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Columns and SuppliesAgilent Restricted
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Tetracyclines in Chicken using Solid Phase Extraction (SPE) and HPLC/UV
Chen-Hao (Andy) Zhai
Agilent TechnologiesOctober 2008
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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%
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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
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MaterialsStandards: 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 # 5982-3036)
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: 0.765 mL trifluoroacetic acid dissolved in water to a final volume of 1000mL.
• Methanol-TFA solution: 5% methanol in 10mM TFA solution
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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# 880975-906). •Flow rate: 1.5ml/min•Column Temperature: 30oC •Injection volume: 100μl•Detector wavelength: 350nm•Mobile phase: 10 mmol/L TFA solution, methanol, acetonitrile, gradient elution
time (minutes) % methanol % acetonitrile % 10mmol TFA
0 1 4 95
7.5 6 24 70
13.5 7 28 65
15 1 4 95
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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. 2
• 200g chicken was homogenized and placed in a clean, sealed container and stored at -18oC
• 5g homogeneous sample (accurate to 0.01 g) was placed into a 50 mL polypropylene centrifugal tube with 20mL 0.1 mol/L Na2EDTA-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 15oC).
• 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 15oC), then filtered.
Compound pKa Log P MW
Minocycline 3.3 /7.2 /9.3 +0.5 457.1849
Oxytetracycline 3.3 / 7.3 /9.1 -0.9 460.1482
Tetracycline 3.3 / 7.7 /9.7 -1.3 444.1533
Demeclocycline 3.3 / 7.2 /9.3 +0.2 464.0986
Chlortetracycline 3.3 / 7.4 /9.3 -0.62 478.1143
Methacycline 3.5 / 7.6 /9.2 -0.3 442.1376
Doxycycline 3.1 / 7.7 /9.3 -0.02 444.1533
Table 1: Physical-Chemical Properties of Selected Tetracyclines
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Chemical Structures of Tetracyclines
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SampliQ OPT Sample Preparation – Chicken MuscleAccurately weigh 5g raw, homogenized chicken meat,
Homogenize 1 min. in 20mL 0.1M Na2EDTA-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
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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 40oC.
• 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
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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
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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 25-500µg/kg. The linearity and LOD are shown in Table 2
Compound Regression equation Correlation coefficient LOD(µg/kg)
Minocycline Y=86.313x-0.1491 0.9996 2.5Oxytetracycline Y=95.965x+0.0261 0.9999 2.5
Tetracycline Y=103.97x-0.4698 0.9999 2.5Demeclocycline Y=68.659x-0.1172 0.9998 5
Chlortetracycline Y=51.752x-0.0284 0.9999 5Methacycline Y=98.243x+1.2567 0.9985 2.5Doxycycline Y=76.408x+1.0756 0.9987 5
Table 2. Linearity and LODs of Tetracyclines
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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.
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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
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Recoveries and RSD’s of Tetracyclines in Chicken
Compound Spiked level (ug/kg)
Recovery (%)
RSD (%)
Minocycline 20 87.6 4.13100 80.8 5.68200 81.3 4.19
Oxytetracycline 20 68.8 6.49100 63.0 4.87200 59.4 4.35
Tetracycline 20 81.0 4.46100 70.0 3.47200 72.3 4.38
Demeclocycline 20 92.0 2.06100 94.8 3.78200 92.9 1.92
Chlortetracycline 20 93.3 3.16100 92.4 4.01200 87.7 2.54
Methacycline 20 93.3 2.89100 91.9 2.51200 86.6 3.39
Doxycycline 20 95.6 4.38100 96.4 1.00200 92.0 3.02
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Conclusion
• Agilent’s 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)
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GB/T 21317-2007, Determination of tetracyclines residues in food of animal origin—LC-MS/MS method and HPLC method
Reference
Part # Description
5982-3013 OPT Polymer - Box, 100x 1ml tubes, 30 mg
5982-3036 OPT Polymer - Box, 50x 3ml tubes, 60 mg
5982-3067 OPT Polymer - Box, 30x 6ml tubes, 150mg
5982-3096 OPT Polymer - 96 Well Plate, 10 mg
880975-906 Agilent Zorbax SB-C8 250×4.6mm 5µm