Columns and Supplies

1
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 (n6) 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 2504.6mm 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
Table 1 Physical-Chemical Properties of
Selected Tetracyclines

• 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
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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 Regression equation Correlation coefficient LOD(µg/kg)
Minocycline Y86.313x-0.1491 0.9996 2.5
Oxytetracycline Y95.965x0.0261 0.9999 2.5
Tetracycline Y103.97x-0.4698 0.9999 2.5
Demeclocycline Y68.659x-0.1172 0.9998 5
Chlortetracycline Y51.752x-0.0284 0.9999 5
Methacycline Y98.243x1.2567 0.9985 2.5
Doxycycline Y76.408x1.0756 0.9987 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, 2-oxytetracycline,
3-tetracycline, 4-demeclocycline,
5-chlortetracycline, 6-demeclocycline,
7-doxycycline
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Recoveries and RSDs of Tetracyclines in Chicken
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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)

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Reference
GB/T 21317-2007, Determination of tetracyclines
residues in food of animal originLC-MS/MS method
and HPLC method
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 2504.6mm 5µm