Identification of Buctopamine and Mebuctopamine, a β2 Receptor Agonist and Its Metabolite, in Swine Hair and Feed Additives

Article abstract of DOI:10.1021/acs.jafc.7b00130

4-[2-(t-Butylamino)-1-hydroxyethyl]phenol (buctopamine, 4), a new β₂ receptor agonist (β₂-agonist), was found to be an adulterant in feed additives for swine in Taiwan, where using β₂-agonists in food-production animals is

Full text of DOI:10.1021/acs.jafc.7b00130

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Article
Identification of Buctopamine and Mebuctopamine, a #2 Receptor
Agonist and Its Metabolite, in Swine Hair and Feed Additives
Ying-Heng Chen, Chia-Ying Yang, Chih Wen Cheng, Yi-Ying Lin, Su Lien Kuo, and Ling-Wei Hsin
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017
Downloaded from http://pubs.acs.org on April 30, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 31
Journal of Agricultural and Food Chemistry
1
2
Identification of Buctopamine and Mebuctopamine, a β2 Receptor Agonist
and Its Metabolite, in Swine Hair and Feed Additives
3
4
5
6
YingꢀHeng Chen,† ChiaꢀYing Yang,† Chih Wen Cheng,§ YiꢀYing Lin,† Su Lien Kuo,§
LingꢀWei Hsin,*,†,‡,#
7
8
†School of Pharmacy, ‡Molecular Probes Development Core, Molecular Imaging Center, and
#Center for Innovative Therapeutics Discovery, National Taiwan University, 17, Xuzhou Road,
Room 936, Taipei 10055, Taiwan.
9
10
§Technical Service Center, National Animal Industry Foundation, 100, Section 2, Heping West
11
12
Road, 5F., Taipei 10070, Taiwan.
13
14
15
16
17
* To whom correspondence should be addressed. Tel.: +886ꢀ2ꢀ3366ꢀ8696. Fax:
+886ꢀ2ꢀ2351ꢀ2086. Eꢀmail: lwhsin@ntu.edu.tw.
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 31
18 Abstract
19
20
4ꢀ[2ꢀ(tertꢀButylamino)ꢀ1ꢀhydroxyethyl]phenol (buctopamine, 4), a new β2 receptor agonist
(β2ꢀagonist), was found to be an adulterant in feed additives for swine in Taiwan, where using
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
β2ꢀagonists in foodꢀproduction animals is prohibited. Buctopamine and its metabolite,
4ꢀ[2ꢀ(tertꢀbutylamino)ꢀ1ꢀhydroxyethyl]ꢀ2ꢀmethoxyphenol (mebuctopamine, 2), were detected
in swine hair specimens. Authentic compounds 2 and 4 were synthesized with 98.6% and
97.7% purity, respectively, as reference standards for analysis, and both compounds were
more hydrophilic than ractopamine and clenbuterol. In a preliminary pharmacological
evaluation, compounds 2 and 4 exhibited moderate human β2 receptor binding affinity and did
not show significant affinities for the human α1, α2, β1, and β3 receptors. After adding
compounds 2ꢀ4 into the β2ꢀagonist library, a multiꢀresidue analysis of 26 β2ꢀagonists by using
triple quadrupole LC/MS/MS for routine screening conducted by regulatory authorities was
established, in which the common limits of quantification for the 26 β2ꢀagonists in swine feed
and hair are 10 and 25 ng/g, respectively. In addition, the illegal use of buctopamine (4) has
been effectively prevented. The results of this study are also useful for controlling the illegal
use of new β2ꢀagonists in foodꢀproduction animals.
Key words: β2ꢀagonist, hair, feed additive, metabolite, identification
2
ACS Paragon Plus Environment

Page 3 of 31
Journal of Agricultural and Food Chemistry
38 INTRODUCTION
39
40
41
42
43
44
β2 receptor agonists (β2ꢀagonists) are sympathomimetic agents that are commonly used
as bronchodilators for the clinical treatment of asthma. When used as feed additives for
foodꢀproduction animals, β2ꢀagonists can promote growth rates, increase muscle mass, and
reduce fat deposition, feed consumption, and therefore, breeding costs.1 However, β2ꢀagonists
produce several undesirable behavioral side effects in livestock, especially pigs, such as
agitation, restlessness, and aggressive behavior.
45
46
47
48
49
50
51
52
53
54
55
The β2ꢀagonist residues that accumulate in animal tissues can cause acute poisoning
symptoms in humans.1ꢀ3 For instance, several outbreaks of food poisoning in humans
involving clenbuterol have occurred in Europe.2,3 In 2006, reports from Shanghai, China,
involved 300 cases of people who were poisoned by eating pork products contaminated by
clenbuterol. Although no deaths occurred,4 these incidents were substantial enough to create
public resistance to using β2ꢀagonists as feed additives for foodꢀproduction animals. In the
United States, both ractopamine and zilpaterol (Chart 1) are used as feed additives for cattle
and swine, whereas, in Australia, ractopamine can only be used in swine, and the use of
zilpaterol is prohibited. In consideration of their potential hazards to human health, the use of
β2ꢀagonists as growth promoters in animal breeding has been banned in more than 120
countries, such as those within the European Union (EU), China, and Russia.1ꢀ3
56
57
58
59
60
61
In Taiwan, the Food and Drug Administration, Ministry of Health and Welfare,
collaborates with the Bureau of Animal and Plant Health Inspection and Quarantine
(BAPHIQ), Council of Agriculture (COA), closely to ensure food safety. The use of
β2ꢀagonists as feed additives is prohibited, and seven β2ꢀagonists are officially listed in the
law, including ractopamine, zilpaterol, clenbuterol, cimaterol, salbutamol, terbutaline, and
tulobuterol (Chart 1). Several new β2ꢀagonists have recently been found to be used in
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 31
62
63
64
65
66
67
68
69
70
71
feedstuffs to elude official control.5ꢀ8 In the spring of 2010, an outbreak of illicit new synthetic
β2ꢀagonist use occurred in Taiwan. Rapid screening of swine hair samples by using a
salbutamol (1, Chart 1) ELISA kit demonstrated strong positive results.9,10 However, the
unknown ingredient was not one of the seven β2ꢀagonists in the prohibited list. Therefore, the
routine inspection conducted by the local authorities utilizing standard LC/MS/MS screening
methods for multiple β2ꢀagonists was incapable of identifying this novel β2ꢀagonist in the feed
and tissue specimens.11 The suspicious material was named “Garlic Essential” and had been
sold to local farmers as a new feed additive for swine. The salesmen claimed it contained the
“No.8 lean meat agent”, which could escape the regular screening control of the local
government.
72
73
74
75
76
77
78
79
80
Here,
we
report
the
identification
of
4ꢀ[2ꢀ(tertꢀbutylamino)ꢀ1ꢀhydroxyethyl]ꢀ2ꢀmethoxyphenol (2, mebuctopamine), a β2ꢀagonist
metabolite found in swine hairs, followed by the identification of the novel β2ꢀagonist
4ꢀ[2ꢀ(tertꢀbutylamino)ꢀ1ꢀhydroxyethyl]phenol (4, buctopamine) in feed additives and swine
hairs. In addition, we have synthesized authentic samples as reference standards for analysis,
established an analytical method for the legal inspection of the residues of multiple
β2ꢀagonists, including 2, colterol (3) and 4 (Chart 1), and determined the lipophilicity and
receptor binding affinities of compounds 2ꢀ4.
81 MATERIALS AND METHODS
82
83
84
85
Chemicals and Samples. Absolute ethanol was purchased from J. T. Baker (Center
Valley, PA, USA). Acetovanillone, sodium heptanesulfonate, and bromine were purchased
from Acros (Geel, Belgium). 4'ꢀHydroxyacetophenone, N,Nꢀdimethylformamide (DMF),
EtOAc, MeCN, MeOH, and silica gel 60 (40–63 ꢀm) for flash column chromatography were
4
ACS Paragon Plus Environment

Page 5 of 31
Journal of Agricultural and Food Chemistry
86
87
88
89
90
91
92
93
94
95
purchased from Merck (Darmstadt, Germany). 4ꢀ(Chloroacetyl)catechol was purchased from
TCIꢀJP (Tokyo, Japan). tꢀButylamine and 10% Pd/C were purchased from Alfa Aesar
(Lancashire, UK). 2ꢀPropanol (IPA) and MgSO4 were purchased from Fisher (Leicestershire,
UK). CHCl3 was purchased from Seedchem (Melbourne, Australia). Na2CO3 was purchased
from SigmaꢀAldrich (Saint Louis, USA). Hydrochloric acid and glacial acetic acid were
purchased from Scharlau (Barcelona, Spain). DMSOꢀd6 was purchased from Cambridge
Isotope Laboratory, Inc. (Tewksbury, MA, USA). Deionized water was prepared with a
MilliꢀQ Plus system at 18.2 Mꢁ (MilliPore, Bedford, MA, USA). Swine hair and feed
additive samples were collected as part of the official control by BAPHIQ, COA, in Taiwan
from local livestock farms.
96
97
Synthesis of Authentic Standards. General procedures － Melting points were
98
99
determined on a MELꢀTEMP II melting point apparatus (Barnstead/ Thermolyne Corp.,
Dubuque, IA, USA) and are uncorrected. NMR spectra were recorded on a Bruker DPXꢀ200
FTꢀNMR spectrometer (Bruker, Billerica, MA, USA). Chemical shifts are expressed in parts
per million (ppm) on the δ scale relative to a tetramethylsilane (TMS) internal standard.
Electrospray ionization (ESI) mass spectrometry and highꢀresolution mass spectrometry
(HRMS) spectra of authentic standards 2ꢀ4 were obtained with Esquire 2000 and Bruker
Daltonik micrOTOF mass spectrometers (Bruker Daltonics Inc., Billerica, MA, USA),
respectively. No attempt was made to optimize yields. Treatment of acetovanillone (5a) and
4'ꢀhydroxyacetophenone (5b) with benzyl bromide and K2CO3 in DMF yielded compounds
6a and 6b as light yellow solids in 99% and 97% yields, respectively (Scheme 1).
100
101
102
103
104
105
106
107
108
109
1-(4-(Benzyloxy)-3-methoxyphenyl)-2-(tert-butylamino)ethanone (7a). Bromine (0.31
mL, 6.00 mmol) was added to a solution of compound 6a (1.28 g, 5.00 mmol) in ethanol (30
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 31
110
111
112
113
114
115
116
117
118
119
120
mL) under nitrogen at room temperature and stirred for 1 h. The reaction mixture was cooled
in an ice bath, and the white crystalline solid was collected by filtration (1.23 g, 3.68 mmol,
74%). This product was directly used in the following reaction without further purification.
tꢀButylamine (1.0 mL, 696 mg, 9.52 mmol) was added to a solution of the bromide (1.00 g,
2.98 mmol) in DMF (5 mL) and stirred at room temperature for 1 h under N2. The mixture
was poured into H2O (30 mL), and the precipitate was collected and dried to obtain compound
1
7a as a light yellow solid (920 mg, 2.81 mmol, 94%). H NMR (200 MHz, CDCl3) δ 1.15 (s,
9, CH3), 2.27 (bs, 1, NH), 3.95 (s, 3, OCH3), 4.08 (s, 2, COCH2), 5.23 (s, 2, OCH2), 6.89 (d, 1,
J = 8.3 Hz, aromatic), 7.29ꢀ7.58 (m, 7, aromatic); 13C NMR (50 MHz, CDCl3) δ 29.0 (3C),
49.0, 50.4, 56.3, 70.9, 110.6, 112.4, 122.0, 127.3 (2C), 128.3, 128.8 (2C), 129.1, 136.3, 149.8,
152.7, 196.7; ESIꢀHRMS calcd for C20H25NO3Na [M+Na]+, 350.1732; found, 350.1733.
121
122
123
124
125
126
127
1-(4-(Benzyloxy)phenyl)-2-(tert-butylamino)ethanone (7b). Compound 7b was
synthesized from 6b following the procedures for the preparation of compound 7a to afford a
1
white solid (75%, two steps). H NMR (200 MHz, CDCl3) δ 1.16 (s, 9, CH3), 2.09 (bs, 1,
NH),4.10 (s, 2, COCH2), 5.13 (s, 2, OCH2), 6.93ꢀ7.07 (m, 2, aromatic), 7.28ꢀ7.52 (m, 5,
aromatic), 7.88ꢀ8.00 (m, 2, aromatic); 13C NMR (50 MHz, CDCl3) δ 29.0 (3C), 49.0, 50.4,
70.3, 114.8 (2C), 127.6 (2C), 128.4, 128.8 (2C), 130.1 (2C), 136.2, 162.9, 196.5; ESIꢀHRMS
calcd for C19H24NO2 [M+H]+, 298.1802; found, 298.1805.
128
129
130
131
132
133
134
4-[2-(tert-Butylamino)-1-hydroxyethyl]-2-methoxyphenol (2, mebuctopamine). An
excess amount of HCl in CH2Cl2 was added to a solution of 7a (450 mg, 1.37 mmol) in
CH2Cl2 (5 mL), and the resulting mixture was evaporated to afford 7a•HCl as a white solid.
Catalytic hydrogenation of 7a•HCl in ethanol (5 mL) using 10% Pd/C (251 mg) under H2
(120 psi) for 2 h followed by filtration through celite and evaporation provided the HCl salt of
the crude product. The crude solid was dissolved in a mixture of NaHCO3(sat) (35 mL) and
EtOAc (22 mL), and then the organic layer was washed with brine, dried over MgSO4,
6
ACS Paragon Plus Environment

Page 7 of 31
Journal of Agricultural and Food Chemistry
135
136
137
138
139
140
141
142
filtered, and evaporated. The solid residue was chromatographed (silica gel, MeOH/CH2Cl2 =
1/50) to afford compound 2 as a light yellow solid (256 mg, 1.07 mmol, 78%). Purity (HPLC)
98.6%; mp 144ꢀ145oC; 1H NMR (200 MHz, CDCl3) δ 1.11 (s, 9, CH3), 2.61 (dd, 1, J = 11.7,
8.9 Hz, CHCH2), 2.85 (dd, 1, J = 11.7, 3.7 Hz, CHCH2), 3.88 (s, 3, OCH3), 4.57 (dd, 1, J =
8.9, 3.7 Hz, CHCH2), 6.75ꢀ6.84 (m, 1, aromatic), 6.87 (d, 1, J = 8.0 Hz, aromatic), 6.94 (d, 1,
J = 1.7 Hz, aromatic) (Figure 1A); 13C NMR (50 MHz, CDCl3) δ 28.9 (3C), 50.4, 50.7, 55.9,
72.2, 108.6, 114.5, 118.7, 134.8, 145.2, 146.9 (Figure 1B); ESIꢀHRMS calcd for
C13H21NO3Na [M+Na]+, 262.1414; found, 262.1420.
143
144
145
146
147
148
149
4-[2-(tert-Butylamino)-1-hydroxyethyl]benzene-1,2-diol (3, colterol). Compound 3
was prepared from 4ꢀ(chloroacetyl)catechol following the literature procedure to afford a light
yellow solid.12 1H NMR (200 MHz, DMSOꢀd6) δ 1.01 (s, 9, CH3), 2.52ꢀ2.55 (m, 2, CHCH2),
4.33 (t, 1, J = 6.3 Hz, CHCH2), 6.55 (d, 1, J = 8.0 Hz, aromatic), 6.65 (d, 1, J = 8.0 Hz,
aromatic), 6.69ꢀ6.78 (m, 1, aromatic); 13C NMR (100 MHz, DMSOꢀd6) δ 28.8 (3C), 49.7,
50.7, 72.2, 113.5, 115.1, 116.7, 135.6, 144.2, 145.0; ESIꢀHRMS calcd for C12H20NO3 [M+H]+,
226.1438; found, 226.1435.
150
151
152
153
154
4-[2-(tert-Butylamino)-1-hydroxyethyl]phenol (4, buctopamine). Compound 4 was
synthesized from 7b following the procedures for the preparation of compound 2 to afford a
yellow solid (66%). An analytical sample was obtained by recrystallization (EtOAc) to grant
1
an ivory white solid. Purity (HPLC) 97.7%; mp 166.5ꢀ167.3oC; H NMR (200 MHz,
DMSOꢀd6) δ 1.00 (s, 9, CH3), 2.48ꢀ2.54 (m, 3, NH and CHCH2), 3.34 (bs, 1, OH), 4.39 (t, 1, J
= 6.3 Hz, CHCH2), 6.65ꢀ6.72 (m, 2, aromatic), 7.12 (dd, 2, J = 8.9, 2.3 Hz, aromatic) (Figure
2A); 13C NMR (50 MHz, DMSOꢀd6) δ 29.0 (3C), 49.5, 50.8, 72.2, 114.7 (2C), 127.0 (2C),
134.9, 156.2 (Figure 2B); ESIꢀHRMS calcd for C12H20NO2 [M+H]+, 210.1494; found,
210.1488.
155
156
157
158
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 31
159
160
161
162
163
164
165
Analysis of Authentic Standards. The chemical purities of authentic standards 2ꢀ4 were
determined with a Shimadzu highꢀperformance liquid chromatography (HPLC) system
(LCꢀ10AT, Shimadzu Corp., Kyoto, Japan) coupled with a diode array detector (Shimadzu®,
SPDꢀM10Avp). The buffer containing the ionꢀpairing reagent (4.23 mM) was prepared by
dissolving sodium 1ꢀheptanesulfonate (565 mg) and glacial acetic acid (6 mL) in deionized
water (600 mL). The buffer was filtered through a 0.45ꢀꢀm membrane filter (PALL Corp.,
166
167
168
169
170
171
Port Washington, NY, USA) and degassed. A C18 column (Gemini® C18 Column, 5ꢀ, 110 Å,
250 x 4.6 mm, Phenomenex, Torrance, CA, USA) was used, and the temperature was
maintained at 30oC using a column oven (COLBOX®, Hipoint Corp., Kaohsiung, Taiwan).
The flow rate was 1.0 mL/min and the detected wavelength was 276 nm. The initial mobile
phase was MeCN/buffer (3:7) held for 15 min before linearly increasing to 9:1 at 25 min,
where it was then held for another 5 min.
172
173
Multi-residue Analysis of β2-agonists in Swine Hairs. A method3,13ꢀ15 has been
174
175
176
177
178
179
180
181
182
established for the simultaneous determination of residues of 26 β2ꢀagonists (including the
seven prohibited β2ꢀagonists and compounds 2ꢀ4) in swine hair and feed additives by HPLC
coupled with a triple quadrupole mass spectrometer (LC/MS/MS, Agilent 6460 Triple Quad
LC/MS, Santa Clara, CA, USA). ESI positive ion scan mode was used. Briefly, the collected
swine hair samples were washed with water and 95% alcohol, dried at 50oC, and then
pulverized in liquid nitrogen using a cryogenic grinder (6870 Freezer/Mill®, Thomas
Scientific, Swedesboro, NJ, USA) before use. NaOH(aq) (1 mol/L, 4 mL) was added to the
accurately weighted and powdered swine hairs (2.0 g), and the system was heated in a water
bath at 65oC for 2 h. After cooling to room temperature, HCl (1 mol/L, 3.5 mL) and NaOAc
8
ACS Paragon Plus Environment

Page 9 of 31
Journal of Agricultural and Food Chemistry
183
184
185
186
187
188
189
190
191
192
buffer (0.25 mol/L, pH = 4.8, 5 mL) were added and mixed well. The mixture was centrifuged
at 4oC (20,000 rpm, 10 min), and the supernatant was then collected for purification. The
resulting solution was passed through an SCX solidꢀphase extraction cartridge (Varian, Inc.,
Palo Alto, CA, USA), and the cartridge was washed with acetic acid (1 mol/L, 1 mL) and
water (5 mL × 2). The Varian® SCX solidꢀphase extraction cartridge was dried for 30 min and
eluted with a solution of NH4OH(conc), MeCN, and EtOAc (1:9:10, 5 mL). The eluent was
dried under a nitrogen flow at 45oC, and the residue was dissolved in a solution of MeCN and
7 mmol/L NH4OAc(aq) (1:9, 1 mL) and then filtered (0.22 ꢀm, PVDF membrane) to afford the
test solution. The test solution (20 ꢀL) was injected into the LC/MS/MS instrument using
multiple reaction monitoring (MRM) mode for analysis. HPLC conditions－The NH4OAc
193
194
195
196
197
198
199
200
201
buffer (7 mmol/L) was prepared by dissolving NH4OAc (270 mg) in deionized water (200 mL)
in a 500ꢀmL volumetric flask. Formic acid (0.5 mL) was then added, followed by adding
deionized water to the volume. Both the MeCN and NH4OAc buffer were filtered through a
0.45ꢀꢀm membrane filter and degassed. An SBꢀC18 column (Zorbex® SBꢀC18 Column, 5ꢀ,
150 x 3.0 mm, Agilent, Santa Clara, CA, USA) was used under ambient temperature for the
analysis. The mobile phase was initially set at 1% MeCN and then linearly increased to 90%
MeCN over 5 min and held for 3 min. The flow rate was 0.8 mL/min. ESIꢀHRMS analysis
was conducted by injection of the test solution (5.0 ꢀL) into the quadrupole timeꢀofꢀflight
(QTOF) LC/MS/MS instrument (Agilent 6500 series, Agilent, Santa Clara, CA, USA).
202
203
204
205
206
Multi-residue Analysis of β2-agonists in Feed Additives. Briefly, specimens of swine
feed additive (2.0 g) were accurately weighed, treated with a solution of 0.2 mol/L phosphoric
acid/MeOH (1:1; 10 mL) for 30 min, and centrifuged (3,000 rpm, 10 min). NaOAc buffer
(0.25 mol/L, pH = 4.8, 5 mL) was added to the supernatant (2 mL), and the solution was
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 31
207
208
209
210
211
212
mixed for 30 seconds. The resulting solution was passed through an SCX solidꢀphase
extraction cartridge, and the cartridge was then washed with acetic acid (1 mol/L, 1 mL) and
water (5 mL × 2). The SCX cartridge was dried for 30 min and eluted with a solution of
NH4OH(conc), MeCN, and EtOAc (1:9:10, 5 mL). The eluent was dried under a nitrogen flow,
and the residue was dissolved by a solution of MeCN and 7 mmol/L NH4OAc(aq) (1:9, 1 mL)
and then filtered (0.22 ꢀm, PVDF membrane) to afford the test solution. The test solution (10
213
214
215
216
217
ꢀL) was injected into the triple quadrupole LC/MS/MS instrument using MRM mode. An
additional 5.0 ꢀL of the test solution was injected into the QTOF LC/MS/MS instrument
using neutral loss mode. Both studies used the same HPLC conditions described in
Multi-residue Analysis of β2-agonists in Swine Hairs for analysis.
218
219
220
221
222
223
224
225
Pharmacological Activity and Physicochemical Properties. The human α1, α2, β1, β2,
and β3 receptor binding affinities of compounds 2ꢀ4 were determined by Eurofins Panlabs
Taiwan Ltd., Eurofins Pharma Discovery Services (http://www.eurofins.com/
biopharmaꢀservices/discovery/), based on literature methods.16ꢀ20 The calculated LogP values
(CLogP) were obtained directly from ChemDraw Ultra version 11.0 (PerkinElmer Informatics,
Waltham, MA, USA). The experimental LogD7.4 were determined by using the shake flask
method following the OECD guideline.21
226 RESULTS AND DISCUSSION
227
228
229
230
Identification of Mebuctopamine (2) in Swine Hairs. Initially, the LC/MS/MS
reconstructed chromatograms and spectra of the swine hair samples from the livestock farms
demonstrated a suspicious ion peak, which had the same m/z as that of the molecular ion of
salbutamol (1) but a different retention time and fragment ion profile (Figure 3). After
10
ACS Paragon Plus Environment

Page 11 of 31
Journal of Agricultural and Food Chemistry
231
ESIꢀHRMS analysis, the exact mass of this unknown ion was determined to be 240.1605, and
therefore, there were five most possible molecular ion formulas for the unknown component:
232
233
234
235
236
237
238
239
240
241
242
243
244
245
[C8H20F2N5O+], [C11H19FN5 ], [C10H23FNO4 ], [C13H22NO3 ], and [C11H24F2NS+]. Based on
+
+
+
the chemical structures of the seven prohibited β2ꢀagonists and the wellꢀknown
structure−activity
relationships
of
β2ꢀagonists,16,17
the
Nꢀisopropylꢀ2ꢀhydroxyꢀ2ꢀphenylethylamine scaffold X should be the common core structure
and essential pharmacophore of various β2ꢀagonists (Chart 1). Therefore, the unknown
compound should possess at least 11 carbon, one nitrogen, and one oxygen atoms to fulfill the
structural requirements of the β2ꢀagonist pharmacophore. Formulas [C8H20F2N5O+] and
+
[C10H23FNO4 ] were ruled out because they have only 8 and 10 carbon atoms, respectively, in
their molecular formulas. There are no oxygen atoms in the molecular ion formulas
+
+
[C11H19FN5 ] and [C11H24F2NS+]. As a result, only [C13H22NO3 ] could be the molecular ion
formula of the unknown component. In addition, the molecular ion formula of compound 1
+
was also [C13H22NO3 ], and therefore, the unknown compound should be an isomer of
compound 1.
246
247
248
249
250
251
252
253
254
255
There were thousands of compounds in the Reaxys database possessing the same
molecular formula as compound 1. After screening with the essential pharmacophore of
β2ꢀagonist X, the number of candidate compounds was reduced to 13. Further analysis of the
LC/MS/MS data of the swine hair specimens and 1 disclosed additional structural information
about the unknown component in the swine hair. There are three major fragment ions in the
mass spectrum of 1, which are formed by i) dehydration to afford ion 1-II (m/z = 222), ii)
dehydration and elimination of isobutene to afford ion 1-III (m/z = 166), and iii) additional
dehydration of 1-III to yield ion 1-IV (m/z = 148) (Figure 4). However, there are only two
major fragment ions for the unknown compound, m/z = 222 and 166, which could be
produced by dehydration and elimination of isobutene, and there is no fragment ion with a
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 31
256
257
258
259
mass of 148. This implied the existence of an Nꢀtertꢀbutyl substitution and the absence of the
benzylic hydroxyl group at the metaꢀposition of the phenyl group in compound 1, which is
essential for the additional dehydration. In consideration of the above information about the
unknown component, compound 2 was chosen for synthesis as a potential candidate.
260
261
262
263
264
265
266
267
268
269
The authentic compound 2 was prepared as shown in Scheme 1 in 98.6% purity and
analyzed by LC/MS/MS using MRM mode (parent ion: m/z = 240; daughter ions: m/z = 222,
166, 148). There are only two major fragment ions in the mass spectrum of 2, which are
formed by i) dehydration to afford ion 2-II (m/z = 222) and ii) dehydration and elimination of
isobutene to yield ion 2-III (m/z = 166). The hydroxymethyl group at the metaꢀposition of
compound 1 was substituted with the methoxy group in 2, and therefore, the additional
dehydration in compound 1 to form the fragment ion, i.e., m/z = 148, was impossible for
compound 2. In addition, the authentic 2 demonstrated the same retention time and mass
fragmentation pattern as the unknown component in the swine hair samples (Figures 3b and
3c), and therefore, compound 2 was confirmed to be the unknown component in swine hairs.
270
271
Identification of Buctopamine (4) in Feed Additives and Swine Hairs.
272
273
274
275
276
277
278
279
Interestingly, no compound 2 was detected in the “Garlic Essential” feed additive when
using authentic 2 as the reference standard. Furthermore, compound 2 exhibited only weak β2
receptor affinity (discussed later). Thus, compound 2 was not the active ingredient
adulterating the swine feed, and it may be a metabolite of another, more potent, β2ꢀagonist. In
the literatures, compound 2 has been reported as a metabolite of colterol (3, Th 1206,
tꢀbutylarterenol, tBA),24,25 transformed by catecholꢀOꢀmethyl transferase (COMT, EC 2.1.1.6)
(Scheme 2).26ꢀ28 Since compound 3 is a known potent β2ꢀagonist, it may be the active
compound added to the “Garlic Essential”. The authentic standard of 3 was then prepared for
12
ACS Paragon Plus Environment

Page 13 of 31
Journal of Agricultural and Food Chemistry
280
LC/MS/MS analysis of the feed additive samples. However, compound 3 was not detected in
the “Garlic Essential” samples, and therefore, the unknown β2ꢀagonist in the feed additive was
not compound 3。
281
282
283
284
285
286
287
288
289
290
291
292
After carefully examining the chemical structures and MS/MS data of compounds 1 and
2, the [M+Hꢀ74] fragment ions resulting from dehydration and elimination of isobutene were
determined to reflect characteristic fragmentation of the pharmacophore of these β2ꢀagonists.
Therefore, the unknown β2ꢀagonist should also demonstrate a similar fragmentation pattern in
its MS/MS spectra. Therefore, the LC/MS/MS analysis of the feed additive specimens using
“neutral loss” mode (M→Mꢀ74) was conducted to screen the potential β2ꢀagonistꢀrelated
substances in the feed additives. A strong parent ion signal with a molecular mass of 210 was
identified, which was 30 and 16 less than those of compounds 2 and 3, respectively (Figure 5).
This difference means that the molecular formula of the unknown component should be a
“CH2O” fragment and one “O” atom less than those of compounds 2 and 3, respectively.
293
294
295
296
297
298
299
300
301
302
303
In a metabolism study, tulobuterol, a β2ꢀagonist possessing pharmacophore X, was
oxidized by aromatic hydroxylase to produce the corresponding catechol metabolite in rat.29
The metaꢀhydroxyl group of compound 3 might also be introduced by the same enzyme, and
therefore, compound 4 (buctopamine, Nꢀtertꢀbutylꢀnorsynephrine, KWD2066) was proposed
to be the unknown component in the “Garlic Essential”. Compound 4 could be metabolized
by aromatic hydroxylase in swine to yield compound 3 (Scheme 2). Compound 4 was first
synthesized in 1945 and exhibited marked depressor action.30 The authentic standard of 4 was
prepared as shown in Scheme 1 in a purity of 97.7%. After LC/MS/MS analysis of the feed
additive samples using MRM mode (parent ion: m/z = 210; daughter ions: m/z = 192, 136, 91),
compound 4 was confirmed to be the unknown β2ꢀagonist in the “Garlic Essential” (Figure 6).
Further analysis of the swine hair samples using 4 as the reference standard disclosed that
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 31
304
305
306
compound 4 also existed in the swine hairs (Figure 6c).
Multi-residue Analysis of β2-agonists
307
308
309
310
311
312
313
An analytical method has been established for the routine simultaneous determination of
residues of 26 β2ꢀagonists, including compounds 2ꢀ4 and the seven prohibited β2ꢀagonists, in
swine hair and feed additives using LC/MS/MS. ESI positive ion scan mode was used. All 26
β2ꢀagonists showed good linear regression in the range of 5.0−30.0 ng/mL, and the correlation
coefficients (r) were not less than 0.995. The intraday and interday recoveries were in the
range of 80.7ꢀ110.8%, with average relative standard deviations (RSD) of 1.06ꢀ10.8%.
Because β2ꢀagonists are not allowed in either swine feed or hair in Taiwan, the detection and
314
315
316
317
318
319
320
321
322
quantitative limits for the β2ꢀagonists in this multiꢀresidue analysis method were therefore not
determined individually. For practical highꢀthroughput screening, common limits of
quantification for the 26 β2ꢀagonists in swine feed and hair are set at 10 and 25 ng/g,
respectively. More than 2000 specimens were collected and analyzed by local authorities in
2010ꢀ2011, with compounds 2 and 4 detected in approximately 6% and 2% of the swine hair
samples, respectively. The concentrations of compound 2 in the positive hair samples were
between 30ꢀ and >100 ng/g. The contents of compound 4 in the feed additive “Garlic
Essential” were in the range of 5.0ꢀ10 mg/g. Currently, this method is used for the analysis of
2000ꢀ3000 swine hair and feed additive specimens each year.
323
324
325
326
327
Pharmacological Activity and Physicochemical Property
The binding affinities of β2ꢀagonists for the human β1ꢀ, β2ꢀ and β3ꢀadrenoceptors and the
bovine β2ꢀadrenoceptor in the literature are shown in Table 1.25,31,32 All β2ꢀagonists have
demonstrated high binding affinity and potent agonistic activity toward the β2 receptor.
14
ACS Paragon Plus Environment

Page 15 of 31
Journal of Agricultural and Food Chemistry
328
Although ractopamine, clenbuterol, and cimaterol have also shown high β1ꢀreceptor affinity,
329
330
331
332
333
334
335
336
337
338
339
ractopamine is only an agonist of the porcine β2 receptor and possesses no agonistic activity
toward the porcine β1 receptor.33 Interestingly, these β2ꢀagonists are full agonists of the human
β1 receptor,31 and therefore, their residues in swine meat and tissue may cause potential
adverse effects that did not exist in pigs.
The β3ꢀreceptor affinities of these β2ꢀagonists are much weaker than their β1ꢀ and
β2ꢀreceptor affinities. In addition, selective β3ꢀagonists lack efficacy for protein synthesis,
which is crucial for muscle formation, and therefore, the β3 receptor should not play a
significant role in livestock production.1
Previously, compounds 3 and 4 were shown to demonstrate moderate to potent bovine
β2ꢀreceptor binding affinity and agonistic efficacy (ꢀlog KD = 6.20ꢀ6.58, Table 1).24,25 In this
study, the binding affinities of compounds 2ꢀ4 for the human α and β receptor subtypes were
340
341
342
343
evaluated. Compounds 2ꢀ4 had negligible binding to the α1 and α2 receptors (Table 2), while
they showed moderate to high β2 receptor binding inhibition in the rank order of 3 > 4 > 2.
Compound 3 also possessed high binding affinity for the β1 receptor, whereas compounds 2
and 4 did not demonstrate significant binding to the β1 and β3 receptors.
344
345
346
347
348
349
350
351
The calculated CLogP and experimental LogD7.4 values of the β2ꢀagonists were
determined for the quantitative evaluation of their lipophilicity.21 Both CLogP and LogD7.4
values demonstrate similar trends for these β2ꢀagonists (Table 2). The differences between the
values from the two methods may be due the protonation of the basic amino groups of the
β2ꢀagonists at physiological conditions (pH = 7.4). Of the compounds in this study,
ractopamine and clenbuterol were the most lipophilic, while salbutamol (1) was the most
hydrophilic. The lipophilicities of compounds 2ꢀ4 were higher than that of 1 and lower than
those of ractopamine and clenbuterol. This can explain why the residues of compounds 2 and
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 31
352
4 existed in the swine hair specimens, while the more hydrophilic 3 was not detected.
353
354
355
356
357
358
359
360
361
In this study, compound 4, a novel β2ꢀagonist adulterating swine feed additives, was
identified. Both compound 4 and its metabolite 2 existed in the swine hair specimens.
Authentic 2 and 4 were synthesized and added to the library containing of 26 β2ꢀagonists for
screenings of multiple residues of β2ꢀagonists using LC/MS/MS conducted by regulatory
authorities. The illegal use of compound 4 as a feed additive has been successfully prevented.
No specimen of swine hair or feed additive has been found to contain either 2 or 4 in Taiwan
since 2013. In comparison to ractopamine and clenbuterol, compounds 2ꢀ4 possess lower β2
receptor affinity and higher hydrophilicity. This work and experience should also be useful
for other countries in preventing the illegal use of new β2ꢀagonists in animal breeding.
362
363
AUTHOR INFORMATION
364
365
Corresponding Author
*L.ꢀW. Hsin. Phone: +886ꢀ2ꢀ3366ꢀ8696. Fax: +886ꢀ2ꢀ2351ꢀ2086. Eꢀmail:
lwhsin@ntu.edu.tw.
366
367
16
ACS Paragon Plus Environment

Page 17 of 31
Journal of Agricultural and Food Chemistry
368
REFERENCES
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
1. Sillence, M. N. Technologies for the control of fat and lean deposition in livestock. Vet. J.
2004, 167, 242ꢀ57.
2. Brambilla, G.; Cenci, T.; Franconi, F.; Galarini, R.; Macri, A.; Rondoni, F.; Strozzi, M.;
Loizzo, A. Clinical and pharmacological profile in a clenbuterol epidemic poisoning of
contaminated beef meat in Italy. Toxicol. Lett. 2000, 114, 47ꢀ53.
3. Liu, C.; Ling, W.; Xu, W.; Chai, Y. Simultaneous determination of 20 betaꢀagonists in pig
muscle and liver by highꢀperformance liquid chromatography/tandem mass spectrometry.
J. AOAC Int. 2011, 94, 420ꢀ7.
4. MartinezꢀNavarro, J. F. Food poisoning related to consumption of illicit betaꢀagonist in
liver. Lancet 1990, 336, 1311.
5. Bocca, B.; Cartoni, C.; Di Mattia, M.; Fiori, M.; Felli, M.; Neri, B.; Russo, M. V.;
Brambilla, G. Feed additives in animal nutrition: Quantification of a new adrenergic drug
by hyphenated techniques. J. Sep. Sci. 2003, 26, 363ꢀ368.
6. Spisso, B. F.; Lopes, C. C.; Marques, M. A. S.; Neto, F. R. A. Determination of
beta(2)ꢀagonists in bovine urine: Comparison of two extraction/cleanꢀup procedures for
highꢀresolution gas chromatographyꢀmass spectrometry analysis. J. Anal. Toxicol. 2000,
24, 146ꢀ152.
7. Nielen, M. W. F.; Elliott, C. T.; Boyd, S. A.; Courtheyn, D.; Essers, M. L.; Hooijerink, H.
H.; van Bennekom, E. O.; Fuchs, R. E. M. Identification of an unknown betaꢀagonist in
feed by liquid chromatography/bioassay/quadrupole timeꢀofꢀflight tandem mass
spectrometry with accurate mass measurement. Rapid Commun. Mass Spectrom. 2003,
17, 1633ꢀ1641.
8. Mazzanti, G.; Daniele, C.; Boatto, G.; Manca, G.; Brambilla, G.; Loizzo, A. New
betaꢀadrenergic agonists used illicitly as growth promoters in animal breeding: chemical
and pharmacodynamic studies. Toxicology 2003, 187, 91ꢀ9.
9. Lei, Y. C.; Tsai, Y. F.; Tai, Y. T.; Lin, C. Y.; Hsieh, K. H.; Chang, T. H.; Sheu, S. Y.; Kuo,
T. F. Development and fast screening of salbutamol residues in swine serum by an
enzymeꢀlinked immunosorbent assay in Taiwan. J. Agric. Food Chem. 2008, 56,
5494ꢀ5499.
10. Sheu, S. Y.; Lei, Y. C.; Tai, Y. T.; Chang, T. H.; Kuo, T. F. Screening of salbutamol
residues in swine meat and animal feed by an enzyme immunoassay in Taiwan. Anal.
Chim. Acta 2009, 654, 148ꢀ53.
11. Sai, F.; Hong, M.; Yunfeng, Z.; Huijing, C.; Yongning, W. Simultaneous detection of
residues of 25 beta(2)ꢀagonists and 23 betaꢀblockers in animal foods by
highꢀperformance liquid chromatography coupled with linear ion trap mass spectrometry.
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 31
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
J. Agric. Food Chem. 2012, 60, 1898ꢀ905.
12. National Research Council of Canada. Use of catecholamines and related compounds as
antiꢀangiogenic agents. WO 2008092257 A1.
13. Shao, B.; Jia, X. F.; Zhang, J.; Meng, J.; Wu, Y. N.; Duan, H. J.; Tu, X. M. Multiꢀresidual
analysis of 16 betaꢀagonists in pig liver, kidney and muscle by ultra performance liquid
chromatography tandem mass spectrometry. Food Chem. 2009, 114, 1115ꢀ1121.
14. Wang, X. F.; Liu, Y. H.; Su, Y. J.; Yang, J. W.; Bian, K.; Wang, Z. N.; He, L. M.
HighꢀThroughput Screening and Confirmation of 22 Banned Veterinary Drugs in
Feedstuffs Using LCꢀMS/MS and HighꢀResolution Orbitrap Mass Spectrometry. J. Agric.
Food Chem. 2014, 62, 516ꢀ527.
15. Guo, C. C.; Shi, F.; Gong, L. P.; Tan, H. J.; Hu, D. F.; Zhang, J. L. Ultraꢀtrace analysis of
12 beta(2)ꢀagonists in pork, beef, mutton and chicken by ultrahighꢀperformance
liquidꢀchromatographyꢀquadrupoleꢀorbitrap tandem mass spectrometry. J. Pharm.
Biomed. Anal. 2015, 107, 526ꢀ534.
16. Greengrass, P.; Bremner, R., Binding characteristics of 3Hꢀprazosin to rat brain
alphaꢀadrenergic receptors. Eur J Pharmacol 1979, 55, 323ꢀ6.
17. Boyajian, C. L.; Leslie, F. M., Pharmacological evidence for alphaꢀ2 adrenoceptor
heterogeneity: differential binding properties of [3H]rauwolscine and [3H]idazoxan in rat
brain. J Pharmacol Exp Ther 1987, 241, 1092ꢀ8.
18. Broadhurst, A. M.; Alexander, B. S.; Wood, M. D., Heterogeneous 3Hꢀrauwolscine
binding sites in rat cortex: two alpha 2ꢀadrenoceptor subtypes or an additional
nonꢀadrenergic interaction? Life Sci 1988, 43, 83ꢀ92.
19. Feve, B.; Elhadri, K.; QuignardꢀBoulange, A.; Pairault, J., Transcriptional
downꢀregulation by insulin of the beta 3ꢀadrenergic receptor expression in 3T3ꢀF442A
adipocytes: a mechanism for repressing the cAMP signaling pathway. Proc Natl Acad Sci
U S A 1994, 91, 5677ꢀ81.
20. McCrea, K. E.; Hill, S. J., Salmeterol, a longꢀacting beta 2ꢀadrenoceptor agonist
mediating cyclic AMP accumulation in a neuronal cell line. Br J Pharmacol 1993, 110,
619ꢀ26.
21. OECD Guideline for the testing of chemicals 107, Partition coefficient (nꢀoctanol/
water)ꢀShakeꢀflask method. 2004.
22. Beale, J. M.; Block, J. H. Wilson and Gisvold’s Textbook of Organic Medicinal and
Pharmaceutical Chemistry. 12 ed.; Lippincott Williams & Wilkins: 2011, 531ꢀ543.
23. Williams, D. A. Foye's Principles of Medicinal Chemistry. 7 ed.; Lippincott Williams &
Wilkins: 2012, 347ꢀ354.
24. AP, I. J.; Aue, G. H.; Bultsma, T.; Linschoten, M. R.; Timmerman, H. Quantitative
18
ACS Paragon Plus Environment

Page 19 of 31
Journal of Agricultural and Food Chemistry
440
evaluation of the beta 2ꢀadrenoceptor affinity of phenoxypropanolamines and
phenylethanolamines. J. Med. Chem. 1985, 28, 1328ꢀ34.
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
25. AP, I. J.; Bultsma, T.; Timmerman, H. Quantitative evaluation of the beta 2ꢀadrenoceptor
intrinsic activity of Nꢀtertꢀbutylphenylethanolamines. J. Med. Chem. 1986, 29, 549ꢀ54.
26. Shargel, L.; Dorrbecker, S. A.; Levitt, M. Physiological Disposition and Metabolism of
NꢀTꢀButylarterenol and Its DiꢀPꢀToluate Ester (Bitolterol) in Rat. Drug Metab. Dispos.
1976, 4, 65ꢀ71.
27. Shargel, L.; Dorrbecker, S. A. Physiological Disposition and Metabolism of
[BitolterolꢀHꢀ3] in Man and Dog. Drug Metab. Dispos. 1976, 4, 72ꢀ78.
28. Park, G. B.; Koss, R. F.; Utter, J.; Edelson, J. Determination of colterol in human plasma
and urine by reversedꢀphase chromatography with amperometric detection. J.
Chromatogr. 1983, 273, 481ꢀ6.
29. Yamamoto, Y.; Higuchi, S.; Fujihashi, T.; Shimizu, S.; Nishide, K. [The metabolic fate of
oꢀchloroꢀaꢀ(teriꢀbutylaminomethyl)ꢀbenzyl alcohol hydrochloride (Cꢀ78). II. Metabolic
products in the rat (author's transl)]. Yakugaku Zasshi 1977, 97, 244ꢀ50.
30. Corrigan, J. R.; Langerman, M. J.; Moore, M. L. Preparation of NꢀSubstituted
1ꢀ(ParaꢀHydroxyphenyl)ꢀ2ꢀAminoethanols. J. Am. Chem. Soc. 1945, 67, 1894ꢀ1896.
31. Baker, J. G. The selectivity of betaꢀadrenoceptor agonists at human beta1ꢀ, beta2ꢀ and
beta3ꢀadrenoceptors. Br J Pharmacol 2010, 160, 1048ꢀ61.
32. Kern, C.; Meyer, T.; Droux, S.; Schollmeyer, D.; Miculka, C. Synthesis and
pharmacological characterization of beta2ꢀadrenergic agonist enantiomers: zilpaterol. J
Med Chem 2009, 52, 1773ꢀ7.
33. Mills, S. E.; Kissel, J.; Bidwell, C. A.; Smith, D. J. Stereoselectivity of porcine
betaꢀadrenergic receptors for ractopamine stereoisomers. J Anim Sci 2003, 81, 122ꢀ9.
463
464
465
466
467
468
469
470
471
472
Notes
1. The authors declare no competing financial interest.
2. This work was supported by the Council of Agriculture, Executive Yuan, Republic of
China (Grant No. 100ASꢀ9.2.3ꢀBQꢀB1(3), 101ASꢀ10.2.3ꢀBQꢀB1(3), and
102ASꢀ10.2.3ꢀBQꢀB1(3)).
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 31
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
Figure captions
Figure 1A. The 1H NMR spectrum (200 MHz, CDCl3) of mebuctopamine (2).
Figure 1B. The 13C NMR spectrum (50 MHz, CDCl3) of mebuctopamine (2).
Figure 2A. The 1H NMR spectrum (200 MHz, DMSOꢀd6) of buctopamine (4).
Figure 2B. The 1H NMR spectrum (50 MHz, DMSOꢀd6) of buctopamine (4).
Figure 3. The LC/MS/MS reconstructed chromatograms and spectra using MRM mode
(multiple reaction monitoring mode; parent ion: m/z 240; daughter ions: m/z 222, 166, 148) of
(a) salbutamol in a standard mixture of 10 β2ꢀagonists; (b) unknown component in swine hair
samples; (c) authentic standard of mebuctopamine (2).
Figure 4. The major fragment ions of salbutamol (1), mebuctopamine (2), colterol (3), and
buctopamine (4) in the mass spectra.
Figure 5. The total ion current (TIC) chromatogram and mass spectrum from the LC/MS/MS
analysis of the feed additive samples using neutral loss mode (parent ion: m/z [M]+; daughter
ions: m/z [Mꢀ74]+).
Figure 6. The LC/MS/MS reconstructed chromatograms and spectra using MRM mode
(parent ion: m/z 210; daughter ions: m/z 192, 136, 91) of (a) buctopamine (4) in a standard
mixture of 26 β2ꢀagonists ; (b) feed additive sample; (c) swine hair sample.
20
ACS Paragon Plus Environment

Page 21 of 31
Journal of Agricultural and Food Chemistry
Table 1. The βꢀreceptor binding affinities and agonistic activities of β2ꢀagonists in the literature.
β1
β2
β2
β3
cAMPc
a
b
a
b
c
a
Compounds
ꢀlog KD
ꢀlog EC50
ꢀlog KD
ꢀlog EC50
ꢀlog KD
ꢀlog KD
production
salbutamol (1)
colterol (3)
buctopamine (4)
ractopamine
zilpaterol
4.68
6.21
6.01
7.72
6.58
6.55
6.20
122
83
3.98
5.82
97
6.97
8.74
6.93
6.24d
7.90
7.26
5.51
6.83
7.63
clenbuterol
cimaterol
6.62
6.57
3.90
5.62
7.29
8.42
5.76
6.59
9.18
8.94
7.29
7.60
7.74
64
5.35
5.28
3.68
4.72
terbutaline
5.73
7.18
50
61
tulobuterol
aHuman β1ꢀ, β2ꢀ and β3ꢀadrenoceptor binding affinities.32 bData obtained from [3H]ꢀcAMP accumulation for cells expressing the human β1ꢀ and
β2ꢀadrenoceptors.32 cβ2ꢀAdrenoceptor affinities and intrinsic activities using a bovine skeletal muscle preparation. The cAMP production values
are in pmol (mg of protein)ꢀ1 minꢀ1.25 dThe binding affinity of zilpaterol is presented as ꢀlog Ki.33
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 31
Table 2. ClogP, LogD7.4, and receptor binding affinities of β2ꢀagonists.
% Inhibition at 1 ꢀMc
CLogPa
LogD7.4
b
Compounds
α1
α2
β1
β2
β3
salbutamol (1)
mebuctopamine (2)
colterol (3)
0.06
1.00
0.55
1.15
2.03
0.95
2.39
−1.14
−0.23
−2
−8
−8
2
1
6
5
−5
15
−8
19
53
28
50
12
buctopamine (4)
ractopamine
zilpaterol
−0.45
0.23
−0.68
0.20
clenbuterol
aThe calculated LogP values (CLogP) were obtained from ChemDraw Ultra version 11.0
(PerkinElmer Informatics, Waltham, USA). bThe experimental LogD7.4 were determined by
using the shake flask method following the OECD guideline.21 cThe receptor binding assays
were determined by measuring the binding inhibition using a single drug concentration (1 
M) by Eurofins Pharma Discovery Services.16ꢀ20
22
ACS Paragon Plus Environment

Page 23 of 31
Journal of Agricultural and Food Chemistry
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 31
Figure 1A. The 1H NMR spectrum (200 MHz, CDCl3) of mebuctopamine (2).
Figure 1B. The 13C NMR spectrum (50 MHz, CDCl3) of mebuctopamine (2).
24
ACS Paragon Plus Environment

Page 25 of 31
Journal of Agricultural and Food Chemistry
9
8
7
6
5
4
3
2
1
ppm
Figure 2A. The 1H NMR spectrum (200 MHz, DMSOꢀ
d
6) of buctopamine (4).
200
180
160
140
120
100
80
60
40
20
ppm
Figure 2B. The 13C NMR spectrum (50 MHz, DMSOꢀ
d6) of buctopamine (4).
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 31
a
b
c
Figure 3. The LC/MS/MS reconstructed chromatograms and spectra using MRM mode
(multiple reaction monitoring mode; parent ion: m/z 240; daughter ions: m/z 222, 166, 148) of
(a) salbutamol in a standard mixture of 10 β2ꢀagonists; (b) unknown component in swine hair
samples; (c) authentic standard of mebuctopamine (2).
26
ACS Paragon Plus Environment

Page 27 of 31
Journal of Agricultural and Food Chemistry
Figure 4. The major fragment ions of salbutamol (1), mebuctopamine (2), colterol (3), and
buctopamine (4) in the mass spectra.
27
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 31
Figure 5. The total ion current (TIC) chromatogram and mass spectrum from the LC/MS/MS
analysis of the feed additive samples using neutral loss mode (parent ion: m/z [M]+; daughter
ions: m/z [Mꢀ74]+).
28
ACS Paragon Plus Environment

Page 29 of 31
Journal of Agricultural and Food Chemistry
a
b
c
Figure 6. The LC/MS/MS reconstructed chromatograms and spectra using MRM mode
(parent ion: m/z 210; daughter ions: m/z 192, 136, 91) of (a) buctopamine (4) in a standard
mixture of 26 β2ꢀagonists; (b) feed additive sample; (c) swine hair sample.
29
ACS Paragon Plus Environment