DRUGS, COSMETICS, FORENSIC SCIENCES

Chlortetracycline, Oxytetracycline, and Tetracycline in Edible Animal

Tissues, Liquid Chromatographic Method: Collaborative Study
MACNEIL ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 79, NO. 2, 1996
JAMES D. MACNEIL, VALERIE K. MARTZ, GARY O. KORSRUD, and CRAIG D.C. SALISBURY
Agriculture and Agri-Food Canada, Health of Animals Laboratory, Saskatoon, SK, S7N 2R3, Canada
HISAO OKA
Aichi Prefectural Institute of Public Health, Laboratory of Food and Drug Chemistry, 7-6 Nagare, Tsujmachi, Kita-Ku
Nagoya 462, Japan
ROBERT L. EPSTEIN
U.S. Department of Agriculture, Agricultural Marketing Service, Science Division, PO Box 96456, Washington, DC 20090
CHARLIE J. BARNES
U.S. Food and Drug Administration, Office of Science, HFV-501, Bldg 328A, BARC-East, Beltsville, MD 20705
Collaborators: G. Alfredsson; C. Barry; B. Bergner; W. Chan; J.M. Diserens; L.P. Ilnicki; E. Klein; B. Koscinski; G. Vasco; T.
Phillippo; H. Mawhinny; E. Mller; M. Petz; H. Oka; R. Patel; G.M. Telling; M. Webb; C. Henry; W.H.H. Farrington

Thirteen laboratories analyzed samples of edible

animal tissues for tetracycline residues. The
method included extraction of analytes into buffer,
elution from a C18 solid-phase extraction (SPE) cartridge, and reversed-phase liquid chromatographic
(LC) analysis, including use of a confirmation column. An additional laboratory, using an alternative
LC assay based on a different sample cleanup,
also analyzed the samples. Results showed the
2 methods are comparable. The LC method for determination of cholortetracycline, oxytetracycline,
and tetracycline in edible animal tissues has been
adopted by AOAC INTERNATIONAL. Results from
13 laboratories indicate that the method under
study provides generally better results at the
higher concentrations tested than at concentrations near the detection limit and that there is less
problem with interferences in muscle tissue than in
kidney. The method can achieve reliable results for
analytes and matrixes studied at concentrations
from 0.1 to 0.6 ppm and above, depending on the
analytematrix combination, with generally better
performance to be expected with muscle than with
kidney. The poorer performance for fortified samples, particularly kidney, was attributed to additional homogenization steps required to prepare
these samples. Recovery of analytes from different
Submitted for publication March 21, 1995.
The recommendation was approved by the Methods Committee on
Drugs and Related Topics and was adopted by the Official Methods Board
of the Association. See Official Methods Board Actions (1995) J. AOAC
Int. 78, 88A, and Official Methods Board Actions (1995) The Referee 19,
June issue.

lots of solid-phase extraction (SPE) cartridges was

an important variable.

Collaborative Study
The study was conducted following Guidelines for Collaborative Study Procedure To Validate Characteristics of a
Method of Analysis of AOAC INTERNATIONAL (1). The
study proposal was submitted to and approved by both the
AOAC Committee on Drugs and Related Topics and the International Union of Pure and Applied Chemistry (IUPAC), Commission VI.1 Food Chemistry, which designated the project
as 65/90.
The method (2) was tested in both the originators and the
associate referees (AR) laboratories and performance data
were collected, on the basis of multianalyst familiarizations and
routine laboratory use over a period of several years. Collaborators were enlisted through various sources, but primarily
through members of the ad hoc Working Group on Methods of
Sampling and Analysis, Committee on Residues of Veterinary
Drugs in Foods, Codex Alimentarius Commission.
Collaborators were provided with an initial set of samples
consisting of 3 samples of fortified pork muscle, prehomogenized; 3 samples of fortified beef kidney, prehomogenized;
6 spiking solutions; 1 container of blank pork muscle, prehomogenized; 1 container of blank beef kidney, prehomogenized;
1 standard solution of tetracycline (TTC), chlortetracycline
(CTC), and oxytetracycline (OTC), concentrations as labeled.
The standard solution was used to provide a check against laboratory standards.
Collaborators were instructed to fortify 3 subsamples of
blank beef with 3 of the coded spiking solutions and to fortify
3 subsamples of blank pork with the remaining 3 coded spiking

solutions. This procedure provided each collaborator with a set

of duplicates, fortified in their own laboratory, that could be
compared with the prehomogenized fortified sample prepared
in the ARs laboratory. Results were used to determine if preparation of homogenized samples in the ARs laboratory produced different results from samples fortified by the collaborators themselves. This exchange was also used to test procedures
and documentation required for shipment of tissue samples
across international borders and to assess the effects of various
lot numbers of SPE cartridges purchased by the participants.
There was no significant difference in results obtained for
samples prepared in the ARs laboratory and those prepared by
the collaborators using samples fortified with spiking solutions.
On the basis of this finding, it was decided that pooled prehomogenized samples would be prepared in the ARs laboratory
for the study to reduce the risk of loss of samples through breakage of vials containing spiking solutions or evaporation and
save considerable time and costs required to seal spiking solutions into glass vials. Minor revisions in shipment scheduling
also were made to reduce the risk of frozen samples being held
over a weekend at a freight facility. Variability found in recoveries obtained with different lot numbers of SPE cartridges, as
well as cartridges obtained from different suppliers, led us to
include in the method a performance specification for cartridges. Use of a second liquid chromatographic (LC) column
for quantitation also is recommended, on the basis of experience gained from this exchange.
Samples were shipped via air express to participants, who
were notified via facsimile transmission of the date and manner
of shipment to facilitate collection on receipt. Participants also
were provided with a form to complete on receipt of samples,
which included information on the state of the samples as received. Participants were instructed to return the forms immediately and to dispose of any samples that were judged unfit
because of thawing and subsequent spoilage. Samples were
packed in insulated containers with dry ice sufficient to maintain the samples frozen for 5 days without refrigeration. Shippers were requested to store samples in a freezer, whenever
possible, during stopovers in transit.
Four sets of tissue samples (pork kidney and muscle and
beef kidney and muscle), consisting of 7 pairs of blind duplicates, were submitted to 16 laboratories, one of which used a
different analytical method for comparative purposes (3). Tissue sets contained fortified and incurred samples with tetracyclines at concentrations from 0.1 to 2.2 g/g, plus blanks.
Laboratories were instructed to report recoveries of the 3 analytes from the SPE cartridges at 0.5 g/g for all 4 tissue matrixes and were provided blank tissue for this purpose. Fortified
samples were prepared from the blank tissue pool and tested for
homogeneity prior to shipment to participants.

Preparation of Incurred Samples

Two steers and 9 hogs were treated to generate incurred residues of CTC, OTC, and TTC. Control animals from the same
herds were used to generate blank tissues. Samples from these
animals were analyzed to determine incurred values, and whole
blank tissues were pretested and selected to avoid interferences.

Blanks were pooled, homogenized, and then retested. The same

blank tissue pool was used for all fortified samples, the true
blank, and the recovery blank. Pools of 500 g blank tissue were
fortified to produce residues at concentrations according to the
protocol approved by AOAC. Aliquots from each pool were
analyzed in triplicate to test for homogeneity. If the coefficient
of variation for these analyses was >7%, the sample was remixed prior to subsampling. Incurred tissues were homogenized and diluted, as required, with blank tissue to achieve the
desired concentration. Samples were tested for homogeneity
prior to bagging.

Treatment of Data
Results were tabulated and reported by each participant.
When sample extracts were reanalyzed with a second LC column to confirm quantitative results, collaborators were requested to review the results from the 2 columns and, on the
basis of their judgement, to select what they believed to be the
correct, or interference-free, result. The tabulated results, along
with all chromatograms, were returned to the ARs laboratory,
where the chromatograms were reviewed by several analysts
with experience in the method. Tabulated results were checked
against analytical records provided for transcription errors.
Where a laboratory reported results for both their initial analysis, and second-column results, the ARs laboratory selected the
final result, on the basis of the same criteria. The results were
corrected for recovery, with recovery data provided by each
participant, and statistical analysis was conducted by using
software provided by AOAC (version of March 4, 1992).
995.09 Chlortetracycline, Oxytetracycline, and
Tetracycline in Edible Animal Tissues, Liquid
Chromatographic Method
First Action 1995
(Applicable to determination of oxytetracycline at
0.11 g/g and tetracycline at 0.14 g/g in bovine muscle tissues, chlortetracycline at 0.21 g/g and oxytetracycline at
0.09 g/g in porcine muscle tissues, oxytetracycline at
0.25 g/g and tetracycline 0.210.92 g/g in bovine kidney
tissues, and chlortetracycline at 0.66 g/g and oxytetracycline
at 0.25 g/g in porcine kidney tissues.)
Caution: See Appendix: Laboratory Safety for Safe Handling of Special Chemical Hazardsmethanol and acetonitrile. Tetracyclines are irritants and tetracycline itself is a possible teratogen. Handle tetracycline standards with care. Prepare
mobile phases in well-ventilated chemical fume hood. Avoid
contact with skin. See safety notes on reagents specified. Dispose of waste solvents in an appropriate manner compatible
with applicable environmental rules and regulations.
Method Performance:
See Table 995.09 for method performance data.

A. Principle
Tetracyclines are extracted from tissue with buffer (pH 4).
Filtered extract is cleaned up on C18 solid-phase extraction col-

umn. Tetracyclines are separated by liquid chromatography

(LC) using C8 column and detected with UV detector set at
350 nm wavelength. Results are corrected for recovery for each
analyte for each analytical run.

B. Apparatus
(a) Liquid chromatograph.Equipped with solvent delivery system capable of providing flow rate 12 mL/min with
low pulsation when using specified LC column and mobile
phase; UV detector set at 350 nm wavelength, 0.005 absorbance unit full scale (AUFS); selectable time constant; manual
injector or autosampler; strip chart recorder. Operating conditions: injection volume 1060 L; time constant, 3.0; chart
speed, 0.5 cm/min; run time, 10 min.
(b) LC columns.(1) For tetracycline determination.
250 4.6 mm id, 5 m C8 reversed-phase deactivated silica
packing (LC column of the same physical dimensions containing similar 10 m particulate material is suitable); flow rate,
1.2 mL/min for 5 m LC column and 2.0 mL/min for 10 m
LC column. Adjust as per system suitability, D. (2) For confirmation of quantitative results.Another column, 250
4.6 mm id, 5 or 10 m C18 reversed-phase deactivated silica
packing (C8 column of the same physical dimensions may be
substituted if retention characteristics are known to be different
from those of the standard analytical column [i.e., made by different manufacturer or with different degree of deactivation].)
(c) Balance.Capable of weighing to 0.001 g.
(d) Buchner funnel.5.5 cm.
(e) Centrifuge.Holding 50 mL polypropylene tubes, capable of providing 2500 g.
(f) Centrifuge tubes.50 mL, polypropylene, disposable.
(g) Automatic dispenser.Graduated to deliver 210 mL.
(h) Filter paper.Glass microfiber, grade GF/B, 5.5 cm.
(i) Erlenmeyer flask.Sidearm, 125 mL.
(j) Volumetric flasks.5, 10, 500, and 1000 mL.
(k) Homogenizer.Equipped with cutting blades to disintegrate and homogenize animal tissue. Recommended treatable
volume capacity of probe, 2500 mL.
(l) Mechanical shaker.Flat bed, 2-speed, oscillating
horizontally.
(m) pH Meter.Capable of measuring 0.05 unit.
(n) Pipet.Pasteur, disposable, 2 mL.
(o) Filtration cartridge.To filter test sample; 13 mm id,
0.45 m porosity, equipped with Luer-lock.
(p) Solid-phase extraction (SPE) apparatus.Equipped
with vacuum block with 1012 ports, vacuum gauge, and
75 mL reservoirs.
(q) Solid-phase extraction (SPE) cartridges.6 mL,
500 mg, C18 packing. Cartridge must meet system suitability,
D.
(r) Vortex mixer.
(s) Vacuum pump.

C. Reagents
(a) McIlvaine buffer.pH 4.0 0.05. Place 28.4 g anhydrous dibasic sodium phosphate (reagent grade) into 1 L volumetric flask and dissolve in distilled H2O. Dilute solution to

volume with H2O and mix. Place 21.0 g citric acid monohydrate into another 1 L volumetric flask and dilute to volume
with distilled H2O. Mix well. Combine 1 L citric acid solution
with 625 mL sodium phosphate solution in 2 L flask. Adjust pH
to 4.0 0.05 by adding dropwise either 0.1M HCl or 0.1M
NaOH. Prepare fresh buffer weekly.
(b) McIlvaine bufferEDTA solution.Adjust McIlvaine
buffer to contain 0.1M disodium ethylenediamine tetraacetate
as follows: To 1.625 L McIlvaine buffer, add 60.5 g disodium
EDTA dihydrate and mix until solid dissolves. Prepare fresh
solution weekly.
(c) Methanolic oxalic acid.Dissolve 1.26 g oxalic acid
dihydrate in methanol (LC grade) in 1 L volumetric flask. Dilute solution to volume with methanol and mix. Prepare fresh
solution daily.
(d) Tetracycline (TC) analytical standards.Certified reference standards of chlortetracycline hydrochloride,
oxytetracycline hydrochloride, and tetracycline hydrochloride.
(1) TC stock standard solutions.1000 g/mL. Weigh 108
0.1 mg of each tetracycline analytical standard into separate
weighing dishes (weights corrected for assayed content) and
transfer with methanol into separate 100 mL volumetric flasks.
Dilute to volume with methanol at room temperature and mix
until dissolved. Prepare fresh TC stock standard solution every
3 months and store at 20C. (2) TC combined stock standard
solution.100 g/mL. Pipet 10 mL each TC stock standard
solution into one 100 mL volumetric flask, dilute to volume
with methanol at room temperature, and mix. (3) TC combined
working standard solution.25 g/mL. Pipet 2.5 mL TC combined stock standard solution into 10 mL volumetric flask, dilute to volume with methanol at room temperature, and mix.
Store solution in refrigerator. Prepare fresh solution weekly.
(e) TC chromatographic standard solutions (0.05, 0.10,
0.25, 0.5, and 1.0 g/mL).Pipet 20, 40, 100, 200, and 400 L
TC combined working standard solution into separate 10 mL
volumetric flasks. Add 6 mL methanolic oxalic acid, (c), to
each flask, bring to volume with distilled H2O at room temperature, and mix. Store solutions in refrigerator. Prepare fresh solutions weekly.
(f) Solvents.Acetonitrile and methanol, LC grade.
(g) LC mobile phase.Dissolve 1.26 g oxalic acid dihydrate (reagent grade) in distilled H2O in 1 L volumetric flask,
dilute to volume with H2O, and mix. (1) For 5 m LC column.Combine 600 mL oxalic acid solution with 300 mL
acetonitrile and 100 mL methanol. (2) For 10 m LC column.Combine 700 mL oxalic acid solution with 200 mL
acetonitrile and 100 mL methanol. Prepare fresh solutions
daily.
Filter and degas LC mobile phases. (Note: Performance of
individual LC columns may vary, depending on manufacturer
and batch. Some adjustment of LC mobile-phase compositions
may be required to meet system suitability specifications.)

D. System Suitability
SPE cartridges should provide 80% recovery for fortified
tissue samples containing 15 ng of each tetracycline (chlortetracycline, oxytetracycline, and tetracycline), at elution condi-

tions specified in the method. Some batches of cartridges tested

that do not meet this requirement may still be acceptable if recoveries are consistent (RSD 10%).
Correction of recovery should be made for each run, based
on fortified sample included in run, when the method is in routine use. Inclusion of 2 fortified samples in each analytical run
for recovery correction is recommended if the method is not
used on a routine basis.
LC system parameters should be adjusted so that injection
of 60 L TC chromatographic standard solution at 0.25 g/mL
produces 3 distinct peaks within 9 min of injection [15 min for
LC column B(2) used for confirmation of quantitative results].
Resolution between oxytetracycline and tetracycline peaks
should be 1.5 (baseline resolution) and at least the same for
chlortetracycline with reference to either oxytetracycline or tetracycline. Retention times for replicate injections of TC chromatographic standard solution should match within 0.05 min.
Detection limits (3 baseline noise) are 1.5 ng for
oxytetracycline and tetracycline and 3 ng for chlortetracycline,
with linear range of 330 ng for samples injected onto LC column. See Figure 995.09 for tetracyclines chromatograms.
Correlation coefficients for standard curves should be
0.995.

E. Preparation of Test Sample

Note: Tissue should be kept frozen until analyzed. The entire extractioncleanup procedure should be completed in one
day.
Weigh 5.00 0.05 g tissue sample into 50 mL
polypropylene centrifuge tube. Fortify 5.00 g known blank tissue with each tetracycline at 0.5 g/g by adding 100 L TC
combined working stock standard solution at 25 g/mL,
C(d)(3).
Add 20 mL McIlvaine bufferEDTA solution to each sample and blend 30 s with homogenizer. Rinse probe twice with
2 mL McIlvaine bufferEDTA solution into centrifuge tube.
Blank tissue should be included in each analytical run to
check for interferences (e.g., coeluting substances).
Cap tubes and shake 10 min on flat-bed shaker at high
speed. Remove tubes from shaker and centrifuge 10 min at
2500 g. Pour supernate into second 50 mL centrifuge tube.
Do not transfer any tissue.
Add 20 mL McIlvaine bufferEDTA solution to the first
tube, cap, and resuspend tissue plug using Vortex mixer. Shake
10 min as above, centrifuge 10 min at 2500 g, and then add
supernate to the supernate in second tube.
Resuspend tissue plug in first tube in 10 mL McIlvaine bufferEDTA solution and repeat all steps, until supernates from all
3 extractions are collected in the second tube. Centrifuge combined supernates 20 min at 2500 g.
Place single GF/B filter paper in Buchner funnel and moisten with McIlvaine bufferEDTA solution. Filter combined supernate through funnel into 125 mL sidearm flask, applying
gentle vacuum to sidearm. [Notes: (1) Prehomogenized tissue
may plug SPE cartridges. If prehomogenized tissue is used,
split extract into 2 equal volumes and filter separately, changing
GF/filters between volumes. Combine filtered extracts before

loading onto SPE cartridge. (2) Too strong vacuum results in

severe foaming. It is essential that vacuum is established before
test sample is poured onto the filter paper. Poor filtration results
in plugging of SPE cartridges.]
Rinse centrifuge tube twice with 2 mL McIlvaine buffer
EDTA solution and filter into sidearm flask.
Prepare set of SPE cartridges on SPE apparatus, connecting
75 mL reservoir to each cartridge. Condition each cartridge
with 20 mL methanol followed by 20 mL H2O and discard eluate.
Add sample extract to 75 mL reservoir, rinse sidearm flask
twice with 2 mL McIlvaine bufferEDTA solution, and add
washes to reservoir. (Note: Do not dry SPE cartridge between
initial methanol conditioning wash and completion of addition
of sample and sample wash. Monitor elutions closely to ensure
that cartridges do not dry. If several columns run low simultaneously, interrupt vacuum to reduce solvent flow when reservoirs are refilled. Flow rate through SPE column is not critical
but should not exceed a steady drip.)
Rinse sidearm flask with 20 mL H2O and add wash to reservoir when sample extract is loaded. Allow cartridge to dry
when H2O rinse is complete and continue to draw air through
cartridge 2 min.
Drain and clean SPE extraction system and place 10 mL
volumetric flasks in position as receiving flasks. Elute tetracyclines from cartridges with 6.0 mL methanolic oxalic acid,
C(c). Carefully monitor final elution; methanolic oxalic acid
passes through cartridges faster than aqueous extracts. Increase
vacuum to maximum for 10 s at end of elution step to remove
residual solvent from cartridge. Dilute eluate to 10 mL with H2O.
Using filtration cartridge, B(o), filter samples and standards
into LC autosampler vials and load into autosampler. Filtered
samples may be injected manually if autosampler is not available.

F. LC Determination
Condition LC column, B(b)(1), with mobile phase 30 min
before any injection of standards or samples.
Inject 60 L of each TC chromatographic standard solution.
Injected standards contain amounts of tetracyclines that are expected to be extracted from tissue containing 0.12.0 g/g
tetracyclines, assuming 100% extraction efficiency.
Measure peak heights and prepare standard curve for each
tetracycline by plotting concentration vs peak height. (Note:
Recorder or detector attenuation may have to be adjusted to
bring 2.0 g/g tissue-equivalent standard [1.0 g/mL TC chromatographic standard solution] on scale.) Follow with injection
of samples. Samples that are off-scale at attenuation setting
used for 0.11.0 g/g tissue-equivalent standards should be reinjected at attenuation used for 2 g/g tissue-equivalent standard.
After analysis, flush LC system 30 min, including LC column, with wateracetonitrilemethanol (7 + 2 + 1, v/v/v), to
remove buffer. If autosampler is used, purge several times
while flushing LC system.

G. Confirmation of Quantitative Results

Samples containing any tetracycline at <0.5 g/g should be
reinjected onto LC confirmation column, B(b)(2), and compared with 0.25 g/mL TC chromatographic standard solution

to ensure that retention times match those of standard and that

analytical responses are the same as those observed upon initial
analysis. Some tissue samples may contain coeluting substances, which interfere in identification and quantitation of
one or more tetracyclines at levels <0.5 g/g.
If comparison of test sample with TC chromatographic
standard, as described, demonstrates presence of interferences
in the original analysis, reanalyze test samples and TC chromatographic standard solutions for appropriate standard curve, using LC confirmation column, B(b)(2).

H. Calculations
Measure peak heights for TC chromatographic standard solutions and test samples, adjusting for attenuation changes as
required. Prepare standard curve of tetracycline tissue-equivalent concentration vs peak height by using data from TC chromatographic standard solutions.
Determine the best fit to data using linear regression as follows:
y = mx + b
where y = peak height; x = tetracycline concentration, g/g; m
= slope of curve; and b = intercept of y.
From measured peak heights of test samples, calculate tetracycline concentrations from regression slope and intercept
values. Correct results for analytical recovery as determined for
fortified recovery sample included in analysis.
Ref.: J. AOAC Int. 79, 405(1996)
Results
Samples were shipped to 16 laboratories, which received the
samples in frozen or semifrozen state and judged them fit. One
participant, however, lost approximately half of the samples
through an accident in the laboratory and reported an incomplete set of results, which were rejected. One laboratory withdrew from the study after samples were shipped. Results from
the remaining 13 laboratories that used the method under study
(2) were then statistically evaluated and compared with the results obtained by another participating laboratory, using an alternative analytical procedure (3).
Statistical evaluation of results from 13 laboratories reporting a complete set of results is given in Tables 15. Table 6
compares the results obtained by the 13 laboratories and those
of the laboratory using alternative methodology.
Discussion
In preparing samples for the study, we noted that kidneys
contained interfering substances that made it difficult to find
truly blank tissue. These interferences seemed to be enhanced
by additional tissue manipulations required to ensure homogeneity. Although these tissues were more prone to generating
interferences than most of those routinely analyzed in our laboratory over the previous 6 years, we decided to proceed on the
basis that the method should be tested on samples that were
representative of those that might be encountered in a labora-

tory submission. The influence of these interferences can be

seen in the results.
To assess recovery performance of SPE cartridges used in
participants laboratories, because recoveries vary widely for
different lot numbers of cartridges even from the same manufacturer, each participant was supplied with blank tissue that
they were to fortify at 0.5 g/g with CTC, OTC, and TTC. Using AOAC software we analyzed these results but expert laboratories, such as the method originators, were not excluded if
they reported only a single result for each tetracycline. As stated
earlier, no 2 laboratories used the same lot number of cartridges. Highest recoveries were obtained for OTC in pork
muscle, while lowest recoveries were obtained for CTC and
TTC in bovine kidney. Five of 12 sets of data gave recoveries
greater than 70% and 4 of 12 gave recovery RSDR values of
<20%. Some participants reported low recoveries, in the range
of 20%, for some analytetissue combinations. These results
were not automatically rejected, but they may account in part
for the relatively large standard deviations calculated for the
recovery-corrected results. On the basis of our own earlier experience, confirmed by the study results, a performance requirement has been included in the method for SPE cartridges.
Recovery data for the 0.5 g/g analyst-fortified samples are
given in Table 1.
Participants were asked to verify results below 0.5 g/g by
using a second LC column and to report the confirmed quantitative results. In some cases, participants reported 2 sets of results, from 2 different columns, and asked us to select. We did
this by using interferences and recovery as our criteria. We did
not reevaluate their chromatograms. All results were recoverycorrected prior to statistical analysis. Participants were asked
not to report results below the claimed detection limits of
0.05 g/g for OTC and TTC, and 0.1 g/g for CTC.
In evaluating data using the AOAC software, results below
the detection limit were treated as zero. Results of laboratories reporting one or both samples of a duplicate pair as zero,
when a positive result should have been obtained, were excluded from statistical evaluation for that sample. Software instructions recommended exclusion of laboratories that did not
report 2 or more results. If a laboratory did not report a duplicate, results of that laboratory were removed as well.
During statistical analyses, if no more than 2 of 9 laboratories had been removed on the basis of the criteria above, outlier
tests (Cochrans, single Grubbs, double Grubbs) were applied
according to the harmonized scheme. Removal of a laboratory
at any point in the outlier cycle resulted in recalculation of statistics and reapplication of outlier tests. Results were summarized in tabular form for each matrix (Tables 25). The numbers
of laboratories removed in these tables include those removed
for reporting zero (false negative) and those removed by outlier tests.
As previously mentioned, there was a problem with
coeluting interferences. In each set, only 2 of 3 possible analytes were present, with no TTC in pork and no CTC in beef.
One true blank was included in each set. It appears that interferences are related in part to freezing, thawing, and homogenizing of tissue, and they were less pronounced in the incurred

and blank tissues, which were processed at least one less time
than fortified tissues.
For pork kidney pair 202/207, 5 of 13 laboratories reported
TTC where none was added and 3 of these results were from
the 7 laboratories with the best recoveries. The laboratory using the alternative procedure also reported TTC at 0.09 g/g in
this sample pair. For the blank pork kidney pair 200/209,
6 laboratories reported false-positive results, including OTC at
concentrations ranging from 0.08 to 0.29 g/g, with a mean
value of 0.13 0.08 g/g (6 samples, 5 laboratories). One
laboratory reported TTC at 0.10 g/g, and one laboratory reported CTC at 0.10 g/g in this sample pair. For the blank pork
muscle pair 306/311, 2 laboratories reported false-positive results, including 3 findings of OTC (0.34 0.21 g/g), 2 of
TTC (0.16 0.10 g/g), and 1 of CTC (0.06 g/g). The worst
interference problems were observed for pork muscle
pair 304/313, with 9 of 13 laboratories reporting TTC at 0.06
0.13 g/g. Laboratory 19, using the alternative metal chelation
method, also reported TTC in sample 304, but not in 313, indicating that the interferences observed for TTC are not unique
to the study method. These results all fall within the range for
which confirmation of quantitative results with a second column is recommended.
In general, interferences from coeluting substance were
more commonly observed in pork kidney samples, with RSDR
values being generally higher than for equivalent concentrations of residues in pork muscle. At concentrations above
1 g/g for OTC, whether incurred or fortified, RSDR values
were 1820%. For 0.30.5 g/g OTC, RSDR values were 20
26%, but RSDR was near 50% at 0.16 g/g, with 5 laboratories
rejected as outliers. As observed for pork muscle, results for
CTC in pork kidney had an increasing RSDR with decreasing
concentration, with RSDR of 43.57% at 0.12 g/g and 8 laboratories rejected. For CTC in pork kidney, method performance
was more acceptable at the levels tested at 0.6 g/g and higher.
Attempts to produce incurred residues in kidney at the United
States tolerance of 4 g/g were, however, not successful, so the
method performance was not assessed at that concentration.
For pork muscle, results on incurred samples for OTC and
CTC, with concentrations in the range 0.200.25 g/g showed
RSDR values for all reporting laboratories in the range 1623%.
Results for OTC and CTC spiked at 0.87 and 0.95 g/g, respectively, gave RSDR values of 16.84 and 23.62%, respectively.
However, variability increased at fortification levels near
0.5 g/g for CTC (29.38%) and OTC (27.07%), although the
number of rejected outliers did not increase. For samples fortified at 0.1 g/g, RSDR values were 24.9832.96% for OTC
with 23 outliers, but for CTC RSDR increased to 4060% with
57 outliers. On the basis of these results, the method appears
reliable to 0.1 g/g for OTC in pork muscle for most laboratories and to 0.2 g/g for CTC in pork muscle, considering the
result for the incurred sample.
For bovine kidney samples, 4 of 13 laboratories reported
CTC in sample pair 404/407 and 3 of 13 reported CTC in
405/409 (both incurred samples). No false-positive results for
CTC were reported for these sample pairs as analyzed by the
alternative method. For the blank bovine kidney pair 406/410,

5 laboratories reported false-positive results. Four laboratories

reported a total of 7 findings of OTC at concentrations of 0.10
0.32 g/g (0.22 0.08 g/g) in this sample pair, 2 laboratories
each reported CTC in one of the paired samples analyzed (0.27
0.004 g/g) and one laboratory reported TTC in one sample
at 0.40 g/g. For the blank bovine muscle pair 506/512, 3 laboratories each reported a single false-positive result, one each for
OTC (0.06 g/g), CTC (0.06 g/g), and TTC (0.15 g/g).
Again, all reported values were within the concentration range
for which confirmation of quantitation with a second column
was recommended.
Overall, bovine kidney gave poorer results than bovine
muscle, particularly at the lower concentrations. This finding
suggests that, as for pork, kidney samples are more prone to
interferences. Results for incurred samples, all near 0.5 g/g,
however, were excellent with RSDR of 13.64% for 0.48 ppm
TTC and RSDR values of 17.02 and 17.62% for OTC at 0.45
and 0.54 g/g, respectively. For fortified tissue, RSDR for TTC
was 21.59% at 0.92 g/g, 18.32% at 0.46 g/g, and 23.00% at
0.21 g/g, but 56.91% with 7 rejected results at 0.11 g/g. For
bovine kidney fortified with OTC, RSDR was 13.36% at
0.95 g/g, 21.36% at 0.48 g/g, and 28.79% at 0.25 g/g, but
59.20% at 0.12 g/g (4 results rejected). On the basis of results
for incurred tissue, the method meets IUPAC guidelines for
RSDR for both OTC and TTC in bovine kidney at concentrations near 0.5 g/g. Results for fortified tissue suggest that most
laboratories will obtain results that may be considered to approach the guideline to about 0.25 g/g for both OTC and TTC
but will encounter difficulties at concentrations near 0.1 g/g.
For bovine muscle, no more than 2 results were rejected in
any of the samples tested. For OTC, RSDR values ranged from
13.70% at 0.5 g/g to 28.42% at 0.11 g/g. TTC results were
similar, ranging from 11.14% RSDR at 0.46 g/g to 26.94% at
0.14 g/g. The results suggest the method may be considered
reliable for the range of concentrations tested for OTC and TTC
in bovine muscle, although some results at lower concentrations are slightly in excess of IUPAC guidelines. IUPAC recommends the following RSDR values for acceptance of a
method: <16% (1 g/g), <23% (0.1 g/g), and <32% (0.01 g/g).
Results for laboratory 19 are summarized and compared
with mean results of the other participants in Table 6. At higher
levels, results from laboratory 19 were higher than the study
means, but the differences may not be statistically significant.
Overall, the interference problems were most evident in falsepositive results for OTC in pork kidney and, to a lesser extent,
for OTC in bovine kidney. In general, muscle samples produced fewer false positives.
The results suggest the method can achieve reliable results
for the analytes and matrixes studied at concentrations from 0.1
to 0.6 g/g and above in most laboratories, depending on the
analytetissue pair. In general, better results may be expected
in muscle than in kidney. The poorer performance observed for
fortified samples, particularly kidney, suggest that in actual
practice, where additional treatment for preparation of homogeneous study samples would not be required, performance
should be more in line with that observed for incurred samples,
which were subjected to fewer homogenization steps. A com-

parison of the study mean results with those obtained by the

method of Farrington and coworkers (3), demonstrates that
similar results may be obtained with the 2 procedures and that
they are similarly affected by interferences.
Recovery from SPE cartridges remains a problem, and users
of the method should pretest each lot of cartridges for recovery,
as stated in the method, and should reject lots that do not yield
reproducible recoveries in the range 60100%. A fortified sample for recovery correction should then be included in each analytical run, and a recovery correction should be applied for all
determinations. In this study, participants reported recoveries
ranging from a low of 20.6% to a high of 126.6% for CTC,
17.8100.0% for TTC, and 33.4124.8% for OTC. Inclusion
of data requiring correction for low recoveries, in particular,
increased the spread in the data, which was reflected in performance statistics. Analysis of data from 7 laboratories reporting recoveries of 40120% resulted in a better calculated RSDR
in 6 of 6 sets of kidney data. We hypothesize that a general
improvement in performance would have been observed if all
participants had obtained recoveries near 80%, as recommended in the method specification for the SPE cartridges.

Collaborators Comments
Several laboratories (72, 87) analyzed all samples with
2 LC columns. Laboratory 72 noted a distinct difference in results from the 2 columns, particularly for kidney samples. In
laboratory 87, the second column was run with a diode array
detector, while the routine column was attached to a standard
variable-wavelength detector and gave different results. Laboratory 30 found more interference for CTC assays when using
a 5 m C18 packing than with a 10 m C8 packing.
Laboratory 88 used the total sample for sets BM1 and BM2,
instead of weighing as instructed. They also homogenized samples with a high-speed blade, followed with 1 min in an ultrasonic bath. They found interferences in the analysis of OTC and
CTC with the C8 column used as the initial screening column,
but the interferences were not a problem on the confirmatory
column.
Laboratory 2 reported that only one GF/B filter was required
in their assays and injected a volume of 50 L instead of the
recommended 60 L. They lost their first set of beef samples;
thus assays were conducted on samples ranging from 1.67 to
3.10 g, with some resultant loss in sensitivity.
Laboratory 25 found an interference in all kidney samples
run on one C8 LC column, but it was not found on a second C8
column. Laboratory 54 found a substance in kidney samples
that interfered with tetracycline analysis.
Conclusions
The method can be applied successfully to analysis of tetracycline residues in edible animal tissues, as tested, at levels of
0.1 to 0.6 g/g and higher, depending on the analytetissue
combination. Performance was less reliable near the claimed
detection limits and the presence of coeluting interferences,
particularly in kidney tissues, requires use of a second analytical column to confirm quantitation. The method appears more

susceptible to interferences when more than one sample homogenization is required, as in preparation of fortified samples
for the study. Selection of SPE cartridges for the method is critical. Cartridges that provide low or inconsistent recoveries
should not be accepted. Method performance was similar with
the method using an alternative cleanup procedure with LC
analysis.
The method should serve as a benchmark for comparison with
other future methods. Improvements in SPE cartridge technology
or use of analyte derivatization techniques may be required to extend the method successfully to concentrations of 0.1 g/g and
below for all analytetissue concentrations of interest.
Recommendation
On the basis of the results of this study, it is recommended
that the liquid chromatographic method for determination of
chlortetracycline, oxytetracycline, and tetracycline in edible
animal tissues be adopted first action.
Acknowledgments
The following collaborators participated in this study:
G. Alfredsson, National Food Administration, Uppsala,
Sweden
C. Barry, Laboratory Services Division, Agriculture Canada, Ottawa, Ontario, Canada
B. Bergner, Chemisches Institut im Amt fr Umweltschutz
der Landeshauptstadt Stuttgart, Stuttgart, Germany
W. Chan, Health of Animals Laboratory, Agriculture Canada, Saskatoon, Saskatchewan, Canada
J.M. Diserens, Food Contaminants, Nestec Ltd. Research
Centre, Lausanne, Switzerland
L.P. Ilnicki, Western Laboratory, U.S. Department of Agriculture, Food Safety and Inspection Services, Alameda, CA
E. Klein, Chemische Landesuntersuchungsanstalt, Sigmaringen, Germany
B. Koscinski and G. Vasco (T. Phillippo), Eastern Laboratory, U.S. Department of Agriculture, Food Safety and Inspection Services, Athens, GA
H. Mawhinny, Animal Research Institute, Queensland, Australia
E. Mller (M. Petz), Institute of Food Chemistry, University
of Wuppertal, Wuppertal, Germany
H. Oka, Laboratory of Food and Drug Chemistry, Aichi Prefectural Institute of Public Health, Nagoya, Japan
R. Patel, Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, Surrey, United Kingdom
G.M. Telling, Unilever Research Colworth Laboratory,
Bedford, United Kingdom
M. Webb (C. Henry), Midwestern Laboratory, U.S. Department of Agriculture, Food Safety and Inspection Services, St.
Louis, MO
Alternative LC method (3):
W.H.H. Farrington, Ministry of Agriculture, Fisheries and
Food, Food Safety Directorate, Food Science Laboratory, Norfolk, United Kingdom

This study is dedicated to the memory of Raymond Ashworth, U.S. Department of Agriculture, Food Safety and Inspection Services, under whose term as Associate Referee the
initial work was begun.
References
(1) AOAC Official Methods Program Manual on Development,
Study, Review and Approval Process for AOAC Official Methods (1993) AOAC International, Arlington, VA
(2) Oka, H., Matsumoto, H., Uno, K., Harada, K., Kadowaki, S.,
& Suzuki, M. (1985) J. Chromatogr. 325, 265274
(3) Farrington, W.H.H., Tarbin, J., Bygrave, J., & Shearer, G.
(1991) Food Addit. Contam. 8, 5564

Table 995.09. Method performance for determination of chlortetracycline, oxytetracycline, and tetracycline in edible
animal tissues by liquid chromatographic method
Sample

Tetracycline, g/g

Mean, g/g

sr

RSDr, %

sR

RSDR, %

ra

Rb

0.09
0.06
0.11
0.17
0.05
0.06
0.06
0.15
0.33
0.09
0.17

60.53
40.38
29.38
23.62
23.00
43.57
28.59
38.76
36.78
26.95
30.11

0.17
0.08
0.25
0.20
0.11
0.08
0.06
0.28
0.31
0.11
0.27

0.25
0.17
0.31
0.48
0.14
0.17
0.17
0.42
0.92
0.25
0.48

0.03
0.03
0.12
0.15
0.04
0.05
0.08
0.07
0.09
0.17
0.24
0.32
0.03
0.03
0.07
0.13
0.06
0.06
0.10
0.07
0.11
0.12
0.09
0.09

32.96
24.98
27.07
16.84
16.68
20.74
49.65
26.26
20.52
18.53
18.88
19.70
27.41
28.42
13.70
14.56
26.83
14.58
59.20
28.79
21.36
13.36
17.02
17.62

0.03
0.03
0.32
0.22
0.06
0.11
0.17
0.17
0.17
0.42
0.25
0.42
0.06
0.03
0.17
0.31
0.14
0.14
0.17
0.14
0.25
0.28
0.25
0.25

0.08
0.08
0.32
0.42
0.11
0.14
0.22
0.20
0.25
0.48
0.67
0.90
0.08
0.08
0.20
0.36
0.17
0.17
0.28
0.20
0.31
0.34
0.25
0.25

0.03
0.03
0.06
0.04
0.04
0.09
0.05
0.08
0.18
0.07

26.94
16.58
23.81
11.14
18.83
56.91
23.00
18.32
21.59
13.64

0.06
0.06
0.18
0.11
0.11
0.22
0.08
0.20
0.48
0.20

0.08
0.08
0.18
0.11
0.11
0.25
0.14
0.22
0.50
0.20

Chlortetracycline
Porcine muscle

Porcine kidney

0.12
0.15
0.44
0.95
0.21
0.12
0.24
0.43
0.92
0.41
0.66

0.15
0.14
0.39
0.73
0.21
0.14
0.22
0.39
0.89
0.33
0.57

0.06
0.03
0.09
0.07
0.04
0.03
0.02
0.10
0.11
0.04
0.10

44.04
24.78
23.11
9.13
19.35
24.02
9.71
24.24
12.17
13.62
16.90
Oxytetracycline

Porcine muscle

Porcine kidney

Bovine muscle

Bovine kidney

0.07
0.09
0.46
0.87
0.20
0.25
0.16
0.32
0.53
1.15
1.86
2.24
0.08
0.11
0.50
1.04
0.23
0.36
0.12
0.25
0.48
0.95
0.45
0.54

0.09
0.11
0.43
0.87
0.21
0.24
0.16
0.26
0.46
0.94
1.27
1.65
0.12
0.12
0.48
0.92
0.24
0.42
0.16
0.26
0.51
0.90
0.51
0.53

0.01
0.01
0.12
0.08
0.02
0.04
0.06
0.06
0.06
0.15
0.09
0.15
0.02
0.01
0.06
0.11
0.05
0.05
0.06
0.05
0.09
0.10
0.09
0.09

16.66
8.87
27.07
9.80
9.94
16.18
35.55
21.32
12.02
16.29
7.06
9.21
16.01
7.76
12.72
11.59
19.30
13.05
33.53
19.20
17.60
11.32
16.76
17.24
Tetracycline

Bovine muscle

Bovine kidney

a
b

r = 2.8 sr.
R = 2.8 sR.

0.14
0.21
0.28
0.46
0.24
0.11
0.21
0.46
0.92
0.48

0.13
0.21
0.26
0.39
0.24
0.15
0.22
0.42
0.85
0.55

0.02
0.02
0.06
0.04
0.04
0.08
0.03
0.07
0.17
0.07

16.50
10.19
23.81
10.31
16.26
49.11
15.39
17.02
20.47
12.03

Table 1. Determination of analyte recoveries at the 0.5 ppm level from analyst-fortified blank tissues a
Matrix
Pork kidney

Pork muscle

Beef kidney

Beef muscle

Analyte

LR

Recovery
range, %

Mean
recovery, %

RSDr, %

RSDR, %

OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC

13
12
13
12
13
13
13
13
12
11
11
12

0
0
0
1
0
0
0
0
1
2
2
1

51.0124.8
28.075.8
21.2126.6
74.0104.8
39.8100.0
39.091.0
33.490.0
17.886.0
20.683.2
57.097
57.079.0
22.092.8

79.78
59.32
59.30
86.73
73.11
66.32
64.05
56.18
56.17
81.74
71.25
65.75

12.82
11.11
39.30
9.74
5.62
10.96
19.92
13.27
26.72
5.44
2.54
12.55

19.76
21.32
39.30
10.88
21.36
22.86
24.80
35.85
31.38
13.00
11.97
24.36

n = number of laboratories; LR = number of laboratories removed; RSDr = repeatability relative standard deviation; RSDR = reproducibility
relative standard deviation.

Table 2. Determination of OTC and CTC residues in blind, coded pork muscle, fortified and incurreda
Sample ID
300/310

301/308

302/307

303/309

304/313

305/312

306/311

Analyte
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC

Incurred,
g/g

Spike,
g/g

LR

Mean,
g/g

sr

sR

RSDr, %

RSDR, %

r, g/g

R, g/g

0.87
0.00
0.15
0.07
0.00
0.44
0.09
0.00
0.95
0.46
0.00
0.12
0.00
0.00
0.00

12
12
11
13
13
10
12
10
8
11
10
12
10
4
11
13
13
6
11
12
12

1
1
2
0
0
3
1
2
5
2
3
1
3
9
2
0
0
7
2
1
1

0.21

0.21
0.24

0.87

0.14
0.09

0.39
0.11

0.73
0.43

0.15

0.02

0.04
0.04

0.08

0.03
0.01

0.09
0.01

0.07
0.12

0.06

0.04

0.05
0.05

0.15

0.06
0.03

0.11
0.03

0.17
0.12

0.09

9.94

19.35
16.18

9.80

24.78
16.66

23.11
8.87

9.13
27.07

44.04

16.68

23.00
20.74

16.84

40.38
32.96

29.38
24.98

23.62
27.07

60.53

0.06

0.11
0.11

0.22

0.08
0.03

0.25
0.03

0.2
0.32

0.17

0.11

0.14
0.14

0.42

0.17
0.08

0.31
0.08

0.48
0.32

0.25

0.20
0.00
0.21
0.25
0.00
0.00

Sample ID: a/b = blind duplicates; n = number of laboratories with data included in calculation; LR = number of laboratories removed; mean =
overall mean of laboratory values; incurred = incurred tissue, pretest value; spike = fortified tissue, pretest value; sr = repeatability standard
deviation; sR = reproducibility standard deviation; RSDr = repeatability relative standard deviation; RSDR = reproducibility relative standard
deviation; r = repeatability value; R = reproducibility value.

Table 3. Determination of OTC and CTC residues in blind, coded pork kidney, fortified and incurred a
Sample ID
200/209

201/211

202/207

203/208

204/212

205/213

206/210

Analyte
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC

Incurred,
g/g

Spike,
g/g
0.00
0.00
0.00
0.16
0.00
0.24
0.53
0.00
0.92
0.32
0.00
0.12
1.15
0.00
0.43

1.86
0.00
0.41
2.24
0.00
0.66

LR

Mean,
g/g

sr

sR

RSDr, %

RSDR, %

r, g/g

R, g/g

8
12
12
8
12
7
13
8
12
12
13
5
13
11
11
11
13
9
13
12
12

5
1
1
5
1
6
0
5
1
1
0
8
0
2
2
2
0
4
0
1
1

0.16

0.22
0.46

0.89
0.26

0.14
0.94

0.39
1.27

0.33
1.65

0.57

0.06

0.02
0.06

0.11
0.06

0.03
0.15

0.10
0.09

0.04
0.15

0.10

0.08

0.06
0.09

0.33
0.07

0.06
0.17

0.15
0.24

0.09
0.32

0.17

35.55

9.71
12.02

12.17
21.32

24.02
16.29

24.24
7.06

13.62
9.21

16.90

49.65

28.59
20.52

36.78
26.26

43.57
18.53

38.76
18.88

26.95
19.70

30.11

0.17

0.06
0.17

0.31
0.17

0.08
0.42

0.28
0.25

0.11
0.42

0.27

0.22

0.17
0.25

0.92
0.2

0.17
0.48

0.42
0.67

0.25
0.9

0.48

Sample ID: a/b = blind duplicates; n = number of laboratories with data included in calculation; LR = number of laboratories removed; mean =
overall mean of laboratory values; incurred = incurred tissue, pretest value; spike = fortified tissue, pretest value; sr = repeatability standard
deviation; sR = reproducibility standard deviation; RSDr = repeatability relative standard deviation; RSDR = reproducibility relative standard
deviation; r = repeatability value; R = reproducibility value.

Table 4. Determination of OTC and TTC residues in blind, coded beef muscle, fortified and incurreda
Sample ID
500/510

501/513

502/508

503/507

504/509

505/511

506/512

Analyte
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC

Incurred,
g/g

Spike,
g/g

LR

Mean,
g/g

sr

sR

RSDr, %

RSDR, %

r, g/g

R, g/g

1.04
0.28
0.00
0.50
0.46
0.00
0.11
0.14
0.00
0.08
0.21
0.00
0.00
0.00
0.00

12
10
12
12
12
11
12
10
12
12
12
12
10
12
11
10
12
12
11
11
12

0
2
0
0
0
1
0
2
0
0
0
0
2
0
1
2
0
0
1
1
0

0.42

0.24
0.24

0.92
0.26

0.48
0.39

0.12
0.13

0.12
0.21

0.05

0.05
0.04

0.11
0.06

0.06
0.04

0.01
0.02

0.02
0.02

0.06

0.06
0.04

0.13
0.06

0.07
0.04

0.03
0.03

0.03
0.03

13.05

19.30
16.26

11.59
23.81

12.72
10.31

7.76
16.50

16.01
10.19

14.58

26.83
18.83

14.56
23.81

13.70
11.14

28.42
26.94

27.41
16.58

0.14

0.14
0.11

0.31
0.18

0.17
0.11

0.03
0.06

0.06
0.06

0.17

0.17
0.11

0.36
0.18

0.2
0.11

0.08
0.08

0.08
0.08

0.36
0.00
0.00
0.23
0.24
0.00

Sample ID: a/b = blind duplicates; n = number of laboratories with data included in calculation; LR = number of laboratories removed; mean =
overall mean of laboratory values; incurred = incurred tissue, pretest value; spike = fortified tissue, pretest value; sr = repeatability standard
deviation; sR = reproducibility standard deviation; RSDr = repeatability relative standard deviation; RSDR = reproducibility relative standard
deviation; r = repeatability value; R = reproducibility value.

Table 5. Determination of OTC and TTC residues in blind, coded beef kidney, fortified and incurreda
Sample ID
400/412

401/413

402/411

403/408

404/407

405/409

406/410

Analyte
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC

Incurred,
g/g

Spike,
g/g
0.25
0.11
0.00
0.12
0.21
0.00
0.95
0.46
0.00
0.48
0.92
0.00

0.45
0.48
0.00
0.54
0.00
0.00
0.00
0.00
0.00

LR

Mean,
g/g

sr

sR

RSDr, %

RSDR, %

r, g/g

R, g/g

11
6
12
9
11
12
12
13
11
13
13
11
13
13
9
12
13
10
9
12
10

2
7
1
4
2
1
1
0
2
0
0
2
0
0
4
1
0
3
4
1
3

0.26
0.15

0.16
0.22

0.90
0.42

0.51
0.85

0.51
0.55

0.53

0.05
0.08

0.06
0.03

0.10
0.07

0.09
0.17

0.09
0.07

0.09

0.07
0.09

0.10
0.05

0.12
0.08

0.11
0.18

0.09
0.07

0.09

19.20
49.11

33.53
15.39

11.32
17.02

17.60
20.47

16.76
12.03

17.24

28.79
56.91

59.20
23.00

13.36
18.32

21.36
21.59

17.02
13.64

17.62

0.14
0.22

0.17
0.08

0.28
0.2

0.25
0.48

0.25
0.2

0.25

0.2
0.25

0.28
0.14

0.34
0.22

0.31
0.5

0.25
0.2

0.25

Sample ID: a/b = blind duplicates; n = number of laboratories with data included in calculation; LR = number of laboratories removed; mean =
overall mean of laboratory values; incurred = incurred tissue, pretest value; spike = fortified tissue, pretest value; sr = repeatability standard
deviation; sR = reproducibility standard deviation; RSDr = repeatability relative standard deviation; RSDR = reproducibility relative standard
deviation; r = repeatability value; R = reproducibility value.

Table 6. Comparison of assay results (in g/g) for coded, blind duplicate samples analyzed by the study method (2)
and the alternative method (3)a
Alternative procedure
Sample ID

Analyte

X(1)

X(2)

Mean

Alternative procedure
Study
mean

Sample ID

Analyte

Matrix: pork kidney

200/209

201/211

202/207

203/208

204/212

205/213

206/210

OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC

0.000
0.000
0.000
0.128
0.000
0.225
0.626
0.105
1.113
0.362
0.000
0.184
1.159
0.000
0.442
1.669
0.000
0.371
2.162
0.000
0.667

0.000
0.000
0.000
0.125
0.000
0.257
0.623
0.068
1.013
0.330
0.000
0.145
1.002
0.000
0.470
1.641
0.000
0.444
1.841
0.000
0.770

301/308

302/307

303/309

304/313

305/312

306/311

OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC

0.258
0.000
0.309
0.290
0.000
0.000
1.106
0.000
0.178
0.109
0.000
0.569
0.132
0.105
1.164
0.568
0.000
0.130
0.000
0.000
0.000

0.232
0.000
0.228
0.278
0.000
0.000
1.140
0.000
0.153
0.090
0.000
0.481
0.116
0.000
0.746
0.454
0.000
0.000
0.000
0.000
0.000

X(2)

Mean

Study
mean

0.280
0.157
0.000
0.123
0.298
0.000
1.065
0.542
0.000
0.633
1.108
0.000
0.648
0.728
0.000
0.688
0.000
0.000
0.000
0.000
0.000

0.26
0.15
0.00
0.16
0.22
0.00
0.90
0.42
0.00
0.51
0.85
0.00
0.51
0.55
0.00
0.53
0.00
0.00
0.00
0.00
0.00

0.517
0.000
0.000
0.296
0.327
0.000
1.187
0.333
0.000
0.565
0.505
0.000
0.110
0.195
0.000
0.111
0.191
0.000
0.000
0.000
0.000

0.42
0.00
0.00
0.24
0.24
0.00
0.92
0.26
0.00
0.48
0.39
0.00
0.12
0.13
0.00
0.12
0.21
0.00
0.00
0.00
0.00

Matrix: beef kidney

0.000
0.000
0.000
0.127
0.000
0.241
0.625
0.087
1.063
0.346
0.000
0.165
1.081
0.000
0.456
1.655
0.000
0.408
2.002
0.000
0.719

0.00
0.00
0.00
0.16
0.00
0.22
0.46
0.00
0.89
0.26
0.00
0.14
0.94
0.00
0.39
1.27
0.00
0.33
1.65
0.00
0.57

400/412

401/413

402/411

403/408

404/407

405/409

406/410

OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC

Matrix: pork muscle

300/310

X(1)

0.245
0.112
0.000
0.127
0.273
0.000
1.022
0.485
0.000
0.619
1.131
0.000
0.633
0.721
0.000
0.633
0.000
0.000
0.000
0.000
0.000

0.315
0.201
0.000
0.118
0.322
0.000
1.107
0.598
0.000
0.647
1.084
0.000
0.663
0.735
0.000
0.742
0.000
0.000
0.000
0.000
0.000

Matrix: beef muscle

0.245
0.000
0.269
0.284
0.000
0.000
1.123
0.000
0.166
0.100
0.000
0.525
0.124
0.053
0.955
0.511
0.000
0.065
0.000
0.000
0.000

0.21
0.00
0.21
0.24
0.00
0.00
0.87
0.00
0.14
0.09
0.00
0.39
0.11
0.00
0.73
0.43
0.00
0.15
0.00
0.00
0.00

500/510

501/513

502/508

503/507

504/509

505/511

506/512

Sample ID: a/b = blind duplicates; mean = overall mean of laboratory values.

OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC
OTC
TTC
CTC

0.557
0.000
0.000
0.322
0.337
0.000
1.331
0.375
0.000
0.651
0.577
0.000
0.118
0.240
0.000
0.109
0.144
0.000
0.000
0.000
0.000

0.477
0.000
0.000
0.269
0.317
0.000
1.042
0.291
0.000
0.478
0.432
0.000
0.102
0.149
0.000
0.112
0.237
0.000
0.000
0.000
0.000

Figure 995.09. Tetracycline chromatograms: oxytetracycline (OTC), tetracycline (TTC), chlortetracycline (CTC).
Chromatographic conditions: mobile phase, 0.01M oxalic acidACNMeOH (65 + 15 + 20, v/v/v); flow rate, 1.5 mL/min;
LC column, 250 4.6 mm id, C8, 5 m; UV detector, 350 nm wavelength, 0.005 AUFS.