230  Kanda et al.: Journal of AOAC International Vol. 98, No.
1, 2015
VETERINARY DRUG RESIDUES
Multi-residue Determination of Polar Veterinary Drugs in
Livestock and Fishery Products by Liquid Chromatography/
Tandem Mass Spectrometry
Maki Kanda, Takayuki Nakajima, Hiroshi Hayashi, Tsuneo Hashimoto, Setsuko Kanai, Chieko Nagano,
Yoko Matsushima, Yukinari Tateishi, Soichi Yoshikawa, Yumi Tsuruoka, Takeo Sasamoto,
and Ichiro Takano
Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan
Residues of 37 polar veterinary drugs belonging to
six families (quinolones, tetracyclines, macrolides,
lincosamides, sulfonamides, and others) in livestock
and fishery products were determined using a
validated LC-MS/MS method. There were two key
points in sample preparation. First, extraction was
performed with two solutions of different polarity.
Highly polar compounds were initially extracted
with Na2EDTA-McIlvaine’s buffer (pH 7.0). Medium
polar compounds were then extracted from the same
samples with acetonitrile containing 0.1% formic
acid. Secondly, cleanup was performed using a
single SPE polymer cartridge. The first extracted
solution was applied to the cartridge. Highly polar
compounds were retained on the cartridge. Then, the
second extracted solution was applied to the same
cartridge. Both highly and medium polar compounds
were eluted from the cartridge. This method satisfied
the guideline criteria for 37 out of 37 drugs in swine
muscle, chicken muscle, bovine muscle, prawn,
salmon trout, red sea bream, milk, and honey; 35 out
of 37 in egg; and 34 out of 37 in flounder. The LOQ
ranged from 0.1 to 5 µg/kg. Residues were detected
in 24 out of 110 samples and analyzed using the
validated method.
V
eterinary drugs are widely used on farms to treat
and prevent diseases. However, over-dosing and
noncompliance with the withdrawal period may
cause drug residues to remain in animal tissues (1, 2).
Drug-contaminated livestock and fishery products may have
a potential risk for the consumer’s health because they can
provoke drug-resistant pathogenic strains of bacteria, allergic
reactions, and toxicity (3, 4). Therefore, it is necessary
to monitor livestock and fishery products for the residual
veterinary drugs using accurate analysis. We have used two
major analytical strategies to measure residual substances,
namely, microbiological screening (5–7) and screening using
LC-MS/MS (8, 9). However, the sensitivity of microbiological
screening was insufficient to detect residual levels of multi-class
veterinary drugs. Moreover, when positive results were found
Received August 13, 2013. Accepted by JB May 26, 2014.
Corresponding author’s e-mail: Maki_1_Kanda@member.metro.
tokyo.jp
DOI: 10.5740/jaoacint.13-272
with microbiological methods, specific chromatographic
analyses were needed to identify the antibiotics. The identifying
process was so complicated that it was difficult to identify each
residual drug.
The accuracy of analysis for the residual drugs has been
required worldwide in recent years. In Japan, the analytic
methodologies used by inspection institutes had to be validated
until December 13, 2013 according to the notice issued by the
Japanese Ministry of Health, Labour, and Welfare (10, 11).
On the other hand, the simultaneous analysis methodologies
for multi-class veterinary drug residues using LC-MS/MS
have already been reported (8, 9, 12–29). However, the
trueness and precision of reported analysis using LC-MS/MS
(8, 9, 12–23, 25, 27) for fluoroquinolones (FQs), tetracyclines
(TCs), penicillins (PCs), 5-hydroxythiabendazole, and clopidol
did not achieve acceptable values according to the “Guidelines
for the Validation of Analytical Methods for Residual
Agricultural Chemicals in Food”. Furthermore, the sensitivity
of some analysis was insufficient to detect residual levels of
multi-class veterinary drugs (12, 17–19, 22, 23, 25, 27). On
the Japanese positive list system, veterinary drugs of which no
established maximum residues limits (MRLs) were given the
default regulatory limit (uniform limit of level) at 10 µg/kg.
Therefore, the analysis of multi-class drugs needs the LOQ for
each drug to be less than 10 µg/kg.
Residues of TCs and FQs have been reported frequently in
analyses performed by national institutions in Japan or in the
European Union (EU; 30, 31). TC residues were found in swine
muscle, fish, and honey. The residues of enrofloxacin were found
in shrimp from Asia. Therefore, we need analytical methods to
accurately measure the residue concentrations of these drugs.
The aim of this study was to determine residues of 37 polar
veterinary drugs belonging to six families [quinolones (QLs),
TCs, macrolides (MLs), lincosamides, sulfonamides (SDs), and
others] in livestock and fishery products using a validated LC-
MS/MS method.
By addressing the following five points, we improved
pretreatment procedures and LC-MS/MS conditions:
(1)  Simple and rapid analysis is desirable to speed up large
amounts of sample inspections.
(2)  Polar veterinary drugs must be simultaneously extracted
from livestock and fishery products. We attempted to use aqueous
solvent on the first extraction and then organic solvent on the
second extraction. Different pretreatment procedures, such as
quick, easy, cheap, effective, rugged, and safe (QuEChERS)
methods (8, 9 12–18, 26) or pressurized liquid extraction (PLE)
were used recently. By using acetonitrile in the QuEChERS
 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015  231
method, extraction of TCs, MLs, and FQs was insufficient
(8, 9, 12, 14, 15, 18, 26). By using other extraction solutions
i.e., acidified acetonitrile (13–16, 26), methanol  (12,  18), or
methanol-acetonitrile (10, 17), extraction of these drugs was
insufficient as well. As shown in Table 1, log P of these drugs
was negative, which means that these drugs were soluble in
the aqueous phase. Actually, a mixture of water and organic
solvent was used (19, 20, 21, 23, 32). Using water at PLE was
significantly more effective for the extraction of QLs, PC V, and
SDs (25–29).
(3) During the measurement by LC-MS/MS, the matrix
interferes with the ionization of the target compounds, which
precludes the quantification. The matrix interference from
livestock and fishery products is removed by a cleanup using
the SPE polymer cartridge.
(4)  To increase the sensitivity, LC conditions (mobile phase,
column, and injection volume) and MS/MS parameters were
modified.
(5) The analytical method developed in this study was
validated in 10 livestock and fishery products: swine muscle,
chicken muscle, bovine muscle, prawn, salmon trout, red sea
bream, flounder, milk, egg, and honey in accordance with the
Japanese guidelines.
Experimental
Samples
Livestock and fishery products (swine muscle, chicken
muscle, bovine muscle, prawn, salmon trout, red sea bream,
flounder, milk, egg, and honey) were purchased from local
supermarkets in Japan and were confirmed to be free of the
targeted analytes in this study. The tissues were minced with an
electric household food processor and stored at –20°C.
Apparatus
(a)  LC system.—LC-20A series (Shimadzu Corp., Kyoto,
Japan).
(b)  MS system.—API 5500 Qtrap mass spectrometer with
an electrospray ionization (ESI) interface and Analyst (Version
1.4.2) software (AB Sciex, Framingham, MA).
(c)  LC column.—Triart C18 column (150 × 2.0 mm, 5 µm
particle size) (YMC Co. Ltd, Kyoto, Japan).
(d)  Mixer.—Vortex-Genie 2 (Scientific Industries Inc.,
Bohemia, NY).
(e)  Ultrasonic machine.—B5510J-DTH (Branson, Danbury,
CT).
(f)  Centrifuge.—AX-320 (Tomy Seiko Co., Tokyo, Japan).
(g)  Microcentrifuge.—5415R (Eppendorf Co. Ltd, Hamburg,
Germany).
(h)  Polypropylene centrifuge tubes.—15 mL and 50 mL
(Corning Inc., Corning, NY).
(i)  Glass volumetric flasks.—50 and 100 mL (SIBATA
Scientific Technology Ltd, Saitama, Japan).
(j)  Polymethylpentene and opaque volumetric flasks.—10 mL
(VITLAB GmbH, Grossostheim, Germany).
(k)  SPE manifold system.—Vacuum manifold system (GL
Sciences Inc., Tokyo, Japan).
(l)  SPE polymer cartridges for the cleanup procedure.—
InertSepTM PLS-3 cartridge, 20 cc/200 mg (GL Sciences Inc.).
Before use, the PLS-3 cartridges were conditioned with 5 mL
acetonitrile, and then 5 mL Na2EDTA-McIlvaine’s buffer
solution (pH 7.0).
(m)  Microtubes.—1.5 mL (Eppendorf Co. Ltd).
(n)  Polypropylene and amber vial tubes.—300 µL (GL
Sciences Inc.).
Reagents
(a)  Water.—Obtained using a Milli-Q system (Millipore
Corp., Billerica, MA).
(b)  Solvent.—Acetonitrile (LC grade), hexane (for pesticide
residue and polychlorinated biphenyl analysis grade) and
methanol (LC grade; Wako Pure Chemical Industries Ltd,
Osaka, Japan).
(c)  Formic acid (99%).—LC-MS grade (Wako Pure
Chemical Industries Ltd).
(d)  Citric acid monohydrate, Na2EDTA, sodium chloride,
and anhydrous magnesium sulfate.—Analytical grade (Wako
Pure Chemical Industries Ltd).
(e)  Disodium hydrogen phosphate dihydrate.—Analytical
grade (Merck KGaA, Darmstadt, Germany).
(f)  Polar extraction solution 1; Na2EDTA-McIlvaine’s buffer
solution (pH 7.0).—Prepared by dissolving 30.92 g disodium
hydrogen phosphate dihydrate, 2.73 g citric acid monohydrate,
and 37.13 g Na2EDTA in water and diluting to 1 L.
(g)  Polar extraction solution 2; Acetonitrile containing 0.1%
formic acid.—Freshly prepared by mixing 0.1 mL of formic
acid with 100 mL of acetonitrile.
(h)  Standard (purity grade).—Marbofloxacin (98.0%),
norfloxacin (98.0%), ciprofloxacin (98.0%), difloxacin (98.0%),
flumequine (98.0%), oxytetracycline (99.0%), erythromycin
A (98.0%), sulfadiazine (99.0%), sulfathiazole (98.0%),
sulfamonomethoxine (99.0%), sulfamethoxazole (99.0%),
sulfadimethoxine (99.0%), 5-hydroxythiabendazole (98.0%),
clopidol (98.0%), and thiabendazole (99.0%) were purchased
from Wako Pure Chemical Industries Ltd Ofloxacin (97.7%),
orbifloxacin (99.6%), and lincomycin A (98.0%) were from
Hayashi Pure Medical Industry (Osaka, Japan). Danofloxacin
(100.0%), enrofloxacin (99.8%), oxolinic acid (98.8%),
nalidixic acid (99.8%), oleandomycin (96.5%), josamycin
(86.8%), sulfamerazine (99.5%), sulfadimidine (99.4%), and
sulfaquinoxaline (99.6%) were from Kanto Chemical Co.
(Tokyo, Japan). Sarafloxacin (97.3%), tetracycline (97.7%),
chlortetracycline (99.1%), doxycycline (98.2%), and tiamulin
(99.9%) were from Sigma-Aldrich (St. Louis, MO). Spiramycin
(96.0%), tilmicosin (98.5%), and tylosin (98.0%) were from Dr.
Ehrenstorfer GmbH (Augsburg, Germany). Pirlimycin (86.6%)
was from Pfizer Japan Inc. (Tokyo, Japan). Mirosamicin
(97.7%) was from Kyoritsu Pharmaceutical Co. (Tokyo, Japan).
(i)  Internal standard (IS).—Demeclocycline (92.3%) was
from Hayashi Pure Medical Industry.
Preparation of Standard Solutions and Calibration
Standards
(a)  Stock standard solutions of 33 individual compounds
except TCs (100 µg/mL).—Stock standard solutions were
prepared individually. The suitable quantity of standard taking
into account the substance purity was weighed in a 50 mL glass
volumetric flask. Clopidol was dissolved in 5 mL acetonitrile,
232  Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Table  1.  log P values of veterinary drugs and made up to 50 mL with methanol. Sulfadimidine and
oxolinic acid were dissolved in acetonitrile, and made up to
Analytes logP 50 mL with acetonitrile. The rest of compounds were dissolved
Quinolones in methanol, and made up to 50 mL with methanol. Stock
Marbofloxacin –0.5 standard solutions were kept in amber glass vials in the dark
at 4°C, under which conditions, they were stable for one year.
Norfloxacin –1.0
(b)  Mixed standard solutions except TCs (1 µg/mL).—An
Ofloxacin –0.4 aliquot (500 µL) of each stock standard solution shown in (a)
Enrofloxacin –0.2 was transferred and mixed together in a 50 mL glass volumetric
Ciprofloxacin –1.1 flask, and made up to 50 mL with methanol. This mixed standard
solution was kept in an amber glass vial in the dark at 4°C,
Danofloxacin –0.3
under which conditions this was stable for 3 months.
Orbifloxacin 0.9 (c)  Stock standard solutions of 4 TCs (1000  µg/mL).—
Sarafloxacin 0.3 Stock standard solutions of TCs (oxytetracycline, tetracycline,
Difloxacin 1.6 chlortetracycline and doxycycline) were prepared individually.
The suitable quantity of standard taking into account
Oxolinic acid 1.7
the substance purity was weighed in a 10 mL opaque
Nalidixic acid 1.4 polymethylpentene volumetric flask (light-shielding). TCs were
Flumequine 2.9 dissolved in methanol and made up to 10 mL with methanol.
Tetracyclines The stock standard solutions were kept in polypropylene vials
in the dark at –20°C, under which conditions they were stable
Oxytetracycline –1.6
for 1 month.
Tetracycline –2.0 (d)  Mixed oxytetracycline and tetracycline standard solution
Chlortetracycline –1.3 (1 µg/mL).—An aliquot (100 µL) of each stock standard solution
Doxycycline –0.7 of oxytetracycline and tetracycline shown in (c) was transferred
and mixed together in a 10 mL opaque polymethylpentene
Demeclocyclinea 0.7
volumetric flask, and made up to 10 mL with acetonitrile
Macrolides containing 0.1% formic acid (ACN/FA) immediately before
Spiramycin 2.1 use. This solution was diluted 10 times with ACN/FA.
Tilmicosin 3.6
(e)  Working standard solutions for 35 veterinary drugs
(except for chlortetracycline and doxycycline) (from 0.001
Mirosamicin 2.0
to 0.1 µg/mL).—Working standard solutions were prepared
Oleandomycin 2.6 immediately before use by serial dilution of each mixed standard
Erythromycin A 2.7 solution shown in (b) and (d) with ACN/FA.
Tylosin 1.0
(f)  Matrix-matched standard solutions for 35 veterinary
drugs (from 0.025 to 50 ng/mL).—Calibration curves for
Josamycin 2.9
35 veterinary drugs (except chlortetracycline and doxycycline)
Lincosamides were obtained from matrix-matched calibration samples. Blank
Lincomycin A 0.2 samples were prepared as described in the Sample Preparation
section. Matrix-matched standard solutions were prepared by
Pirlimycin 1.7
mixing an aliquot (500 µL) of blank solution and the appropriate
Sulfonamides
volume of working standard solutions shown in (e), and then
Sulfadiazine –0.1 made up to 1 mL with ACN/FA, e.g., a 0.025 ng/mL solution
Sulfathiazole 0.1 was made by mixing an aliquot (500 µL) of blank solution and
the working standard solution (0.001 µg/mL, 25 µL), and then
Sulfamerazine 0.1
made up to 1 mL.
Sulfadimidine 0.3 (g)  Mixed chlortetracycline and doxycycline standard
Sulfamonomethoxine 0.8 solution (1 µg/mL).—An aliquot (100 µL) of each stock
Sulfamethoxazole 0.9 standard solution of chlortetracycline and doxycycline shown
in (c) was transferred and mixed together in a 10 mL opaque
Sulfaquinoxaline 1.7
polymethylpentene volumetric flask, and made up to 10  mL
Sulfadimethoxine 1.6 with ACN/FA immediately before use. This solution was diluted
Others 10 times with ACN/FA.
Thiabendazole 2.5 (h)  Working standard solutions for chlortetracycline and
doxycycline (from 0.001 to 0.1 µg/mL).—Working standard
5-hydroxythiabendazole 2.1
solutions were prepared immediately before use by serial
Clopidol 2.6 dilution of the mixed standard solution shown in (g) with
Tiamulin 5.6 ACN/FA.
a
 The internal standard material for the quantification of chlortetracycline (i)  IS.—Demeclocycline was the IS for the quantification of
and doxycycline. chlortetracycline and doxycycline. Demeclocycline (10.9 mg)
was accurately weighed in a 10 mL opaque polymethylpentene
 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015  233
volumetric flask, dissolved in methanol, and made up to 10 mL
with methanol. The stock IS solution (1000 µg/mL) was kept
in polypropylene vials in the dark at –20°C, under which
conditions the solution was stable for 1 month. Working IS
solutions (from 0.01 to 1 µg/mL) were prepared immediately
before use by serial dilution of the stock IS solution with
ACN/FA.
(j)  IS calibration standard solutions for chlortetracycline
and doxycycline (from 0.025 to 50 ng/mL).—Calibration
curves for chlortetracycline and doxycycline were obtained
from IS calibration samples. IS calibration standard solutions
were prepared by mixing the working IS solution shown in (i)
(0.01 µg/mL, 100 µL) and the appropriate volume of working
solutions shown in (h), and made up to 1 mL with ACN/FA,
e.g., a 0.025 ng/mL solution was made by mixing the working
IS standard solution (0.01 µg/mL, 100 µL) together with the
working standard solution for chlortetracycline and doxycycline
(0.001 µg/mL, 25 µL), and then brought to 1 mL volume.
LC Separation Conditions
(a)  Mobile phase.—The 0.05% formic acid solution was
prepared by mixing 0.5 mL of formic acid with 1 L water. (A)
The 0.05% formic acid solution and (B) acetonitrile were mixed
using the pump in gradient mode as follows: 5% B (3  min);
5–90% B (12 min); 90% B (5 min); 90-5% B (0.1 min); and
5% B (5 min).
(b)  Flow rate.—0.3 mL/min.
(c)  Column temperature.—40°C.
(d)  Injection volume.—2 µL.
MS/MS Conditions
(a)  Ionization mode.—Positive-ion ESI.
(b)  Ion spray voltage.—5500 V.
(c)  Source temperature.—650°C.
(d)  Entrance potential.—10 V.
(e)  Curtain gas pressure.—20 psi (nitrogen).
(f)  Collision gas pressure.—7 psi (nitrogen).
(g)  Ion source gas pressure 1.—80 psi (nitrogen).
(h)  Ion source gas pressure 2.—40 psi (nitrogen).
(i)  Acquisition function.—Selected reaction monitoring
(SRM); the SRM program is shown in Table 2.
Sample Preparation
The schematic procedure of sample preparation is shown in
Figure  1. For the sample preparation, glass vessels were not
used, because silica in the glass could make an interference
signal during LC-MS/MS analysis of TCs.
Thoroughly minced sample (5.0 g) was poured in 50 mL
polypropylene centrifuge tubes (A). IS was spiked at a level of
10 µg/kg. Na2EDTA–McIlvaine’s buffer (pH 7.0, 20 mL) was
added. The tube (A) was vortexed for 1 min. A 5 mL amount of
hexane was added. The tube (A) was vortexed again for 1 min,
ultrasonicated for 10 min, and then centrifuged at 9 600 × g for
20 min at 4°C. The hexane layer was discarded by pipetting.
Hexane washing was used at all sample types to ease the
operations.
As shown in “First extraction” of Figure 1, the
Na2EDTA–McIlvaine’s buffer layer was transferred into new
50 mL polypropylene centrifuge tubes containing 1 mL of 25%
NaCl solution (B). The tube (B) was vortexed for 1 min., and
then centrifuged at 9600 × g for 10 min at 4°C. The supernatant
was loaded to the conditioned PLS-3 cartridge at approximately
1 mL/min. The target compounds were retained on the cartridge,
while the solution containing the matrix of food was passed
through the cartridge. The cartridge was washed with 5 mL of
water, and then vacuum-dried for 3 min at a pressure of 10 mm Hg.
In addition, the second extraction from the remaining substance
in tube (A) was performed as shown in the “Second extraction”
stage of Figure 1. The characteristics of the remaining matrixes
were varied and depended on the different type of samples, as
well as the pellets or the insoluble matrix floating on the top
of the hexane layer. The following procedure was used for all
sample types. Water (2 mL) was added to the tube (A) and then
(A) was vortexed. Subsequently, 10  mL ACN/FA was added.
The tube (A) was vortexed again for 1 min, and ultrasonicated
for 10 min. Magnesium sulfate was added for dehydration (3 g
each for bovine muscle, swine muscle, chicken muscle, prawn,
milk, and honey). A 4 g amount of magnesium sulfate was added
for salmon, red sea bream, and flounder; 5g was added for egg.
Then tube (A) was vigorously shaken for 1 min, and centrifuged
at 1800 × g for 10 at 4°C.
As shown as the black arrow in Figure 1, the organic phase
was used as the elution solution for the PLS-3 cartridge
previously loaded with Na2EDTA–McIlvaine’s buffer layer.
The eluate from the cartridge was collected into an opaque
polymethylpentene volumetric flask.
The eluate was made up to 10 mL with ACN/FA. An aliquot
(1 mL) was transferred to a microtube, diluted to 2-fold with
ACN/FA, and centrifuged at 16 000 × g for 5 min at 4°C. The
supernatant was transferred into an amber polypropylene vial
tube. The resultant solution was analyzed by LC-MS/MS twice
on the same day. Each quantitative value was taken as a mean
of two measurements.
Single-Laboratory Validation Tests with Spiked Samples
The method was validated according to the guidelines of the
Japanese Ministry of Health, Labour, and Welfare. Selectivity
was confirmed by analyzing blank samples. Trueness,
repeatability (RSDr), and within-run reproducibility (RSDWR)
were determined by means of the recoveries using samples
spiked with 37 veterinary drugs and demeclocycline at levels
of 10 or 100 µg/kg, performed with two samples per day over
five different days. LOQs and LODs were estimated from the
repeatability data of the blank samples spiked with 0.1, 0.25,
0.5, 1, 2.5, and 5 µg/kg for each of the 37 veterinary drugs
examined. LOQs were calculated as 10 times the SD, and LODs
were calculated as 3 times the SD using the Analyst software
(AB Sciex).
Results and Discussion
LC-MS/MS Parameters
The MS scans of the 37 veterinary drugs revealed that the
most abundant molecular ion was the protonated molecule
[M+H]+. As each [M+H]+ is a precursor ion, a further
MS/MS scan was performed after the collision energy was
increased. Two fragment ions (corresponding to quantitative
234  Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Table  2.  SRM parameters

Retention time, Declustering Collision Collision cell exit
Analytes Transition, m/z min potential, V energy, eV potential, V Ion ratio, %c

Quinolones
a
Marbofloxacin 363.0→320.1 5.9 81 21 20 52.2
363.0→121.9 79 10

Norfloxacin 320.0→276.2a 6.0 96 23 12 34.7

320.0→230.9 53 12
a
Ofloxacin 362.0→318.0 6.0 46 29 16 84.2

362.0→261.0 41 16
a
Ciprofloxacin 332.0→288.1 6.1 81 25 24 110

332.0→231.1 57 18
a
Danofloxacin 358.0→82.0 6.2 81 70 23 57.9

358.0→255.0 65 23
a
Enrofloxacin 360.2→316.0 6.3 116 29 10 66.5
360.2→245.0 36 8
a
Orbifloxacin 396.0→295.1 6.4 116 35 18 41.4

396.0→351.9 27 18
a
Sarafloxacin 385.9→341.9 6.6 116 27 22 86.7

385.9→298.8 39 22
a
Difloxacin 400.0→356.0 6.6 116 29 20 85.0

400.0→299.1 45 16
a
Oxolinic acid 261.9→243.9 7.7 61 27 18 16.9

261.9→215.9 41 18
a
Nalidixic acid 232.9→215.0 8.5 56 21 16 96.6

232.9→186.9 35 14
a
Flumequine 262.0→201.9 8.6 66 41 14 42.4

262.0→126.1 67 20
Tetracyclines

Oxytetracycline 461.1→426.1a 6.1 50 27 26 59.5

461.1→443.1 19 24
a
Tetracycline 445.1→410.1 6.3 36 29 24 15.8

445.1→225.9 77 16
a
Chlortetracycline 479.1→443.9 6.9 91 29 28 74.4

479.1→462.0 23 26
a
Doxycycline 445.1→428.1 7.0 50 25 24 9.6

445.1→154.0 37 12
b
Demeclocycline 465.1→429.9a 6.6 76 31 22 56.7

465.1→288.9 45 18
Macrolides

Spiramycin 843.4→174.0a 6.5 6 45 22 28.6

843.4→142.2 45 10
a
Tilmicosin 869.4→174.0 7.0 1 57 40 20.0

869.4→696.4 57 16
a
Mirosamicin 728.3→158.0 7.3 6 37 12 16.2

728.3→116.0 61 12
a
Oleandomycin 688.4→158.2 7.4 116 35 10 52.2

688.4→544.3 23 36
 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015  235

Table 2.  (continued)
Retention time, Declustering Collision Collision cell exit
Analytes Transition, m/z min potential, V energy, eV potential, V Ion ratio, %c

Erythromycin A 734.3→158.1a 7.6 86 39 10 80.3

734.3→576.3 27 34

Tylosin 916.4→174.0a 7.7 1 51 14 10.3

916.4→101.0 85 12
a
Josamycin 828.3→109.2 8.6 11 67 14 68.8

828.3→174.0 43 16
Lincomycins

Lincomycin A 407.1→126.2a 5.6 121 37 12 12.0

407.1→359.1 27 22
a
Pirlimycin 411.0→112.1 6.6 101 31 8 18.9

411.0→363.2 25 18
Sulfonamides

Sulfadiazine 251.0→155.9a 5.8 81 21 14 73.7

251.0→107.9 33 18
a
Sulfathiazole 255.9→155.9 6.0 56 21 14 52.3

255.9→107.9 33 16
a
Sulfamerazine 264.9→155.9 6.4 101 25 14 88.2

264.9→107.9 37 18
a
Sulfadimidine 278.9→155.9 6.8 56 23 12 58.3

278.9→107.9 25 16
a
Sulfamonomethoxine 281.0→107.9 7.0 191 35 10 42.9

281.0→155.9 17 12
a
Sulfamethoxazole 253.9→155.9 7.4 76 23 12 69.0

253.9→107.9 33 16
a
Sulfaquinoxaline 300.9→155.9 7.9 66 23 14 52.4

300.9→107.9 39 10
a
Sulfadimethoxine 310.9→155.9 7.9 81 29 14 39.3

310.9→107.9 39 10
Others

5-hydroxythiabendazole 217.8→190.9a 5.5 51 37 14 38.5

217.8→146.8 47 22
a
Clopidol 191.9→100.9 5.7 171 37 16 25.6

192.9→155.9 33 14
a
Thiabendazole 201.9→174.9 6.0 161 37 14 70.8

201.9→131.0 45 12
a
Tiamulin 494.3→192.2 8.1 86 29 14 63.3

494.3→119.0 61 12
a
 Ion used for quantification.
b
 The internal standard material for the quantification of chlortetracycline and doxycycline.
c
 The relative ion abundance ratio of the selected product ions for the standard solution, 10 ng/mL of each compound.
236 Kanda et al.: Journal of aoaC InternatIonal Vol. 98, no. 1, 2015

Highly polar veterinary drugs

(A) (A) (A)
medium polar veterinary drugs

Vortex (1 min) Vortex (1 min) Hexane Discard

by pipetting
Ultrasonicate (10 min)
Centrifuge
(9600 xg,20 min, 4oC)
Sample (5g) +Hexane (5 mL)
+I.S.
Na2EDTA–McIlvain’s
buffer (pH 7, 20 mL)

Load to
PLS-3 (200 mg, 20 mL)
PLS
First extraction

(B) (B)
se
ha
sp
ou Vortex (1 min) Wash
ue
Aq Centrifuge
with water (5 mL)
(9600 xg,10 min, 4oC) Vaccum-dry (3 min)
Discard
+25% NaCl sol. (1 mL) the passed through
solution
Collect
Second extraction the eluate
solution

Re
m
ain (A)
in (A) (A) (A)
g
m Supernatant
at
ri x
Vortex (1 min) Vortex (1 min) Violently shake (1 min)
Ultrasonicate (10 min) Centrifuge Using the second
(1800 xg,10 min, 4oC) extracted solution
as the elution solution
for PLS-3
+Water (2 mL) +Acetonitrile containing + MgSO4
0.1% formic acid (10 mL) (3, 4 or 5 g)

Analyse
Aliquot Supernatant
by LC/MS/MS

Make up to 10 mL Transfer to Dilute by 2-fold Pour into

a microtube an amber
Centrifuge
polypropylene
(16000 xg, 5 min , 4oC)
vial tube

Figure 1. Schematic representation of the sample preparation procedure for the analysis of 37 veterinary drugs in livestock and fishery
products.

and confirmative ions) were monitored for each of the 37 metal ion impurities remaining in the C18 column, which made
veterinary drugs (Table 2). Several MS parameters including their peaks broad. The novel organic hybrid silica base column
ion-spray voltage, source temperature, declustering potential, (YMC-Triart) has been reported to reduce metal ion impurities
entrance potential, and four gas pressures were systematically and achieve good chromatographic retention and separation of
varied according to the manual of flow injection analysis, and metal chelating and hydrophilic compounds. Using the column,
we selected the conditions that yielded the best sensitivity, as the peak shapes of TCs and QLs were better, and the sensitivities
listed in the Experimental section. In particular, we noted the were improved by a factor of 3. Thiabendazole, sulfathiazole,
curtain gas, ion source gas 1 and 2 conditions that measured sulfamerazine, and clopidol diluted with an organic solvent
macrolides with high sensitivity. Because MS scans of some of were poorly retained on the column because the organic solvent
penicillins showed that the most abundant molecular ion was may act as a part of the mobile phase. We minimized the drug
–
the deprotonated molecule [M-H] , it was excluded from the injection volume to 2 µL, which resulted in the peak widths at
analytes in this study. half height ranging from 0.1 to 0.44 min and the tailing factors
ranging from 0.85 to 1.21.
LC Conditions
Extraction and Cleanup Procedure
LC conditions to determine multi-class veterinary drugs
in livestock and fishery products were previously reported The extraction and cleanup procedure was developed using
by our laboratory (9), in which a gradient mixture of 0.1% 11 veterinary drugs, norfloxacin, ciprofloxacin, chlortetracycline,
formic acid in 10 mM ammonium acetate and acetonitrile as doxycycline, 5-hydroxythiabendazole, clopidol, erythromycin
the mobile phase and a C18 column were used. However, the A, spiramycin, lincomycin A, oxolinic acid, and sulfadimidine.
sensitivities of TCs and QLs were low under these conditions. Among these drugs, norfloxacin, ciprofloxacin, chlortetracycline,
Because the ionization mode of these drugs was positive-ion doxycycline, 5-hydroxythiabendazole, and clopidol did
ESI, the ammonium ion which lowered the sensitivity of not achieve acceptable values following the guidelines of
+
[M+H] was excluded from the mobile phase. The peak shapes Japanese Ministry of Health, Labour, and Welfare (10, 11) by
of FQs and thiabendazole were split. The peak shapes of TCs, using our reported QuEChERS methods (8, 9), because these
sulfathiazole, sulfamerazine, clopidol, and oxolinic acid were compounds were soluble in the aqueous phase. Erythromycin
poor. The tailing factors of these drugs were 0.3–0.6. TCs and A and spiramycin represent the macrolides class. Lincomycin
QLs which are strong metal chelating compounds interact with A represents the lincosamides. Oxolinic acid and sulfadimidine
 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015  237

Acetonitrile containing 0.1% formic acid Na2EDTA-MacIlvain Buffer

120%
(a)
100%

80%

60%

40%

20%

Extracted Ratio (%) a

0%
First extraction Second extraction
using Na2EDTA-MacIlvain Buffer using Acetonitrile containing 0.1% formic acid
120%
(b)
100%
80%
60%
40%
20%
0%
n

id

e
in
ol
in

in

sin
ne
e

in
ci

in
l

ac
yc
c

yc
zo
xa

cli

id
xa

cl

lo
pi

om
m

ic
a

cy

m
ty
flo

cy
clo
o

nd

ir a

in
rf l

di
xy

nc
tra
ro

ol
be

lf a
no

sp
do

Li
cip

ox
r te
a

su
hi

lo
yt

ch
ox
dr
hy
5-

Figure  2.  Effect on the extracted ratios of 11 veterinary drugs from swine muscle, twice extraction by the same solvent (a), by the different
solvents (b). Mean of 5 replications.

had higher accuracy than other drugs on LC-MS/MS. These follows. Eleven drugs spiked into a swine muscle were extracted
compounds served as indicators, showing that the LC-MS/MS with Na2EDTA-McIlvaine’s buffer. The extraction solution was
measurements are stable. After spiking 50 µL of a 1 µg/mL loaded onto the PLS-3 cartridge, and was eluted with ACN/FA.
standard solution of these drugs into a minced swine muscle, the This eluate was analyzed by LC-MS/MS. The recovery rates (a)
following studies were performed. At this time, the drugs were were calculated. Na2EDTA-McIlvaine’s buffer spiked with 11
quantified by using matrix-matched calibration standard curves. drugs was loaded onto the PLS-3 cartridge. The recovery rates
Veterinary drugs were extracted from the sample using from the PLS-3 cartridge (b) were calculated. The extraction
an ultrasonic machine (33–35). This procedure allowed the rates by Na2EDTA-McIlvaine’s buffer were corrected (a) using
simultaneous handling of many samples and lowered the risk of (b). The pH of the buffer was set as 7.0 because the retention
contamination. The sufficient extraction ability was confirmed of drugs was better than at pH 4. Na2EDTA was added to the
using the incurred swine muscle containing chlortetracycline. buffer because the extraction of TCs and QLs were better with
As extraction solvents, we compared ACN/FA used on our buffer containing Na2EDTA which had the ability to chelate
modified QuEChERS method (9) and Na2EDTA-McIlvaine’s divalent cations (8, 13, 16, 19, 22). As shown in Figure 2a, the
buffer used on our antibiotic extraction (5–7). The extracted rates extraction rate of each drug, i.e., norfloxacin, ciprofloxacin,
of 11 drugs by Na2EDTA-McIlvaine’s buffer were calculated as chlortetracycline, doxycycline, and lincomycin A was better with

Non-combination of the eluted solution Combination of the eluted solution

120%
(a)
100%
Recovery Ratio (%) a

80%

60%

40%

20%

0%

15
14 (b)
13
12
Rate of matrix effect a

11
10
9
8
7
6
5
4
3
2
1
0
n

id

e
in
ol
in

in

sin
ne
e

in
ci

in
l

ac
yc
c

yc
zo
xa

cli

id
xa

cl

lo
pi

om
m

ic
a

cy

m
ty
flo

cy
clo
o

nd

ir a

in
rf l

di
xy

nc
tra
ro

ol
be

lf a
no

sp
do

Li
cip

ox
r te
a

su
hi

lo
yt

ch
ox
dr
hy
5-

Figure  3.  Effect of the two conditions eluting from the SPE polymer cartridge on the recovery rate of 11 veterinary drugs (a), the rate of
matrix effect (b). Mean of 5 replications.
238  Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

(a) Swine muscle spiked with 10 µg/kg 37 drugs (b) Blank swine muscle (a) Swine muscle spiked with 10 µg/kg 37 drugs (b) Blank swine muscle
5x104 1x103 5x105 1x104
Marbofloxacin Oxolinic acid

0 0
0 0
5x104 1x103
Norfloxacin 5x105 1x104
Nalidixic acid

0 0
15x104 1x103 0 0
Ofloxacin 5x104 1x103
Flumequine

0 0
5x104 1x103 0
0
Ciprofloxacin
5x104 1x103
Oxytetracycline

0 0

Intensity
1x104
1x103
Danofloxacin
Intensity

0 0
5x104 1x103
Tetracycline

0
0
10x104 1x103
Enrofloxacin
0 0
1x104 1x103
Chlortetracycline

0 0
10x104 1x103
Orbifloxacin
0 0
5x104 1x103
Doxycycline
0 0
5x104 1x103
Sarafloxacin
0 0
1x104 1x103
Demeclocycline
0 0
10x104 1x103
Difloxacin

0 0
5 6 7 8 9 5 6 7 8 9

0 0
Retention time (min)
5 6 7 8 9 5 6 7 8 9

Retention time (min)

(a) Swine muscle spiked with 10 µg/kg 37 drugs (b) Blank swine muscle (a) Swine muscle spiked with 10 µg/kg 37 drugs (b) Blank swine muscle
4x103 1x103 2x105 2x103
Spiramycin Sulfadiazine

0 0
0 0
4x103 1x103
Tilmicosin 2x105 1x103
Sulfathiazole

0 0
5x104 1x103 0 0
Mirosamaycin
1x105 1x103
Sulfamerazine

0 0
3x104 1x103
Oleandomycin 0 0

1x105 1x103
Sulfadimidine

0 0

3x104 1x103
Erythromycin A
Intensity
Intensity

0 0
2x104 1x103
Sulfamonomethoxine

0 0
1x104 1x103
Tylosin
0 0

1x105 1x103
Sulfamethoxazole
0 0

5x104 1x103
Josamycin

0 0

1x105 1x103
0 0 Sulfaquinoxaline
5x105 1x104
Lincomycin A

0 0

0 3x105 1x103
0
Sulfadimethoxine
1x105 1x103
Pirlimycin

0 0
0 0 5 6 7 8 9 5 6 7 8 9
5 6 7 8 9 5 6 7 8 9
Retention time (min)
Retention time (min)

Figure  4.  Chromatograms obtained in the MRM mode (quantification transition) for swine muscle spiked with 10 mg/kg of 37 veterinary
drugs (a), and for corresponding blank swine muscle (b).
 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015  239
(a) Swine muscle spiked with 10 µg/kg 37 drugs (b) Blank swine muscle
(2)  The second extracted solution was re-used as the elution
5-hydroxythiabendazole
solution. The eluate was diluted by 2-fold with ACN/FA, and
analyzed by LC-MS/MS.
1x105 2x103
On (1) and (2) conditions, the recovery rates of 11 veterinary
Clopidol
drugs were the same (Figure 3a). However, the matrix effects
were dramatically different. The matrix effect was defined as
Intensity

3x105
0
1x103
the ratio of the slope of the matrix-matched calibration curve
Thiabendazole
and the standard solution calibration curve. On the condition
of (1), strong matrix enhancements were found for norfloxacin,
0 0 ciprofloxacin, chlortetracycline, doxycycline, spiramycin, and
3x105 1x103
Tiamulin
lincomycin A. In contrast, the matrix enhancements were not
observed under (2) conditions. Because the pork fatty acids and
0
5 6 7 8 9
0
5 6 7 8 9
phospholipids were reported to be retained by the SPE polymer
Retention time (min) cartridge (36, 37), the interfering matrix was considered to be
Figure 4.  (continued)  Chromatograms obtained in the MRM mode cleaned-up when the second extraction solution was passed
(quantification transition) for swine muscle spiked with 10 mg/kg through the SPE polymer cartridge (Figure 3b). Finally, we
of 37 veterinary drugs (a), and for corresponding blank swine
muscle (b). chose the extraction and cleanup procedure shown in Figure 1.

Na2EDTA-McIlvaine’s buffer than with ACN/FA. The second Instrument Performance

extraction using Na2EDTA-McIlvaine’s buffer did not improve
recovery rates. The first extraction using Na2EDTA-McIlvaine’s Figure 4a shows the SRM chromatograms obtained from
buffer and second extraction step using ACN/FA improved swine muscle spiked with 10 µg/kg of 37 veterinary drugs and
recovery rates to over 70%, except for chlortetracycline and demeclocycline. No matrix effect was observed on peak shape
doxycycline, which were unstable in solution. Therefore, in all samples. The retention time determined for the spiked
polar veterinary drugs were extracted with two different polar
samples was not significantly different from that determined for
solvents, Na2EDTA-McIlvaine’s buffer (pH 7.0) and ACN/FA.
the standards. The relative ion abundance ratios of the selected
Subsequently, we evaluated the two conditions to elute the
compounds from the SPE polymer cartridge which retained the product ions for each compound are shown in Table 2 together
compounds first-extracted by Na2EDTA-McIlvaine’s buffer. with those of the standard solutions. All of the relative ion
(1)  A new ACN/FA (10 mL) was used as the elution solution. abundance ratios of the spiked samples were within 20% of those
The resultant eluate and the second extracted solution were of the standard solutions, which satisfied the permitted tolerance
mixed and analyzed by LC-MS/MS.  required in the EU guidelines (38). These results indicated that

swine muscle chicken muscle bovine muscle prawn salmon traut red sea bream flounder milk egg honey

2.2

2.0

1.8

1.6

1.4
The slope ratio a

1.2

1.0

0.8

0.6

0.4

0.2
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Analytes

Figure  5.  Slope ratio between matrix-matched and solvent calibrations. The compliance interval covering the range between
0.8 and 1.2 for the tolerable matrix effect was plotted. Veterinary drug code: (1) marbofloxacin; (2) norfloxacin; (3) ofloxacin;
(4) ciprofloxacin; (5) danofloxacin; (6) enrofloxacin; (7) orbifloxacin; (8) sarafloxacin; (9) difloxacin; (10) oxolinic acid; (11) nalidixic
acid; (12) flumequine; (13) oxytetracycline; (14) tetracycline; (15) chlortetracycline; (16) doxycycline; (17) demeclocycline (the internal
standard material for the quantification of chlortetracycline and doxycycline); (18) spiramycin; (19) tilmicosin; (20) mirosamycin; (21)
oleandomycin; (22) erythromycin A (23) tylosin; (24) josamycin; (25) lincomycin A; (26) pirlimycin; (27) sulfadiazine; (28) sulfathiazole;
(29) sulfamerazine; (30) sulfadimidine; (31) sulfamonomethoxine; (32) sulfamethoxazole; (33) sulfaquinoxaline; (34) sulfadimethoxine;
(35) 5-hydroxythiabendazole; (36) clopidol; (37) thiabendazole; (38) tiamulin.
240  Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Table  3.  Validation results of veterinary drugs

Swine muscle Chicken muscle

Trueness, % (RSDra, %; RSDWRb, %) Trueness, % (RSDra, %; RSDWRb, %)

Analytes 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg

Quinolones

Marbofloxacin 82 (6; 6) 89 (4; 7) 0.5 50 80 (5; 5) 86 (6; 6) 0.5 10c

Norfloxacin 74 (6; 7) 77 (4; 7) 2 20 74 (6; 9) 76 (10;10) 2 20

c
Ofloxacin 82 (6; 6) 93 (3; 7) 0.2 10 82 (6; 7) 91 (5; 10) 0.2 50

Enrofloxacin 87 (7; 7) 92 (4; 6) 0.5 82 (6; 6) 87 (7;10) 0.2

d d
50 50
Ciprofloxacin 72 (7; 6) 81 (3; 6) 1 73 (7; 5) 76 (7; 6) 1

Danofloxacin 85 (9; 9) 84 (5; 7) 2 100 77 (10;13) 76 (4;11) 2 200

Orbifloxacin 89 (8; 9) 98 (7; 8) 0.5 20 87 (6; 5) 92 (8; 8) 0.5 10c

c
Sarafloxacin 80 (9; 9) 88 (4; 7) 0.5 10 81 (6; 8) 89 (3; 8) 0.5 10

Difloxacin 88 (5; 9) 96 (5; 5) 1 20 80 (9; 9) 92 (7; 6) 0.5 10c

Oxolinic acid 94 (5; 7) 101 (3; 4) 0.5 20 89 (4; 6) 100 (5; 5) 0.5 30
c
Nalidixic acid 93 (6; 9) 101 (4; 6) 0.5 10 89 (3; 3) 95 (3; 4) 0.5 10c

Flumequine 91 (5; 5) 97 (3; 5) 0.2 500 88 (5; 5) 96 (3; 4) 0.2 500

Tetracyclines

Oxytetracycline 79 (7;10) 77 (4; 8) 1 72 (3; 9) 75 (4; 6) 1

e e
Tetracycline 79 (11; 9) 80 (4; 8) 1 200 78 (4; 8) 72 (4; 5) 1 200
Chlortetracycline 92 (9;12) 93 (4; 7) 2 101 (10;13) 95 (6;12) 2

Doxycycline 83 (9; 9) 81 (2; 6) 0.5 50 96 (9; 9) 88 (5;10) 1 50

Demeclocyclinef 76 (12;10) 69 (3;11) 0.5 59 (9;14) 75 (5; 8) 1

Macrolides

Spiramycin 85 (8; 9) 87 (13;10) 1 200 94 (15;12) 83 (9;13) 1 200

Tilmicosin 92 (10;11) 94 (4; 5) 1 100 82 (8;14) 100 (5; 8) 1 70

Mirosamycin 87 (7; 10) 96 (2; 9) 0.2 50 83 (6; 10) 96 (3; 3) 0.2 40

Oleandomycin 94 (6; 6) 98 (3; 3) 0.5 100 87 (7; 6) 100 (8; 7) 0.5 200

Erythromycin A 100 (7; 8) 98 (4; 4) 0.5 50 91 (4; 3) 98 (2; 5) 0.5 50

Tylosin 82 (6; 6) 91 (4; 7) 0.2 50 73 (9;10) 80 (8; 9) 0.2 50

Josamycin 88 (3; 3) 90 (4; 4) 0.2 40 82 (4; 6) 91 (3; 6) 0.2 40

Lincosamides

Lincomycin A 92 (4; 4) 92 (3; 8) 0.5 200 85 (5; 5) 94 (4; 4) 0.5 200

c
Pirlimycin 78 (6; 5) 77 (6; 7) 0.2 10 75 (6; 7) 79 (5; 6) 0.2 10c

Sulfonamides

Sulfadiazine 98 (4; 7) 102 (5; 8) 0.2 100 96 (4; 5) 109 (5; 6) 0.2 100

Sulfathiazole 96 (4; 7) 109 (6;10) 0.5 100 94 (6; 6) 108 (6;10) 0.5 100

Sulfamerazine 96 (8; 8) 109 (7; 8) 0.5 100 89 (8;10) 101 (10; 7) 0.2 10c

Sulfadimidine 97 (7; 7) 104 (5; 6) 0.5 100 91 (5; 5) 105 (4; 5) 0.2 100

Sulfamonomethoxine 95 (10;11) 108 (3; 4) 2 20 91 (6; 6) 101 (4; 4) 2 100

Sulfamethoxazole 95 (4; 4) 102 (3; 4) 0.5 20 92 (4; 5) 98 (5; 4) 0.5 20

Sulfaquinoxaline 92 (7; 10) 97 (3; 9) 1 10 c 86 (6; 7) 95 (4; 5) 1 50

Sulfadimethoxine 84 (5; 4) 96 (3; 7) 0.2 200 89 (4; 6) 99 (2; 2) 0.2 50

Others

Thiabendazole 84 (7; 6) 96 (2; 4) 0.2 83 (5; 5) 98 (7; 6) 0.2

g g
100 50
5-hydroxythiabendazole 77 (5; 6) 86 (2; 5) 0.1 81 (3; 6) 93 (3; 3) 0.1

Clopidol 89 (4; 6) 99 (4; 4) 1 200 89 (3; 3) 99 (4; 3) 0.5 5000

Tiamulin 84 (6; 6) 87 (4; 7) 0.2 40 79 (7; 5) 86 (4; 5) 0.2 100

 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015  241

Table 3.  (continued)
Bovine muscle Prawn

Trueness, % (RSDra, %; RSDWRb, %) Trueness, % (RSDra, %; RSDWRb, %)

Analytes 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg

Quinolones

Marbofloxacin 85 (4; 8) 101 (4; 4) 1 100 79 (6; 9) 89 (3; 5) 1 10c

c
Norfloxacin 73 (6; 6) 81 (4; 6) 2 10 74 (6; 5) 85 (3; 6) 1 10c

Ofloxacin 84 (8; 10) 95 (5; 7) 0.2 10c 80 (7; 7) 90 (5; 8) 0.2 10c

Enrofloxacin 84 (3; 5) 95 (2; 6) 1 86 (4; 6) 99 (3; 3) 0.5

d d
50 10
Ciprofloxacin 71 (6; 6) 80 (2; 3) 1 74 (7; 6) 87 (3; 4) 2

Danofloxacin 79 (8; 7) 83 (4; 6) 5 200 78 (4; 8) 85 (1; 7) 2 100

Orbifloxacin 88 (5; 8) 101 (3; 5) 0.5 20 90 (2; 4) 97 (3; 4) 0.5 10c

c
Sarafloxacin 81 (4; 5) 89 (4; 5) 1 10 86 (6; 8) 91 (4; 8) 1 10c

Difloxacin 88 (4; 3) 97 (3; 5) 1 10c 92 (4; 8) 98 (4; 5) 0.5 10c

Oxolinic acid 89 (5; 7) 100 (2; 3) 0.5 100 92 (5; 8) 98 (2; 3) 0.5 30
c
Nalidixic acid 88 (5; 8) 96 (2; 3) 0.5 10 91 (3; 7) 96 (1; 3) 0.5 10c

Flumequine 86 (4; 7) 97 (2; 6) 0.2 500 90 (5; 9) 96 (2; 4) 0.2 10c

Tetracyclines

Oxytetracycline 70 (5; 6) 73 (3; 6) 1 77 (9;14) 78 (3; 6) 2 200

Tetracycline 71 (6; 7) 71 (5; 6) 1 200

e
75 (7; 6) 78 (5; 8) 1 10c

Chlortetracycline 106 (5; 9) 96 (2;11) 2 92 (7; 8) 84 (5; 7) 2 10c

Doxycycline 90 (5; 7) 90 (4;10) 1 100 81 (7;10) 77 (3; 6) 1 10c

f
Demeclocycline 61 (9;11) 69 (3;12) 1 64 (6; 7) 75 (5; 9) 1

Macrolides

Spiramycin 87 (9; 8) 85 (8;10) 1 200 86 (7; 8) 90 (5;10) 1 200

Tilmicosin 91 (9; 8) 99 (2; 3) 0.2 100 93 (5;10) 96 (3; 7) 0.2 10c

c
Mirosamycin 86 (4; 8) 97 (4; 5) 0.2 10 82 (4; 6) 91 (2; 7) 0.2 10c

Oleandomycin 91 (5; 7) 103 (3; 5) 1 50 95 (2; 5) 101 (1; 3) 0.2 10c

Erythromycin A 93 (5; 7) 102 (3; 5) 1 50 99 (5; 5) 102 (2; 2) 1 200

Tylosin 81 (5; 7) 90 (1; 6) 1 50 82 (6; 9) 92 (4; 5) 0.1 100

Josamycin 85 (5; 7) 96 (2; 4) 1 10c 91 (4; 7) 94 (3; 4) 0.5 10c

Lincosamides

Lincomycin A 94 (15;12) 83 (9;13) 2 200 92 (2; 5) 98 (2; 5) 0.5 100

Pirlimycin 71 (3; 3) 76 (3; 4) 0.2 100 82 (4; 9) 87 (2; 4) 0.2 10c

Sulfonamides

Sulfadiazine 103 (4; 5) 118 (4; 4) 1 100 95 (4; 6) 101 (4; 4) 0.2 10c

Sulfathiazole 100 (5; 6) 114 (3; 5) 1 100 91 (4; 6) 101 (2; 8) 1 10c

Sulfamerazine 93 (6; 6) 102 (2; 5) 0.5 100 93 (3; 5) 99 (2; 4) 0.2 10c

Sulfadimidine 92 (3; 4) 104 (1; 4) 0.5 100 93 (4; 5) 100 (2; 2) 0.2 10c

Sulfamonomethoxine 92 (4; 6) 99 (2; 2) 2 10 96 (6; 6) 100 (3; 3) 1 10c

c
Sulfamethoxazole 89 (5; 8) 99 (2; 3) 0.5 10 96 (3; 5) 97 (2; 2) 0.5 10c

Sulfaquinoxaline 82 (5; 8) 94 (3; 4) 0.5 100 91 (5; 10) 92 (2; 4) 0.2 10c

Sulfadimethoxine 85 (5; 7) 98 (3; 3) 0.2 50 94 (4; 7) 96 (2; 5) 0.2 10c

Others

Thiabendazole 83 (7; 6) 93 (4; 8) 0.2 85 (5; 7) 90 (5; 7) 0.2

g g
100 20
5-hydroxythiabendazole 71 (3; 3) 79 (4; 7) 0.1 88 (4; 6) 95 (3; 5) 0.1

Clopidol 92 (3; 6) 105 (2; 8) 0.5 200 93 (4; 8) 100 (4; 3) 0.2 10c

Tiamulin 76 (4; 6) 90 (3; 8) 0.2 10c 80 (4; 7) 83 (3; 5) 0.1 10c

242  Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Table 3.  (continued)
Salmon trout Red sea bream

Trueness, % (RSDra, %; RSDWRb, %) Trueness, % (RSDra, %; RSDWRb, %)

Analytes 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg

Quinolones

Marbofloxacin 82 (5; 5) 87 (1; 5) 1 10c 85 (4;10) 92 (2; 6) 1 10c

c
Norfloxacin 72 (5; 6) 75 (3; 3) 2 10 77 (5;11) 81 (3; 6) 1 10c

Ofloxacin 87 (5; 6) 96 (3; 5) 1 10c 88 (3; 8) 95 (3; 6) 0.5 10c

Enrofloxacin 89 (5; 7) 92 (5; 8) 1 88 (4; 6) 99 (3; 7) 0.5

c,d c,d
10 10
Ciprofloxacin 75 (7; 9) 80 (2; 6) 5 75 (5; 6) 88 (4; 6) 2

Danofloxacin 84 (8;11) 84 (4; 5) 2 100 84 (9; 7) 93 (4;14) 5 100

c
Orbifloxacin 92 (6; 5) 93 (2; 5) 0.2 10 93 (4; 5) 98 (1; 5) 0.2 10c

Sarafloxacin 82 (4; 6) 89 (3; 4) 1 30 86 (5; 5) 94 (2; 5) 1 10c

Difloxacin 92 (5; 5) 94 (3; 3) 0.5 10c 92 (5; 4) 95 (3; 5) 1 10 c

Oxolinic acid 92 (4; 8) 95 (4; 4) 0.5 100 95 (2; 3) 98 (1; 2) 0.2 60

c
Nalidixic acid 89 (6; 5) 93 (3; 4) 0.5 10 92 (3; 4) 97 (2; 2) 0.5 10c

Flumequine 92 (5; 8) 94 (4; 5) 0.2 500 92 (3; 4) 97 (1; 5) 0.2 40

Tetracyclines

Oxytetracycline 76 (7; 9) 79 (6; 7) 1 200 78 (5; 9) 79 (4; 9) 2 200

c
Tetracycline 77 (7; 8) 76 (4; 5) 1 10 74 (6;11) 76 (4; 8) 1 10c
c
Chlortetracycline 103 (3; 5) 97 (4; 4) 2 10 106 (7;10) 98 (3; 4) 1 10c

Doxycycline 93 (6; 5) 87 (2; 5) 0.5 10c 113 (9; 8) 105 (5; 9) 0.5 50
f
Demeclocycline 64 (8;15) 71 (4; 6) 1 54 (10;12) 67 (3; 7) 1

Macrolides

Spiramycin 93 (8; 9) 102 (5; 6) 1 200 90 (7;10) 91 (7;11) 1 200

c
Tilmicosin 90 (5; 8) 96 (3; 3) 0.2 10 90 (6; 8) 96 (2; 9) 0.5 10c
c
Mirosamycin 87 (6; 6) 90 (4; 4) 0.2 10 94 (3; 3) 103 (3; 7) 0.2 10c

Oleandomycin 93 (5; 5) 99 (4; 3) 1 10c 99 (3; 4) 100 (3; 9) 0.2 50

Erythromycin A 97 (4; 5) 99 (4; 8) 0.2 200 102 (4; 5) 103 (4;10) 0.2 60

Tylosin 89 (4; 4) 95 (4; 6) 0.2 100 90 (4; 6) 96 (2; 5) 0.2 100

Josamycin 86 (4; 4) 92 (3; 6) 0.5 10c 92 (4; 5) 94 (1; 5) 0.5 50

Lincosamides

Lincomycin A 90 (4; 3) 94 (2; 4) 2 100 96 (3; 8) 99 (2; 4) 0.2 50

c
Pirlimycin 81 (5; 5) 84 (4; 4) 0.2 10 80 (5; 6) 86 (4; 8) 0.2 10c

Sulfonamides

Sulfadiazine 89 (7; 9) 92 (4; 7) 0.2 100 96 (5; 5) 101 (2; 3) 0.1 10c
c
Sulfathiazole 93 (5; 7) 97 (4; 5) 0.5 10 82 (4; 8) 87 (2; 5) 0.5 10c

Sulfamerazine 93 (3; 8) 95 (3; 4) 0.1 10c 97 (4; 6) 99 (3; 8) 0.2 10c

c
Sulfadimidine 81 (5;11) 90 (5; 5) 0.2 10 96 (2; 5) 97 (3; 5) 0.2 10c

Sulfamonomethoxine 92 (3; 7) 96 (2; 5) 0.2 100 81 (5; 8) 87 (4;12) 2 100

Sulfamethoxazole 78 (6; 9) 81 (4; 4) 0.5 10c 95 (4; 4) 101 (1; 6) 0.5 10c

Sulfaquinoxaline 78 (6; 9) 86 (4; 6) 0.5 10c 74 (4; 6) 77 (1; 6) 0.2 10c

Sulfadimethoxine 82 (5; 5) 90 (3; 4) 0.2 100 83 (3; 4) 90 (1; 5) 0.2 10c

Others

Thiabendazole 75 (5; 6) 79 (5; 7) 0.2 86 (6; 7) 94 (1; 3) 0.2

g g
20 20
5-hydroxythiabendazole 82 (5; 5) 90 (3; 4) 0.1 85 (4; 6) 89 (2; 5) 0.1

Clopidol 94 (6; 5) 97 (4; 5) 0.2 10c 95 (4; 7) 101 (2; 5) 2 10c

Tiamulin 82 (5; 5) 87 (1; 5) 1 10c 77 (4; 6) 85 (2; 6) 0.1 10c

 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015  243

Table 3.  (continued)
Flounder Milk

Trueness, % (RSDra, %; RSDWRb, %) Trueness, % (RSDra, %; RSDWRb, %)

Analytes 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg

Quinolones

Marbofloxacin 78 (8; 7) 88 (4; 3) 1 10c 91 (2; 4) 92 (4; 9) 1 75

h
Norfloxacin 67 (7; 7) 78 (5; 4) 2 10c 84 (4; 5) 89 (2; 5) 2 10c

Ofloxacin 78 (5; 5) 90 (5; 4) 0.5 10c 93 (3; 5) 93 (5; 6) 0.5 10c

Enrofloxacin 83 (6; 6) 91 (4; 5) 0.5 93 (4; 5) 91 (4; 5) 1

c,d d
10 50
Ciprofloxacin 71 (5; 6) 80 (4; 4) 1 87 (5;10) 86 (2; 7) 2

Danofloxacin 31 (17; 27)h 57 (6; 7) h 5 100 86 (8; 9) 85 (4; 9) 2 50

c
Orbifloxacin 85 (3; 5) 96 (4; 3) 0.2 10 89 (6; 6) 93 (4; 4) 0.5 20

Sarafloxacin 80 (4; 4) 89 (3; 4) 1 10c 88 (6; 6) 92 (2; 9) 5 10c

Difloxacin 86 (5; 5) 96 (4; 5) 0.5 10c 94 (3; 3) 92 (5; 7) 1 10c

Oxolinic acid 89 (5; 6) 100 (2; 5) 0.5 50 95 (2; 7) 97 (3; 2) 0.5 10c

Nalidixic acid 90 (5; 4) 100 (1; 4) 0.5 10c 91 (3; 6) 98 (2; 4) 0.2 10c

Flumequine 89 (5; 5) 97 (2; 2) 0.2 600 89 (2; 9) 94 (4; 5) 0.2 100

Tetracyclines

Oxytetracycline 72 (6; 7) 77 (4; 4) 1 200 94 (5; 8) 93 (3; 3) 2

c e
Tetracycline 73 (5; 6) 76 (4; 6) 2 10 89 (4; 4) 93 (3; 8) 1 100
c
Chlortetracycline 102 (4; 9) 95 (2; 2) 1 10 98 (4; 6) 92 (2; 4) 2
c
Doxycycline 112 (6;10) 103 (3; 4) 1 10 98 (4; 5) 94 (4; 5) 1 10c
f
Demeclocycline 63 (9; 9) 74 (3; 7) 1 88 (9;10) 93 (6; 6) 1

Macrolides

Spiramycin 84 (7;10) 95 (5; 5) 1 200 89 (6; 6) 89 (3; 4) 1 200

c
Tilmicosin 89 (7; 8) 93 (5; 9) 0.2 10 88 (5;10) 90 (3; 7) 0.5 50

Mirosamycin 89 (6; 6) 95 (4; 5) 0.2 10c 91 (4; 7) 91 (3; 4) 0.1 10c

Oleandomycin 89 (6; 6) 95 (4; 3) 0.2 10c 94 (5; 7) 93 (1; 6) 0.2 50

Erythromycin A 97 (3; 3) 102 (3; 4) 0.2 200 96 (3; 4) 93 (3; 4) 0.2 40

Tylosin 87 (5; 6) 95 (4; 3) 0.2 100 88 (7; 7) 88 (3; 3) 0.2 50

Josamycin 86 (4; 4) 95 (3; 3) 0.5 10c 86 (2; 4) 87 (3; 7) 0.5 10c

Lincosamides

Lincomycin A 86 (5; 5) 96 (3; 5) 0.5 100 88 (3; 4) 91 (2; 3) 0.5 150

c
Pirlimycin 75 (5; 6) 82 (3; 3) 0.2 10 93 (5; 8) 92 (5; 8) 0.2 300

Sulfonamides

Sulfadiazine 92 (7; 7) 102 (4; 5) 0.2 10c 92 (3; 7) 98 (3; 4) 0.2 70

Sulfathiazole 74 (7; 7) 89 (4; 5) 0.5 10c 95 (4; 8) 97 (3; 7) 0.5 90

c
Sulfamerazine 93 (7; 7) 99 (5; 5) 0.2 10 92 (4; 9) 91 (5; 7) 0.2 10c

Sulfadimidine 90 (6; 5) 97 (3; 5) 0.2 10 c 93 (5; 5) 96 (5; 5) 0.2 25

Sulfamonomethoxine 74 (7; 5) 85 (3;12) 2 100 89 (4; 6) 98 (4; 5) 1 10c

c
Sulfamethoxazole 86 (4; 7) 98 (2; 4) 0.5 10 96 (4; 5) 93 (3; 3) 0.2 10c

Sulfaquinoxaline 64 (6; 8) h 75 (2; 3) 0.2 10c 91 (4; 8) 95 (4; 5) 0.5 10

Sulfadimethoxine 72 (5; 5) 84 (3; 5) 0.2 100 93 (4; 5) 94 (3; 3) 0.1 20

Others

Thiabendazole 82 (5; 5) 93 (3; 5) 0.2 87 (4; 5) 92 (3; 3) 0.2

g g
20 100
5-hydroxythiabendazole 81 (4; 4) 94 (3; 4) 0.1 81 (4; 9) 89 (5; 9) 0.1

Clopidol 91 (5; 5) 100 (2; 2) 2 10c 90 (4; 4) 93 (4; 4) 1 20

c
Tiamulin 75 (6; 5) 85 (2; 5) 0.1 10 77 (5; 5) 80 (5;11) 0.1 10c
244  Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Table 3.  (continued)
Egg Honey
a b
Trueness, % (RSDr , %; RSDWR , %) Trueness, % (RSDr , %; RSDWRb, %)
a

Analytes 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg
Quinolones
c
Marbofloxacin 79 (4; 6) 75 (4; 9) 0.5 10 96 (5; 5) 101 (2; 2) 1 10c
c
Norfloxacin 80 (4; 5) 81 (4; 6) 2 10 92 (6; 6) 95 (1; 4) 1 10c
Ofloxacin 88 (4; 5) 91 (3; 5) 0.2 10c 98 (4; 5) 102 (2; 3) 1 10c
Enrofloxacin 90 (4; 5) 91 (5; 7) 0.5 92 (5; 6) 98 (4; 4) 2
10c,d 10c,d
Ciprofloxacin 81 (7; 7) 82 (2; 7) 1 95 (6; 4) 100 (2; 4) 5
c
Danofloxacin 89 (5; 9) 87 (6; 7) 5 10 96 (5; 4) 102 (3; 4) 1 10c
Orbifloxacin 86 (5; 5) 85 (4;12) 0.5 10c 92 (3; 6) 100 (4; 4) 0.5 10c
c
Sarafloxacin 83 (3; 3) 88 (3; 4) 1 10 91 (5; 7) 98 (3; 3) 2 10c
c
Difloxacin 90 (4; 7) 94 (3; 4) 0.5 10 96 (3; 3) 100 (3; 4) 2 10c
Oxolinic acid 90 (3; 5) 96 (2; 2) 2 10c 97 (3; 5) 100 (2; 4) 1 10c
c
Nalidixic acid 90 (3; 2) 93 (2; 6) 0.2 10 96 (3; 3) 100 (3; 3) 0.2 10c
c
Flumequine 76 (3; 5) 84 (3; 4) 0.2 10 95 (3; 3) 100 (3; 4) 0.2 10c
Tetracyclines
Oxytetracycline 76 (3; 4) 75 (2; 4) 1 95 (6; 5) 96 (2; 2) 1
e e
Tetracycline 84 (6; 7) 79 (4; 7) 1 400 92 (5; 5) 92 (2; 6) 2 300
Chlortetracycline 99 (3; 7) 90 (3; 5) 1 85 (6;10) 83 (4; 4) 2
c
Doxycycline 113 (5; 8) 112 (4; 7) 0.5 10 103 (9; 9) 101 (3; 4) 0.5 10c
Demeclocyclinef 60 (7; 7) 57 (4;10) 2 88 (6; 9) 97 (3;10) 2
Macrolides
c
Spiramycin 94 (7;10) 90 (4; 6) 1 10 96 (7; 7) 98 (5; 5) 1 10c
Tilmicosin 92 (5; 7) 96 (7; 3) 1 10c 94 (4; 6) 101 (4; 6) 1 10c
c
Mirosamycin 92 (4; 4) 96 (1; 3) 0.1 10 96 (3; 4) 103 (4; 4) 0.1 50
c
Oleandomycin 93 (2; 6) 98 (2; 3) 0.5 10 94 (4; 5) 102 (4; 4) 0.2 10c
Erythromycin A 98 (2; 4) 98 (2; 4) 0.2 90 101 (3; 5) 98 (2; 5) 0.2 10c
Tylosin 91 (4; 6) 91 (3; 5) 0.2 200 97 (4; 4) 102 (4; 3) 0.2 10c
c
Josamycin 84 (2; 3) 89 (1; 3) 0.5 10 90 (6; 6) 95 (2; 4) 0.5 10c
Lincosamides
c
Lincomycin A 81 (4; 8) 78 (4; 7) 0.2 100 96 (3; 5) 99 (2; 3) 0.5 10
Pirlimycin 81 (4; 7) 82 (4; 8) 0.2 10c 95 (3; 3) 96 (4; 7) 0.2 10c
Sulfonamides
c
Sulfadiazine 72 (9;16) 57 (12;24) 0.2 20 97 (4; 6) 99 (2; 3) 0.2 10
Sulfathiazole 97 (4; 3) 100 (3; 4) 1 10c 92 (6; 6) 98 (5; 5) 0.5 10c
c
Sulfamerazine 91 (4; 5) 90 (3; 6) 0.2 10 92 (6; 4) 100 (3; 3) 0.2 10c
Sulfadimidine 94 (3; 6) 96 (4; 3) 0.2 10 92 (4; 5) 98 (4; 4) 0.2 10c
c
Sulfamonomethoxine 92 (3; 5) 93 (2; 5) 0.5 10 95 (4; 6) 100 (3; 4) 2 10c
Sulfamethoxazole 91 (2; 4) 94 (2; 4) 0.2 10c 92 (5; 5) 100 (2; 3) 0.2 10c
Sulfaquinoxaline 90 (3; 4) 90 (2; 3) 0.2 10 90 (3; 5) 98 (2; 3) 0.2 10c
Sulfadimethoxine 90 (2; 3) 91 (1; 3) 0.1 1000 92 (4; 4) 100 (2; 3) 0.1 10c
 Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015  245
Table 3.  (continued)
Egg
a b
Trueness, % (RSDr , %; RSDWR , %)
Analytes 10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg
Others
Thiabendazole 89 (5; 4) 95 (3; 3) 0.2
5-hydroxythiabendazole 88 (4; 3) 90 (3; 3) 0.1
h
Clopidol 29 (12; 30) 25 (26; 36) h 2 10c
Tiamulin 77 (4; 5) 84 (2; 3) 0.1
a
 RSDr of repeatability.
b
  RSDWR of within-run reproducibility.
c
  The 10 mg/kg default regulatory limit; MRLs for some analytes in livestock and fishery products have not been defined.
d
  MRL is the sum of enrofloxacin and ciprofloxacin.
e
  MRL is the sum of oxytetracycline, tetracycline, and chlortetracycline.
f
  The internal standard material for the quantification of chlortetracycline and doxycycline.
g
  MRL is the sum of thiabendazole and 5-hydroxythiabendazole.
h
  Did not satisfy the criteria of the Japanese guideline.
the matrix did not significantly affect the fragmentation patterns
of each precursor ion of 37 veterinary drugs to two product ions.
Linearity of Calibration
The matrix-matched calibration curves of 35 veterinary
drugs, except for chlortetracycline and doxycycline, were
obtained for a series of standard solutions containing each
matrix at five concentrations by plotting the peak area against
the concentration. Chlortetracycline and doxycycline were
unstable in the resultant solution. Therefore demeclocycline
was used as an IS to more accurately measure the concentrations
of both chlortetracycline and doxycycline. Chlortetracycline
and doxycycline calibration curves were obtained for a series
of standard solutions at five concentrations by plotting the
peak area against the concentration, corrected by 0.01 µg/mL
demeclocycline. All of the correlation coefficient (r) values
were over 0.999, and deviations in individual points from
the calibration curves were lower than 20%. Accordingly,
satisfactory linearity was obtained in the range examined for
each compound.
To evaluate the matrix effect, slopes derived from the
standard solution and matrix-matched calibration curves
derived from each livestock and fishery product were compared
at the same range as described above. The slope ratios of the
matrix-matched/standard solution calibration curves were
obtained for each of the 37 veterinary drugs (Figure 5). The slope
ratios ranging from 0.8 to 1.2 were considered to be tolerable,
whereas the ratio higher than 1.2 or lower than 0.8 implied a
strong matrix effect (39). A significant matrix effect was noticed
on marbofloxacin in bovine muscle and prawn; danofloxacin
in chicken muscle, prawn, flounder, and honey; orbifloxacin
in prawn, difloxacin in bovine muscle; tetracycline in swine
muscle and prawn; chlortetracycline in prawn; demeclocycline
in prawn; spiramycin in swine muscle, chicken muscle, bovine
muscle, prawn, salmon trout, flounder, and honey; tilmicosin in
swine muscle, chicken muscle, bovine muscle, red sea bream,
flounder, and honey; pirlimycin in honey; sulfadiazine in bovine
Honey
Trueness, % (RSDr , %; RSDWRb, %)
a
10 μg/kg 100 μg/kg LOQ, μg/kg MRL, μg/kg
93 (6; 6) 99 (2; 3) 0.1
100g 20g
97 (4; 6) 100 (2; 3) 2
93 (4; 7) 99 (3; 3) 0.2 10c
1000 76 (5; 6) 82 (3; 8) 0.1 10c
muscle and honey; sulfathiazole in bovine muscle, prawn,
flounder, and honey; sulfamerazine in honey; sulfadimidine
in honey; sulfamonomethoxine in bovine muscle and honey;
sulfaquinoxaline in salmon trout, sulfadimethoxine in salmon
trout; 5-hydroxythiabendazole in prawn; thiabendazole in
bovine muscle and prawn. Therefore, we consider that the
matrix-matched standard calibration curves were adequate for
the quantification of each of the 37 veterinary drugs in livestock
and fishery products.
Method Validation
Validation was carried out following the guidelines of the
Japanese Ministry of Health, Labour, and Welfare (5, 6). The
developed method in this study was validated by means of
recovery tests using swine muscle, chicken muscle, bovine
muscle, prawn, salmon trout, red sea bream, flounder, milk, egg,
and honey samples which were spiked with 50 µL of working
standard solutions (1 µg/mL or 10 µg/mL) in two replicates for
5 separate days. As shown in Table 3, the overall recovery of
the 37 drugs ranged from 25 to 118%. The RSDr ranged from 1
to 26%. The RSDWR ranged from 2 to 36%. In this method, the
numbers of analytes that satisfied the guidelines criteria were
37 out of 37 in swine muscle, chicken muscle, bovine muscle,
prawn, salmon trout, red sea bream, milk, and honey samples,
35 out of 37 in egg, and 34 out of 37 in flounder. Only two
analytes (sulfadiazine and clopidol) in egg and three analytes
(norfloxacin, danofloxacin, and sulfaquinoxaline) in flounder
were not sufficiently recovered. Selectivity was confirmed
by analyzing blank samples, and no interfering peaks were
observed at the same retention times of the target analytes.
Figure  4b shows the SRM chromatograms obtained from the
blank swine muscle.
LODs and LOQs
As shown in Table 3, LOQs for the 37 veterinary drugs ranged
from 0.1 to 5 µg/kg, which was less than the 10 µg/kg default
246  Kanda et al.: Journal of AOAC International Vol. 98, No. 1, 2015
regulatory limit, set by the positive list system for agricultural
chemical residues in foods in Japan. LODs ranged from 0.03 to
2 µg/kg.
Survey of Livestock and Fishery Products
To demonstrate the applicability of the developed method in
this study for the determination of 37 veterinary drug residues,
110 samples (20 swine muscle, 15 chicken muscle, 13 bovine
muscle, 10 prawn, 10 salmon trout, 7 red sea bream, 10 flounder,
5 milk, 10 egg, and 10 honey), purchased from retail outlets in
Japan, were tested. When the peak was detected, the ion ratios
were compared with those of the standard solutions at comparable
concentrations. Because the relative ion abundance ratios were
within 20% recommended by EU guidelines (38), the identity of
residual drugs was accurate. No analyte was detected in bovine
muscle, prawn, red sea bream, milk, or egg. In swine muscle,
TCs were detected in ten samples. In four samples, more than one
TC was found; oxytetracycline (2.5 µg/kg) and chlortetracycline
(3.9 µg/kg), oxytetracycline (1.3 µg/kg) and doxycycline
(1.4 µg/kg), tetracycline (1.7 µg/kg) and chlortetracycline
(10.9 µg/kg), chlortetracycline (18.7 µg/kg) and doxycycline
(4.0 µg/kg). On the other hand, oxytetracycline was found in
four samples (1.3, 3.6, 4.5, and 9.5 µg/kg), tetracycline in one
sample (6.4 µg/kg), and doxycycline in one sample (0.8 µg/kg).
In chicken muscle, three veterinary drugs were detected in four
samples. Clopidol (3.4 µg/kg), oxytetracycline (8.5 µg/kg),
and enrofloxacin (20.9  µg/kg) were contained in one sample.
Oxytetracycline was contained in one sample (10.1 µg/kg).
Enrofloxacin was found in two samples (1.0, and 1.0 µg/kg). In
salmon trout, the oxytetracycline residue was detected in two
samples (4.5 and 27.0  µg/kg). In flounder, the oxytetracycline
residue was detected in three samples (9.1, 20.5, and 23.7 µg/kg).
In honey, the norfloxacin residue was detected in two samples
(1.1 and 1.5 µg/kg), mirosamycin residue in two samples (3.0
and 36.3 µg/kg), and tylosin residue in one sample (4.4 µg/kg).
All values were lower than the MRLs or regulatory default limits
of 10 µg/kg for livestock and fishery products. The residues of
veterinary drugs were found in 24 of 110 samples (22%).
Conclusions
We developed a novel method to determine 37 polar
veterinary drugs in 10 livestock and fishery products using
LC-MS/MS. The sample preparation for 10 samples takes only
2 h and no evaporation step is needed. Polar veterinary drugs
were efficiently extracted from livestock and fishery products
with two different polar solvents, Na2EDTA-McIlvaine’s buffer
(pH  7.0) and ACN/FA. Among the compounds examined,
highly polar veterinary drugs were initially extracted from
samples with Na2EDTA-McIlvaine’s buffer, and then medium
polar veterinary drugs were extracted by a second extraction
step with ACN/FA.
We re-used the second extracted solution as an elution
solution from the SPE polymer cartridge which retained the
compounds first-extracted by Na2EDTA-McIlvaine’s buffer.
The matrix extracted with Na2EDTA-McIlvaine’s buffer was
reduced by being passed through the SPE polymer cartridge.
The matrix extracted by the second extraction step was retained
on the same SPE polymer cartridge. Strong matrix effects were
reduced by this cleanup procedure.
TCs and QLs were measured with good sensitivity and
excellent peak shapes using the novel hybrid column, a mobile
phase consisting of a mixture of 0.05% formic acid and
acetonitrile, and a minimum injection volume. By preparing the
gas pressure on MS/MS parameters, macrolides were measured
with high sensitivity. LOQs of the 37 veterinary drugs were
lower than the MRLs.
Using this method, the numbers of analytes that were
validated in accordance with the Japanese Ministry of Health,
Labour, and Welfare guideline were 37 analytes out of 37 in
swine muscle, chicken muscle, bovine muscle, prawn, salmon
trout, red sea bream, milk, and honey, 35 in egg, and 34 in
flounder. FQs, TCs, and 5-hydroxythiabendazole, which could
not be determined using previously reported methods, were
successfully analyzed using our novel method.
This method was successfully applied on 110 commercially
available livestock and fishery products. Veterinary drug
residues were found in 24 samples. It is necessary to continue
monitoring for the residues of 37 veterinary drugs in livestock
and fishery products using this method. The method developed
in this study provides high-quality performance and ease of
implementation for the routine monitoring of 37 polar veterinary
drugs in livestock and fishery products.
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