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Columns and Supplies Agilent Restricted Month ##, 200X Page 1 Tetracyclines in Chicken using Solid Phase Extraction (SPE) and HPLC/UV Chen-Hao (Andy) Zhai.

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Presentation on theme: "Columns and Supplies Agilent Restricted Month ##, 200X Page 1 Tetracyclines in Chicken using Solid Phase Extraction (SPE) and HPLC/UV Chen-Hao (Andy) Zhai."— Presentation transcript:

1 Columns and Supplies Agilent Restricted Month ##, 200X Page 1 Tetracyclines in Chicken using Solid Phase Extraction (SPE) and HPLC/UV Chen-Hao (Andy) Zhai Agilent Technologies October 2008

2 Columns and Supplies Agilent Restricted Month ##, 200X Page 2 Abstract A method for the determination of residual Tetracyclines in chicken has been established: Tetracyclines were extracted from chicken with EDTA-McIlvaine buffer Clean up was performed using Agilent SampliQ OPT solid phase extraction cartridges Eluate was analyzed by HPLC with DAD Limits of detection (LOD) were between 2.5 and 5 ug/kg Calibration curves were linear over the range of 25 to 500 ug/kg Recoveries based on solution standards were between 52.0% and 99.0% with RSD (n=6) values between 1.0% and 6.5%

3 Columns and Supplies Agilent Restricted Month ##, 200X Page 3 Introduction Tetracyclines is the name used for a class of antimicrobials which all contain a hydronaphthacene backbone. Effective against a wide range of gram negative and gram positive micro-organisms. Most governments now regulate the amount of residual tetracyclines found in food, particularly animal tissues

4 Columns and Supplies Agilent Restricted Month ##, 200X Page 4 Materials Standards: All reagents and solvents were HPLC or analytical grade Tetracycline standards were purchased from Sigma-Aldrich or NICPBP Stock solution: (0.1mg/mL) prepared in methanol and kept in the freezer (-20C) Working solutions prepared using the stock solution diluted with a mixture of methanol / 10mM trifluoroacetic acid solution (1/19) Working solutions prepared daily SPE cartridges: Agilent SampliQ OPT 3mL, 60mg (Part # ) Solutions: McIlvaine buffer, mix 1000mL 0.1M citric acid with 625mL 0.2M disodium hydrogen phosphate (pH adjusted to 4.0 ± 0.05 with NaOH or HCL as needed) Na2EDTA-McIlvaine buffer (0.1M), mix 60.5g Na2EDTA.2H2O into 1625 mL McIlvaine buffer. 10mM TFA solution: mL trifluoroacetic acid dissolved in water to a final volume of 1000mL. Methanol-TFA solution: 5% methanol in 10mM TFA solution

5 Columns and Supplies Agilent Restricted Month ##, 200X Page 5 Materials and Methods – HPLC Chromatography System: Agilent 1200 HPLC, quarternary pump, autosampler, thermostatic column compartment, diode array detector HPLC conditions: Column: Agilent Zorbax SB-C8 250×4.6mm 5µm (Part# ). Flow rate: 1.5ml/min Column Temperature: 30 o C Injection volume: 100μl Detector wavelength: 350nm Mobile phase: 10 mmol/L TFA solution, methanol, acetonitrile, gradient elution time (minutes)% methanol% acetonitrile% 10mmol TFA

6 Columns and Supplies Agilent Restricted Month ##, 200X Page 6 Methods – Sample Preparation The physical-chemical properties of the seven tetracyclines compounds are shown in Table 1 and the chemical structures are shown in Fig. 1 The method used for the preparation of the chicken tissue is shown schematically in Fig g chicken was homogenized and placed in a clean, sealed container and stored at -18 o C 5g homogeneous sample (accurate to 0.01 g) was placed into a 50 mL polypropylene centrifugal tube with 20mL 0.1 mol/L Na 2 EDTA-Mcllvaine buffer solution and vortexed for 1 minute followed by a 10 minute ultrasonic extraction in an ice bath. Sample was then centrifuged at a speed of 3000 r/min for 5 minutes (below 15 o C). The supernatant was removed and saved in a clean tube. The extraction was repeated twice with 20mL and 10mL successively and the combined supernatants were brought to 50 mL with buffer, mixed well, and centrifuged at a rotate speed of 4000 r/min for 10 min (below 15 o C), then filtered. CompoundpKaLog PMW Minocycline 3.3 /7.2 / Oxytetracycline 3.3 / 7.3 / Tetracycline 3.3 / 7.7 / Demeclocycline 3.3 / 7.2 / Chlortetracycline 3.3 / 7.4 / Methacycline 3.5 / 7.6 / Doxycycline 3.1 / 7.7 / Table 1: Physical-Chemical Properties of Selected Tetracyclines

7 Columns and Supplies Agilent Restricted Month ##, 200X Page 7 Chemical Structures of Tetracyclines

8 Columns and Supplies Agilent Restricted Month ##, 200X Page 8 SampliQ OPT Sample Preparation – Chicken Muscle Accurately weigh 5g raw, homogenized chicken meat, Homogenize 1 min. in 20mL 0.1M Na 2 EDTA-McIlvaine buffer Bring total supernatant to 50mL final vol. Centrifuge at 4000 rpm for 10 min. repeat 2x, 20mL and 10mL successively Filter, take 10mL for SPE Ultrasonic extraction in ice bath for 10 min. Centrifuge 3000rpm 5min transfer supernatant to centrifuge tube B

9 Columns and Supplies Agilent Restricted Month ##, 200X Page 9 Methods – SPE Purification The procedure used for the SPE extraction is shown in Figure 3 Agilent SampliQ OPT cartridges were preconditioned with 5mL methanol then 5mL 10mM TFA solution. 10 mL extract (equivalent to 1 g sample) was passed through the SampliQ OPT cartridge at a speed of 1mL/min. After it effused completely, the cartridge was washed with 3 mL water (pH adjusted to 4.5 with TFA) and the entire effluent was discarded. The cartridge was dried under a negative pressure (below 2.0 kPa) for 3 minutes Sample is eluted with 10ml of 10mmol oxalic acid in methanol (Oxalic acid acts as a chelating agent and maintains the pH where tetracyclines are most stable) Eluent was collected and dried under nitrogen below 40 o C. Residue was dissolved and made to a constant volume of 0.5mL using the methanol-TFA solution, filtered through a 0.45 µm filter membrane, and analyzed

10 Columns and Supplies Agilent Restricted Month ##, 200X Page 10 SampliQ OPT Process for 3mL Cartridge Condition 5mL methanol Equilibrate 5mL 10mM TFA (pH 2.16) Wash 3mL water (adjust pH to 4.5 w/ TFA) Elute with 8mL 10mM Oxalic acid* in MeOH Dry 3 minutes under vacuum Recommended flow through cartridge: not faster than 1mL per minute Load 10mL extract (equivalent to 1g meat) Dry, reconstitute in 5% MeOH 10mM TFA Filter

11 Columns and Supplies Agilent Restricted Month ##, 200X Page 11 Results - Linearity and Limits of Detection Stock solutions were diluted to different concentrations and analyzed by HPLC Linear regressions were calculated for the tetracyclines based on the areas and the solution concentrations Limit of detection (LOD) was the injection concentration which signal to noise ratio was between 2 and 3 Linear range was between µg/kg. The linearity and LOD are shown in Table 2 CompoundRegression equationCorrelation coefficientLOD(µg/kg) MinocyclineY=86.313x OxytetracyclineY=95.965x TetracyclineY=103.97x DemeclocyclineY=68.659x ChlortetracyclineY=51.752x MethacyclineY=98.243x DoxycyclineY=76.408x Table 2. Linearity and LODs of Tetracyclines

12 Columns and Supplies Agilent Restricted Month ##, 200X Page 12 Recovery and Reproducibility The precision of the method was determined as recoveries of spiked tetracycline standards in chicken at 50µg/kg, 100µg/kg and 200µg/kg levels The analysis was performed in replicates of 6 at each level The chromatograms of the blank and spiked standard are shown in Figure 4 and Figure 5. The recovery and reproducibility data are shown in Table 3.

13 Columns and Supplies Agilent Restricted Month ##, 200X Page 13 Figure 4. Chromatogram of chicken blank Figure 5. Chromatogram of a chicken sample spiked with: 1-minocycline, 2- oxytetracycline, 3-tetracycline, 4-demeclocycline, 5-chlortetracycline, 6-demeclocycline, 7-doxycycline

14 Columns and Supplies Agilent Restricted Month ##, 200X Page 14 Recoveries and RSDs of Tetracyclines in Chicken

15 Columns and Supplies Agilent Restricted Month ##, 200X Page 15 Conclusion Agilents SampliQ OPT, a polymeric sorbent with combined hydrophilic and lipophilic characteristics that allows retention of both polar and non-polar compounds, provides a simplified and effective single cartridge method for the purification and enrichment of multiple tetracycline compounds in chicken Recovery and reproducibility (routinely below 5%) based on solution standards are acceptable for tetracycline residue determination in chicken under the Chinese regulation Impurities from chicken were minimal and did not interference with any of the tetracyclines analyzed LODs of the 7 tetracyclines were much lower than the MRL (100µg/kg)

16 Columns and Supplies Agilent Restricted Month ##, 200X Page 16 GB/T , Determination of tetracyclines residues in food of animal originLC-MS/MS method and HPLC method Reference Part #Description OPT Polymer - Box, 100x 1ml tubes, 30 mg OPT Polymer - Box, 50x 3ml tubes, 60 mg OPT Polymer - Box, 30x 6ml tubes, 150mg OPT Polymer - 96 Well Plate, 10 mg Agilent Zorbax SB-C8 250×4.6mm 5µm


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