Professional Documents
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Collaborative Study
The objective of this study was to evaluate the performance
of the determinative procedure for tetracyclines in raw bovine
milk under the auspices of the CVM Method Trial program.
Because this multiresidue procedure detects 3 drugs approved
in the United States as well as 4 other tetracyclines, it was
evaluated at a target level of 30 ng/mL.
Test Samples
Control milk was collected from cows that had been antibiotic-free for at least 30 days. Incurred milk was produced by
dosing cows orally with single boluses (2.55 mg/lb body
weight) of one of the tetracyclines in the spring and summer of
1991. The fresh milk was assayed at the time of collection for
residues. Milk containing residues between 15 and 150 ng/mL
was frozen and stored at 40C or less in preparation for this
trial. To provide participating laboratories with blind, incurred
samples, a bag of milk containing residue at the appropriate
level was quickly thawed in tepid water; blended with other
incurred milks, when necessary, to produce incurred milk containing multiple residues; subdivided into coded 50 mL disposable tubes; and refrozen. Results from previous experiments
indicated that there is little or no difference in the concentration
determined for OTC and CTC in milk assayed fresh and milk
assayed after being stored at 60C for several weeks. To ensure that there were no major changes due to refreezing milk, a
sample of each subdivided milk was reassayed in the authors
laboratory (laboratory 1) prior to sample shipment. The analyst
was unaware of the samples identity at the time of analysis.
Incurred milk samples were analyzed a third time in the
authors laboratory at the completion of the collaborative study
to monitor whether there were any changes that occurred between initial shipment of the milk samples (May 1992) and receipt of data from the last collaborator to finish the study
(June 1993).
Experimental Design and Procedures
The method trial consisted of the following 3 phases, each
of which had to be successfully completed before moving to the
next phase.
A. Principle
Tetracyclines are specifically adsorbed from milk extracts
via their chelation to copper ions reversibly bound to iminodiacetic acid epoxy-activated resin. Sample is defatted, acidified, and centrifuged. Clear supernate is applied to chelating
minicolumn that has been previously charged with copper ions.
Column is washed, and tetracyclines are specifically eluted
B. Apparatus
(a) Liquid chromatograph.Equipped with quaternary
LC pump with automatic helium solvent sparging [binary LC
pump may be used, see C(j) for modifications of LC mobile
phases and I(a) for LC operating conditions]; UV detector set
at 355 nm (Note: Use a full-scale setting equivalent to 0.02
0.05 absorption unit. The 150 ng/mL tetracycline chromatographic solution should give a full-scale deflection); manual
injector or autosampler equipped with 2 mL loop; and integrator or computer-assisted data capture and analysis system.
(b) LC column.PLRP-S, 150 4.6 mm id, 5 m particle
size, 100 , equipped with guard column containing same
packing material. Available from Polymer Laboratories, Amherst, MA. Substitutions not recommended.
(c) Refrigerated centrifuge.Capable of operating at 10C.
Equipped with fixed-angle rotor holding 18 mm diameter tubes,
and with rotor holding disposable 15 mL centrifuge tubes.
(d) Minicolumns.Disposable, polypropylene, with 10 mL
reservoir, containing frit at the bottom, graduated every 0.2 mL for
the lower 2 mL (Bio-Rad, Hercules, CA, or equivalent).
(e) Rack.To hold minicolumns (optional). Commercial
or made from wire test tube racks.
(f) Centrifugal ultrafilters.Capacity ca 2.5 mL. Used to
remove proteins of molecular weight 30 000 daltons, without
significantly reducing tetracycline concentration. Immediately
before use, wash ultrafilters by centrifuging 15 min at 1500
g with 2 mL H2O. Shake retentate and filtrate chambers to remove all H2O.
(g) Syringe filter.0.2 m Nylon-66. Alternative to (f).
See H, Ultrafiltration.
(h) Filters.Glass LC solvent filtration apparatus with
0.2 m Nylon-66 filters.
(i) Volumetric flasks.1 L, 100 mL, and 5 mL. Class A.
(j) Automatic pipets.Adjustable, 10200 L and 0.05
1.00 mL, with accuracy of delivery of 12% RSD.
(k) Centrifuge tubes.15 mL disposable polypropylene
with screw-top cap.
C. Reagents
All chemicals must be reagent grade, unless otherwise
specified.
(a) Water.LC grade. Deionize distilled H2O and then irradiate with UV to remove trace organic impurities. Use throughout
the method. Quality of H2O is critical for LC analysis.
(b) Solvents.Methanol and acetonitrile; LC grade.
(c) McIlvaineEDTANaCl buffer.Prepare McIlvaine
buffer as follows: Place 12.9 g citric acid monohydrate and
10.9 g Na2HPO4 in 1 L volumetric flask and dilute to volume
with H2O. Store McIlvaine buffer in refrigerator.
Prepare McIlvaineEDTANaCl buffer as follows: Add
37.2 g Na2EDTA 2H2O and 29.2 g NaCl to 1 L volumetric
D. System Suitability
Check LC system suitability with every set of samples by
running TC chromatographic standard solutions with gradient
as in I(a). LC system must meet the following criteria: (1) Absence of peaks in region of tetracyclines during runs made with
no injection or with only buffer injected. (2) Ready detection
(signal-to-noise ratio >10) of 10 ng chlortetracycline injected
E. Preparation of Controls
For controls, use whole raw bovine milk (fresh or previously
frozen, showing no signs of souring or curdling).
(a) Negative control.Milk free of tetracycline residues.
(b) Positive control.Fortify milk to 15, 30, and 60 ng
TC/mL by adding, respectively, 75, 150, and 300 L TC combined working stock solution, C(d)(2), to 5 mL aliquots of milk
sample.
Negative and positive controls should be analyzed initially
as part of familiarization with method. Afterward, single negative and positive controls should be analyzed on day of analysis
as part of method quality assurance.
F. Preparation of Minicolumn
Minicolumns can be prepared simultaneously in groups;
1014 test samples can be extracted in 8 h. Prepare minicolumn as follows: (1) Swirl bottle containing metal chelate resin,
C(f), to obtain even suspension. (2) Transfer 2 aliquots of
0.7 mL metal chelate resin to minicolumn, B(d), using automatic pipet, B(j), equipped with blue tip of which lower 2
3 mm was removed with sharp razor to increase bore size. (3)
Open bottom outlet of minicolumn and let shipping buffer
drain out. If necessary, add or remove resin so that bed volume
is 1.01.2 mL. Wash resin 3 with 2 mL H2O and then add
2 mL 10 mM CuSO4, C(h). Wash minicolumn again 2 with
2 mL H2O. Bed volume should be 1.01.2 mL, with ca 0.7 mL
blue top from Cu2+ adsorption. One-third bottom of minicolumn should remain white. Minicolumn operates by gravity
feed. Same column may be used 6 times.
G. Extraction
Perform following steps:
(1) Transfer 5.0 0.1 mL test milk sample (whole raw
milk, fresh or previously frozen, showing no signs of souring
or curdling) into 15 mL disposable centrifuge tube and centrifuge 15 min at 1500 g at 10C to separate cream.
(2) Transfer lower (skim) layer into clean 15 mL centrifuge
tube using 9 in. Pasteur pipet. Alternatively, while milk is still
cold, puncture through solid fat layer on opposite sides with
Pasteur pipet and decant skim milk through holes. Discard fat.
Add 10 mL sodium succinate buffer, C(g), to defatted milk; cap
tube; mix contents by inverting tube several times; and centrifuge 30 min at 1500 g at 10C.
(3) Apply clear supernate directly onto minicolumn from
F. If reservoir is not large enough, apply supernate in 2 batches.
Let solution filter through. Avoid disturbing column bed excessively. After no liquid is visible above resin, proceed with next
step. Do not let minicolumn dry out.
(4) Wash minicolumn sequentially with 2 mL sodium succinate buffer, 2 mL H2O, 2 mL methanol, and then 2 mL H2O.
Note: Next 2 steps are critical for good recoveries. Use gravity flow only.
(5) Carefully apply 0.70 0.05 mL McIlvaineEDTA
NaCl buffer, C(c), onto minicolumn. Drip buffer around sides
of column without disturbing column bed. Discard clear flowthrough.
(6) Elute tetracyclines from column with additional 2.5
0.05 mL McIlvaineEDTANaCl buffer. Collect eluted solution (should be blue) in test tube and refrigerate until analysis
or collect it directly in upper (retentate) chamber of centrifugal
ultrafilters, B(f), and perform ultrafiltration as in H. Minicolumn should be white at this point.
(7) Clean minicolumn with additional 23 mL McIlvaine
EDTANaCl buffer. Wash column 3 with 2 mL H2O and then
with 510 mL ethanol, C(i). Cap minicolumns with excess
20% ethanol and store in refrigerator. Before next use, mix contents of column on Vortex mixer or invert column several times
to resuspend metal chelate resin thoroughly. When reusing
minicolumn, open top of column and start from step F(3). Do
not reuse columns that have been exposed to sour milk or excessive amounts (>5 g) of tetracyclines. Columns are reusable
at least 2 months, if stored properly.
H. Ultrafiltration
Note: Eluates collected in G(6) are not stable and develop
precipitate, which can clog and effectively destroy LC column.
For this reason, it is strongly recommended that samples be
further deproteinized prior to LC analysis.
Cap and invert ultrafilter containing eluate, G(6), several
times to ensure homogeneity of solution. Centrifuge samples
3090 min at 5000 g in fixed-angle rotor. Stop centrifugation
when 1 mL filtrate is in bottom chamber.
Note: This step may be omitted if extract from G(6) collected in test tube is refrigerated until LC analysis and then filtered through 0.2 m syringe filter, B(g), immediately before
injection onto LC column.
I. LC Determination
Inject equal volumes of the 4 TC chromatographic standard
solutions and filtered samples onto LC system. Monitor UV
absorbance at 355 nm. Follow procedure outlined below:
(a) Mobile phase gradient.See Table 995.04C for specific operating conditions. Inject filtered sample while mobile
phase is 100% solvent A at 1 mL/min flow rate. After 1 min,
linearly change mobile phase over 5 min to solvent Amethanolacetonitrile (70 + 8 + 22). Maintain this composition
11 min at 1 mL/min flow rate, before returning linearly over
2 min at 1.5 mL/min to 100% solvent A.
Reequilibrate LC column 4 min (to stabilize retention
times) at initial conditions before injecting next sample.
Systems without automatic helium sparging may experience degassing problems, which typically appear as large,
broad peaks around elution time of oxytetracycline. If degassing problems occur during gradient chromatography, add small
amounts of acetonitrile (25%) to solvent A and increase equili-
bration time between runs. It may be necessary to prepare combined TC working standard solution, C(d)(2), with H2O instead
of methanol. Tetracyclines in aqueous solutions are not stable;
therefore, aqueous TC combined standard solution must be prepared on day of analysis.
Store LC column in H2Oacetonitrile solution (1 + 1). Before and after storage, flush LC column and chromatographic
system with LC grade H2O (ca 10-15 min at 1 mL/min) to prevent precipitation of oxalic acid caused by high concentrations
of organic solvent.
(b) Sample injection.Use 2.0 mL sample loop. Sample
injection size may vary. Depending on detector sensitivity, inject 0.51.0 mL filtered sample. For accurate quantitation, inject identical volumes each time. Use H2O to flush autosampler
to prevent salt precipitation. Inject a TC chromatographic
standard solution every 510 sample injections as a check of
the retention time.
Make 12 blank gradient runs each day prior to injecting TC
chromatographic standard solution or test samples to ensure
absence of potential background interferences near the retention times of tetracyclines.
(c) Peak identification.Tetracyclines elute in the following order: minocycline, oxytetracycline, tetracycline, demeclocycline, chlortetracycline, methacycline, and doxycycline.
Note: Retention times tend to shift slightly with increases of
age of column and number of injections.
Define very tight windows for peak identification because
tetracyclines elute closely together. All putative residue peaks
should have retention times within 0.05 min of retention times
observed in bracketing standards.
Some metabolites may interfere with analysis of other parent compounds. Oral administration of tetracycline to cows
may result in appearance of both tetracycline and earlier peaks
(e.g., epitetracycline), which elute very near oxytetracycline.
Occasionally, an endogenous peak may appear between
oxytetracycline and tetracycline. This may be caused by high
concentrations of riboflavin in milk, the retention time of which
is ca 0.1 min earlier than that of tetracycline. To reduce interference of riboflavin, double the volume of sodium succinate
buffer wash of minicolumn in extraction procedure (this only
slightly decreases recovery of oxytetracycline or tetracycline).
(d) Extract stability.Tetracyclines are not stable at room
temperature under acidic conditions (i.e., in McIlvaine
EDTANaCl buffer). Tetracycline and chlortetracycline degrade 50% within 24 h. Degradation products tend to elute
earlier than parent compound and usually migrate with
oxytetracycline. To avoid this problem, perform all centrifugation steps at 10C. Refrigerate sample extracts or analyze them
within 4 h of preparation. Minicolumn eluate solutions may be
refrigerated 2 days or frozen 1 week with only slight
changes in tetracycline concentrations.
J. Calculations
Prepare standard curve for each tetracycline from standard
chromatograms. Calculate concentrations of tetracyclines by
using linear regression as follows:
C = mP + b
where C = tetracycline concentration of injected extract
(ng/mL) and P = tetracycline peak area or peak height. (Correlation coefficients for each TC standard curve should be
0.995.)
Calculate tetracycline concentration in original milk sample
by dividing concentration determined for injected sample by 2
(concentration factor used in minicolumn procedure, which reduces volume of sample from 5 to 2.5 mL).
Care should be taken with integration. Baselines determined
by automated data systems should be checked for each chromatogram (see Figure 995.04 for appropriate baseline construction).
Ref.: J. AOAC Int. 76, 329(1993); J. AOAC Int. 79, 29(1996)
Collaborators Comments
For most participants, phase I (system suitability) was the
longest part of the trial. Some LC systems were unable to mix
the 100% aqueous oxalic acid buffer with organic modified
mobile phase [either 100% 70 + 8 + 22 buffer or 30% acetonitrilemethanol (22 + 8)] without forming air bubbles. For these
laboratories, adding 25% acetonitrile to the initial buffer
seemed to alleviate this problem. In a few laboratories, it was
necessary to increase the equilibration time between runs to
stabilize retention times.
Most laboratories completed phase II (assay familiarization) quickly. Laboratories 5 and 7 did not have a refrigerated
centrifuge or an LC autosampler. They omitted the ultrafiltration step and injected extracts manually after regular filtration.
Degradation of residues was minimized by keeping extracts in
the refrigerator until analysis. Laboratory 5 also placed its centrifuge inserts in a freezer prior to use, which helped keep the
milk cool during centrifugation steps.
Laboratory 2 had very low recoveries, especially of Mino,
from their fortification of control milk samples shipped to them
prior to phase II. Their recoveries improved significantly when
they fortified control raw milk from their own laboratory. During phase III, laboratory 2 used its own milk to prepare the
known fortified samples, although it analyzed the same unknown samples as did the other participants.
Laboratory 7 experienced difficulties with LC during
phase III. One set of fortified samples was lost. Also, this laboratory experienced severe loss of resolution between Metha and
Doxy after the first days analyses. On the advice of the authors,
the analyst omitted Metha from the standards and continued the
trial with only the other 6 tetracyclines. In this laboratory only,
unknown samples containing either Metha or Doxy residues
were identified as Doxy. Other laboratories also reported loss
of resolution between Metha and Doxy, although not as severe
as that experienced by laboratory 7. This laboratory was also
the only one that had difficulties determining OTC and TC,
getting low recoveries for the fortified residues and altogether
missing 2 incurred OTC and 1 incurred TC residues. This may
have been due to the problems with the LC system, because an
incorrect gradient or incomplete reequilibration between runs
will result in the early eluting tetracyclines (OTC, TC, and especially Mino) eluting with the solvent front and essentially
disappearing.
A number of laboratories found that decreasing the attenuation for the last 3 eluting tetracyclines (CTC, Metha, and
Doxy) improved the appearance of chromatograms. A couple
of laboratories found that purchased LC grade water was preferable to house LC water.
Results and Discussion
In addition to the authors laboratory, 10 laboratories initially agreed to participate in this collaborative study. Six
months after standards had been shipped, 1 laboratory still had
not begun phase I of the trial. This laboratory was replaced. Of
10 laboratories, 7 successfully completed all phases of the trial.
Because of instrumental and personnel problems, 2 laboratories were unable to complete the trial within 1 year of its initiation. A third laboratory completed the work, but its data were
omitted because the analyst did not follow the method in the
preparation and inclusion of sufficient standards. In particular,
the analyst did not inject a standard following the sample extracts. Retention times in this laboratory showed considerable
drift, resulting in erroneous peak identification.
No interferences were detected in control milk (data not
shown). Recoveries for known fortified samples are reported in
Tables 24. Results from laboratory 9 for Mino analysis were
found to be outliers by the single Grubbs or Cochrans test
(Tables 2 and 4, respectively). Mean recoveries for all drugs
fortified at 30 ng/mL were between 60 and 80% (Table 3),
meeting CVM individual residue method guidelines for accuracy (60110%) when the target level is below 100 ppb (8).
Recoveries do not meet the 80110% limits set by the Commission of the European Communities (9). Repeatability for all
residues at 30 and 60 ng/mL was below 20%, meeting CVM
guidelines, and was below 15% for all but 1 residue (Mino at
30 ng/mL), also meeting European requirements.
A total of 144 unknown samples containing 224 drug residues were analyzed (Table 5). Excluding results of laboratory 7, only 1 residue (Mino in Laboratory 9) was not identified. Only 1 laboratory (Laboratory 4) incorrectly identified an
unknown control milk sample as containing a residue. This
laboratory had a series of 5 consecutive LC system runs that
had extra residues in them. Neither the analyst nor the authors
have an explanation for these results. This problem was not
seen in any other laboratory nor by laboratory 4 on any other
day. The most common error was identification of epi-tetracycline (a TC degradation product) as OTC. This error only occurred in samples that would already be identified as residue
positive and, therefore, would not result in false positive determinations.
Recoveries and precision of OTC, TC, and CTC in fortified
blind sample (Table 5, last sample) were nearly identical to
those found in samples the participants fortified themselves.
Intralaboratory relative repeatability (RSDr) was below 15%
for all but 3 unknown incurred residues and below 20% for all
except the incurred Mino sample.
Four laboratories reported values of <10 ng/mL for the incurred Mino sample. This was according to the authors instruction, because the lowest point on the standard curve for this
procedure is equivalent to 10 ng residue/mL milk. To approximately calculate repeatability and reproducibility for this sample, the authors estimated numerical values for the <10 results from the raw data submitted by collaborators.
Mino analyses were least reproducible and the only analyses
to which outlier tests were applied. This may be due to changes
in milk character affecting extraction of Mino residues from
milk rather than actual degradation of the drug itself. Shipment
on dry ice may be a factor. All laboratories prepared their own
fortified samples from control milk shipped to them. Laboratory 2 initially had consistently very low recoveries of Mino
when using the shipped milk. When they substituted their own
control milk in preparing fortified samples, recoveries of Mino
were acceptable. (These are the data reported in this paper.)
Other laboratories did not have consistent problems with fortified Mino recoveries, though there were occasional problems,
such as with laboratory 9 (outlier; Tables 24). It may not be
entirely coincidence that the shipment time from the authors
laboratory to laboratory 2 was the longest and shipment time to
laboratory 9 was the second longest. Other experiments have
shown that milk quality may affect recovery of Mino residues
with this procedure. The authors found that Mino fortified into
milk that had soured or that had been stored as little as a month
at regular freezer temperature (20C or higher) was recovered
very poorly (unpublished data). There was no problem with
milk stored at 60C or lower. The factor or variable causing
erratic Mino recovery has not been completely characterized, although the authors suspect pH of milk may be important.
Residue concentrations found by the authors laboratory before sample shipment and at the end of the study are compared
in Table 6. The similar results show that, except for Mino, there
was no significant change in the incurred residue concentrations over the lengthy course of this study.
Recommendation
Metal chelate affinity chromatography is a convenient,
class-specific extraction method for determination of tetracyclines in milk. Milk samples prepared with this method are free
of chromatographic interferences, enabling low detection limits and ready peak identification. This method has additional
advantages in that numerous samples may be processed at one
time and that almost no hazardous waste is generated by the
extraction procedure.
Results from this interlaboratory validation meet CVM
guidelines for accuracy and precision for residue analysis at this
target level. This method also meets European recommendations for precision for most of the residues tested, although it
does not meet accuracy recommendations. It is possible that
modifying the method to incorporate an internal standard such
as DMC or allowing some other recovery correction would enable it also to meet European accuracy limits. This method
should be a useful tool for regulatory and other laboratories
Acknowledgments
We thank the Division of Animal Research of CVM and, in
particular, H.F. Righter, for dosing animals and collecting milk
samples containing incurred residues; Michael Smedley for
many useful discussions on conducting interlaboratory studies;
Michael H. Thomas of the authors laboratory and W.A. Moats
of ARS, Beltsville, MD, for many useful discussions on tetracycline analysis. We also thank the following collaborators for
their participation in the study:
William H.H. Farrington, Ministry of Agriculture, Fisheries
and Food Safety Directorate, Norwich, Norfolk, United Kingdom
Roberta Wagner, Baltimore District Office, U.S. Food and
Drug Administration, Baltimore, MD
Sandy Cross, Denver District Office, U.S. Food and Drug
Administration, Denver, CO
Byron L. Wilson and Eric Shepherd, Brooke Army Medical
Center, Regional Veterinary Laboratory, Fort Sam Houston, TX
Paula Lansdon and Alyson Hahn, Bureau of Clinical Laboratory, Montgomery, AL
Jack Carmany and Min Li, California State Department of
Agriculture, Chemistry Laboratory Services, Sacramento, CA
Daulat Singh, Michigan Department of Agriculture, Laboratory Division, East Lansing, MI
References
(1) Farrington, W.H.H., Tarbin, J., & Bygrave, J. (1990) in
EuroResidue: Residues of Veterinary Drugs in Food, N.
Haagsma, A. Ruiter, & P.B. Czedik-Eysenberg (Eds), University of Utrecht, The Netherlands, pp. 179184
(2) Farrington, W.H.H., Tarbin, J., Bygrave, J., & Shearer, G.
(1991) Food Addit. Contam. 8, 5564
(3) Thomas, M.H. (1989) J. Assoc. Off. Anal. Chem. 72, 564567
(4) Moats, W.A. (1986) J. Chromatogr. 358, 253259
(5) Moats, W.A., & Leskinen, L. (1988) J. Assoc. Off. Anal.
Chem. 71, 776778
(6) Carson, M.C. (1993) J. AOAC Int. 76, 329334
(7) Guidelines for Collaborative Study Procedure To Validate
Characteristics of a Method of Analysis (1989) J. Assoc.
Off. Anal. Chem. 72, 694704; along with AOAC Spreadsheet Instructions, Revision 3/5/91
(8) General Principles for Evaluating the Safety of Compounds
Used in Food-Producing Animals (1986) U.S. Food and
Drug Administration, Washington, DC
(9) Commission of the European Communities (1987) Off. J.
Eur. Comm. L 223, 1836
Table 995.04A. Method performance for determination of multiple tetracycline residues in fortified milk by metal
chelate affinityliquid chromatographya
Mean recovery, %
Antibiotic
sr
RSDr, %
sR
RSDR, %
Minocycline
Oxytetracycline
Tetracycline
Demeclocycline
Chlortetracycline
Methacycline
Doxycycline
69.0
75.2
73.6
71.0
61.7
58.9
64.9
11.4
9.41
9.30
11.6
15.6
10.3
14.5
17
12
13
16
25
18
22
32
26
26
32
44
29
41
16.8
14.4
16.2
11.6
15.6
10.6
14.5
24
19
22
16
25
18
22
47
40
45
32
44
30
41
16.2
12.1
15.1
9.13
8.45
8.53
8.66
24
16
20
13
13
14
13
45
34
42
26
24
24
24
6.97
9.98
9.68
7.79
11.0
9.48
9.25
9.7
13
13
11
17
16
14
20
28
27
22
31
26
26
69.2
77.5
74.8
72.3
64.2
60.3
65.2
12.4
6.32
6.48
8.16
4.72
6.36
7.34
18
8.2
8.7
11
7.4
11
11
35
18
18
23
13
18
21
Minocycline
Oxytetracycline
Tetracycline
Demeclocycline
Chlortetracycline
Methacycline
Doxycycline
a
b
72.0
78.2
74.1
69.1
63.5
58.6
64.8
4.11
6.75
6.38
6.20
6.25
5.84
6.76
5.7
8.6
8.6
9.0
9.8
10
10
12
19
18
17
18
16
19
Known triplicates prepared by collaborators; based on data submitted by 8 laboratories, except as noted.
Based on data from 7 laboratories.
Table 995.04B. Method performance for determination of multiple tetracycline residues in milk containing unknown
incurred and fortified tetracycline residues by metal chelate affinityliquid chromatography a
Antibiotic
Minocycline
Oxytetracycline
Oxytetracycline
Oxytetracycline
b
Oxytetracycline (40)
Tetracycline
Tetracycline
Tetracycline (80)
Demeclocycline
Chlortetracycline (25)
Chlortetracycline
Chlortetracycline
c
Methacycline
Doxycycline
a
b
c
Mean, ng/mL
sr
RSDr, %
sR
RSDR, %
11.6
18.6
18.8
30.0
31.4
23.1
45.3
54.1
37.0
14.5
34.1
37.4
38.2
30.1
2.74
2.50
3.03
4.47
2.55
3.73
3.44
3.94
4.39
1.87
4.06
3.98
5.51
3.60
24
13
16
15
8.1
16
7.6
7.3
12
13
12
11
14
12
7.7
7.0
8.5
13
7.1
10
9.6
11
12
5.2
11
11
15
10
9.25
3.97
6.45
10.3
3.79
8.62
13.3
8.70
5.93
2.94
6.28
6.02
7.05
6.25
80
21
34
34
12
37
29
16
16
20
18
16
18
21
26
11
18
29
11
24
37
24
17
8.2
18
17
20
18
Gradient
0.00
01
16
617
1719
1920
2024
Day 2
Day 3
Control milk
15 ng/mL fortified
30 ng/mL fortified
60 ng/mL fortified
Blind sample 7
Blind sample 8
Blind sample 9
Blind sample 10
Blind sample 11
Blind sample 12
Control milk
15 ng/mL fortified
30 ng/mL fortified
60 ng/mL fortified
Blind sample 13
Blind sample 14
Blind sample 15
Blind sample 16
Blind sample 17
Blind sample 18
Table 2. Residue recovery from control milk fortified with 7 tetracyclines, each at 15 ng/mL
Residue recovery, %
Laboratory
1
Mean
RSDr, %
RSDR, %
Replicate
Mino
OTC
TC
DMC
CTC
Metha
Doxy
1
2
3
1
2
3
1
2
3
a a
3
4
5
1
2
3
1
2
3
1
2
3
1
2
3
69.4
90.7
64.4
72
70
74
49.0
58.1
35.4
40
74
51
87.1
84.8
86.5
73
70
97
68.1
65.1
9.3
7.5
44.9
62.7
b
69.0b
21.0
b
16.6b
38.6
b
24.3b
73.9
86.4
61.3
80
75
80
78.4
85.7
73.8
70
81
69
82.3
90.8
98.5
87
63
74
41.9
42.9
82.0
90.0
62.0
75.2
74.3
85.7
60.8
73
76
78
73.0
81.6
65.1
63
84
80
84.8
88.2
90.9
80
68
91
29.8
35.2
66.4
90.0
73.9
73.6
71.7
84.5
69.0
73
66
72
68.5
80.6
54.4
46
83
73
69.0
83.1
85.3
80
54
83
70.4
59.0
71.2
73.3
63.3
71.0
67.4
74.8
57.9
68
69
73
58.3
72.9
47.8
17
72
85
67.2
82.8
68.7
60
53
62
62.8
41.7
49.5
62.8
45.0
61.7
57.1
64.3
52.9
62
59
62
57.3
71.9
51.2
49
83
63
57.4
66.4
62.9
40
42
63
74.2
41.6
63.5
51.4
58.9
55.5
77.4
45.1
63
64
70
63.5
78.8
54.2
57
81
90
62.6
72.2
72.8
73
37
66
79.1
41.6
52.5
72.2
64.7
64.9
12.5
12.6
16.4
25.4
17.5
22.4
19.1
22.1
14.7
23.9
18.1
20.2
Laboratory 5 reported results for 5 replicates of fortified milk sample extractions. To have equivalent weighting with other participants, only the
last 3 sets of results were included in the statistical analysis.
Results excluding laboratory 9 on the basis of the single Grubbs outlier test.
Table 3. Residue recovery from control milk fortified with 7 tetracyclines, each at 30 ng/mL
Residue recovery, %
Laboratory
1
Mean
RSDr, %
RSDR, %
a
Replicate
Mino
OTC
TC
DMC
CTC
Metha
Doxy
1
2
3
1
2
3
1
2
3
b a
3
4
5
1
2
3
1
2
3
1
2
3
1
2
3
72.7
81.1
66.9
67
77
74
74.5
66.6
61.1
78
68
66
86.3
81.3
78.6
67
73
81
93.1
57.0
13.9
43.6
62.9
69.2
18.0
23.5
73.4
82.9
66.7
73
72
75
80.4
83.0
77.2
91
77
77
84.3
93.8
100.6
83
73
83
55.2
44.7
79.3
83.7
73.0
77.5
8.2
15.6
69.1
79.3
64.7
71
80
78
73.4
78.6
71.5
88
78
77
87.5
86.4
89.2
77
71
84
38.1
31.8
73.5
78.5
95.4
74.8
8.7
20.2
63.0
77.4
67.7
73
69
78
67.0
75.3
60.5
67
63
64
72.3
82.6
86.4
67
59
76
89.3
76.3
61.1
78.0
89.0
72.3
11.3
12.6
61.2
69.1
61.2
70
68
73
55.6
61.5
54.1
62
55
61
73.7
79.0
82.9
60
57
63
72.5
63.0
55.4
66.7
52.3
64.2
7.4
13.2
52.8
58.2
53.8
59
60
62
58.9
67.6
55.8
61
64
64
61.2
73.0
83.4
50
45
60
68.1
49.3
63.4
55.1
60.3
10.6
14.2
54.1
68.0
55.2
64
66
68
66.0
72.6
62.8
56
57
70
69.1
79.1
86.1
67
50
60
72.5
55.3
63.0
73.4
63.4
65.2
11.3
13.3
Laboratory 5 reported results for 5 replicates of fortified milk sample extractions. To have equivalent weighting with the other participants, only
the last 3 sets of results were included in the statistical analysis.
Table 4. Residue recovery from control milk fortified with 7 tetracyclines, each at 60 ng/mL
Residue recovery, %
Laboratory
1
Mean
RSDr, %
RSDR, %
Replicate
Mino
OTC
TC
DMC
CTC
Metha
Doxy
1
2
3
1
2
3
1
2
3
a a
3
4
5
1
2
3
1
2
3
1
2
3
1
2
3
79.7
66.9
72.8
76
78
73
76.6
63.2
60.4
72
74
77
86.6
78.9
78.5
60
66
65
65.4
70.8
15.1
2.5
55.5
65.8
b
72.0b
17.0
b
5.7b
30.7
b
9.7b
95.1
69.1
75.5
79
78
79
81.9
76.2
79.3
78
72
85
85.1
91.7
97.9
72
70
66
58.5
63.7
78.9
89.7
76.3
78.2
81.5
64.5
68.5
75
81
77
75.3
71.5
75.6
75
74
84
86.7
84.7
88.7
67
68
64
68.9
45
77.1
80.7
69.9
74.1
77.4
64.9
68.5
68
73
77
67.0
66.3
65.7
59
65
79
76.1
81.9
84.1
60
59
65
65.1
73.2
54.6
70.7
68.6
69.1
75.8
57.2
61.8
67
73
75
55.3
52.4
58.3
63
57
62
74.7
80.4
84.2
53
50
53
58.5
81.7
56.2
56.4
53.8
63.5
69.8
52.2
55.8
56
60
60
61.0
60.0
61.8
70
55
64
64.6
73.8
79.7
53
46
48
44.4
53.8
54.1
46.5
58.6
79.6
56.5
60.1
60
66
66
66.0
64.1
67.8
67
57
68
71.2
81.7
84.9
60
55
57
48.4
56.2
66.6
73.7
57.8
64.8
8.6
8.6
9.0
9.8
10.0
10.4
12.8
13.1
11.3
17.3
16.2
14.3
Laboratory 5 reported results for 5 replicates of fortified milk sample extractions. To have equivalent weighting with the other participants, only
the last 3 sets of results were included in the statistical analysis.
Results with laboratory 9 excluded on the basis of Cochrans test for outliers.
Replicate
Mino
OTC
TC
DMC
CTC
Metha Doxy
Mino
Control milk;
residue concentration, ng/mL
1
2
1
2
1
2
OTC
TC
DMC
CTC
Metha Doxy
Mino
OTC
24.2
30.1
11.6
13.7
TC
DMC
CTC
Metha Doxy
36.5
40.1
30.8
31.2
21.6
21.2
22.4
19.9
<10
30.5
19.1
<10
30.9
19.7
<10
33.2
14.4
2
1
2
20.9
<10
19.3
14.1
34.0
38.6
28.0
23.7
21.7
19.8
6
7
9
<10
21.4
17.5
2
1
2
1
<10
21.1
17.5
25.9
22.6
18.0
33.5
18.4
11.0
9.7
18.3
<10
(11.6)b
(23.6)
(80.0)
27.2
30.1
11.9
20.7
18.8
18.6
13.4
21.4
Mean, ng/mL
RSDr, %
RSDR, %
Mixture of incurred OTC and TC milk;
residue concentration, ng/mL
1
2
3
4
5
6
Incurred TC milk;
residue concentration, ng/mL
1
2
1
2
1
2
35.0
36.4
35.6
30.6
34.6
34.1
23.7
25.6
30.0
29.0
28.8
27.8
23.1
24.2
21.9
21.2
22.4
21.3
41.0
44.6
44.9
41.4
38.9
33.7
40.4
39.2
42.4
41.3
32.4
31.3
18.4
14.0
55.4
50.8
55.1
60.2
48.2
53.2
1
2
1
2
32.7
25.6
40.5
35.7
27.2
16.1
32.1
28.1
19.9
21.5
14.8
19.3
19.3
36.1
48.4
38.9
32.0
34.2
26.5
43.7
36.9
7.1
11.6
<10
12.8
10.6
42.6
35.1
58.7
54.2
31.9
23.1
24.5
30.1
25.6
<10
33.2
35.9
24.1
20.5
33.1
33.8
<10
38.4
Table 5. (continued)
Mixture of incurred OTC and TC milk;
residue concentration, ng/mL
Laboratory
Replicate
Mino
OTC
TC
DMC
CTC
1
2
13.8
8.8
1
2
26.8
30.3
30.0
14.9
34.5
22.5
22.4
23.1
16.2
37.4
Mean, ng/mL
RSDr, %
RSDR, %
Metha Doxy
Mino
OTC
TC
DMC
CTC
9.0
35.0
28.2
34.6
26.6
17.9
19.3
18.8
16.2
34.4
33.5
31.9
37.0
11.9
16.0
30.4
25.6
34.1
11.9
18.5
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
2
3
4
5
6
7
9
Mean, ng/mL
RSDr, %
RSDR, %
Recovery, %
a
b
c
d
10.1
42.6
38.9
44.5
44.9
31.9
32.5
35.0
28.6
44.6
44.8
32.2
38.2
39.1
33.7
27.6
39.1
37.4
10.6
16.1
Incurred TC milk;
residue concentration, ng/mL
Metha Doxy
OTCc
TC
DMC
CTC
18.7
19.9
<10
48.5
52.0
45.3
7.6
29.4
Mino
, not detected.
Raw data was used to estimate numerical values for results listed as <10.
EpiTC, a TC metabolite, elutes very near OTC.
NR, laboratory 7 unable to resolve Metha and Doxy; omitted from calculations.
41.0
41.7
39.6
37.1
35.0
32.4
29.0
38.7
42.1
50.5
36.3
51.2
d
NRd
NR
32.9
27.9
38.2
14.4
18.4
Metha Doxy
75.2
58.6
31.7
36.9
35.4
29.6
32.6
33.6
30.7
29.7
38.0
32.5
30.2
30.3
26.0
23.0
30.7
32.2
31.4
8.1
12.0
78.6
57.2
58.5
65.5
62.0
55.7
57.4
43.4
40.1
64.5
53.2
51.8
57.8
42.3
39.7
54.4
61.5
54.1
7.3
16.1
67.6
17.8
17.7
18.1
16.4
12.6
13.9
13.3
13.7
19.0
15.2
7.1
13.1
14.2
14.0
12.9
13.5
14.5
12.8
20.3
58.1
Replicate
Mino
OTC
TC
DMC
CTC
Metha
Doxy
36.5
40.1
33.2
34.2
41.0
44.6
41.0
37.9
40.4
39.2
42.9
40.5
42.6
38.9
41.1
40.6
41.0
41.7
37.2
32.9
Control milk
April 1992
August 1993
1
2
1
2
1
2
1
2
24.2
30.1
13.1
9.4
April 1992
August 1993
1
2
1
2
21.6
21.2
19.4
19.2
1
2
1
2
35.0
36.4
36.4
33.9
23.7
25.6
28.6
28.6
1
2
1
2
23.1
24.2
20.2
22.2
Incurred TC milk
April 1992
August 1993
1
2
1
2
55.4
50.8
49.0
54.2
Incurred CTC milk
April 1992
August 1993
1
2
1
2
April 1992
August 1993
1
2
1
2
Table 6. (continued)
Control milk fortified with OTC (40 ng/mL), TC (80 ng/mL), and CTC (25 ng/mL)
April 1992
August 1993
31.7
(79.2)a
36.9
(92.2)
28.0
(70.0)
28.5
(71.2)
57.2
(71.5)
58.5
(73.1)
56.8
(71.0)
58.6
(73.2)
17.8
(71.2)
17.7
(70.8)
14.5
(58.0)
15.4
(61.6)
Figure 995.04. Sample chromatograms. (A) Extract of control milk fortified to 30 ng each TC/mL: 1, minocycline; 2,
oxytetracycline; 3, tetracycline; 4, demeclocycline; 5, chlortetracycline; 6, methacycline; 7, doxycycline. (B) Extract of
milk from cow treated with tetracycline: 8, epitetracycline. (C) Extract of control milk.