MP 178

Fast and Simlutaneous LC/MS/MS Analysis for Veterinary Drugs in Meat Combined with STQ method
Natsuyo ASANO1, Eishi IMOTO1, Mami OKAMOTO1, Mikie SHIMA2, Jun WATANABE1
1 Shimadzu Corporation, MS Business Unit, Kyoto, Japan., 2 AiSTI SCIENCE Co., LTD., Wakayama, Japan.
Sample pretreatment

1. Introduction
Veterinary drugs are used for therapeutic and growth promotion purposes foranimals or fishes. To provide assurance that food from animals is safe in regard toveterinary medicine residues, regulatory authorities have established Maximum
Residue Limits (MRL) for certain drugs in target tissues and animal species.
Veterinary drugs analysis commonly uses liquid chromatography coupled to mass
spectrometer which is fast, highly sensitive and highly selective. This work describes
the application of high-throughput LC-MS/MS system utilizing fast polarity switching.
Faster, easier and high precision total workflow was investigated with QuEChERS
method combined with solid-phase extraction cartridge to enhance purification
efficiency.

ODS Column
Cleanup by ST-L400

QuEChERS methodExtraction

Sample: 5 g(x100,000)

Sum of ① and ②

STD mixture (2 ng/μL): 25 μL

Beta-trenbolone

2.01.5Adjusting to 20 mL with ACN

Soaking Water (15 min)Beta-trenbolone

Alpha-trenbolone

1.0

Fractionation (1 mL)

1.00.09.0

Homogenized (13,000 rpm, 1 min)Smart-SPE C18-50 mg+PSA-30 mg

3.0

9.5

10.0

10.5

0.0min(x100,000)

Elution (2%Formic acid-ACN：
Water = 8:2 (v/v), 0.5mL)

2.0

Chicken, pork and beef were selected for recovery tests of veterinary drugs.
Evaluation of analytical system and recovery test used 129 veterinary drugs spikedin meat (1.25 ppb in vial). Solid phase extraction Technique with QuEChERS
method (STQ method) were processed using fully automated solid phase extraction
system (ST-L400, AiSTI SCIENCE, Japan). LC and MS conditions are shown in
Table1. ODS column and Biphenyl column were used to evaluate the peak shapeof drugs.

0.5

Smart-SPE C18-30 mg

Adjusting to 2 mL with Water

1.25 ppb in a vial

AnalyticalColumn
Solvent A
Solvent B

Method 2
Method 1
YMC-Triart C18 [Metal Free]
Restek RaptorTM Biphenyl(2.1 mmI.D.x 150 mmL., 3 μm) (2.1 mmI.D.x 100 mmL., 2.7 μm)
0.5 mM ammonium formate+
0.1% formic acid – Water
0.1% formic acid – Water
0.1% formic acid – Acetonitrile 0.1% Formic – MeOH

Homogenized(13,000 rpm, 1 min)Centrifuge (3,500 rpm, 5 min)Extract ACN Supernatant ・・・②Time (min)Gradient
ProgramFlow Rate
Column Temp

0.0
1.0
6.0
10.0
15.0
15.1
18.0%B

11540
100
1001STOP
0.2 mL/min
40 ℃[MS] LCMS-8060
Ionization： ESI (Positive/Negative)
Nebulizer Gas： 2 L/minInterface temp
： 300 ℃Drying Gas： 10 L/min

Time (min)

%B

0.0
12.50
14.50
14.60
17.5

2100100
2STOP

0.4 mL/min
40 ℃

Fig2. Fully automated solid phase extraction system(ST-L400, AiSTI SCIENCE)

3. Results

Comparison of ODS column and Biphenyl column
In the Fig3 below, both ODS column and Biphenyl column measured all of 129
veterinary drugs from standard sample (10 ppb) within 18 minutes. MRM
chromatogram of alpha- and beta- trenbolone, and sulfatroxazole and sulfisoxazole
using ODS column and biphenyl column are shown in Fig4. Biphenyl column achieved
good peak separation for alpha-trenbolone and beta-trenbolone. Conversely, ODS
column sufficiently separated sulfatroxazole and sulfisoxazole. Adequate selection ofcolumn enables to establish accurate quantitative analytical system.
(x1,000,000)

ODS column

2.5

0.0
2.5

(x1,000,000)

5.0

7.5

10.0

： 250 ℃
： 400 ℃
： 10 L/min

15.0 minBiphenyl column

2.5

DL temp
HB tempHeating Gas

12.5

0.0
2.5

5.0

7.5

10.0

12.5

7.5

8.0

8.5

9.0

0.0min5.5

6.0

6.5minFig4. MS Chromatogram of alpha- and beta- trenbolone, and sulfatroxazole andsulfisoxazole using ODS column and biphenyl column (STD, 10 ppb)

Recoveries of Veterinary Drugs in Chicken, Pork and Beef

Table1. LC and MS conditions[LC] NexeraTM X2 System

Sulfatroxazole

1.0

0.0

5% Formic acid ACN: 10 mL

11.0 minSulfisoxazole

Centrifuge (3,500 rpm, 5 min)2. Methods and Pretreatment

10.5

1.0

Water: 0.5 mLResidue

10.0

1.5

Vortex (1 min)Extract ACN Supernatant ・・・①

9.5

2.0 (x100,000)
Sulfisoxazole
SulfatroxazoleNaCl: 0.5 g, MgSO4: 2 g

Fig1. LC-MS/MS system (Nexera X2+LCMS-8060, Shimadzu Corporation.)

Alpha-trenbolone

0.5

ACN: 10 mL

15.0 minFig3. MS Chromatogram of 129 veterinary drugs (10 ppb) using ODS and Biphenyl column

Purified extract from chicken, pork and beef were assayed using LC-MS/MS using
ODS column. The peak area of standard and post-spiked sample were comparedfor matrix effects. The peak area of pre-spiked sample and post-spiked sample
were compared for recovery rates. The concentration of standard, pre-spiked andpost-spiked sample were diluted at the concentration of 1.25 ppb. 94 drugs wereobtained from each sample. The results indicated that 82% of the compounds inchicken, 85% of the compounds in pork and 84% of the compounds in beef wererecovered from 70 to 120% (n=3). Table2 shows the typical result of matrix effects
and recovery test from each samples. Stable and good recoveries were achieved
with fully automated STQ method.
Table2. Typical results of matrix effects and recovery tests (n=3)

CompoundChicken

PorkBeefMatrix
RecoveryMatrix
Recovery
MatrixRecovery
Effect (%)(%)
Effect (%)(%)
Effect (%)(%)
Altrenogest
Azaperone
Bromacil
Carazolol
Carprofen
Dicyclanil
Ethopabate
Famphur
Flubendazole
Flunixin
Josamycin
Mafoprazine

87101
94
99
100
94
98
106
101
103
93
94

79
83
90
78
76
84
86
79
86
83
76
84

78
97
92
90
104
91
99
89
97
96
90
96

92
86
92
98
71
83
104
93
98
81
90
93

80
99
115
101
84
74
92
94
10191
115
104

Chicken

Pork

Beef

MatrixRecovery
MatrixRecovery
MatrixRecoveryEffect (%)(%)
Effect (%)(%)
Effect (%)(%)

3.0 (x100,000)

2.52.0Compound

Biphenyl Column

8287
8788102898591
8185
7787

Mebendazole
Meloxicam
Menbutone
Miloxacin
MorantelNifurstyrenate
Oxibendazole
Praziquantel
Prifinium
PyrantelPamoate
Robenidine
Sulfabenzamide
Sulfabromomethazine Na
Sulfachlorpyrida
zine
Sulfadimethoxine
Sulfadimidine
Sulfadoxine
Sulfaethoxypyrida
zine
Sulfamerazine
Sulfamethoxazole
Sulfamonomethoxine
Sulfapyridine
Sulfaquinoxaline
Sulfathiazole
Sulfatroxazole
Sulfisoxazole
Thiamphenicol
Tiamulin
Tripelennamine
Valnemulin
Warfarin
Xylazine

100103
92
119
98
100
96
101
9698
1061108686
94
74807784828688
8284101
8191
9496109
91
9295
100
91
898891
10989919794
899591
9292

9597
93
9410994
93
95
9998
8393

91
8295
88
92
8794
89
91
98
7189

97

101

107858685103

78869296

79

95
99
97

92
87
84

97
92
91

99
92
96

98
100
99

8880
84

106

76104

90

103

77

94
101

8199

94
87

9298

72
98

9476

93

72

99

100

88

90

95
96
89
112
93
105
93
93108
101
102

8691
77
80
86
85
84
8686
81
85

95
99
82105
10480
92
91
100
99
91

91
98
8681
86
113
97
93
100
86
96

72
92
71
90
92
78101889886879280
1038980107901039495
83

5. Conclusion
・129 veterinary drugs were detected using ODS column and Biphenyl column within 18 min
・ 82% of the compounds in chicken, 85% of the compounds in pork and 84% of thecompounds in beef were recovered between 70 to 120%.
・Fully automated solid phase extraction achieved minimized matrix effect with sufficient
recovery rates
The product and application are Research Use Only. Not for use in human clinical diagnostics or in vitro diagnostic procedures.