ISSN 2319-7315 A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF TADALAFIL IN FORMULATIONS Gudipati Edukondalu, Mahaboob.Subhani. D. Nunna.Bhaskar Raju, Ashok Kumar varma, Rambabu Kuchi* Dept of P.G Chemistry, D. N. R College, Bhimavaram, West Godavari (D.T) Andra Pradesh, India