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Identification of ractopamine glucuronides and determination of bioactive ractopamine residues and its metabolites in food animal urine by ELISA, LC-MS/MS and GC-MS abc
Xiao-Fei Jiang a
b
d
, Yue-Hui Zhu & Xiao-Yun Liu
School of Chemistry and Life Science, Guizhou Normal College, Guiyang 550018, China
b
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Guangzhou 510650, China c
College of Chemical and Environmental Engineering, Chongqing Three Gorges University, 404000, Chongqing, China d
Guangzhou Accurate and Correct Test Co., Ltd., 510663, Guangzhou, China Accepted author version posted online: 15 Oct 2013.Published online: 15 Oct 2013.
To cite this article: Food Additives & Contaminants: Part A (2013): Identification of ractopamine glucuronides and determination of bioactive ractopamine residues and its metabolites in food animal urine by ELISA, LC-MS/MS and GC-MS, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2013.855327 To link to this article: http://dx.doi.org/10.1080/19440049.2013.855327
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Identification of ractopamine glucuronides and determination of bioactive ractopamine residues and its metabolites in food animal urine by ELISA, LC-MS/MS and GC-MS
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School of Chemistry and Life Science, Guizhou Normal College, Guiyang 550018,
b
M an us
China;
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South
China Botanical Garden, Guangzhou 510650, China; c
College of Chemical and Environmental Engineering, Chongqing Three Gorges
University, 404000, Chongqing, China.
Guangzhou Accurate and Correct Test Co., Ltd., 510663, Guangzhou, China.
pt
ed
d
ce
* Corresponding authors. Tel. /Fax: +86 20 37252958. E-mail address: [email protected] (X.Y. Liu) or [email protected] (Y.H. Zhu).
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a
t
Xiao-Fei Jiang a,b,c, Yue-Hui Zhub, *, and Xiao-Yun Liud,*
1
Abstract Ractopamine glucuronides have been identified in cattle urine sample by LC-MS/MS. An ELISA method, which was capable of specifically determining (1R, 3R)-ractopamine stereoisomer and its glucuronide metabolites, had more than 100% recovery with an acceptable coefficient of variation in the inter-assay and intra-assay
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variation tests for RR ractopamine. The concentration levels of parent ractopamine
ractopamine in cattle and sheep urine showed similar depletion trends, in which the
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concentration curves increased and reached a climax during the feeding period, and then dropped fast when entering the withdrawal period. Data from the three methods
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had very good pair-wise correlations. In the cattle urine samples, the correlation coefficient (R2) for parent ractopamine between the ELISA and the LC-MS/MS or
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GC-MS results were 0.93 or 0.92, R2 values for parent ractopamine and total ractopamine data measured by LC-MS/MS and GC-MS were 0.9651 and 0.9677,
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respectively. All R2 values for data gained from sheep urine samples were > 0.95. Our study indicated that the close levels of RR ractopamine stereoisomer in cattle and sheep urine samples may imply the presence of a similar depletion pattern in other livestock, and thus would facilitate an accurate detection and management of ractopamine usage in food safety.
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and ractopamine glucuronide metabolites as the main components of total
Keywords: (1R, 3R)-ractopamine; glucuronide metabolites; depletion pattern;
livestock; ELISA; mass spectrometry
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Introduction Ractopamine, one of β-adrenergic agonists, is a leanness-enhancing agent in farm animals. The beneficial aspects include its efficiency in lifting daily weight gain and protein synthesis rates in swine. Ractopamine residues may pose human health risks (Barnes 1997). The use of ractopamine in swine is banned by most countries in the
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world, such as the EU and China, meanwhile, a few countries including the USA
1995; European Food Safety Authority 2009). The conflicting situation of
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ractopamine raises the needs to monitor its presence and metabolism so as to provide information for official regulation and the import or export of food animals.
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The management of illegal use or contamination of β-agonists in food animal production has become an important issue of concern. Nowadays, novel methods and
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devices have been developed to detect ractopamine with high efficiency (Huang et al.
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2011; Shelver et al. 2010; Lin et al. 2013; Liu et al. 2009; Zhai et al. 2011). On the other hand, conventional analytical methods for ractopamine such as HPLC, ELISA and LC-MS/MS are still proving to be powerful approaches to provide comprehensive information on the metabolism of ractopamine (Freire et al. 2013; Lehner et al. 2004; Liu et al. 2009; Elliott et al. 1998). Lehner et al. (2004) screened ractopamine incurred horse urine samples by ELISA and identified the glucuronidation, sulfated and
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allow it as a component of feed additive in cattle, turkeys and pigs (Maistro et al.
methylated forms of ractopamine metabolites by GC-MS. Previous studies showed that the main form of ractopamine metabolites are the glucuronide conjugates (Lehner et al. 2004; Dalidowicz et al. 1992; Elliott et al. 1998; Smith et al. 2000). 1R,
3R-stereoisomer (RR), as one of the four ractopamine stereoisomers (also including RS, SR and SS), was indicated to be the functional and the most bioactive stereoisomer responsible for leanness-enhancing effect of ractopamine (Ricke et al.
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1999; Shappell et al. 2000). The commonly used commercial ractopamine is a racemic mixture of the four stereoisomers, and previous assays generally have dealt with all isomers of the dosed ractopamine as a whole, and paid little attention to the depletion patterns of conjugated metabolites (Qiang et al. 2007; Vulić et al. 2012; Lu et al. 2012). Shelver and Smith used RR and RS ractopamine stereoisomers to prepare antibody,
however,
the
antibody
showed
strong
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monoclonal
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ractopamine
antibody had excellent sensitivity to enzyme-hydrolyzed urine samples and
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immunoassays based on the monoclonal antibody yielded comparable results with those data from HPLC and surface plasmon resonance (SPR) biosensor analysis
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(Shelver et al. 2002, 2003). However, when detecting samples without enzymatic hydrolysis, the results for parent ractopamine (free ractopamine) derived from ELISA
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and HPLC methods had low correlation (R2 value = 0.58 for sheep urine samples)
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(Shelver et al. 2002). Another earlier study showed results determined by an ELISA method based on a polyclonal antibody and LC-MS/MS had only fairly good agreement (R2 = 0.73) in ractopamine dosed bovine urine samples (Elliott et al. 1998). Currently, there has been no in-depth examination of the depletion patterns of bioactive ractopamine residues and ractopamine glucuronide metabolites. We supposed that a combination of confirmatory mass spectrometric assays with immune
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stereoselectivity towards both RR and SR stereoisomers (Shelver et al. 2000). The
screening assay would help to disclose the depletion patterns of RR ractopamine residue and ractopamine glucuronide metabolites. In the current study, we exploited an LC-MS/MS method to directly analyze and
identify ractopamine glucuronides in cattle urine samples. We also used an ELISA kit in which the monoclonal antibody enabled us to detect the physiologically active RR ractopamine isomer. By using the three complementary methods including ELISA,
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LC-MS/MS and GC-MS in incurred cattle and sheep urine samples, this study compared the concentration variations of parent ractopamine stereoisomer, ractopamine glucuronide metabolites and total ractopamine during the feeding and withdrawal period of a total of 14 days. The results obtained may help to indicate the metabolic mechanism of ractopamine stereoisomers and the administration of its
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Materials and methods
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Chemicals and Reagents
Ractopamine hydrochloride (ractopamine) was used for animal treatment,
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ractopamine and its glucuronide conjugates were used for method validation, and isoxuprine hydrochloride was used as an internal standard, which together with
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β-glucuronidase and other chemicals were purchased from Sigma-Aldrich
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(Milwaukee, KI) and were reagent grade or higher. Ractopamine glucuronide conjugates were generous gifts from Dr. Smith (Smith et al. 1993). The derivatization reagent N, O-bis (trimethylsilyl) trifluoroacetamide + 1% trimethylchlorosilane (BSTFA + 1% TMCS) was obtained from Pierce (Rickford, IL).
Sampling of Incurred Urine
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rational use in food-producing livestock.
Urine samples containing ractopamine residues were obtained from Dr. Shelver
(USDA-ARS, Biosciences Research Laboratory, Fargo, ND). Briefly, control animal (cattle or sheep) urine (n = 3) across different collection days was mixed to generate a composite control urine. The drug incurred urine samples were collected from cattle or sheep fed a diet containing 20 mg/kg ractopamine for 8 or 7 consecutive days, respectively. Urine samples were collected during the feeding and withdrawal periods.
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Pooled urine (pH 7.2) was buffered with 1:1 (V: V) volume of 100 mM phosphate buffer. Aliquots of the samples were stored at -20 °C until analyzed.
Enzyme-linked Immunosorbent Assay (ELISA) Ractopamine ELISA test kit obtained from Immunalysis (Pomona, CA; private
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communication) was used for screening the urine samples by following the
antibody was immobilized with 96-well polystyrene plate, and the antigen consisting
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of ractopamine was coupled to bovine thyroglobulin. In this study, urine specimens in most cases were pre-diluted 1:10 with phosphate buffer (100 mM, pH 7.2) before
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analysis, in order that the determined ractopamine concentrations were less than 20% B/B0 from the ELISA calibration curve. Aliquots of 100 µl calibration solutions or
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specimens were added into each plate well. Goat anti-mouse HRP-IgG of 1: 20000
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was finally used. The absorbance of the samples was read at 450 nm and 650 nm with a Tecan model Infinite 200 PRO Microplate Readers (Tecan Inc, San Jose, CA). The calibration curves for ractopamine were plotted by measuring control urines with ractopamine concentrations of 0, 0.1, 1, 2, 5, 10, 50 and 100 ng/mL. The selectivity of the ELISA kit was checked beforehand, where the quantification of ractopamine were not affected by the presence of clenbuterol and cycloclenbuterol. Moreover, the
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manufacturer’s protocol. Briefly, a (1R, 3R)-ractopamine-specific monoclonal
absorbance variation due to the presence of other three ractopamine stereoisomers was less than 5% of absorbance measured with only (1R, 3R)-ractopamine, indicating the monoclonal antibody had specific stereo-selectivity towards binding RR ractopamine and the ELISA kit had excellent selectivity.
Sample Preparations for Chromatographic Analysis
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β-glucuronidase hydrolysis An aliquot of urine samples (1 mL) was mixed thoroughly with 400 µl sodium acetate (1 M, pH 5.0) and 1000 Units β-glucuronidase incubated at 65 °C for 3 hours. Cooled the tubes, following by adding 10 µl methanol solution of isoxuprine at 10 µg/mL. Centrifuged at 2500 × g for 10 min and the supernatants were used for
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Solid-phase extraction (SPE) cleanup
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The SPE procedure was employed to remove ractopamine metabolites and other unwanted compounds in the samples. A SPE mixed mode C18 column model Clin II,
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691-0353T (SPEWare Corp, San Pedro, CA) was operated to purify urine sample following a previously described procedure (Liu et al. 2009). The eluted ractopamine
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in methylene chloride/isopropanol/ammonium hydroxide (78:20:2, v/v/v, 3 mL) was
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dried under nitrogen stream and reconstituted in 50 µl methanol for further chromatographic or ELISA analysis, respectively.
Identification of Metabolites and Confirmation by LC-MS/MS The operating and tuning procedures, following a previously reported method with minor modifications (Liu et al. 2009), were run on an Agilent Technologies 1200
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ractopamine determination.
series liquid chromatography pump coupled to a 6410 triple quadrupole mass spectrometer (MS), ionized using Electrospray ionization (ESI) in the positive mode. Aliquot (5 µl) of ractopamine standard solution, control or incurred urine samples were injected into a Zorbax Eclipse XDB C18 (4.6 × 50 mm, 1.8 μm; Agilent) and a guard column (4.6 × 12.5 mm, 1.8 μm). The columns were thermostatically controlled at 40 °C, and the flow rate was 0.5 mL/min. The binary gradient system consisted of
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A, 20 mM ammonium formate (pH 6.4), and B, methanol, and the composition of the mobile phase (85% A) for the entire program remained constant. The calibration curve for ractopamine was made with 0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25 and 50 ng/mL of ractopamine as well as ractopamine glucuronides in the control urine. Data were acquired, calculated and reported by the Agilent Masshunter Quantitative Analysis
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(QQQ) software.
conjugate were injected into MS equipment directly. The precursor ion for
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ractopamine was m/z 302.2 and the ion transition of isoxuprine was from m/z 302.1 to m/z 284.2. The ion m/z 162.1 and m/z 478.4 were used for LC-MS/MS quantification
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of ractopamine and the main glucuronide conjugates (identified in this study, Figure 1), respectively. Validations of both ractopamine and ractopamine glucuronides for the
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LC-MS/MS method were conducted, in which the linearity from 0.1 to 50 ng/mL was
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obtained with an average coefficient R2 > 0.99 and > 0.97, and they could be detected at the lowest concentrations at 0.1 ng/mL and 0.5 ng/mL, respectively, indicating the method had excellent sensitivity. The intraday and interday precision and accuracy were determined in control urine with fortified ractopamine or ractopamine glucuronides at 1 ng/mL and 25 ng/mL, respectively.
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To optimize mass spectrometric conditions, ractopamine and its glucuronide
GC-MS confirmation Confirmation of ractopamine and ractopamine glucuronides in cattle and sheep
urine samples following β-glucuronidase hydrolysis, SPE and trimethylchlorosilane (TMCS) derivatization was accomplished by GC-MS method as described by Hughes (2002), with modifications. An Agilent 7890 GC coupled to a 5975C mass selective detector (MSD), operating in electron ionisation mode was used for analysis. The GC
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column was a DB-5MS (15-m length, 0.25 mm i.d., 0.25 µm film thickness, J&W Scientific), and the injection temperature was 280 °C. The standard and urine samples were hydrolyzed and SPE extracted as above method, and the extracts were finally reconstituted in 10 µl ethyl acetate and 40 µl BSTFA + 1% TMCS. The extracts were then transferred to autosampler vial, capped, and heated at 85 °C for 45 min. The
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purge flow rate was 50 mL/min for 0.5 min, and the carrier gas was helium. The
mL/min. The initial GC oven temperature was 60 °C ramped at 20 °C/min to 280 °C.
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The transfer line was held at 280 °C, the ion source at 230 °C, and the quadrupole at 150 °C. The dwell time for all ions was 50 ms. Ractopamine was identified at the
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retention time of 12.8 min, and three ions were selected from the full scan spectrum, m/z 250, 179 and 234. Isoxuprine was at the retention time of 11.46 min, and the ions
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selected for monitoring were m/z 178 and 267. The calibration curve for ractopamine
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(and ractopamine glucuronides) was determined at 0, 0.5, 1, 5, 10, 25, 50 and 100 ng/mL, and the validations of quantification were conducted similarly as LC-MS/MS method. The results were analyzed and reported with the Agilent DrugQuant ChemStation software package.
Results and discussion
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injection mode was splitless, the injection volume was 2 µl with the constant flow at 2
Identification of ractopamine glucuronide metabolites by LC-MS/MS Ractopamine standard has two ion transitions, m/z 302.2→164.1 and m/z
302.2→121.2, as previously described for ractopamine detection by LC-MS/MS (Elliott et al. 1998; Liu et al. 2009). In this study, in order to maximize the detection possibility of ractopamine metabolites, the incurred cattle urine sample with only proper dilution but no clean-up treatment was injected directly into the LC-MS/MS
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system. Parent ractopamine was identified based on the retention time and full-scan ESI (+)-MS spectra compared with those of standard ractopamine, an ion peak at m/z 302.2 was used to monitor and quantify the parent ractopamine residue (Figure 1A). Representative ion chromatogram from LC-MS/MS analysis of an incurred cattle urine sample showed a new peak at 2.74 min (Figure 1B). The peak was further
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measured by ESI (+)-MS and showed a characteristic ion signal at m/z 478.4. Its mass
mono-glucuronide acid conjugate at a phenolic hydroxyl group of ractopamine,
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(Lehner et al. 2004), and was designated as ractopamine-O-glucuronide herein (Figure 1C). Other different peaks of the incurred sample were also analyzed and showed no
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characteristic ion signals (data not shown). Glucuronidation of ractopamine was showed to be the major type of modification compared to sulfation and methylation of (Lehner
et
al.
2004;
Shelver
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ractopamine
et
al.
2002),
for
example,
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mono-glucuronides conjugated to the phenols at C-10 and C-10’ could be up to 73% total conjugate metabolites in turkey (Smith et al. 2000). All kind of conjugated metabolites could be detected by ELISA and ESI (+)-MS. In the current work, we determined ractopamine glucuronide metabolites by using three complementary methods.
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spectral data was compared with a previous report and was consistent with that of
Validation of methods We employed an ELISA kit in which the monoclonal antibody was specifically
raised to bind (1R, 3R)-ractopamine stereoisomer and its conjugated metabolites. The calibration curves for RR ractopamine and ractopamine glucuronides were prepared in buffered control cattle urine, respectively (Figure 2). Eight concentration levels of standard chemicals were set up from 0.01 up to 100 ng/mL. The linearity of the
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ELISA method developed to detect RR ractopamine and ractopamine glucuronides was from 0.1 to 50 ng/mL, with R2 = 0.97 and 0.95, respectively. The sensitivity for RR ractopamine and ractopamine glucuronides was also determined with IC50 values at 3 ± 0.11 ng/mL (n = 5) and 1 ± 0.89 ng/mL (n = 3), respectively. The limit of detection and limit of quantification for both of them were 0.5 and 1 ng/mL,
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respectively. Shelver and Smith had found (RR, SS) diastereoisomer glucuronides
showed 4% cross-activity when measured by a polyclonal antibody, they further
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developed an ELISA method in which the monoclonal antibody was specifically sensitive to detect RR and SR ractopamine stereoisomers (Shelver et al. 2000, 2002).
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The linearity and sensitivity of the ELISA method used in this study was as good as other sensitive methods like HPLC-FLD and SPR biosensors (Lu et al. 2012; Vulić et
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al. 2012; Zhai et al. 2011; Shelver et al. 2003).
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The analytical precision and recovery of the ELISA method were comparable with those obtained by the other two methods in current study, LC-MS/MS and GC-MS (Table 1). Two fortification concentrations (1 ng/mL and 25 ng/mL for ELISA and LC-MS/MS, 2 ng/mL and 50 ng/mL for GC-MS. respectively) of RR ractopamine, ractopamine or its glucuronides were set up. In the inter-assay tests, the recovery of RR ractopamine for ELISA was larger than 100% with the coefficient of variation
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conjugates of ractopamine showed no detectable reactivity and (RS, SR) glucuronides
(CV) usually < 10.8%, the recovery values of total ractopamine for the LC-MS/MS and GC-MS methods had an average recovery of 111.4 ± 12% with CV < 15.3% and about 113% recovery with CV < 13.2%, respectively. In the intra-assay variation tests, the average recovery of RR ractopamine for ELISA was 100 ± 1.8% with acceptable CV values, and both the values of total ractopamine for LC-MS/MS and GC-MS were more than 98.6% with acceptable CV values. We also developed the three methods to 11
determine ractopamine glucuronides (Figure 1). The average recoveries were all more than 100%, however, the CV values (from 6.8% up to 16.8%) were generally higher than those from SPE purified urine samples. In addition, compared with the LC-MS/MS method, the linearity of both ractopamine and ractopamine glucuronides for GC-MS method was from 1 to 100 ng/mL, with the sensitivity of 1 ng/mL and the
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Depletion patterns monitored by ELISA, LC-MS/MS and GC-MS
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The SPE cleaned cattle urine samples (n = 6) without enzymatic hydrolysis were firstly analyzed by the three methods, which allowed us to examine the depletion of
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parent ractopamine (free ractopamine). We plotted the data in order to display how administrated ractopamine depleted in the food animals. As shown in the bottom panel
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of Figure 3A, the determined concentrations of parent ractopamine by LC-MS/MS
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and GC-MS methods are much closer and smaller than those determined by ELISA, especially during the feeding period (8 days for cattle). This difference was similar to the previous results reported by Shelver et al. (2002), in which the concentrations of parent ractopamine measured by HPLC were higher than that by ELISA at several sampling points. Our results obtained from the three methods showed roughly the same depletion trend, in which the levels peaked at around the 6th day of the feeding
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average coefficient values R2 = 0.998 and 0.96, respectively.
period, and then the concentration of parent ractopamine dropped quickly to the lowest point of the concentration curve, usually on the fourth day of the withdrawal period (6 days for cattle). The trend of a rapid decline of ractopamine residues during the withdrawal days was consistent with previous reports (Elliott et al. 1998; Shelver et al. 2002; Smith et al. 2002). We also tested hydrolyzed cattle urine samples by LC-MS/MS and GC-MS
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(Figure 3A), the determined concentrations of total ractopamine changed along sampling days with the similar pattern of parent ractopamine, only that the amount increased up to about 100 times due to the mass release of the conjugated metabolites, which indicated that ractopamine was largely metabolized. The observation is consistent with previous studies (Shelver et al. 2002; Smith et al. 2002). However, our
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data for total ractopamine is 2-4 times lower than that reported by Shelver et al.
than that used by Shelver et al. (2002), for example, we incubated the urine sample
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with β-glucuronidase for 3 h rather than 18 h used by Shelver et al (2002).
We further quantified the ratio of ractopamine glucuronides to the total
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ractopamine in unpurified and unhydrolyzed urine samples. As shown in Figure 4A, the concentrations of RR ractopamine and its glucuronides determined by ELISA
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were about 50% of total ractopamine during the feeding period (see Figure 3A), and
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RR glucuronides of all stereoisomers (Figure 4A) measured by the LC-MS/MS (or GC-MS) made up 75 – 90% of total ractopamine residues (Figure 3A). The explanation for this difference is evident because the chromatographic-MS methods do not differentiate the stereoisomers of ractopamine as the ELISA method did in this study.
To verify the depletion pattern of ractopamine and its glucuronide metabolites,
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(2002). This difference may be due to the hydrolytic efficiencies which were lower
we also detected both unhydrolyzed and hydrolyzed sheep urine samples (n = 4) treated with the same dose by similar procedures. Both the feeding and withdrawal period of sheep treatments were 7 days. The concentrations of both parent ractopamine (parent RR stereoisomer in our ELISA assays) and total ractopamine went up as well as dropped with a smoother curve trend relative to those depletion curves for cattle samples (Figure 3B, Figure 4B).
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The concentration levels of ractopamine glucuronides from cattle and sheep urine samples were close (Figure 4). The concentrations ranged from parent ractopamine to total ractopamine in sheep urine samples (roughly one total ractopamine value ≈ 5 times of parent ractopamine at the maximum level) which was much narrower than that in cattle urine samples (roughly one total ractopamine value
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≈ 75 times of parent ractopamine). This suggests that the extent of ractopamine
ractopamine detected in sheep and cattle urine were larger than Shelver’s results, in
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which parent ractopamine was generally less than 10% of total ractopamine residue (Shelver et al. 2002; Smith et al. 2002). The parent RR ractopamine concentration
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levels by ELISA during the feeding period were relatively stable (see the bottom panels of Figure 3A and 3B), which might suggest that the RR ractopamine
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stereoisomer accumulated in food animals under a similar metabolic profile. On the
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other hand, the concentrations of parent ractopamine by LC-MS/MS and GC-MS measurements exceeded the levels measured by ELISA, the maxima peaks of the former two methods were about 2-3 times higher in sheep urine samples (Figure 3B), rather than 2-3 times lower in cattle urine samples (Figure 3A). There were two explanations for the response difference. One was animal species-specific metabolism difference of the four ractopamine stereoisomers. In the current study, relative to the
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metabolism among animals could be tremendous. The unmetabolized extent of
levels of RR ractopamine stereoselectively measured by ELISA, the levels of three other ractopamine stereoisomers in cattle were much lower than that in sheep. The other reason was that the detection ranges of LC-MS/MS and GC-MS were not limited to RR stereoisomer as the ELISA method did in this study.
Correlation analyses of depletion results by the three methods
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Due to the wide range of data variations as seen from Figure 3 and Figure 4, the second-order polynomial regression model was used to analyze the correlations of results obtained by the three methods. All trend lines were forced through the origin. Our results show that the data of parent ractopamine by ELISA and LC-MS/MS or GC-MS with only SPE cleanup were highly correlated (Figure 5A). The correlation
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coefficient (R2) of the functional equation y = -0.0064x2 + 0.7991x was 0.9201
cattle urine samples, indicating good agreement between the two assay methods. The
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R2 value of 0.93 was slightly higher between the results by ELISA and LC-MS/MS (0.04-28.61 ng/mL) assays (Figure 5A). These results were comparable with the result
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reported by Shelver et al. in which the R2 value was 0.95 for parent ractopamine by ELISA and HPLC (Shelver et al. 2002). However, as shown in Figure 3, the
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regression between LC-MS/MS and GC-MS assays was more highly correlated with a
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R2 value of 0.9651, which demonstrated the two methods had very close reliability with regard to the detection of ractopamine residues. This conclusion was also affirmative when testing total ractopamine which contained parent ractopamine and conjugated metabolites released by β-glucuronidase hydrolysis, as showed in Figure 6A, where R2 = 0.9677.
Moreover, the correlations of results derived from sheep urine sample were
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between the results by ELISA (0.72-70.73 ng/mL) and GC-MS (0.22-24.32 ng/mL) in
generally better than those of cattle samples (Figure 5B, Figure 6B). The regression between LC-MS/MS and GC-MS assays was excellent, regardless of data measured for parent ractopamine and total ractopamine, in which R2 values were 0.9807 (Figure
5B) and 0.9868 (Figure 6B), respectively. For parent ractopamine determined in unhydrolyzed cattle urine samples, the result by ELISA and HPLC in Shelver and colleague’s study was not well correlated (R2 = 0.58) (Shelver et al. 2002; Smith et al.
15
2002), while in our study the correlations between the data by ELISA and LC-MS/MS (and also GC-MS) were quite good. These results suggested LC-MS/MS and GC-MS were well complementary to the ELISA method in this study. In summary, we identified the glucuronide conjugates of ractopamine, a leanness-promoting agent in food animals. We demonstrated the capability of an
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ELISA method to detect (1R, 3R)-ractopamine stereoisomer, the biologically active
well as ractopamine glucuronide, the main form of ractopamine metabolites, were
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conducted by ELISA, LC-MS/MS and GC-MS assays. The depletion trends of ractopamine residues and its metabolites were found to be that the concentrations of
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ractopamine residues or its glucuronides increased and reached a maximum during the mid-late feeding period and then dropped quickly when entering the post-feeding
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period. This study is helpful in providing insights into the administration of
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ractopamine, especially the misuse or abuse of ractopamine in those countries and districts where the use of ractopamine for food animals is banned, and the further understanding of the metabolism of ractopamine would better serve the detection and monitoring of ractopamine usage in the fields related to meat food safety. In the future, it is essential to examine the depletion mechanisms of ractopamine residues and its metabolites in other matrices such as muscle, serum, hair, liver and even retinal
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isomer of ractopamine. The determination of parent ractopamine, total ractopamine as
tissues for better meat food safety.
Acknowledgments The authors thank Dr. Shelver (USDA-ARS, Biosciences Research Laboratory, Fargo, ND) for kindly providing the incurred urine samples.
16
References Barnes PJ. 1997. Effect of β-agonists on airway effector cells. Lung Biol Health Dis. 106:35–64. Dalidowicz JE, Thomson TD, Babbitt GE. 1992. Ractopamine hydrochloride, a phenethanolamine repartitioning agent: Metabolism and residues. In Xenobiotics
Elliott CT, Thompson CS, Arts CJM, Crooks SRH, vanBaak MJ, Verheij ER, Baxter
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GA. 1998. Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the β-agonist. Analyst.
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123:1103-1107.
European Food Safety Authority. 2009. Safety evaluation of ractopamine: scientific
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EFSA J. 1041:1-52.
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opinion of the panel on additives and products or substances used in animal feed.
Freire EF, Borges KB, Tanimoto H, Nogueira RT, Bertolini LCT, de Gaitani CM. . 2013. Monitoring of ractopamine concentration in the mixture of this feed additive with vitamin mineral complex and with swine feed by HPLC. Food Addit Contam., Part A. 30: 796-803
Huang JD, Lin Q, Zhang XM, He XR, Xing XR, Lian WJ, Zuo MM, Zhang QQ. 2011.
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Eds., American Chemical Society, pp 234-243.
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and Food-Producing Animals, Hutson DH. Hawkins DR, Paulson GD, Struble CB,
Electrochemical immunosensor based on polyaniline/poly (acrylic acid) and Au-hybrid graphene nanocomposite for sensitivity enhanced detection of salbutamol. Food Res Int. 44:92-97.
Hughes CG. 2002. Confirmation and quantitation of ractopamine in equine urine. Equine Pharmacology Department, Gluck Equine Research Center, University of Kentucky, Lexington.
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Lehner AF, Hughes CG, Harkins JD, Nickerson C, Mollett B, Dirikolu L, Bosken J, Camargo F, Boyles J, Troppmann A, Karpiesiuk W, Woods WE, Tobin T. 2004. Detection and confirmation of ractopamine and its metabolites in horse urine after paylean® administration. J Anal Toxicol. 28(4):226-237. Lin KC, Hong CP, Chen SM. 2013. Simultaneous determination for toxic ractopamine
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and salbutamol in pork sample using hybrid carbon nanotubes. Sens Actuators B
Liu XY. He XW, Moore C, Wang GH, Coulter C. 2009. Highly sensitive and specific
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liquid chromatography-tandem mass spectrometry method for testing ractopamine in cattle and sheep urine. J Anal Toxicol. 33(6): 289-293.
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Lu X, Zheng H, Li XQ, Yuan XX, Li H, Deng LG, Zhang H, Wang WZ, Yang GS, Meng M, Xie RM, Aboul-Enein HY. 2012. Detection of ractopamine residues in
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pork by surface plasmon resonance-based biosensor inhibition immunoassay.
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Food Chem. 130(4):1061-1065.
Maistro S, Chiesa E, Angeletti R, Brambilla G. 1995. Beta blockers to prevent clenbuterol poisoning. Lancet. 346:180. Qiang ZY, Shentu FQ, Wang B, Wang JP, Chang JY. Shen JZ. 2007. Residue depletion of ractopamine and its metabolites in swine tissues, urine, and serum. J Agric Food Chem. 55:4319–4326.
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Chem. 177:428–436.
Ricke EA, Smith DJ, Feil VJ, Larsen GL, Caton JS. 1999. Effects of ractopamine HCI stereoisomers on growth, nitrogen retention, and carcass composition in rats. J Anim Sci. 77:701-707. Shappell NW, Feil VJ, Smith DJ, Larsen GL, McFarland DC. 2000. Response of C12 mouse and turkey skeletal muscle cells to the beta-adrenergic agonist ractopamine. J Anim Sci. 78: 699-708.
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Shelver WL, Smith DJ. 2000. Development of an immunoassay for the β-adrenergic agonist ractopamine. J Immunoassay. 21:1-23. Shelver WL, Smith DJ. 2002. Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals. J Agric Food Chem. 50:2742-2747.
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Shelver WL, Smith DJ. 2003. Determination of ractopamine in cattle and sheep urine
ELISA. J Agric Food Chem. 51:3715−3721.
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Shelver WL, Smith DJ, Berry ES. 2000. Production and characterization of a
Food Chem. 48:4020-4026.
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monoclonal antibody against the beta-adrenergic agonist ractopamine. J Agric
Shelver WL, Thorson JF, Hammer CJ, Smith DJ. 2010. Depletion of urinary zilpaterol
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58(7):4077-4083.
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residues in horses as measured by ELISA and UPLC-MS/MS. J Agric Food Chem.
Smith DJ. 1998. The pharmacokinetics, metabolism, and tissue residues of β-adrenergic agonists in livestock. J Anim Sci. 76:173. Smith DJ, Feil VJ, Huwe JK, Paulson GD. 1993. Metabolism and disposition of ractopamine hydrochloride by turkey poults. Drug Metab Disp. 21:624-633. Smith DJ, Feil VJ, Paulson GD. 2000. Identification and metabolism of turkey biliary
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samples using an optical biosensor analysis: comparative study with HPLC and
metabolites of ractopamine HCl and the metabolism and disposition of synthetic [14C]-ractopamine glucuronides in turkeys. Xenobiotica. 30:427-440.
Shelver WL, Smith DJ. 2002. Tissue residues of ractopamine and urinary excretion of ractopamine and metabolites in animals treated for 7 days with dietary ractopamine. J Anim Sci. 80(5): 1240-1249. Vulić A, Pleadin J, Perši N, Milić D, Radeck W. 2012. UPLC-MS/MS determination
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of ractopamine residues in retinal tissue of treated food-producing pigs. J Chromatogr B. 895-896:102–107. Zhai FL, Huang YQ, Li CY, Wang XC, Lai KQ. 2011. Rapid determination of ractopamine in swine urine using surface-enhanced Raman spectroscopy. J Agric
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Food Chem. 59:10023–10027.
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Figure 1. Identification of ractopamine and its glucuronide metabolites by
LC-MS/MS. Cattle urine samples were injected without β-glucuronidase hydrolysis and SPE cleanup. (A) Full-scan by ESI (+)-MS of parent ractopamine (m/z 302.2) in incurred cattle urine sample. (B) Representative chromatograph of incurred cattle urine sample (dashed line) showed the new peak different from negative control urine (solid line). (C) ESI (+)-MS spectra for the new peak (m/z 478.4) with identified structures showed.
21
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Figure 2. Calibration curves of RR ractopamine (RAC) (A) and ractopamine glucuronide (B) for ELISA. B and B0, the average absorbance at the concentration indicated and at a zero concentration, respectively. Datum represents three independent measurements.
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Figure 2
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ractopamine (lower) in (A) cattle urine (n = 6) and (B) sheep urine (n = 4) measured
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by ELISA, LC-MS/MS and GC-MS. Total ractopamine (parent ractopamine + glucuronide metabolites) was determined from urine sample with enzymatic hydrolysis and SPE extraction, parent ractopamine was measured from un-hydrolyzed SPE sample, dot represented average value of measurements. Feeding duration indicated before the vertical dashed line was 8 days for cattle (A) and 7 days for sheep (B), and withdrawal period after the dashed line was 6 days for cattle (A)and 7 days
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Figure 3. Comparisons of analyses of total ractopamine (upper) and parent
for sheep, respectively.
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Figure 4. Comparisons of analyses of ractopamine glucuronide conjugates in (A)
cattle urine (n = 6) and (B) sheep urine (n = 4) measured by ELISA, LC-MS/MS and
GC-MS. Ractopamine glucuronides were determined from diluted samples without SPE and enzymatic hydrolysis treatments, dot represented average value of measurements. Feeding duration was 8 days for cattle (A) and 7 days for sheep (B), and withdrawal period of 6 and 7 days, respectively.
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Figure 5. Pairwise correlations of parent ractopamine results measured by the
three methods. Cattle urine sample (A) and sheep urine sample (B). □, parent ractopamine results by ELISA and GC-MS; ▲, ELISA and LC-MS/MS; ○, LC-MS/MS and GC-MS.
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Figure 6. Regression of total ractopamine results by LC-MS/MS on results by
GC-MS method. Cattle urine sample (A) and sheep urine sample (B).
26
Table 1. Percent Recovery, Inter-assay Variation, and Intra-assay Variation of the ELISA, LC-MS/MS and GC-MS Methods Fortification
recovery
CV
average
recovery CV (%)
(ng/mL)
(%)
(%)
(ng/mL)
(%)
1.07
113.5
6.4
25.00 (RR RAC)
25.86
127.3
10.8
1.00 (RAC Glu)b
1.32
118.2
12.6
25.00 (RAC Glu)
26.54
116.8
98.2
1.3
23.59
101.8
3.5
1.15
106.5
9.5
24.68
109.5
10.5
(n = 5)
1.25
123.8
15.3
1.01
98.6
2.5
25.00 (RAC)
24.64
98.9
8.8
23.96
99.3
6.3
1.00 (RAC Glu )
1.17
115.8
13.4
1.06
99.7
8.9
25.00 (RAC Glu )
25.89
118.7
12.7
25.06
106.2
10.8
M
1.00 (RAC)
GC-MS
b
14.3
an
(n = 5)
0.98
us
1.00 (RR RAC) a
LC-MS/
(n = 5)
(n = 5)
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a
(n = 10)
2.00 (RAC)
2.85
114.5
13.2
1.86
98.8
4.1
50.00 (RAC)
51.41
113.7
9.8
50.41
105.6
2.8
2.00 (RAC Glu )
2.16
120.5
16.8
1.93
99.7
6.8
50.00 (RAC Glu )
52.13
118.4
14.8
49.86
112.5
9.5
RR RAC, (1R, 3R)-ractopamine.
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(n = 10)
t
average
ELISA
MS
Intra-assay variation
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(ng/mL)
Inter-assay variation
RAC Glu, ractopamine glucuronides.
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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Identification of ractopamine glucuronides and determination of bioactive ractopamine residues and its metabolites in food animal urine by ELISA, LC-MS/MS and GC-MS abc
Xiao-Fei Jiang a
b
d
, Yue-Hui Zhu & Xiao-Yun Liu
School of Chemistry and Life Science, Guizhou Normal College, Guiyang 550018, China
b
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Guangzhou 510650, China c
College of Chemical and Environmental Engineering, Chongqing Three Gorges University, 404000, Chongqing, China d
Guangzhou Accurate and Correct Test Co., Ltd., 510663, Guangzhou, China Accepted author version posted online: 15 Oct 2013.Published online: 15 Oct 2013.
To cite this article: Food Additives & Contaminants: Part A (2013): Identification of ractopamine glucuronides and determination of bioactive ractopamine residues and its metabolites in food animal urine by ELISA, LC-MS/MS and GC-MS, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2013.855327 To link to this article: http://dx.doi.org/10.1080/19440049.2013.855327
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Identification of ractopamine glucuronides and determination of bioactive ractopamine residues and its metabolites in food animal urine by ELISA, LC-MS/MS and GC-MS
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School of Chemistry and Life Science, Guizhou Normal College, Guiyang 550018,
b
M an us
China;
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South
China Botanical Garden, Guangzhou 510650, China; c
College of Chemical and Environmental Engineering, Chongqing Three Gorges
University, 404000, Chongqing, China.
Guangzhou Accurate and Correct Test Co., Ltd., 510663, Guangzhou, China.
pt
ed
d
ce
* Corresponding authors. Tel. /Fax: +86 20 37252958. E-mail address: [email protected] (X.Y. Liu) or [email protected] (Y.H. Zhu).
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a
t
Xiao-Fei Jiang a,b,c, Yue-Hui Zhub, *, and Xiao-Yun Liud,*
1
Abstract Ractopamine glucuronides have been identified in cattle urine sample by LC-MS/MS. An ELISA method, which was capable of specifically determining (1R, 3R)-ractopamine stereoisomer and its glucuronide metabolites, had more than 100% recovery with an acceptable coefficient of variation in the inter-assay and intra-assay
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variation tests for RR ractopamine. The concentration levels of parent ractopamine
ractopamine in cattle and sheep urine showed similar depletion trends, in which the
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concentration curves increased and reached a climax during the feeding period, and then dropped fast when entering the withdrawal period. Data from the three methods
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had very good pair-wise correlations. In the cattle urine samples, the correlation coefficient (R2) for parent ractopamine between the ELISA and the LC-MS/MS or
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GC-MS results were 0.93 or 0.92, R2 values for parent ractopamine and total ractopamine data measured by LC-MS/MS and GC-MS were 0.9651 and 0.9677,
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respectively. All R2 values for data gained from sheep urine samples were > 0.95. Our study indicated that the close levels of RR ractopamine stereoisomer in cattle and sheep urine samples may imply the presence of a similar depletion pattern in other livestock, and thus would facilitate an accurate detection and management of ractopamine usage in food safety.
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and ractopamine glucuronide metabolites as the main components of total
Keywords: (1R, 3R)-ractopamine; glucuronide metabolites; depletion pattern;
livestock; ELISA; mass spectrometry
2
Introduction Ractopamine, one of β-adrenergic agonists, is a leanness-enhancing agent in farm animals. The beneficial aspects include its efficiency in lifting daily weight gain and protein synthesis rates in swine. Ractopamine residues may pose human health risks (Barnes 1997). The use of ractopamine in swine is banned by most countries in the
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world, such as the EU and China, meanwhile, a few countries including the USA
1995; European Food Safety Authority 2009). The conflicting situation of
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ractopamine raises the needs to monitor its presence and metabolism so as to provide information for official regulation and the import or export of food animals.
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The management of illegal use or contamination of β-agonists in food animal production has become an important issue of concern. Nowadays, novel methods and
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devices have been developed to detect ractopamine with high efficiency (Huang et al.
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2011; Shelver et al. 2010; Lin et al. 2013; Liu et al. 2009; Zhai et al. 2011). On the other hand, conventional analytical methods for ractopamine such as HPLC, ELISA and LC-MS/MS are still proving to be powerful approaches to provide comprehensive information on the metabolism of ractopamine (Freire et al. 2013; Lehner et al. 2004; Liu et al. 2009; Elliott et al. 1998). Lehner et al. (2004) screened ractopamine incurred horse urine samples by ELISA and identified the glucuronidation, sulfated and
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allow it as a component of feed additive in cattle, turkeys and pigs (Maistro et al.
methylated forms of ractopamine metabolites by GC-MS. Previous studies showed that the main form of ractopamine metabolites are the glucuronide conjugates (Lehner et al. 2004; Dalidowicz et al. 1992; Elliott et al. 1998; Smith et al. 2000). 1R,
3R-stereoisomer (RR), as one of the four ractopamine stereoisomers (also including RS, SR and SS), was indicated to be the functional and the most bioactive stereoisomer responsible for leanness-enhancing effect of ractopamine (Ricke et al.
3
1999; Shappell et al. 2000). The commonly used commercial ractopamine is a racemic mixture of the four stereoisomers, and previous assays generally have dealt with all isomers of the dosed ractopamine as a whole, and paid little attention to the depletion patterns of conjugated metabolites (Qiang et al. 2007; Vulić et al. 2012; Lu et al. 2012). Shelver and Smith used RR and RS ractopamine stereoisomers to prepare antibody,
however,
the
antibody
showed
strong
t
monoclonal
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ractopamine
antibody had excellent sensitivity to enzyme-hydrolyzed urine samples and
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immunoassays based on the monoclonal antibody yielded comparable results with those data from HPLC and surface plasmon resonance (SPR) biosensor analysis
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(Shelver et al. 2002, 2003). However, when detecting samples without enzymatic hydrolysis, the results for parent ractopamine (free ractopamine) derived from ELISA
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and HPLC methods had low correlation (R2 value = 0.58 for sheep urine samples)
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(Shelver et al. 2002). Another earlier study showed results determined by an ELISA method based on a polyclonal antibody and LC-MS/MS had only fairly good agreement (R2 = 0.73) in ractopamine dosed bovine urine samples (Elliott et al. 1998). Currently, there has been no in-depth examination of the depletion patterns of bioactive ractopamine residues and ractopamine glucuronide metabolites. We supposed that a combination of confirmatory mass spectrometric assays with immune
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stereoselectivity towards both RR and SR stereoisomers (Shelver et al. 2000). The
screening assay would help to disclose the depletion patterns of RR ractopamine residue and ractopamine glucuronide metabolites. In the current study, we exploited an LC-MS/MS method to directly analyze and
identify ractopamine glucuronides in cattle urine samples. We also used an ELISA kit in which the monoclonal antibody enabled us to detect the physiologically active RR ractopamine isomer. By using the three complementary methods including ELISA,
4
LC-MS/MS and GC-MS in incurred cattle and sheep urine samples, this study compared the concentration variations of parent ractopamine stereoisomer, ractopamine glucuronide metabolites and total ractopamine during the feeding and withdrawal period of a total of 14 days. The results obtained may help to indicate the metabolic mechanism of ractopamine stereoisomers and the administration of its
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Materials and methods
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Chemicals and Reagents
Ractopamine hydrochloride (ractopamine) was used for animal treatment,
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ractopamine and its glucuronide conjugates were used for method validation, and isoxuprine hydrochloride was used as an internal standard, which together with
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β-glucuronidase and other chemicals were purchased from Sigma-Aldrich
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(Milwaukee, KI) and were reagent grade or higher. Ractopamine glucuronide conjugates were generous gifts from Dr. Smith (Smith et al. 1993). The derivatization reagent N, O-bis (trimethylsilyl) trifluoroacetamide + 1% trimethylchlorosilane (BSTFA + 1% TMCS) was obtained from Pierce (Rickford, IL).
Sampling of Incurred Urine
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rational use in food-producing livestock.
Urine samples containing ractopamine residues were obtained from Dr. Shelver
(USDA-ARS, Biosciences Research Laboratory, Fargo, ND). Briefly, control animal (cattle or sheep) urine (n = 3) across different collection days was mixed to generate a composite control urine. The drug incurred urine samples were collected from cattle or sheep fed a diet containing 20 mg/kg ractopamine for 8 or 7 consecutive days, respectively. Urine samples were collected during the feeding and withdrawal periods.
5
Pooled urine (pH 7.2) was buffered with 1:1 (V: V) volume of 100 mM phosphate buffer. Aliquots of the samples were stored at -20 °C until analyzed.
Enzyme-linked Immunosorbent Assay (ELISA) Ractopamine ELISA test kit obtained from Immunalysis (Pomona, CA; private
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communication) was used for screening the urine samples by following the
antibody was immobilized with 96-well polystyrene plate, and the antigen consisting
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of ractopamine was coupled to bovine thyroglobulin. In this study, urine specimens in most cases were pre-diluted 1:10 with phosphate buffer (100 mM, pH 7.2) before
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analysis, in order that the determined ractopamine concentrations were less than 20% B/B0 from the ELISA calibration curve. Aliquots of 100 µl calibration solutions or
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specimens were added into each plate well. Goat anti-mouse HRP-IgG of 1: 20000
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was finally used. The absorbance of the samples was read at 450 nm and 650 nm with a Tecan model Infinite 200 PRO Microplate Readers (Tecan Inc, San Jose, CA). The calibration curves for ractopamine were plotted by measuring control urines with ractopamine concentrations of 0, 0.1, 1, 2, 5, 10, 50 and 100 ng/mL. The selectivity of the ELISA kit was checked beforehand, where the quantification of ractopamine were not affected by the presence of clenbuterol and cycloclenbuterol. Moreover, the
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manufacturer’s protocol. Briefly, a (1R, 3R)-ractopamine-specific monoclonal
absorbance variation due to the presence of other three ractopamine stereoisomers was less than 5% of absorbance measured with only (1R, 3R)-ractopamine, indicating the monoclonal antibody had specific stereo-selectivity towards binding RR ractopamine and the ELISA kit had excellent selectivity.
Sample Preparations for Chromatographic Analysis
6
β-glucuronidase hydrolysis An aliquot of urine samples (1 mL) was mixed thoroughly with 400 µl sodium acetate (1 M, pH 5.0) and 1000 Units β-glucuronidase incubated at 65 °C for 3 hours. Cooled the tubes, following by adding 10 µl methanol solution of isoxuprine at 10 µg/mL. Centrifuged at 2500 × g for 10 min and the supernatants were used for
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Solid-phase extraction (SPE) cleanup
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The SPE procedure was employed to remove ractopamine metabolites and other unwanted compounds in the samples. A SPE mixed mode C18 column model Clin II,
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691-0353T (SPEWare Corp, San Pedro, CA) was operated to purify urine sample following a previously described procedure (Liu et al. 2009). The eluted ractopamine
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in methylene chloride/isopropanol/ammonium hydroxide (78:20:2, v/v/v, 3 mL) was
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dried under nitrogen stream and reconstituted in 50 µl methanol for further chromatographic or ELISA analysis, respectively.
Identification of Metabolites and Confirmation by LC-MS/MS The operating and tuning procedures, following a previously reported method with minor modifications (Liu et al. 2009), were run on an Agilent Technologies 1200
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ractopamine determination.
series liquid chromatography pump coupled to a 6410 triple quadrupole mass spectrometer (MS), ionized using Electrospray ionization (ESI) in the positive mode. Aliquot (5 µl) of ractopamine standard solution, control or incurred urine samples were injected into a Zorbax Eclipse XDB C18 (4.6 × 50 mm, 1.8 μm; Agilent) and a guard column (4.6 × 12.5 mm, 1.8 μm). The columns were thermostatically controlled at 40 °C, and the flow rate was 0.5 mL/min. The binary gradient system consisted of
7
A, 20 mM ammonium formate (pH 6.4), and B, methanol, and the composition of the mobile phase (85% A) for the entire program remained constant. The calibration curve for ractopamine was made with 0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25 and 50 ng/mL of ractopamine as well as ractopamine glucuronides in the control urine. Data were acquired, calculated and reported by the Agilent Masshunter Quantitative Analysis
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(QQQ) software.
conjugate were injected into MS equipment directly. The precursor ion for
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ractopamine was m/z 302.2 and the ion transition of isoxuprine was from m/z 302.1 to m/z 284.2. The ion m/z 162.1 and m/z 478.4 were used for LC-MS/MS quantification
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of ractopamine and the main glucuronide conjugates (identified in this study, Figure 1), respectively. Validations of both ractopamine and ractopamine glucuronides for the
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LC-MS/MS method were conducted, in which the linearity from 0.1 to 50 ng/mL was
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obtained with an average coefficient R2 > 0.99 and > 0.97, and they could be detected at the lowest concentrations at 0.1 ng/mL and 0.5 ng/mL, respectively, indicating the method had excellent sensitivity. The intraday and interday precision and accuracy were determined in control urine with fortified ractopamine or ractopamine glucuronides at 1 ng/mL and 25 ng/mL, respectively.
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To optimize mass spectrometric conditions, ractopamine and its glucuronide
GC-MS confirmation Confirmation of ractopamine and ractopamine glucuronides in cattle and sheep
urine samples following β-glucuronidase hydrolysis, SPE and trimethylchlorosilane (TMCS) derivatization was accomplished by GC-MS method as described by Hughes (2002), with modifications. An Agilent 7890 GC coupled to a 5975C mass selective detector (MSD), operating in electron ionisation mode was used for analysis. The GC
8
column was a DB-5MS (15-m length, 0.25 mm i.d., 0.25 µm film thickness, J&W Scientific), and the injection temperature was 280 °C. The standard and urine samples were hydrolyzed and SPE extracted as above method, and the extracts were finally reconstituted in 10 µl ethyl acetate and 40 µl BSTFA + 1% TMCS. The extracts were then transferred to autosampler vial, capped, and heated at 85 °C for 45 min. The
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purge flow rate was 50 mL/min for 0.5 min, and the carrier gas was helium. The
mL/min. The initial GC oven temperature was 60 °C ramped at 20 °C/min to 280 °C.
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The transfer line was held at 280 °C, the ion source at 230 °C, and the quadrupole at 150 °C. The dwell time for all ions was 50 ms. Ractopamine was identified at the
an
retention time of 12.8 min, and three ions were selected from the full scan spectrum, m/z 250, 179 and 234. Isoxuprine was at the retention time of 11.46 min, and the ions
M
selected for monitoring were m/z 178 and 267. The calibration curve for ractopamine
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(and ractopamine glucuronides) was determined at 0, 0.5, 1, 5, 10, 25, 50 and 100 ng/mL, and the validations of quantification were conducted similarly as LC-MS/MS method. The results were analyzed and reported with the Agilent DrugQuant ChemStation software package.
Results and discussion
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injection mode was splitless, the injection volume was 2 µl with the constant flow at 2
Identification of ractopamine glucuronide metabolites by LC-MS/MS Ractopamine standard has two ion transitions, m/z 302.2→164.1 and m/z
302.2→121.2, as previously described for ractopamine detection by LC-MS/MS (Elliott et al. 1998; Liu et al. 2009). In this study, in order to maximize the detection possibility of ractopamine metabolites, the incurred cattle urine sample with only proper dilution but no clean-up treatment was injected directly into the LC-MS/MS
9
system. Parent ractopamine was identified based on the retention time and full-scan ESI (+)-MS spectra compared with those of standard ractopamine, an ion peak at m/z 302.2 was used to monitor and quantify the parent ractopamine residue (Figure 1A). Representative ion chromatogram from LC-MS/MS analysis of an incurred cattle urine sample showed a new peak at 2.74 min (Figure 1B). The peak was further
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measured by ESI (+)-MS and showed a characteristic ion signal at m/z 478.4. Its mass
mono-glucuronide acid conjugate at a phenolic hydroxyl group of ractopamine,
us
(Lehner et al. 2004), and was designated as ractopamine-O-glucuronide herein (Figure 1C). Other different peaks of the incurred sample were also analyzed and showed no
an
characteristic ion signals (data not shown). Glucuronidation of ractopamine was showed to be the major type of modification compared to sulfation and methylation of (Lehner
et
al.
2004;
Shelver
M
ractopamine
et
al.
2002),
for
example,
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mono-glucuronides conjugated to the phenols at C-10 and C-10’ could be up to 73% total conjugate metabolites in turkey (Smith et al. 2000). All kind of conjugated metabolites could be detected by ELISA and ESI (+)-MS. In the current work, we determined ractopamine glucuronide metabolites by using three complementary methods.
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spectral data was compared with a previous report and was consistent with that of
Validation of methods We employed an ELISA kit in which the monoclonal antibody was specifically
raised to bind (1R, 3R)-ractopamine stereoisomer and its conjugated metabolites. The calibration curves for RR ractopamine and ractopamine glucuronides were prepared in buffered control cattle urine, respectively (Figure 2). Eight concentration levels of standard chemicals were set up from 0.01 up to 100 ng/mL. The linearity of the
10
ELISA method developed to detect RR ractopamine and ractopamine glucuronides was from 0.1 to 50 ng/mL, with R2 = 0.97 and 0.95, respectively. The sensitivity for RR ractopamine and ractopamine glucuronides was also determined with IC50 values at 3 ± 0.11 ng/mL (n = 5) and 1 ± 0.89 ng/mL (n = 3), respectively. The limit of detection and limit of quantification for both of them were 0.5 and 1 ng/mL,
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respectively. Shelver and Smith had found (RR, SS) diastereoisomer glucuronides
showed 4% cross-activity when measured by a polyclonal antibody, they further
us
developed an ELISA method in which the monoclonal antibody was specifically sensitive to detect RR and SR ractopamine stereoisomers (Shelver et al. 2000, 2002).
an
The linearity and sensitivity of the ELISA method used in this study was as good as other sensitive methods like HPLC-FLD and SPR biosensors (Lu et al. 2012; Vulić et
M
al. 2012; Zhai et al. 2011; Shelver et al. 2003).
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The analytical precision and recovery of the ELISA method were comparable with those obtained by the other two methods in current study, LC-MS/MS and GC-MS (Table 1). Two fortification concentrations (1 ng/mL and 25 ng/mL for ELISA and LC-MS/MS, 2 ng/mL and 50 ng/mL for GC-MS. respectively) of RR ractopamine, ractopamine or its glucuronides were set up. In the inter-assay tests, the recovery of RR ractopamine for ELISA was larger than 100% with the coefficient of variation
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conjugates of ractopamine showed no detectable reactivity and (RS, SR) glucuronides
(CV) usually < 10.8%, the recovery values of total ractopamine for the LC-MS/MS and GC-MS methods had an average recovery of 111.4 ± 12% with CV < 15.3% and about 113% recovery with CV < 13.2%, respectively. In the intra-assay variation tests, the average recovery of RR ractopamine for ELISA was 100 ± 1.8% with acceptable CV values, and both the values of total ractopamine for LC-MS/MS and GC-MS were more than 98.6% with acceptable CV values. We also developed the three methods to 11
determine ractopamine glucuronides (Figure 1). The average recoveries were all more than 100%, however, the CV values (from 6.8% up to 16.8%) were generally higher than those from SPE purified urine samples. In addition, compared with the LC-MS/MS method, the linearity of both ractopamine and ractopamine glucuronides for GC-MS method was from 1 to 100 ng/mL, with the sensitivity of 1 ng/mL and the
t cr ip
Depletion patterns monitored by ELISA, LC-MS/MS and GC-MS
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The SPE cleaned cattle urine samples (n = 6) without enzymatic hydrolysis were firstly analyzed by the three methods, which allowed us to examine the depletion of
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parent ractopamine (free ractopamine). We plotted the data in order to display how administrated ractopamine depleted in the food animals. As shown in the bottom panel
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of Figure 3A, the determined concentrations of parent ractopamine by LC-MS/MS
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and GC-MS methods are much closer and smaller than those determined by ELISA, especially during the feeding period (8 days for cattle). This difference was similar to the previous results reported by Shelver et al. (2002), in which the concentrations of parent ractopamine measured by HPLC were higher than that by ELISA at several sampling points. Our results obtained from the three methods showed roughly the same depletion trend, in which the levels peaked at around the 6th day of the feeding
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average coefficient values R2 = 0.998 and 0.96, respectively.
period, and then the concentration of parent ractopamine dropped quickly to the lowest point of the concentration curve, usually on the fourth day of the withdrawal period (6 days for cattle). The trend of a rapid decline of ractopamine residues during the withdrawal days was consistent with previous reports (Elliott et al. 1998; Shelver et al. 2002; Smith et al. 2002). We also tested hydrolyzed cattle urine samples by LC-MS/MS and GC-MS
12
(Figure 3A), the determined concentrations of total ractopamine changed along sampling days with the similar pattern of parent ractopamine, only that the amount increased up to about 100 times due to the mass release of the conjugated metabolites, which indicated that ractopamine was largely metabolized. The observation is consistent with previous studies (Shelver et al. 2002; Smith et al. 2002). However, our
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data for total ractopamine is 2-4 times lower than that reported by Shelver et al.
than that used by Shelver et al. (2002), for example, we incubated the urine sample
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with β-glucuronidase for 3 h rather than 18 h used by Shelver et al (2002).
We further quantified the ratio of ractopamine glucuronides to the total
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ractopamine in unpurified and unhydrolyzed urine samples. As shown in Figure 4A, the concentrations of RR ractopamine and its glucuronides determined by ELISA
M
were about 50% of total ractopamine during the feeding period (see Figure 3A), and
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RR glucuronides of all stereoisomers (Figure 4A) measured by the LC-MS/MS (or GC-MS) made up 75 – 90% of total ractopamine residues (Figure 3A). The explanation for this difference is evident because the chromatographic-MS methods do not differentiate the stereoisomers of ractopamine as the ELISA method did in this study.
To verify the depletion pattern of ractopamine and its glucuronide metabolites,
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(2002). This difference may be due to the hydrolytic efficiencies which were lower
we also detected both unhydrolyzed and hydrolyzed sheep urine samples (n = 4) treated with the same dose by similar procedures. Both the feeding and withdrawal period of sheep treatments were 7 days. The concentrations of both parent ractopamine (parent RR stereoisomer in our ELISA assays) and total ractopamine went up as well as dropped with a smoother curve trend relative to those depletion curves for cattle samples (Figure 3B, Figure 4B).
13
The concentration levels of ractopamine glucuronides from cattle and sheep urine samples were close (Figure 4). The concentrations ranged from parent ractopamine to total ractopamine in sheep urine samples (roughly one total ractopamine value ≈ 5 times of parent ractopamine at the maximum level) which was much narrower than that in cattle urine samples (roughly one total ractopamine value
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≈ 75 times of parent ractopamine). This suggests that the extent of ractopamine
ractopamine detected in sheep and cattle urine were larger than Shelver’s results, in
us
which parent ractopamine was generally less than 10% of total ractopamine residue (Shelver et al. 2002; Smith et al. 2002). The parent RR ractopamine concentration
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levels by ELISA during the feeding period were relatively stable (see the bottom panels of Figure 3A and 3B), which might suggest that the RR ractopamine
M
stereoisomer accumulated in food animals under a similar metabolic profile. On the
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other hand, the concentrations of parent ractopamine by LC-MS/MS and GC-MS measurements exceeded the levels measured by ELISA, the maxima peaks of the former two methods were about 2-3 times higher in sheep urine samples (Figure 3B), rather than 2-3 times lower in cattle urine samples (Figure 3A). There were two explanations for the response difference. One was animal species-specific metabolism difference of the four ractopamine stereoisomers. In the current study, relative to the
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metabolism among animals could be tremendous. The unmetabolized extent of
levels of RR ractopamine stereoselectively measured by ELISA, the levels of three other ractopamine stereoisomers in cattle were much lower than that in sheep. The other reason was that the detection ranges of LC-MS/MS and GC-MS were not limited to RR stereoisomer as the ELISA method did in this study.
Correlation analyses of depletion results by the three methods
14
Due to the wide range of data variations as seen from Figure 3 and Figure 4, the second-order polynomial regression model was used to analyze the correlations of results obtained by the three methods. All trend lines were forced through the origin. Our results show that the data of parent ractopamine by ELISA and LC-MS/MS or GC-MS with only SPE cleanup were highly correlated (Figure 5A). The correlation
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coefficient (R2) of the functional equation y = -0.0064x2 + 0.7991x was 0.9201
cattle urine samples, indicating good agreement between the two assay methods. The
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R2 value of 0.93 was slightly higher between the results by ELISA and LC-MS/MS (0.04-28.61 ng/mL) assays (Figure 5A). These results were comparable with the result
an
reported by Shelver et al. in which the R2 value was 0.95 for parent ractopamine by ELISA and HPLC (Shelver et al. 2002). However, as shown in Figure 3, the
M
regression between LC-MS/MS and GC-MS assays was more highly correlated with a
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R2 value of 0.9651, which demonstrated the two methods had very close reliability with regard to the detection of ractopamine residues. This conclusion was also affirmative when testing total ractopamine which contained parent ractopamine and conjugated metabolites released by β-glucuronidase hydrolysis, as showed in Figure 6A, where R2 = 0.9677.
Moreover, the correlations of results derived from sheep urine sample were
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between the results by ELISA (0.72-70.73 ng/mL) and GC-MS (0.22-24.32 ng/mL) in
generally better than those of cattle samples (Figure 5B, Figure 6B). The regression between LC-MS/MS and GC-MS assays was excellent, regardless of data measured for parent ractopamine and total ractopamine, in which R2 values were 0.9807 (Figure
5B) and 0.9868 (Figure 6B), respectively. For parent ractopamine determined in unhydrolyzed cattle urine samples, the result by ELISA and HPLC in Shelver and colleague’s study was not well correlated (R2 = 0.58) (Shelver et al. 2002; Smith et al.
15
2002), while in our study the correlations between the data by ELISA and LC-MS/MS (and also GC-MS) were quite good. These results suggested LC-MS/MS and GC-MS were well complementary to the ELISA method in this study. In summary, we identified the glucuronide conjugates of ractopamine, a leanness-promoting agent in food animals. We demonstrated the capability of an
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ELISA method to detect (1R, 3R)-ractopamine stereoisomer, the biologically active
well as ractopamine glucuronide, the main form of ractopamine metabolites, were
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conducted by ELISA, LC-MS/MS and GC-MS assays. The depletion trends of ractopamine residues and its metabolites were found to be that the concentrations of
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ractopamine residues or its glucuronides increased and reached a maximum during the mid-late feeding period and then dropped quickly when entering the post-feeding
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period. This study is helpful in providing insights into the administration of
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ractopamine, especially the misuse or abuse of ractopamine in those countries and districts where the use of ractopamine for food animals is banned, and the further understanding of the metabolism of ractopamine would better serve the detection and monitoring of ractopamine usage in the fields related to meat food safety. In the future, it is essential to examine the depletion mechanisms of ractopamine residues and its metabolites in other matrices such as muscle, serum, hair, liver and even retinal
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isomer of ractopamine. The determination of parent ractopamine, total ractopamine as
tissues for better meat food safety.
Acknowledgments The authors thank Dr. Shelver (USDA-ARS, Biosciences Research Laboratory, Fargo, ND) for kindly providing the incurred urine samples.
16
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Elliott CT, Thompson CS, Arts CJM, Crooks SRH, vanBaak MJ, Verheij ER, Baxter
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GA. 1998. Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the β-agonist. Analyst.
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123:1103-1107.
European Food Safety Authority. 2009. Safety evaluation of ractopamine: scientific
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EFSA J. 1041:1-52.
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opinion of the panel on additives and products or substances used in animal feed.
Freire EF, Borges KB, Tanimoto H, Nogueira RT, Bertolini LCT, de Gaitani CM. . 2013. Monitoring of ractopamine concentration in the mixture of this feed additive with vitamin mineral complex and with swine feed by HPLC. Food Addit Contam., Part A. 30: 796-803
Huang JD, Lin Q, Zhang XM, He XR, Xing XR, Lian WJ, Zuo MM, Zhang QQ. 2011.
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and Food-Producing Animals, Hutson DH. Hawkins DR, Paulson GD, Struble CB,
Electrochemical immunosensor based on polyaniline/poly (acrylic acid) and Au-hybrid graphene nanocomposite for sensitivity enhanced detection of salbutamol. Food Res Int. 44:92-97.
Hughes CG. 2002. Confirmation and quantitation of ractopamine in equine urine. Equine Pharmacology Department, Gluck Equine Research Center, University of Kentucky, Lexington.
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Lehner AF, Hughes CG, Harkins JD, Nickerson C, Mollett B, Dirikolu L, Bosken J, Camargo F, Boyles J, Troppmann A, Karpiesiuk W, Woods WE, Tobin T. 2004. Detection and confirmation of ractopamine and its metabolites in horse urine after paylean® administration. J Anal Toxicol. 28(4):226-237. Lin KC, Hong CP, Chen SM. 2013. Simultaneous determination for toxic ractopamine
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and salbutamol in pork sample using hybrid carbon nanotubes. Sens Actuators B
Liu XY. He XW, Moore C, Wang GH, Coulter C. 2009. Highly sensitive and specific
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liquid chromatography-tandem mass spectrometry method for testing ractopamine in cattle and sheep urine. J Anal Toxicol. 33(6): 289-293.
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Lu X, Zheng H, Li XQ, Yuan XX, Li H, Deng LG, Zhang H, Wang WZ, Yang GS, Meng M, Xie RM, Aboul-Enein HY. 2012. Detection of ractopamine residues in
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pork by surface plasmon resonance-based biosensor inhibition immunoassay.
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Food Chem. 130(4):1061-1065.
Maistro S, Chiesa E, Angeletti R, Brambilla G. 1995. Beta blockers to prevent clenbuterol poisoning. Lancet. 346:180. Qiang ZY, Shentu FQ, Wang B, Wang JP, Chang JY. Shen JZ. 2007. Residue depletion of ractopamine and its metabolites in swine tissues, urine, and serum. J Agric Food Chem. 55:4319–4326.
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Chem. 177:428–436.
Ricke EA, Smith DJ, Feil VJ, Larsen GL, Caton JS. 1999. Effects of ractopamine HCI stereoisomers on growth, nitrogen retention, and carcass composition in rats. J Anim Sci. 77:701-707. Shappell NW, Feil VJ, Smith DJ, Larsen GL, McFarland DC. 2000. Response of C12 mouse and turkey skeletal muscle cells to the beta-adrenergic agonist ractopamine. J Anim Sci. 78: 699-708.
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Shelver WL, Smith DJ. 2000. Development of an immunoassay for the β-adrenergic agonist ractopamine. J Immunoassay. 21:1-23. Shelver WL, Smith DJ. 2002. Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals. J Agric Food Chem. 50:2742-2747.
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Shelver WL, Smith DJ. 2003. Determination of ractopamine in cattle and sheep urine
ELISA. J Agric Food Chem. 51:3715−3721.
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Shelver WL, Smith DJ, Berry ES. 2000. Production and characterization of a
Food Chem. 48:4020-4026.
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monoclonal antibody against the beta-adrenergic agonist ractopamine. J Agric
Shelver WL, Thorson JF, Hammer CJ, Smith DJ. 2010. Depletion of urinary zilpaterol
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58(7):4077-4083.
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residues in horses as measured by ELISA and UPLC-MS/MS. J Agric Food Chem.
Smith DJ. 1998. The pharmacokinetics, metabolism, and tissue residues of β-adrenergic agonists in livestock. J Anim Sci. 76:173. Smith DJ, Feil VJ, Huwe JK, Paulson GD. 1993. Metabolism and disposition of ractopamine hydrochloride by turkey poults. Drug Metab Disp. 21:624-633. Smith DJ, Feil VJ, Paulson GD. 2000. Identification and metabolism of turkey biliary
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samples using an optical biosensor analysis: comparative study with HPLC and
metabolites of ractopamine HCl and the metabolism and disposition of synthetic [14C]-ractopamine glucuronides in turkeys. Xenobiotica. 30:427-440.
Shelver WL, Smith DJ. 2002. Tissue residues of ractopamine and urinary excretion of ractopamine and metabolites in animals treated for 7 days with dietary ractopamine. J Anim Sci. 80(5): 1240-1249. Vulić A, Pleadin J, Perši N, Milić D, Radeck W. 2012. UPLC-MS/MS determination
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of ractopamine residues in retinal tissue of treated food-producing pigs. J Chromatogr B. 895-896:102–107. Zhai FL, Huang YQ, Li CY, Wang XC, Lai KQ. 2011. Rapid determination of ractopamine in swine urine using surface-enhanced Raman spectroscopy. J Agric
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Food Chem. 59:10023–10027.
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Figure 1. Identification of ractopamine and its glucuronide metabolites by
LC-MS/MS. Cattle urine samples were injected without β-glucuronidase hydrolysis and SPE cleanup. (A) Full-scan by ESI (+)-MS of parent ractopamine (m/z 302.2) in incurred cattle urine sample. (B) Representative chromatograph of incurred cattle urine sample (dashed line) showed the new peak different from negative control urine (solid line). (C) ESI (+)-MS spectra for the new peak (m/z 478.4) with identified structures showed.
21
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Figure 2. Calibration curves of RR ractopamine (RAC) (A) and ractopamine glucuronide (B) for ELISA. B and B0, the average absorbance at the concentration indicated and at a zero concentration, respectively. Datum represents three independent measurements.
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Figure 2
22
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ractopamine (lower) in (A) cattle urine (n = 6) and (B) sheep urine (n = 4) measured
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by ELISA, LC-MS/MS and GC-MS. Total ractopamine (parent ractopamine + glucuronide metabolites) was determined from urine sample with enzymatic hydrolysis and SPE extraction, parent ractopamine was measured from un-hydrolyzed SPE sample, dot represented average value of measurements. Feeding duration indicated before the vertical dashed line was 8 days for cattle (A) and 7 days for sheep (B), and withdrawal period after the dashed line was 6 days for cattle (A)and 7 days
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Figure 3. Comparisons of analyses of total ractopamine (upper) and parent
for sheep, respectively.
23
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Figure 4. Comparisons of analyses of ractopamine glucuronide conjugates in (A)
cattle urine (n = 6) and (B) sheep urine (n = 4) measured by ELISA, LC-MS/MS and
GC-MS. Ractopamine glucuronides were determined from diluted samples without SPE and enzymatic hydrolysis treatments, dot represented average value of measurements. Feeding duration was 8 days for cattle (A) and 7 days for sheep (B), and withdrawal period of 6 and 7 days, respectively.
24
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Figure 5. Pairwise correlations of parent ractopamine results measured by the
three methods. Cattle urine sample (A) and sheep urine sample (B). □, parent ractopamine results by ELISA and GC-MS; ▲, ELISA and LC-MS/MS; ○, LC-MS/MS and GC-MS.
25
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Figure 6. Regression of total ractopamine results by LC-MS/MS on results by
GC-MS method. Cattle urine sample (A) and sheep urine sample (B).
26
Table 1. Percent Recovery, Inter-assay Variation, and Intra-assay Variation of the ELISA, LC-MS/MS and GC-MS Methods Fortification
recovery
CV
average
recovery CV (%)
(ng/mL)
(%)
(%)
(ng/mL)
(%)
1.07
113.5
6.4
25.00 (RR RAC)
25.86
127.3
10.8
1.00 (RAC Glu)b
1.32
118.2
12.6
25.00 (RAC Glu)
26.54
116.8
98.2
1.3
23.59
101.8
3.5
1.15
106.5
9.5
24.68
109.5
10.5
(n = 5)
1.25
123.8
15.3
1.01
98.6
2.5
25.00 (RAC)
24.64
98.9
8.8
23.96
99.3
6.3
1.00 (RAC Glu )
1.17
115.8
13.4
1.06
99.7
8.9
25.00 (RAC Glu )
25.89
118.7
12.7
25.06
106.2
10.8
M
1.00 (RAC)
GC-MS
b
14.3
an
(n = 5)
0.98
us
1.00 (RR RAC) a
LC-MS/
(n = 5)
(n = 5)
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a
(n = 10)
2.00 (RAC)
2.85
114.5
13.2
1.86
98.8
4.1
50.00 (RAC)
51.41
113.7
9.8
50.41
105.6
2.8
2.00 (RAC Glu )
2.16
120.5
16.8
1.93
99.7
6.8
50.00 (RAC Glu )
52.13
118.4
14.8
49.86
112.5
9.5
RR RAC, (1R, 3R)-ractopamine.
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(n = 10)
t
average
ELISA
MS
Intra-assay variation
cr ip
(ng/mL)
Inter-assay variation
RAC Glu, ractopamine glucuronides.
27
