Targeted Veterinary Drug Screening
in Food Matrices Using EMR—Lipid
Authors
Yanan Yang, Dan-Hui Dorothy Yang,
and Joan Stevens,
Agilent Technologies, Inc.
QuEChERS Kit and an Agilent 6470A
Triple Quadrupole LC/MS System
Application Note
Abstract
Veterinary drugs are used to treat animal diseases and improve animal growth.
Due to bioaccumulation of these drugs and their metabolites in adipose tissue
and subsequent entry into the food chain, strict regulations for maximum residue
levels are in place. The combination of an Agilent EMR—Lipid QuEChERS sample
preparation kit and an Agilent 6470A LC/MS system provides a superior solution
for the screening and quantitation of 105 veterinary drugs in beef, beef liver, pork,
and salmon.
Introduction Table 1. Spike Levels

Level Concentration
Governmental Agencies, such as FDA-USDA, WHO, and
1 0.1 ng/g
the United Nation Food and Agriculture Organization, have
2 0.5 ng/g
set maximum residue levels (MRLs) for veterinary drugs in
3 1 ng/g
different food matrices to limit the use of veterinary drugs
4 2 ng/g
in food producing animals [1,2]. Sensitive screening and
5 5 ng/g
quantitation of veterinary drugs at MRL is essential for
adherence to those regulations. 6 10 ng/g
7 20 ng/g
The Agilent Bond Elut Enhanced Matrix Removal 8 50 ng/g
(EMR—Lipid) QuEChERS kit is specifically designed and 9 100 ng/g
formulated to remove fatty acid components from food
matrices. It has been demonstrated that the extensive
removal of fatty materials does not negatively impact analyte Table 2. Agilent 1290 Infinity HPLC Conditions
recovery in complex matrices, thus improving detection
Parameter Value
sensitivity [3,4]. Sample preparation with an EMR—Lipid
QuEChERS kit enables a fast, robust, and effective analysis of Column Agilent PoroShell 120, EC-C18, 2.1 × 150 mm, 2.7 µm
(p/n 693775-902)
fatty samples.
Temperatures Column: 40 °C
The stringent requirements for targeted veterinary drug Sampler: 6 °C
screening require a sensitive and selective instrument Injection volume 2 µL
capable of delivering robust results. The Agilent 6470A Mobile phase A) Water, 0.5 mM NH4F + 0.1 % formic acid
B) ACN + 0.1 % formic acid
Tandem Quadrupole system (LC/TQ), features innovative
Flow rate 0.4 mL/min
technologies such as quadrupole prefilters, tapered
Gradient Time (min) %B
and curved hexapole collision cell, and a high-energy
0 2
dynode detector (HED). These improvements enhance 0.5 2
ion transmission efficiencies for precursor ions, eliminate 3 35
cross‑talk while promoting rapid collision-cell evacuation, and 8 45
allow for better sensitivities. These lead to enhanced linear 15 98
18 98
dynamic ranges (LDRs) and limits of quantitation (LOQs).
18.1 2
Stop time 19 minutes
Methods and Experiments Post time 2 minutes

Beef, beef liver, organic pork, and salmon were purchased

from local grocery stores, and were prepared per EMR—Lipid Table 3. Agilent Jet Stream Source and Agilent 6470A
Protocols as previously described [3]. One-hundred five Triple Quadrupole Parameters
veterinary drugs (Table 4) sourced from Ultra Scientific
Parameter Value
(N. Kingstown, RI, USA) were spiked into the matrices at nine
Drying gas temperature 250 °C
levels ranging 0.1–100 ng/g (Table 1). Agilent 1290 Infinity
Drying gas flow 10 L/min
UHPLC conditions and parameters for the 6470A LC/TQ
Sheath gas temperature 350 °C
equipped with an Agilent Jet Stream ESI source are listed in
Tables 2 and 3. Sheath gas flow 11 L/min
Nebulizer pressure 35 psi
Analytes were detected with dynamic MRM acquisition with Capillary voltage 3,500 V(+), 3,500 V(–)
two optimized MRM transitions per compound. Fast polarity Nozzle voltage 500 V(+), 1,000 V(–)
switching was fully used to detect all compounds in a single Delta EMV 200 V(+), 200 V(–)
run. Data analysis was carried out with Agilent MassHunter Cycle time 500 ms
Quantitative Analysis software (version B.07).

2
Table 4. Compound List of 105 Veterinary Drugs

17-alpha-19-Nortestosterone Halofuginone Prednisolone

17-beta-Estradiol Hexestrol Propionylpromazine
17-Methyltestosterone HMMNI Hydroxydimetridazole (Dimetridazol-OH) Ractopamine
Amino-Mebendazole Hydroxy-Ipronidazole Rifaximin
Betamethasone Hydroxymetronidazole Robenidine
Boldenone (Dehydrotestosterone) Ibuprofen Ronidazole
Brombuterol Ipronidazole Salbutamol (Albuterol)
Carprofen Isopyrin Salinomycin
Chlorbrombuterol (Bromoclenbuterol) Isoxsuprine (Isolait) Spiramycin I
Chlormadinone acetate Josamycin Stanozolol
Chlormadinone Ketoprofen Sulfachloropyridazine
Chlorpromazine Levamisole Sulfadiazine (Silvadene)
Cimaterol Lincomycin Sulfadimethoxine
Cimbuterol Mabuterol Sulfadimidine (Sulfamethazine)
Clenbuterol Maduramycin Sulfadoxine
Clenbuterolhydroxymethyl Mapenterol (Methylmabuterol) Sulfamerazine
Clencyclohexerol Marbofloxacin Sulfamethoxazole
Clenpenterol Mebendazole Sulfamethoxypyridazine (Midicel)
Clenproperol Mebendazole-hydroxy Sulfanilamide
Clopidol Medroxyprogesterone Sulfathiazole
DCL Diclazuril Mefenamic acid Terbutaline
Decoquinate Megestrol acetate Testosterone
Dexamethasone Melengestrol acetate Thiabendazole
Diclofenac Meloxicam Tilmicosin
Dienestrol Methylprednisolone Tolfenamic acid
Diethylstilbestrol Metronidazole Toltrazuril
Dimetridazole Monensin Trenbolone
Dinitolmide Nandrolone Triclabendazole
Erythromycin Narasin Triclabendazole sulfoxide
Ethynyl estradiol Nicarbazin Trimethoprim
Febantel Oxfendazole Tulobuterol
Fenbendazole Oxibendazole Tylosin
Fenoterol (Th 1165a) Oxolinic acid Zearalanone (Zanone)
Flumequine Phenylbutazone Zeranol
Flunixin Praziquantel Zilpaterol

3
 Results and Discussion cleaner matrices for better analytical sensitivity. Figure 1
shows the response of 105 analytes at 1.0 ng/g in beef and
pork, the two most complex matrices in the study.
Increased method performance
The EMR—Lipid QuEChERS sample preparation kit helps
remove most of the lipids in the food matrices, thus providing

×103
A
8.0
Beef
7.5
7.0
6.5
6.0
5.5
5.0
4.5
Counts

4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Acquisition time (min)
×103
7.5 B
Pork
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Counts

3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Acquisition time (min)

Figure 1. Excellent performance of 105 veterinary drugs in complex matrices (beef and pork). Signals overlaid in the two most complex matrices at
1.0 ng/g spike, with 2 µL injection.

4
 Veterinary drugs screen and accuracy Precision
Most of the veterinary drugs were detected at concentrations The %RSD calculation was based on six replicate injections
as low as 0.1 ng/g in the four matrices. Figure 2 shows the of 105 analytes from 0.1 ng/g to 20 ng/g depending on the
number of compounds that could be quantified with accuracy matrices at the lowest limit of quantitation (LLOQ). The
between 80–120 % for at least four out of six replicates. The results shown in Figure 3 clearly demonstrate the precise
signal-to-noise ratio (S/N) of the analyte chromatograms was quantitative capability of the 6470A Triple Quadrupole.
well above 10, but, as expected, the quantitation accuracy
at the lower spike level was affected by the contribution
from the matrix baseline to cause the accuracy outside the
80–120 %.

80
73 Pork

70 Beef
Salmon
58
60 Beef liver
52 52
50
No. of analytes

40
40
33
30 28

20
14
9
10 6 5 5 7 7
3 4 3 4 3
1 2 1 0 1 1 2 2 2
0
0.1 ng/g 0.5 ng/g 1 ng/g 2 ng/g 5 ng/g 10 ng/g 20 ng/g
LLOQ

Figure 2. Number of compounds that could be quantified at the LLOQ level with accuracy of 80–120 %
in four matrices.

80
Pork
70
70 Beef
Salmon
60
60 Beef liver

49
50
No. of analytes

40 38
36
31
30
24
19
20
15
13
11 11
10 8
6 6
4 5
2 3 1 2 1 0 0
0
0-5 5-10 10-15 15-20 20-25 >25
%RSD

Figure 3. Measurement precision of six replicates at LLOQ levels. Most of the analytes have %RSD less than 10 %.

5
Calibration curves and dynamic range ×106
Beef
The calibration curves shown in Figure 4 were generated 1.1 Clenbuterolhydroxymethyl
1.0 R2 = 0.9984
from 0.1–100 ng/g with linear fitting and 1/x weighing. 0.9
More than 96 % of the analytes gave linear responses with 0.8

Responses
0.7
R2 >0.99. Several analytes showed quadratic fitting, possibly 0.6
due to material loss at lower concentration levels or partial 0.5
saturation at the higher spike levels. 0.4
0.3
0.2
0.1
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105
Concentration (ng/mL)
×10 6

1.1 Beef liver

Clenbuterolhydroxymethyl
1.0 R2 = 0.9988
0.9
0.8
0.7

Responses
0.6
0.5
0.4
0.3
0.2
0.1
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105
Concentration (ng/mL)

×106
Pork
1.0 Clenbuterolhydroxymethyl
0.9 R2 = 0.9994
0.8
0.7
Responses

0.6
0.5
0.4
0.3
0.2
0.1
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105
Concentration (ng/mL)
×106
1.2 Salmon
Clenbuterolhydroxymethyl
1.1
R2 = 0.9989
1.0
0.9
0.8
Responses

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105
Concentration (ng/mL)

Figure 4. Calibration curves of clenbuterolhydroxymethyl in four matrices.

More than 96 % of analytes gave R2 >0.99.

6
 Veterinary drugs detected in blank matrices Conclusions
Some of the veterinary drugs were detected in products
purchased from local grocery markets. Figure 5 shows Analysis and detection of veterinary drugs can be achieved
examples of the analytes detected in beef liver and salmon. in animal tissue at 1.0 ng/g with a 2.0 µL injection. A
There are several other veterinary drugs that were detected at combination of an Agilent EMR—Lipid QuEChERS kit
concentrations less than 1.0 ng/g. In ground beef, robenidine and an Agilent 6470A LC/TQ system provide enhanced
and ketoprofen were detected at 1.0 ng/g and 2.2 ng/g removal of lipid content from the animal matrices, and
respectively (graphs not shown). However, in organic pork sensitive detection at promulgated MRLs. The complete
none of the veterinary drugs analyzed in this study were Agilent solution, including powerful data analysis software,
detected at a concentration above 0.5 ng/g. facilitates method development and validation, and provides
a powerful tool kit for veterinary drug screening and
quantitation in animal tissues.

Beef liver

×104 ×103
Ractopamine 9.8 ng/g Monensin 1.7 ng/g
6.5 2.6
302.2 → 284.2 2.4 693.5 → 675.3
6.0
5.5 302.2 → 107.1 2.2 693.5 → 461.3
5.0 2.0
4.5 1.8
4.0 1.6
Counts
Counts

3.5 1.4
3.0 1.2
2.5 1.0
2.0 0.8
1.5 0.6
1.0 0.4
0.5 0.2
0 0
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 15.5 15.6 15.7 15.8 15.9 16.0 16.1 16.2 16.3 16.4
Acquisition time (min) Acquisition time (min)

Salmon
×102 Robenidine 2.6 ng/g ×103
9 334.1 → 155.0 1.7
334.1 → 111.0 1.6
8 1.5 Ketoprofen 2.2 ng/g
1.4 255.1 → 77.1
7 1.3
1.2 255.1 → 209.1
6 1.1
Counts

1.0
Counts

5 0.9
4 0.8
0.7
3 0.6
0.5
2 0.4
0.3
1 0.2
0.1
0 0
8.15 8.25 8.35 8.45 8.55 8.65 8.75 8.85 8.95 9.05 9.15 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6
Acquisition time (min) Acquisition time (min)

Figure 5. Chromatographs of veterinary drugs in beef liver and salmon purchased from local grocery stores. Their concentrations are calculated based on the
spike-in concentration in the same matrix.

7
References
1. https://www.fsis.usda.gov/Oa/codex/system.
htm?redirecthttp=true
2. CODEX CAC/MRL 2-2015 on http://www.fao.org/fao-
who-codexalimentarius/standards/veterinary-drugs-mrls/
en/
3. L. Zhao, D. Lucas, Agilent Technologies Application Note,
publication number 5991-6096EN.
4. Joan Stevens, Agilent Technologies Application Note,
publication number 5991-6771EN.

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© Agilent Technologies, Inc., 2017

Printed in the USA
May 5, 2017
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