23.1.17 (p) Solid-phase extraction (SPE) apparatus.

—With vacuum
AOAC Official Method 995.09 block with 10–12 ports, vacuum gauge, and 75 mL reservoirs.
Chlortetracycline, Oxytetracycline, (q) SPE cartridges.— 6 mL, 500 mg, C18 packing. Column
and Tetracycline in Edible Animal Tissues must meet system suitability requirements, D. Varian Bond Elut
Liquid Chromatographic Method columns, or equivalent, have been found satisfactory.
First Action 1995
(r) Vortex mixer.
Final Action 1999
(s) Vacuum pump.
(Applicable to determination of oxytetracycline and tetracycline
in bovine muscle tissues, chlortetracycline and oxytetracycline in C. Reagents
porcine muscle tissues, oxytetracycline and tetracycline in bovine (a) McIlvaine buffer.—pH 4.0 ± 0.05. Place 28.4 g anhydrous
kidney tissues, and chlortetracycline and oxytetracycline in porcine Na2HPO4 (reagent grade) into 1 L volumetric flask and dissolve in
kidney tissues.) distilled H2O. Dilute to volume with H2O and mix. Place 21.0 g
See Table 995.09 for the results of the interlaboratory study citric acid monohydrate into another 1 L volumetric flask and dilute
supporting acceptance of the method. to volume with distilled H2O and mix well. Combine 1 L citric acid
A. Principle solution with 625 mL Na2HPO4 solution in 2 L flask. Adjust pH to
Tetracyclines are extracted from tissue with pH 4 buffer. Filtered 4.0 ± 0.05 by adding dropwise either 0.1M HCl (8.3 mL HCl/L) or
extract is cleaned up on C18 solid-phase extraction column. 0.1M NaOH (4.0 g/L). Prepare weekly.
Tetracyclines are separated by liquid chromatography using C8 (b) McIlvaine buffer–EDTA solution.—Adjust McIlvaine buffer,
column and measured with UV detector at 350 nm. Results are (a), to contain 0.1M disodium ethylenediamine–tetracetate as
corrected for recovery for each analyte for each analytical run. follows: To 1.625 L McIlvaine buffer, add 60.5 g disodium EDTA
dihydrate and mix until dissolved. Prepare weekly.
B. Apparatus
(c) Methanolic oxalic acid.—Dissolve 1.26 g oxalic acid
(a) Liquid chromatograph.—With solvent delivery system dihydrate (reagent grade) in methanol (LC grade) in 1 L volumetric
providing flow rate 1–2 mL/min with low pulsation using specified flask. Dilute to volume with methanol and mix. Prepare daily.
LC col umn and mo bile phase; UV de tec tor at 350 nm,
(d) Tetracyclines (TC) analytical standards.—USP reference
0.005 absorbance unit full scale (AUFS); selectable time constant;
standards of chlortetracycline hy drochloride, oxy tetracy cline
manual injector or autosampler; strip chart recorder. Operating
hy drochloride, and tetracy cline hy drochloride. (1) TC stock
conditions: injection volume 10–60 µL; time constant, 3.0; chart
standard solutions.—1000 µg/mL. Weigh 108 ± 0.1 mg each
speed, 0.5 cm/min; run time, 10 min.
tetracy cline standard into separate weighing dishes (weights
(b) LC columns.—(1) For tetracyclines determination.—250 × corrected for assayed content) and transfer with methanol into
4.6 mm id, 5 µm C8 reversed-phase deactivated silica packing (LC separate 100 mL volumetric flasks. Dilute to volume with methanol
column of the same physical dimensions containing similar 10 µm at room temperature and mix until dissolved. Prepare TC stock
particulate material is suitable); flow rate, 1.2 mL/min for 5 µm LC standard solution every 3 months and store at –20°C. (2) TC
column and 2.0 mL/min for 10 µm LC column. (2) For confirmation combined stock standard solution.—100 µg/mL. Pipet 10 mL each
of quantitative results.—Another column, 250 × 4.6 mm id, 5 or TC stock standard solution into one 100 mL volumetric flask, dilute
10 µm C18 reversed-phase deactivated silica packing. (C8 column to volume with methanol at room temperature, and mix. (3) TC
of the same physical dimensions may be substituted if retention combined working standard solution.—25 µg/mL. Pipet 2.5 mL TC
characteristics are known to be different from those of the standard combined stock standard solution into 10 mL volumetric flask,
analytical column [i.e., made by different manufacturer or with dilute to volume with methanol at room temperature, and mix. Store
different degree of deactivation].) solution in refrigerator. Prepare weekly.
(c) Balance.—Readability to ±0.001 g. (e) TC chromatographic standard solutions.—0.05, 0.10, 0.25,
(d) Büchner funnel.—5.5 cm. 0.5, and 1.0 µg/mL. Pipet 20, 40, 100, 200, and 400 µL TC combined
(e) Cen tri fuge.—Holding 50 mL poly propy lene tubes; working standard solution into separate 10 mL volumetric flasks.
providing 2500 × g. Add 6 mL methanolic oxalic acid, (c), to each flask, bring to volume
(f) Centrifuge tubes.—50 mL, poly propy lene, disposable. with distilled H2O at room temperature, and mix. Store solutions in
(g) Automatic dispenser.—Graduated to de liver 2–10 mL. refrigerator. Prepare weekly.
(h) Filter paper.—Glass microfiber, grade GF/B, 5.5 cm. (f) Solvents.—Acetonitrile and methanol, LC grade.
(i) Erlenmeyer flask.—Sidearm, 125 mL. (g) LC mobile phase.—Dissolve 1.26 g oxalic acid dihydrate
(j) Volumetric flasks.—5, 10, 500, and 1000 mL. (reagent grade) in distilled H2O in 1 L volumetric flask, dilute to
(k) Homogenizer.—With cutting blades to disintegrate and volume with H2O, and mix. (1) For 5 µm LC column.—Combine
homogenize animal tissue. Recommended volume capacity of 600 mL oxalic acid solution with 300 mL acetonitrile and 100 mL
probe, 2–500 mL. methanol. (2) For 10 µm LC column.—Combine 700 mL oxalic acid
(l) Me chan i cal shaker.—Flat bed, 2-speed, os cil lat ing solution with 200 mL acetonitrile and 100 mL methanol.
horizontally. Prepare daily. Filter and de-gas LC mobile phases.
(m) pH Meter.—Measuring ±0.05 unit. (Note: Per formance of in dividual LC col umns may vary,
(n) Pipet.—Pasteur, disposable, 2 mL. depending on manufacturer and batch. Some adjustment of LC
(o) Filtration cartridge.—To filter tissue supernates; 13 mm mobile phase compositions may be required to meet system
id, 0.45 µm porosity, with Luer-lock. suitability specifications.)

 2005 AOAC INTERNATIONAL

Table 995.09. Interlaboratory study results for determination chlortetracycline, oxytetracycline, and tetracycline in edible animal
tissues by liquid chromatographic method

Tetracycline, Mean,
a b
Sample µg/g µg/g sr RSDr, % sR RSDR, % r R HorRat
Chlortetracycline
Porcine muscle 0.12 0.15 0.06 44.04 0.09 60.53 0.17 0.25 2.77
0.15 0.14 0.03 24.78 0.06 40.38 0.08 0.17 1.91
0.44 0.39 0.09 23.11 0.11 29.38 0.25 0.31 1.64
0.95 0.73 0.07 9.13 0.17 23.62 0.20 0.48 1.48
0.21 0.21 0.04 19.35 0.05 23.00 0.11 0.14 1.15
Porcine kidney 0.12 0.14 0.03 24.02 0.06 43.57 0.08 0.17 2.00
0.24 0.22 0.02 9.71 0.06 28.59 0.06 0.17 1.45
0.43 0.39 0.10 24.24 0.15 38.76 0.28 0.42 2.15
0.92 0.89 0.11 12.17 0.33 36.78 0.31 0.92 2.29
0.41 0.33 0.04 13.62 0.09 26.95 0.11 0.25 1.48
0.66 0.57 0.10 16.90 0.17 30.11 0.27 0.48 1.78
Oxytetracycline
Porcine muscle 0.07 0.09 0.01 16.66 0.03 32.96 0.03 0.08 1.39
0.09 0.11 0.01 8.87 0.03 24.98 0.03 0.08 1.10
0.46 0.43 0.12 27.07 0.12 27.07 0.32 0.32 1.52
0.87 0.87 0.08 9.80 0.15 16.84 0.22 0.42 1.04
0.20 0.21 0.02 9.94 0.04 16.68 0.06 0.11 0.82
0.25 0.24 0.04 16.18 0.05 20.74 0.11 0.14 1.06
Porcine kidney 0.16 0.16 0.06 35.55 0.08 49.65 0.17 0.22 2.37
0.32 0.26 0.06 21.32 0.07 26.26 0.17 0.20 1.39
0.53 0.46 0.06 12.02 0.09 20.52 0.17 0.25 1.17
1.15 0.94 0.15 16.29 0.17 18.53 0.42 0.48 1.19
1.86 1.27 0.09 7.06 0.24 18.88 0.25 0.67 1.30
2.24 1.65 0.15 9.21 0.32 19.70 0.42 0.90 1.40
Bovine muscle 0.08 0.12 0.02 16.01 0.03 27.41 0.06 0.08 1.18
0.11 0.12 0.01 7.76 0.03 28.42 0.03 0.08 1.28
0.50 0.48 0.06 12.72 0.07 13.70 0.17 0.20 0.78
1.04 0.92 0.11 11.59 0.13 14.56 0.31 0.36 0.92
0.23 0.24 0.05 19.30 0.06 26.83 0.14 0.17 1.35
0.36 0.42 0.05 13.05 0.06 14.58 0.14 0.17 0.79
Bovine kidney 0.12 0.16 0.06 33.53 0.10 59.20 0.17 0.28 2.71
0.25 0.26 0.05 19.20 0.07 28.79 0.14 0.20 1.47
0.48 0.51 0.09 17.60 0.11 21.36 0.25 0.31 1.70
0.95 0.90 0.10 11.32 0.12 13.36 0.28 0.34 0.83
0.45 0.51 0.09 16.76 0.09 17.02 0.25 0.25 0.95
0.54 0.53 0.09 17.24 0.09 17.62 0.25 0.25 1.01
Tetracycline
Bovine muscle 0.14 0.13 0.02 16.50 0.03 26.94 0.06 0.08 1.26
0.21 0.21 0.02 10.19 0.03 16.58 0.06 0.08 0.83
0.28 0.26 0.06 23.81 0.06 23.81 0.18 0.18 1.24
0.46 0.39 0.04 10.31 0.04 11.14 0.11 0.11 0.62
0.24 0.24 0.04 16.26 0.04 18.83 0.11 0.11 0.96
Bovine kidney 0.11 0.15 0.08 49.11 0.09 56.91 0.22 0.25 2.57
0.21 0.22 0.03 15.39 0.05 23.00 0.08 0.14 1.15
0.46 0.42 0.07 17.02 0.08 18.32 0.20 0.22 1.03
0.92 0.85 0.17 20.47 0.18 21.59 0.48 0.50 1.34
0.48 0.55 0.07 12.03 0.07 13.64 0.20 0.20 0.77
a
r = 2.8 × sr
b
R = 2.8 × sR.

 2005 AOAC INTERNATIONAL

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

D. System Suitability
Weigh 5.00 ± 0.05 g tissue into 50 mL polypropylene centrifuge
SPE cartridges should provide ≥80% recovery for fortified tissues tube. Fortify 5.00 g known blank tissue with each tetracycline at
containing 15 ng each tet ra cy cline (chlortetracycline, 0.5 µg/g by adding 100 µL TC combined stock standard solution
oxytetracycline, and tetracycline), using elution conditions as 25 µg/mL, C(d)(3).
specified. Some batches of cartridges tested that do not meet this Add 20 mL McIlvaine buffer–EDTA solution to each tube portion
requirement may still be acceptable for use if recoveries are and blend 30 s with homogenizer. Rinse probe twice with 2 mL
consistent (RSD ≤ 10%). McIlvaine buffer–EDTA solution into centrifuge tube.
Correction of recovery should be made for each run, based on Include blank tis sue in each an a lyt i cal run to check for
fortified analyte included in run, when the method is in routine use. interferences (e.g., co-eluting substances).
Inclusion of ≥2 fortified test solutions in each analytical run for
Cap tubes and shake 10 min on flat-bed shaker at high speed.
recovery correction is recommended if the method is not used on a
Remove tubes from shaker and centrifuge 10 min at 2500 × g. Pour
routine basis. % Recovery = (measured concn/fortified concn)
supernate into second 50 mL centrifuge tube. Do not transfer any tissue.
×100.
Add 20 mL McIlvaine buffer–EDTA solution to the first tube, cap,
Adjust LC system pa ram e ters so in jec tion of 60 µL TC and resuspend tissue plug using Vortex mixer. Shake 10 min as
chromatographic standard solution 0.25 µg/mL produces 3 distinct above, centrifuge 10 min at 2500 × g, and then add supernate to the
peaks within 9 min of injection (15 min for LC column B(2) used for supernate in second tube.
con fir ma tion of quan ti ta tive re sults). Res o lu tion be tween
Re suspend tis sue plug in first tube in 10 mL McIlvaine
oxytetracycline and tetracycline peaks should be ≥1.5 (baseline
buffer–EDTA solution and repeat all steps, until supernates from all
resolution) and at least the same for chlortetracycline with reference
3 extractions are collected in the second tube. Centrifuge combined
to either oxytetracycline or tetracycline. (Resolution = RS = (t2 –
supernates 20 min at 2500 × g.
t1)/0.5 (w1 – w2) where t1 and t2 are the retention times of the two
peaks and w1 and w2 are the baseline widths of the two peaks.) Place single GF/B filter paper in Büchner funnel and moisten with
Retention times for replicate injections of TC chromatographic McIlvaine buffer–EDTA solution. Fil ter combined supernate
standard solution should match within 0.05 min. through funnel into 125 mL sidearm flask, applying gentle vacuum
to sidearm.
Detection limits (3 × baseline noise) are 1.5 ng for oxytetracycline
and tetracycline, and 3 ng for chlortetracycline, with linear range of [Notes: (1) Tissues which have been previously homogenized and
3–30 ng for analytes injected onto LC column. See Figure 995.09 for stored frozen prior to thawing for analysis may release materials
tetracyclines chromatograms. which may plug SPE cartridges. This is not normally observed with
tissues which are homogenized immediately prior to analysis. If
E. Preparation of Test Solution such plugging of the cartridge occurs, split extract into 2 equal
(Note: Tissue should be kept frozen until analyzed. The entire volumes and filter separately, changing GF/filters, B(h), between
extraction–clean-up procedure should be completed in one day.) volumes. Combine filtered extracts before loading onto SPE

 2005 AOAC INTERNATIONAL

cartridge. (2) Too strong vacuum results in severe foaming. It is Follow with injection of test solutions. Tests which are off-scale at
essential that vacuum is established before test solution is poured attenuation setting used for 0.1–1.0 µg/g tissue-equivalent standards
onto the filter paper. Poor filtration results in plugging of SPE should be re-injected at attenuation used for 2 µg/g tissue-equivalent
cartridges.] standard.
Rinse centrifuge tube twice with 2 mL McIlvaine buffer–EDTA
After analysis, flush LC system ≥30 min, including LC column,
solution and filter into sidearm flask.
with water–acetonitrile–methanol (7 + 2 + 1, v/v/v), to remove
Prepare set of SPE cartridges on SPE apparatus, connecting
buffer. If an autosampler is used, rinse the loop/injector several
75 mL reservoir to each cartridge. Condition each cartridge with
times during the LC system flush.
20 mL methanol followed by 20 mL H2O and discard eluate.
Add test extract to 75 mL reservoir, rinse sidearm flask twice G. Confirmation of Quantitative Results
with 2 mL McIlvaine buffer–EDTA solution, and add washings to
reservoir. Test solutions containing <0.5 µg/g any tetracycline should be
[Note: Do not dry SPE cartridge between initial methanol re-injected onto LC confirmation column, B(b)(2), and compared
conditioning wash and completion of addition of test solution and with 0.25 µg/mL TC chromatographic standard solution to ensure
wash. Monitor elutions closely to ensure that cartridges do not dry. If that retention times match those of standard and that analytical
several columns run low simultaneously, interrupt vacuum to reduce responses are the same as those observed upon initial analysis. Some
solvent flow when reservoirs are refilled. Flow rate through SPE tissues may contain co-eluting substances which interfere in
column is not critical, but should not exceed a steady drip. ] identification and quantitation of one or more tetracyclines at levels
Rinse sidearm flask with 20 mL H2O and add washing to reservoir <0.5 µg/g.
when test extract is loaded. Let cartridge dry when H2O rinse is If comparison of test solution with TC chromatographic standard,
complete and continue to draw air through cartridge ≥2 min. as described, demonstrates presence of interferences in the original
Drain and clean SPE extraction system and place 10 mL analysis, re-analyze test portions and TC chromatographic standard
volumetric flasks in position as receiving flasks. Elute tetracyclines solutions with appropriate standard curve, using LC confirmation
from cartridges with 6.0 mL methanolic oxalic acid, C(c). Carefully column, B(b)(2).
monitor final elution; methanolic oxalic acid solution passes
through cartridges faster than aqueous extracts. Increase vacuum to H. Calculations
maximum for 10 s at end of elution step to remove residual solvent Measure peak heights for TC chromatographic standard solutions
from cartridge. Dilute eluate to 10 mL with H2O. and test solutions, adjusting for attenuation changes as required.
Using filtration cartridge, B(o), filter test solutions and standards Pre pare stan dard curve of tet ra cy cline tis sue-equivalent
into LC autosampler vials and load into autosampler. Filtered test concentration vs peak height using data from TC chromatographic
solutions may be injected manually, if autosampler is not available. standard solutions.
F. LC Determination Determine the best fit of data using linear regression as follows:
Condition LC column, B(b)(1), with mobile phase 30 min before
any injection of standards or test solutions. y = mx + b
Inject 60 µL each TC chromatographic standard solution. Injected
standards contain amounts of tetracyclines that are expected to be
where y = peak height; x = tetracycline concentration, µg/mL; m =
extracted from tissue containing 0.1–2.0 µg/g tetracyclines, slope of curve; and b = intercept of y.
assuming 100% extraction efficiency.
From measured peak heights of analyte, calculate tetracycline
Measure peak heights and prepare standard curve for each
concentrations using regression slope and intercept values. Correct
tetracycline by plotting concentration vs. peak height.
results for analytical recovery as determined for fortified recovery
[Note: Recorder or detector attenuation may have to be adjusted
test solution included in analysis.
to bring 2.0 µg/g tissue-equivalent standard (1.0 µg/mL TC
chromatographic standard solution) on scale.] Reference: J. AOAC Int. 79, 405(1996).

 2005 AOAC INTERNATIONAL