Tetracyclines in Chicken using Solid Phase
Extraction (SPE) and HPLC/UV
Chen-Hao (Andy) Zhai
Agilent Technologies
October 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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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 # 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
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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.
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Table 1: Physical-Chemical Properties of
Selected Tetracyclines
Compound
pKa
Log P
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
Doxycycline
3.1 / 7.7 /9.3 -0.02 444.1533
-0.3
MW
442.1376
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Chemical Structures of Tetracyclines
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SampliQ OPT Sample Preparation – Chicken Muscle
Accurately weigh 5g raw, homogenized chicken meat,
Homogenize 1 min. in 20mL 0.1M Na2EDTA-McIlvaine buffer
Ultrasonic extraction in ice bath for 10 min.
Centrifuge 3000rpm 5min transfer supernatant to
centrifuge tube B
repeat 2x, 20mL and
10mL successively
Bring total supernatant to 50mL final vol.
Centrifuge at 4000 rpm for 10 min.
Filter, take 10mL for SPE
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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)
Load 10mL extract (equivalent to 1g meat)
Recommended flow
through cartridge: not
faster than 1mL per
minute
Wash 3mL water (adjust pH to 4.5 w/ TFA)
Dry 3 minutes
under vacuum
Elute with 8mL 10mM Oxalic acid* in MeOH
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
Table 2. Linearity and LODs of Tetracyclines
Compound
Minocycline
Oxytetracycline
Tetracycline
Demeclocycline
Chlortetracycline
Methacycline
Doxycycline
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Regression equation Correlation coefficient
Y=86.313x-0.1491
Y=95.965x+0.0261
Y=103.97x-0.4698
Y=68.659x-0.1172
Y=51.752x-0.0284
Y=98.243x+1.2567
Y=76.408x+1.0756
0.9996
0.9999
0.9999
0.9998
0.9999
0.9985
0.9987
LOD(µg/kg)
2.5
2.5
2.5
5
5
2.5
5
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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, 2oxytetracycline, 3-tetracycline, 4-demeclocycline, 5-chlortetracycline, 6-demeclocycline,
7-doxycycline
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Recoveries and RSD’s of Tetracyclines in Chicken
Compound
Minocycline
Oxytetracycline
Tetracycline
Demeclocycline
Chlortetracycline
Methacycline
Doxycycline
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Spiked level
(ug/kg)
20
100
200
20
100
200
20
100
200
20
100
200
20
100
200
20
100
200
20
100
200
Recovery
(%)
87.6
80.8
81.3
68.8
63.0
59.4
81.0
70.0
72.3
92.0
94.8
92.9
93.3
92.4
87.7
93.3
91.9
86.6
95.6
96.4
92.0
RSD
(%)
4.13
5.68
4.19
6.49
4.87
4.35
4.46
3.47
4.38
2.06
3.78
1.92
3.16
4.01
2.54
2.89
2.51
3.39
4.38
1.00
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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Reference
GB/T 21317-2007, Determination of tetracyclines residues
in food of animal origin—LC-MS/MS method and HPLC
method
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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
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