May 09, 2018
Chemical extraction of sulfachloropyridazine from feather samples
- Ekaterina Pokrant1,
- Francisca Medina1,
- Aldo Maddaleno1,
- Betty San Martin2,
- Javiera Cornejo1
- 1Preventive Medicine Department, Faculty of Veterinary and Animal Sciences, University of Chile;
- 2Laboratory of Veterinary Pharmacology, Faculty of Veterinary and Animal Sciences, University of Chile
External link: https://doi.org/10.1371/journal.pone.0200206
Protocol Citation: Ekaterina Pokrant, Francisca Medina, Aldo Maddaleno, Betty San Martin, Javiera Cornejo 2018. Chemical extraction of sulfachloropyridazine from feather samples. protocols.io https://dx.doi.org/10.17504/protocols.io.pr4dm8w
Manuscript citation:
Pokrant E, Medina F, Maddaleno A, Martín BS, Cornejo J (2018) Determination of sulfachloropyridazine residue levels in feathers from broiler chickens after oral administration using liquid chromatography coupled to tandem mass spectrometry. PLoS ONE 13(7): e0200206. doi: 10.1371/journal.pone.0200206
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 25, 2018
Last Modified: May 09, 2018
Protocol Integer ID: 11804
Keywords: Broiler chickens, feather, Sulfachloropyridazine, residues, mas spectrometry
Abstract
Analytical methodology for the detection of sulfachloropyridazine (SCP) in samples of feathers via LC-MS/MS was implemented based on techniques previously published by other authors:
1- Hindle R. A validated atmospheric pressure chemical ionization method for analysing sulphonamides in pork muscle. 2013. Available at http://www.youngin.com/application/0411-0045EN-E.pdf. Accessed October 12, 2017.
2- Renew JE, Huang CH. Simultaneous determination of fluoroquinolone, sulfonamide, and trimethoprim antibiotics in wastewater using tandem solid phase extraction and liquid chromatography–electrospray mass spectrometry. J Chromatogr A 2004; 1042: 113-21.
3- Shao B, Dong D, Wu y, Hu J, Meng J, Tu X, Xu S. Simultaneous determination of 17 Sulfonamide residues in porcine meat, kidney and liver by solid phase extraction and liquid chromatography-tandem mass spectrometry. Anal Chim Acta 2005; 546:174-81.
4- Pang G, Cao YZ, Zhang JJ, Jia GQ, Fan CL, Li XM, Liu YM, Li ZY, Shi YQ. Determination of sulfonamides in honey by liquid chromatography- tandem mass spectrometry. J AOAC Int 2005; 88:1304-11.
5- Stubbings G, Bigwood T. The development and validation of a multi-class liquid chromatography tandem mass spectrometry (LC– MS/MS) procedure for the determination of veterinary drug residues in animal tissue using a QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) approach. Anal Chim Acta 2009; 637:68-78.
6- Bedendo GC, Jardim IC, Carasek E. A simple hollow fiber renewal liquid membrane extraction method for analysis of sulphonamides in honey samples with determination by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2010; 1217:6449-54.
7- Yu H, Tao Y, Chen D, Wang Y, Huang L, Peng D, et al. Development of a high-performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulphonamides in the foods of animal origin. J Chromatogr B 2011; 879:2653-62.
The method is based on a solid-liquid extraction with organic solvents. The clean up is carried out throught aromatic sulfonic acid (Bakerbond spe™) disposable extraction columns of 6 mL. The analyte is concentrated using a water bath at 40-50°C under a mild nitrogen flow. For the instrumental analysis, a Symmetry C8 analytical column of 3.5μm and 2.1 x 100mm (Waters®) was fitted in an Agilent 1290 infinity series liquid-chromatograph equipment, coupled to an API 3200 (AB Sciex, Darmstadt, Germany) triple-quadrupole mass-spectrometer. The analytical data was then integrated using the Analyst® version 1.5 software package (SCIEX, Framingham, Massachusetts).
Materials
MATERIALS
1.5 mL Eppendorf tubes
Falcon Tube (50 mL)Fischer Scientific
BAKERBOND spe™ Aromatic Sulfonic Acid (C6H5SO3H) Disposable Extraction ColumnsJ.T. BakerCatalog #7090-29
Sulfamethazine-(phenyl-13C6) hemihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #32519
SulfachloropyridazineMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9882
1 ml syringe Catalog #JD+01T2713
10 mL syringe Catalog #JD+10L2125-WEI
Glass woolMerck Millipore (EMD Millipore)Catalog #1040860250
Millex Syringe FilterMerck Millipore (EMD Millipore)Catalog #SLGVX13NK
STEP MATERIALS
Ethyl AcetateJ.T. BakerCatalog #9280-03
BAKERBOND spe™ Aromatic Sulfonic Acid (C6H5SO3H) Disposable Extraction ColumnsJ.T. BakerCatalog #7090-29
Ethyl Acetate J.T. BakerCatalog #9280-03
n-HexaneMerck Millipore (EMD Millipore)Catalog #1037014000
Methanol J.T. BakerCatalog #9093-03
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Methanol J.T. BakerCatalog #9093-03
Ammonia solution 25%Merck Millipore (EMD Millipore)Catalog #1054322500
Formic Acid 98-100%Merck Millipore (EMD Millipore)Catalog #100264
Methanol J.T. BakerCatalog #9093-03
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Ethyl AcetateJ.T. BakerCatalog #9280-03
BAKERBOND spe™ Aromatic Sulfonic Acid (C6H5SO3H) Disposable Extraction ColumnsJ.T. BakerCatalog #7090-29
Ethyl Acetate J.T. BakerCatalog #9280-03
n-HexaneMerck Millipore (EMD Millipore)Catalog #1037014000
Methanol J.T. BakerCatalog #9093-03
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Methanol J.T. BakerCatalog #9093-03
Ammonia solution 25%Merck Millipore (EMD Millipore)Catalog #1054322500
Formic Acid 98-100%Merck Millipore (EMD Millipore)Catalog #100264
Methanol J.T. BakerCatalog #9093-03
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Protocol materials
Methanol J.T. BakerCatalog #9093-03
10 mL syringe Catalog #JD+10L2125-WEI
1.5 mL Eppendorf tubes
Falcon Tube (50 mL)Fischer Scientific
SulfachloropyridazineMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9882
Millex Syringe FilterMerck Millipore (EMD Millipore)Catalog #SLGVX13NK
Ethyl AcetateJ.T. BakerCatalog #9280-03
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Ethyl Acetate J.T. BakerCatalog #9280-03
n-HexaneMerck Millipore (EMD Millipore)Catalog #1037014000
Ammonia solution 25%Merck Millipore (EMD Millipore)Catalog #1054322500
Methanol J.T. BakerCatalog #9093-03
BAKERBOND spe™ Aromatic Sulfonic Acid (C6H5SO3H) Disposable Extraction ColumnsJ.T. BakerCatalog #7090-29
n-HexaneMerck Millipore (EMD Millipore)Catalog #1037014000
Methanol J.T. BakerCatalog #9093-03
Methanol J.T. BakerCatalog #9093-03
BAKERBOND spe™ Aromatic Sulfonic Acid (C6H5SO3H) Disposable Extraction ColumnsJ.T. BakerCatalog #7090-29
Methanol J.T. BakerCatalog #9093-03
Formic Acid 98-100%Merck Millipore (EMD Millipore)Catalog #100264
Ethyl Acetate J.T. BakerCatalog #9280-03
Methanol J.T. BakerCatalog #9093-03
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Ethyl AcetateJ.T. BakerCatalog #9280-03
BAKERBOND spe™ Aromatic Sulfonic Acid (C6H5SO3H) Disposable Extraction ColumnsJ.T. BakerCatalog #7090-29
Formic Acid 98-100%Merck Millipore (EMD Millipore)Catalog #100264
Water Merck Millipore (EMD Millipore)Catalog #1153334000
1 ml syringe Catalog #JD+01T2713
Glass woolMerck Millipore (EMD Millipore)Catalog #1040860250
Ammonia solution 25%Merck Millipore (EMD Millipore)Catalog #1054322500
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Sulfamethazine-(phenyl-13C6) hemihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #32519
Ethyl AcetateJ.T. BakerCatalog #9280-03
Ethyl Acetate J.T. BakerCatalog #9280-03
n-HexaneMerck Millipore (EMD Millipore)Catalog #1037014000
BAKERBOND spe™ Aromatic Sulfonic Acid (C6H5SO3H) Disposable Extraction ColumnsJ.T. BakerCatalog #7090-29
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Methanol J.T. BakerCatalog #9093-03
Methanol J.T. BakerCatalog #9093-03
Ammonia solution 25%Merck Millipore (EMD Millipore)Catalog #1054322500
Formic Acid 98-100%Merck Millipore (EMD Millipore)Catalog #100264
Methanol J.T. BakerCatalog #9093-03
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Safety warnings
Protect hands, eyes and face from organic solvents and liquid nitrogen during al the extraction steps with all the necessary materials.
Before start
Feather samples must be cryogenically treated with liquid nitrogen and then ground in an industrial Robot Coupe® R4 table-top cutter food processor (Burgundy, France) to ensure their homogeneity.
Weigh 2 ± 0.02 g of feather sample in a 50-mL polypropylene tube.
Weigh 2 ± 0.02 g of feather sample in a 50-mL polypropylene tube.
Fortifie each sample with the internal standard solution, Sulfamethazine-(phenyl-13C6) hemihydrate (SMZ-13C6) and the positive controls with the analyte Sulfamethazine .
Fortifie each sample with the internal standard solution, Sulfamethazine-(phenyl-13C6) hemihydrate (SMZ-13C6) and the positive controls with the analyte Sulfamethazine .
Samples must be rested before extraction.
Samples must be rested before extraction.
00:15:00
Add 40 mL of ethyl acetate.
Add 40 mL of ethyl acetate.
Ethyl AcetateJ.T. BakerCatalog #9280-03
Stirr the samples on a vortex-mixer.
Stirr the samples on a vortex-mixer.
00:15:00
Sonicate the sample.
Sonicate the sample.
00:05:00
Centrifuge at 1,800 g
Centrifuge at 1,800 g
00:10:00
Filter the supernatant through glass wool, using a syringe of 10 mL. Pass the filtrate to a fresh 50-mL polypropylene tube.
Filter the supernatant through glass wool, using a syringe of 10 mL. Pass the filtrate to a fresh 50-mL polypropylene tube.
Concentrate down to 15 mL using a water bath at 40-50 °C under a mild nitrogen flow.
Concentrate down to 15 mL using a water bath at 40-50 °C under a mild nitrogen flow.
Condition Aromatic sulfonic acid (Bakerbond spe™) disposable extraction columns with 6 mL of hexane and 6 mL of ethyl acetate.
Condition Aromatic sulfonic acid (Bakerbond spe™) disposable extraction columns with 6 mL of hexane and 6 mL of ethyl acetate.
BAKERBOND spe™ Aromatic Sulfonic Acid (C6H5SO3H) Disposable Extraction ColumnsJ.T. BakerCatalog #7090-29
Ethyl Acetate J.T. BakerCatalog #9280-03
n-HexaneMerck Millipore (EMD Millipore)Catalog #1037014000
FIlter the samples through the columns.
FIlter the samples through the columns.
Wash the column with 2 mL of water and 2 mL of methanol.
Wash the column with 2 mL of water and 2 mL of methanol.
Methanol J.T. BakerCatalog #9093-03
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Elute the column with 10 mL of a mixture of methanol and ammonia solution (97/3)
Elute the column with 10 mL of a mixture of methanol and ammonia solution (97/3)
Methanol J.T. BakerCatalog #9093-03
Ammonia solution 25%Merck Millipore (EMD Millipore)Catalog #1054322500
Evaporate the samples using a water bath at 40-50°C under a mild nitrogen flow.
Evaporate the samples using a water bath at 40-50°C under a mild nitrogen flow.
Reconstitute the sample with 300 µL of a mixture of mobile phase A and B (15/85)
Reconstitute the sample with 300 µL of a mixture of mobile phase A and B (15/85)
Note
Mobile phase A: 0.1% formic acid in methanol (pH 2.9 ± 0.3).
Mobile phase B: 0.1% formic acid in water (pH 2.7 ± 0.2).
Formic Acid 98-100%Merck Millipore (EMD Millipore)Catalog #100264
Methanol J.T. BakerCatalog #9093-03
Water Merck Millipore (EMD Millipore)Catalog #1153334000
Stirr the reconstituted solution on a vortex-mixer.
Stirr the reconstituted solution on a vortex-mixer.
00:05:00
Sonicate the reconstituted solution.
Sonicate the reconstituted solution.
00:05:00
Transfer the reconstituted solution into an Eppendorf tube and centrifuge at 17,000 g
Transfer the reconstituted solution into an Eppendorf tube and centrifuge at 17,000 g
00:10:00
Filter the sample through 13 mm millex filters with 0.22 µm polyvinylidene fluoride (PVDF) membranes and transfer into a glass vials.
Filter the sample through 13 mm millex filters with 0.22 µm polyvinylidene fluoride (PVDF) membranes and transfer into a glass vials.