Food Anal.
Methods
DOI 10.1007/s12161-015-0238-z
Analysis of Multiple -Agonist and -Blocker Residues in Porcine
Muscle Using Improved QuEChERS Method and UHPLC-LTQ
Orbitrap Mass Spectrometry
Zhaohui Zhang 1 & Hua Yan 1 & Fengyun Cui 1 & Huan Yun 1 & Xiaohui Chang 1 &
Jianhui Li 1 & Xin Liu 1 & Lijun Yang 2 & Qiaoru Hu 2
Received: 4 February 2015 / Accepted: 22 June 2015
# Springer Science+Business Media New York 2015
Abstract The objective of the present study is to develop and
optimize a simple, high-throughput method for determining
14 -agonists (i.e., banbuterol, brombuterol, cimaterol,
cimbuterol, clenbuterol, clenpeterol, clorpenaline,
isoxsuprine, mabuterol, mapenterol, ractopamine, salbutamol,
terbutaline, and tulobuterol) and two -blockers (propranolol
and penbutolol) in porcine muscle. The samples were
pretreated by a modified quick, easy, cheap, efficient, rugged,
and safe (QuEChERS) method, and analysis was carried out
in a reversed phase HSS T3 C18 column using gradients of
acetonitrile and 5 mmol L1 ammonium acetate (0.1 % formic
acid) solution for elution. Ultra-high performance liquid chro-
matography coupled with high-resolution mass spectrometry
(UHPLC-LTQ Orbitrap MS, resolution 60,000) was used for
qualification and quantification of the 16 target compounds.
Under optimized conditions, the limits of detection and quan-
tification obtained ranged from 0.17 to 1.67 g kg1 and from
0.56 to 5.00 g kg1, respectively. Recoveries for spiking
levels of 5.0, 10.0, and 20.0 g kg1 ranged from 62.4 to
121.9 %, from 60.4 to 104.3 %, and from 66.5 to 121.3 %,
respectively. The relative standard deviations obtained were
lower than 20 % for all spiking levels assayed. The proposed
method was applied successfully in sample analysis, and sat-
isfactory results were obtained.
* Zhaohui Zhang
Zhangzhh@bjciq.gov.cn
1
Beijing Inspection and Quarantine Testing Center, Beijing Entry-Exit
Inspection and Quarantine Bureau, Beijing 100026, China
2
Weihai Entry-Exit Inspection and Quarantine Bureau,
Weihai, Shandong Prov 264200, China
Keywords -Agonist . -Blocker . QuEChERS . Porcine
muscle . UHPLC . LTQ Orbitrap XL
Introduction
-Agonists are a group of synthetic compounds that share
some similarity in their chemical structures. The most com-
monly used -agonists include clenbuterol, ractopamine, and
salbutamol. -Agonists can promote growth and increase
muscle leanness by inducing redistribution of fat in the muscle
tissues of certain food animal species, such as swine and cat-
tle. -Blockers are used during animal transport to prevent
sudden death. However, the residues of these compounds
can cause acute poisoning in humans, with symptoms includ-
ing muscular tremors, cardiac palpitations, nervousness, head-
ache, and muscular pain reported in clenbuterol residue-
induced food poisoning events (Martinez-Navarro 1990;
Pulce et al. 1991). On the grounds of safety, the Chinese gov-
ernment banned the use of ractopamine in animal feeds sev-
eral years ago. Nonetheless, some countries, such as the USA,
Australia, Canada, Japan, and Mexico, continue to apply
ractopamine as an animal feed additive. In 2012, the Codex
Alimentarius Commission established new ractopamine limits
for swine and bovine tissues: 10 g kg1 for muscle and fat,
40 g kg1 for liver, and 90 g kg1 for kidney (Codex
Alimentarius Commission 2012).
To detect residues of -agonists and -blockers in porcine
muscle and other biosamples, various analytical methods, in-
cluding enzyme immunoassay (Vanoosthuyze et al. 1997),
liquid chromatography with UV detection, and GC-MS, have
been employed (Silva-Forsberg et al. 1997; Hooijerink et al.
1994; Batjoens et al. 1996). However, derivatization by GC-
MS is time consuming. Compared with GC-MS, LC-MS has
the advantage of no requirement of derivatization procedures.

Ultra-high performance liquid chromatography coupled with
triple quadrupole MS (UHPLC-MS/MS) is a very sensitive
and selective technique for -agonist analysis (Shao et al.
2009; De Wasch et al. 1998; Nielen et al. 2008; Yang et al.
2012). Nielen et al. (Nielen et al. 2008) used HPLC-MS/MS
for multi-residue analysis of -agonists in bovine and porcine
urine, feed, and hair. Shao et al. (Shao et al. 2009) developed a
solid-phase extraction (SPE) method for cleanup coupled with
UHPLC-MS/MS to analyze 16 -agonists in swine liver, kid-
ney, and muscle. Yang et al. (Yang et al. 2012) developed a
UHPLC-MS/MS method to detect -agonist residues in milk.
Fan et al. (Fan et al. 2012, 2013) reported an HPLC-based
method coupled with linear ion trap MS to simultaneously
determine the residues of 25 -agonists and more than 20 -
blockers in animal feeds and urine samples.
To ensure the excellent high-throughput performance of
LC-MS instruments, sample preparation is an important step
in residue analysis of -agonists. Enzymatic hydrolysis is
generally used to pretreat animal tissue samples, and SPE is
used for cleanup. Reports show that quick, easy, cheap, effi-
cient, rugged, and safe (QuEChERS) method has been suc-
cessfully used to analyze a diverse range of pesticides in plant
tissues (Chai et al. 2012; Koesukwiwat et al. 2010; Cajka et al.
2008; Wilkowska and Biziuk 2011) as well as multiple classes
of veterinary drugs in food of animal origin (Stubbings and
Bigwood 2009; Kinsella et al. 2009; Villar-Pulido et al. 2011;
Lopes et al. 2012; Yan et al. 2013; Yogendrarajah et al. 2013).
Pesticides or veterinary drugs are extracted by acetonitrile,
and water is removed from the extract by salting out with
sodium chloride and magnesium sulfate. Cleanup is per-
formed by matrix dispersive SPE, which involves mixing of
the extract in a mixer with a sorbent, such as primary second-
ary amine (PSA), instead of passing it through an SPE col-
umn. QuEChERS is widely used in many labs and is more
than a promising technique, i.e., it has displaced cartridge-
based SPE in many labs. Len et al. (Len et al. 2012) devel-
oped a UPLC-HRMS screening method in full-scan mode
coupled with QuEChERS sample preparation for multi-drug
residues in bovine urine. Quatitation and QC procedure were
discussed. However, the sample matrix was single limited to
urine. Guo et al. (Guo et al. 2015) proposed data-dependent
MS2 mode by HRMS for -agonist residue analysis in cattle
and poltry meat followed by USFDA method validation
guideline.
In the present study, QuEChERS method was applied in the
pretreatment of -agonists and -blockers in porcine muscle
tissues for the first time. UHPLC combined with full-scan
high-resolution LTQ Orbitrap MS was then performed to an-
alyze 16 target compounds. At 60,000 resolution power, the
Orbitrap MS instrument presents advantages of resolving an-
alyte ions from co-eluting isobaric matrix compounds and
detecting low levels of analytes. Several validation parame-
ters, including accuracy, precision, linearity, limits of
Food Anal. Methods
detection (LODs), and limits of quantification (LOQs), were
examined, and the proposed methodology was successfully
utilized in real samples.
Experimental
Chemicals and Reagents
Banbuterol hydrochloride, brombuterol hydrochloride,
cimaterol, cimbuterol, clenbuteral hydrochloride, clenpeterol
hydrochloride, clorpenaline hydrochloride, isoxsuprine hy-
drochloride, mabuterol hydrochloride, mapenterol hydrochlo-
ride, penbutolol hydrochloride, propranolol hydrochloride,
ractopamine hydrochloride, salbutamol, terbutaline, and
tulobuterol hydrochloride were obtained from Dr.
Ehrenstorfer (Augsburg, Germany). Chemical structures of
the 16 compounds are shown in Fig. 1. Individual stock stan-
dard solutions (100 mg L1) were prepared by dissolving
10 mg of each compound in 100 mL methanol and then stored
at 20 C. A composite working standard solution
(10 mg L1) was prepared by mixing an appropriate volume
of each stock solution and diluting with methanol. Calibration
standard solutions were prepared daily by diluting the work-
ing standard solution with acetonitrile0.1 % formic acid
aqueous solution (30:70, v/v).
HPLC-grade acetonitrile, methanol, and ethyl acetate were
obtained from Fisher Scientific (Fair Lawn, NJ, USA). Formic
acid and ammonium acetate (purity >99 % for analysis) were
obtained from TEDIA (Fairfield, OH, USA). Cleanert Pestic
Carb (GCB, 120400 mesh), Cleanert PSA (4060 m),
Cleanert NH2 (4060 m), Cleanert Alumina-N (150 mesh),
Cleanert C18 (4060 m), and Florisil (60100 mesh) were
supplied by Bonna-Agela Technologies (Tianjin, China). So-
dium chloride and anhydrous magnesium sulfate were pur-
chased from Sinopharm Chemical Reagent Co., Ltd. (Shang-
hai, China). Purified water from a Milli-Q Elix ultra-pure wa-
ter system (Millipore, Beaford, MA, USA) was used during
analysis.
Instrumentation
Separation of the sample was performed on an UHPLC Ther-
mo Scientific Accela System (Thermo Scientific, San Jos,
USA). The UHPLC instrument was equipped with a degasser,
an auto sampler with a cooled sample tray, a column oven, and
a quaternary pump. Elution was performed at a stable temper-
ature of 30 C using a Water Acquity HSS T3 column
(150 mm2.1 mm i.d., 1.8 m particle size). Separation was
performed using 5 mmol L1 NH4Ac0.1 % formic acid as
solvent A and acetonitrile as solvent B at 0.25 mL min1 flow
rate in a gradient run. The elution program was as follows:
The initial mobile phase was composed of 80 % solvent A and
Food Anal. Methods
20 % solvent B, a linear gradient of solvent B from 20 to 50 %
in 0 to 6 min, an increase in solvent B from 50 to 90 % from
6.0 to 6.1 min, elution held for 2.5 min. The analytical column
was then equilibrated at initial conditions for 4 min. The total
run-to-run time was 12 min, and the injection volume was
10 L.
MS analysis was carried out using a Thermo LTQ Orbitrap
XL mass spectrometer (Thermo Fisher Scientific, San Jos,
USA). The instrument was operated using electrospray ioni-
zation in positive ion mode. The ionization source parameters
were electrospray voltage 4.5 kV, sheath gas flow rate
0.45 L min1, auxiliary gas flow rate 2.4 L min1, capillary
temperature 350 C, capillary voltage 47 V, and tube lens
offset 130 V. Instrument calibration using a calibration solu-
tion was performed externally prior to each sequence. Accu-
rate mass spectra of [M+H]+ ions were recorded from 100 m/z
to 1000 m/z, with the mass resolution power of the mass an-
alyzer set to 60,000 (m/m).
Sample Preparation
Extraction and cleanup were based on the QuEChERS method
(Anastassiades et al. 2003) with modifications as follows:
3.0 g homogenized tissue sample was weighed into a 50-mL
centrifuge tube. Then, 5 mL of H2O was added to the sample,
and the mixture was vortex mixed for 30 s. Afterward, 10 mL
of acetonitrile1 % HAc was added to the tube, which was
then shaken by hand for 30 s and extracted by ultrasonication
for 10 min. Then, 3 g MgSO4 and 0.5 g of NaCl were added to
the tube, and the mixture was vortex mixed for 1 min. Samples
were centrifuged at 8000 rpm for 10 min at 15 C. A 6 mL
aliquot of the upper acetonitrile layer was transferred into a
15-mL tube containing 150 mg of C18 sorbent. The mixture
was vortex mixed for 1 min and centrifuged at 1500 rpm for
10 min at 15 C. Then, 4 mL of the extract was transferred into
a 10-mL glass tube and dried by N2 gas in 45 C water bath.
Samples were reconstituted with 0.8 mL of acetonitrile0.1 %
aqueous formic acid solution (30:70 v/v) and then filtered
through a 0.2-m nylon syringe filter (Whatman, Florham
Park, NJ, USA). Filtered samples were finally transferred into
auto sampler vials for analysis.
Method Validation Procedure
For quantitation of the 14 -agonists and 2 -blockers in
porcine muscles, solvent calibration curves and matrix-
matched calibration curves using blank porcine muscles were
constructed. LODs and LOQs were estimated according to the
US EPAs Office of Pesticide Programs (US 2000), where
LOD =t0.99 S (for 6 degrees of freedom) and LOQ = 3
LOD. Recoveries were measured in spiked samples at three
concentration levels (5.0, 10.0, and 20.0 g kg1) based on six
replicates. The spiked samples were vortex mixed and left to
stand for 30 min for sufficient stabilization. The spiked sam-
ples were analyzed, and recoveries were calculated by com-
paring the measured concentration with the nominal concen-
trations. The accuracy and precision of the proposed method
were described by means of absolute recovery and repeatabil-
ity (n=6 replicates per concentration level).
Results and Discussion
Optimization of UHPLC Conditions
The UHPLC gradient, mobile phase, and analytical column
were evaluated to achieve the best separation performance.
Two eluent compositions were tested: the first using a gradient
of acetonitrile0.1 % aqueous formic acid solution and the
second using a gradient of methanol0.1 % aqueous formic
acid solution. The separation performance and sensitivity of
the two mobile phases under the same gradient condition were
compared (Fig. 2). The acetonitrile0.1 % formic acid system
gave better separation performance than the methanol0.1 %
aqueous formic acid system. Specifically, clorpenaline, clen-
buterol, tulobuterol, isoxsuprine, brombuterol, and mabuterol
(peaks 5, 6, 10, 11, 12, and 13, respectively) were only partly
separated under the methanol0.1 % aqueous formic acid sys-
tem but completely separated under the acetonitrile0.1 %
formic acid system. The retention times of the compounds
under these two mobile phase systems are listed in Table 1.
Based on the results obtained, the acetonitrile0.1 % aqueous
formic acid solution gradient was selected as the mobile phase
for further experiments.
Theoretically, the retention of a compound on a reversed
phase column depends on its polarity, compounds with high
polar may be co-eluted with matrix. The log P values (parti-
tion coefficient) of the 16 compounds analyzed in this study
ranged from 0.9493 to 4.2465 (Table 1) (calculated by
ChemOffice software). In order to promote the retention of
high polar compounds on reversed phase column, the Water
Acquity BEH C18 (150 mm2.1 mm i.d., 1.7 m) and Water
Acquity HSS T3 (150 mm2.1 mm i.d., 1.8 m) columns
were compared. The advantage of the T3 column is that T3
end capping of octadecyl chains promotes more retention in
high polar compounds than traditional trimethyl silane treat-
ment and end capping. Compared with the Acquity BEH C18
column, all compounds were more strongly retained on the
HSS T3 column, especially compounds with high polarity,
such as salbuteral and cimaterol (log P values 0.9728 and
0.9493, respectively). The retention times of salbuteral and
cimaterol were 1.93 min on the BEH C18 column and
2.07 min on the HSS T3 column. As a result, the Water
Acquity HSS T3 column was selected as the analytical
column.
 Food Anal. Methods

OH HO
H NC OH
N OH H
OH NC N
H2N
NH N
HO HO H
H2N
OH
Salbutamol Terbutaline Cimbuterol
Cimaterol
OH
H
Cl Cl N O N
OH
OH O OH
H2N H
O N
N
NH H N O
Cl
O
Clorprenaline Clenbuterol Banbuterol Penbutolol

Br
OH OH OH
H HO
N H H2N
HO N N
NH OH Br H
Cl O

Ractopamine Tulobuterol Isoxsuprine Brombuterol

F F OH OH H
H H
F3C N N
F OH Cl N
H2N H O OH
N H2N H2N
Cl Cl Cl
Mabuterol Mapenterol Clenpeterol Propranolol

Fig. 1 Chemical structures of the 16 compounds

LTQ Orbitrap MS Detection wherein compounds from the matrix with masses very close
to that of the analyte may be co-eluted with the analyte. When
Resolution power is a key parameter that affects the correct the two masses are not fully resolved by the MS detector, the
assignment of masses for analytes during residue analysis, measured mass profile represents the sum of the two

(A) (B)
100 1 100 1
50
50
0
0
100 2 100 2
50
50
0
0
100 3 5 6 100 5 6
Relative abundance

4 4
50 50 3
0 0
100 7 8 100 7 8
50 50
0
0 100
100 10 11 10
9 11
9 50
50
0 0
100 13 14 100 12, 13 14
12
50 50
0
0
100 16 100 16
50 15 50 15
0 0
0 2 4 6 8 0 2 4 6 8
Time (min) Time (min)

Fig. 2 UHPLC LTQ Orbitrap MS chromatograms of the mixed standard penbutolol, 9 ractopamine, 10 tulobuterol, 11 isoxsuprine, 12
(20 ng mL1) in different mobile phases: a acetonitrile0.1 % formic acid brombuterol, 13 mabuterol, 14 mapenterol, 15 clenpeterol, 16
and b methanol0.1 % formic acid. 1 Salbutamol, 2 cimaterol, 3 propranolol
terbutaline, 4 cimbuterol, 5 clorpenaline, 6 clenbuterol, 7 banbuterol, 8
Food Anal. Methods

Table 1 Names, formulas, exact masses, log P, and retention times of 16 compounds

No. Compound Formula Theoretical Actual Mass accuracy Log P Retention time (min) Retention time (min)
name [M+H]+ [M+H]+ (ppm) ACN MeOH

1 Salbutamol C13H21NO3 240.15942 240.15936 0.25 0.9728 1.62 2.12

2 Cimaterol C12H17N3O 220.14444 220.14438 0.27 0.9493 1.62 2.12
3 Terbutaline C12H19NO3 226.14377 226.14397 0.88 1.1577 1.64 2.12
4 Cimbuterol C13H19N3O 234.16009 234.16013 0.17 1.1672 2.22 3.77
5 Clorpenaline C11H16ClNO 214.09932 214.09930 0.093 2.2770 3.66 7.08
6 Clenbuterol C12H18Cl2N2O 277.08690 277.08685 0.18 2.2503 4.45 7.26
7 Banbuterol C18H29N3O5 368.21800 368.21768 0.87 1.3953 4.87 8.35
8 Penbutolol C18H29NO2 292.22711 292.22739 0.96 3.4383 7.89 8.93
9 Ractopamine C18H23NO3 302.17507 302.17517 0.33 4.2465 3.54 6.62
10 Tulobuterol C12H18ClNO 228.11497 228.11504 0.31 2.4949 4.35 8.32
11 Isoxsuprine C18H23NO3 302.17507 302.17517 0.33 2.9732 5.22 8.25
12 Brombuterol C12H18Br2N2O 366.98382 366.98352 0.82 2.9719 5.01 8.25
13 Mabuterol C13H19Cl2F3N2O 311.11325 311.11316 0.29 2.6132 5.40 8.19
14 Mapenterol C14H20F3N2O 325.1289 325.12909 0.58 3.0993 6.15 8.47
15 Clenpeterol C13H20Cl2N2O 291.10255 291.10233 0.76 2.7364 5.29 8.32
16 Propranolol C16H21NO2 260.16451 260.16455 0.15 2.6470 6.11 8.58

individual profiles. High-resolution MS increases the selectiv-
ity of a monitored mass trace and discriminates matrix inter-
ferences (Kaufmann et al. 2010). High-resolution power (50,
000) is necessary to discriminate interferences from the matrix
at the same nominal mass (Kellmann et al. 2009). In this study,
measurements were performed at four resolution power set-
tings of 7500, 15,000, 30,000, and 60,000. When the resolu-
tion power was set to 60,000, good mass accuracies (from
0.093 to 0.96 ppm, Table 1) were obtained for the target com-
pounds. Accurate mass measurements, along with character-
istic retention times, were used to confirm the target com-
pounds. Quantification was performed using the extracted
ion chromatograms of m/z values corresponding to protonated
molecules with 5 ppm accurate mass windows
Optimization of the Sample Extraction Procedure
Sample preparation is a critical step in the simultaneous ex-
traction of multiple residues. In the original QuEChERS ex-
traction method (Len et al. 2012), pesticide residues in plant-
origin food were extracted using organic solvents, such as
acetonitrile and ethyl acetate, and liquidliquid partitioning
through addition of MgSO4NaCl mixture. The samples
were then cleaned by dispersive-SPE (d-SPE). Different

Fig. 3 Comparison of the 120

recoveries of 16 target ACN
compounds with different ACN - 1% FA
extraction solvents at 10 g kg1 100
ACN -1% HAc
ACN - 1% NH4OH
spiking level: (116 represent the
same compounds as in Fig. 2)
80
Recovery (%)

60

40

20

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
 Food Anal. Methods

Fig. 4 Comparison of the 160

Al2O3 C18
recoveries of 16 target
Florisil GCB
compounds with different 140 NH2 PSA
cleanup sorbents at 10 g kg1
spiking level (116 represent the
same compounds as in Fig. 2) 120

100

Recovery (%)
80

60

40

20

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

parameters, such as water content, extraction solvent, compo- with 1 % HAc was selected as the extraction solvent for sub-
sition of salt, and type of cleanup sorbents, influence the ex- sequent experiments.
traction efficiency.
First, the influence of water content on sample extraction
Selection of the Cleanup Sorbent
was examined. As the water content in porcine muscle tissue
sample is low, addition of anhydrous MgSO4 could create
To evaluate the performance of the cleanup sorbent, a blank
colloids that hinder vortex mixing, and the volume of the
matrix solution (acetonitrile1 % HAc extraction solution)
acetonitrile supernatant obtained after centrifugation is lower
was prepared following the procedure described in BSample
than the added volume. Thus, addition of a proper volume of
Preparation^ section. The matrix was spiked with the standard
water to the tissue sample is necessary because doing so im-
proves the dispersity of the sample and increases the contact Table 2 Slopes of solvent-based and matrix-matched standards, LODs,
area of muscle particles with acetonitrile, thereby improving and LOQs of 16 target compounds
extraction efficiency. In this study, the effects of volumes of
No. Compound Slope LOD (g kg1) LOQ (g kg1)
added water from 0 to 8.0 mL on separation efficiency were
compared, and the best result was obtained when 5 mL of H2O Solvent Matrix
was added to porcine muscle tissue prior to acetonitrile
addition. 1 Salbutamol 23482 10082 1.67 5.00
Second, different extraction solvents were compared. A 2 Cimaterol 7261 3012 0.76 2.54
suitable extraction solvent must have high extraction capabil- 3 Terbutaline 16700 14534 1.67 5.00
ity for target analytes and can be salted out by further addition 4 Cimbuterol 26310 19182 1.63 4.89
of MgSO4 and NaCl. Methanol was discarded because it can- 5 Clorpenaline 58213 50392 1.07 3.58
not be salted out. Ethyl acetate exhibited poor recoveries (be- 6 Clenbuterol 40420 36262 0.40 1.20
low 20 %) for salbutamol, terbutaline, cimaterol, cimbuterol, 7 Banbuterol 118540 104765 0.17 0.56
and banbuterol. The recoveries were improved when acetoni- 8 Penbutolol 227451 192336 0.38 1.28
trile was used. 9 Ractopamine 68352 61180 0.24 0.81
The type of acid or base added to the acetonitrile solution 10 Tulobuterol 49982 44621 0.42 1.41
was further studied. The effects of formic acid, acetic acid, and 11 Isoxsuprine 32990 19743 0.54 1.79
aqueous ammonia were evaluated, and the experimental re- 12 Brombuterol 62320 55748 0.19 0.65
sults are shown in Fig. 3. The lowest recoveries were obtained 13 Mabuterol 86943 81016 0.50 1.65
when formic acid was used. Compared with formic acid, re- 14 Mapenterol 117303 113195 0.90 2.99
coveries for most -agonists except cimaterol (below 30 %) 15 Clenpeterol 44200 38336 0.45 1.50
improved when aqueous ammonia was used. The best results 16 Propranolol 132924 125090 0.94 3.13
were obtained when acetic acid was used. Thus, acetonitrile
Food Anal. Methods

1.0
respectively, when C18 was used. When Florisil was used, the
recoveries of the 16 target compounds ranged from 55.7 %
0.8
(banbuterol) to 140 % (salbutamol). When C18 sorbent was
used, the recoveries ranged from 73.0 to 119.8 %. Based on
0.6
Slope ratio

the results obtained, C18 was selected as the cleanup sorbent

in subsequent experiments.
0.4

Method Performance Characteristics

0.2

0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Compound
Fig. 5 Matrix/solvent slope ratios for 16 target compounds (116
represent the same compounds as in Fig. 2)
solution to obtain a matrix-spiked solution. Exactly 150 mg of
C18, NH2, Alumina-N, PSA, GCB, and Florisil were weighed
separately into d-SPE tubes (three replicates). Then, 5 mL of
the spiked matrix solution was added to each tube, which was
subsequently vortex mixed for 30 s and centrifuged. A 4 mL
aliquot of the upper layer was transferred into a 10-mL cen-
trifuge tube and dried under a N2 stream in a 45 C water bath.
The recoveries of each group were compared, and the exper-
imental results are shown in Fig. 4. The lowest average recov-
ery (72.4 %) was obtained when NH2 was used, and the
highest average recovery (95.9 %) was obtained when C18
was used. The recoveries of cimaterol and cimbuterol were
significantly influenced by the sorbents. The recoveries of
these compounds were lower than 50 % when PSA, Alumi-
na-N, GCB, and NH2 were used but reached 85 and 91 %,
Matrix Effects
To evaluate matrix effects, solvent-based and matrix-matched
standards were analyzed at concentrations ranging from 1 to
100 g L1, and the slopes for each compound were obtained
(Table 2). Matrix/solvent slope ratios for each compound were
calculated to evaluate matrix effects (Fig. 5). A signal en-
hancement or suppression effect was acceptable if the slope
ratio fell between 0.8 and 1.2. Slope ratios higher than 1.2 or
lower than 0.8 indicate strong enhancement or suppression
matrix effects. The experimental results showed that signifi-
cant matrix suppression effects were observed for salbuterol,
cimaterol, cimbuterol, and isoxsuprine, whereas acceptable
matrix effects were observed for the rest of the compounds.
Therefore, the use of matrix-matched standards is necessary
for quantification of real samples.
Linearity, LODs, and LOQs
The linearity of the method was evaluated using matrix-
matched calibration curves obtained as described in BMethod

Table 3 Recoveries and RSDs of -agonists and -blockers in spiked porcine muscle samples (n=6)

Spiked level 5 g kg1 10 g kg1 20 g kg1

No. Compound Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%)

1 Salbutamol 95.8 7.7 104.3 9.83 83.9 19.0

2 Cimaterol 121.9 9.0 88.7 18.8 121.3 1.5
3 Terbutaline 76.4 9.0 71.4 10.5 83.5 12.1
4 Cimbuterol 62.4 16.5 69.2 15.6 66.5 13.4
5 Clorprenaline 84.6 5.5 86.2 5.34 77.5 10.4
6 Clenbuterol 77.6 10.7 78.8 2.64 70.4 12.1
7 Banbuterol 82.4 4.9 82.2 2.82 79.2 12.5
8 Penbutolol 76.8 12.5 66.7 3.95 89.8 11.9
9 Ractopamine 81.0 10.2 77.0 5.61 82.0 8.6
10 Tulobuterol 84.8 1.4 84.8 3.84 82.1 16.8
11 Isoxsuprine 93.2 7.8 103.8 8.27 84.8 14.3
12 Brombuterol 82.6 11.9 78.1 6.33 82.4 6.2
13 Mabuterol 79.7 13.9 73.0 6.60 81.4 9.0
14 Mapenterol 80.3 12.9 70.6 4.69 85.8 6.4
15 Clenpeterol 70.9 13.1 60.4 9.51 83.3 8.9
16 Propranolol 72.5 3.7 70.5 6.43 88.5 5.40
 Food Anal. Methods

Fig. 6 a Extracted ion (D) OH H2

100 (A) N
chromatogram corresponding to OH

Relative Abundance
the positive finding of 80
ractopamine in a porcine tissue HO
sample, b accurate mass spectrum 60
corresponding to the detected 40 C18H24NO3+ m/z 302.1751
peak, c HCD fragmental ion mass
20 -H2O
spectrum corresponding to m/z
302.17508, d proposed 0 H2
dissociation mechanism of 0 2 4 6 8 10 12 N O
ractopamine 302.17508 H
100 (B)
Relative Abundance
HO
80
C18H22NO2+ m/z 284.1645
60
OH
40 -
300.92041 305.19385
20 303.17850
299.22015 306.19724 H2
0 N
300 302 304 306 308
HO
(C) 121.06498
100
Relative Abundance

164.10698 C10H14NO+ m/z 164.1070

80
107.04945 -NC2H5
60
40
20 218.11743
208.88300 284.16425 H2O
0
50 100 150 200 250 300 C8H9O+ m/z 121.0646
m/z

Validation Procedure^ section. The calibration curves were
linear in the range of 0.520 g kg1. The correlation coeffi-
cients (R2) of the matrix calibration curves were higher than
0.9900 for all compounds. LODs and LOQs ranged from 0.17
to 1.67 g kg1 and from 0.56 to 5.00 g kg1, respectively
(Table 2). These results demonstrate that the sensitivity of the
proposed method is high enough for application in the quan-
titative analysis of trace residues of -agonists and -blockers
in real tissue samples.
Accuracy and Precision
The recovery of the method was studied by spiking at three
concentration levels from 5.0 to 20.0 g kg1. Tests for each
concentration were repeated six times, and the results obtained
are listed in Table 3. The recoveries for spiking levels of 5.0,
10.0, and 20.0 g kg1 ranged from 62.4 to 121.9 %, from
60.4 to 104.3 %, and from 66.5 to 121.3 %, respectively. The
repeatability of the proposed method is illustrated by RSDs in
Table 3. The RSDs for spiking levels of 5.0, 10.0, and
20.0 g kg1 ranged from 1.42 to 16.5 %, from 2.64 to
18.8 %, and from 1.54 to 10.0 %, respectively.
Application of the Method
The proposed method was applied to 246 samples, including
chicken, bovine, sheep, and porcine tissues, collected from
local markets in Beijing. Matrix-matched calibration was used
during quantification. A reagent blank and a spiked sample at
10 g kg1 were set up for quality control. The retention times
of detected ions in real samples were compared with those of
corresponding calibration standards in the same batch to con-
firm the identity of the detected analytes. Positive results were
found in one sample (clenbuterol, 1.76 g kg1) and three
samples (ractopamine, 1.84, 2.37, and 2.85 g kg1, respec-
tively). Figure 6 shows the extracted ion chromatogram cor-
responding to ractopamine and its accurate mass spectrum.
Higher energy collisional dissociation (HCD) fragmentation
was applied to determine the fragment ions of ractopamine;
fragment ions with m/z 284.16425, 164.10698, and
121.06498 were found. Highly valuable structural information
and dissociation mechanism of ractopamine was deduced
from the HCD fragmental information, which gives unambig-
uous confirmation of the positive sample.
Conclusions
In this work, an improved QuEChERS method coupled with
UHPLC-LTQ Orbitrap MS was developed for the rapid sam-
ple pretreatment and simultaneous determination of 14 -
agonists and 2 -blockers in porcine muscle samples. The
method was validated, and good linearity, accuracy, and pre-
cision were demonstrated. The QuEChERS method has
Food Anal. Methods
potential use as a generic sample preparation method for mul-
tiple residue analysis of animal tissues.
Acknowledgments This work was supported by AQSIQ industrial
public service scientific research project of the Ministry of Science and
Technology of P. R. China (201210029), AQSIQ industrial public service
scientific research project of P.R. China (201310143), and Science and
Technology Planning Project of General Administration of AQSIQ of
China (2014IK107).
Compliance with Ethics Requirements Zhaohui Zhang has received
research grants from AQSIQ industrial public service scientific research
project of the Ministry of Science and Technology of P.R. China
(201210029).
Hua Yan has received research grants from Science and Technology
Planning Project of General Administration of AQSIQ of China
(2014IK107).
Lijun Yang has received research grants from AQSIQ industrial public
service scientific research project of P.R. China (201310143).
Conflict of Interest Fengyun Cui declares that she has no conflict of
interest.
Huan Yun declares that she has no conflict of interest.
Xiaohui Chang declares that she has no conflict of interest.
Jianhui Li declares that he has no conflict of interest.
Xin Liu declares that she has no conflict of interest.
Qiaoru Hu declares that she has no conflict of interest.
This article does not contain any studies with human or animal
subjects.
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