Bioanalytical

DETERMINATION OF SALBUTAMOL, CLENBUTEROL,AND BROMBUTEROL IN URINE BY A HIGHLYSENSITIVE CHEMILUMINESCENCE ENZYMEIMMUNOASSAY

Yuping Wu,1,2 Fei Xu,1 Haiyang Jiang,1 Xiaoqi Tao,1

Kui Zhu,3 Wenli Liu,1 Yifang Cui,1 Xiaoyong Huang,1 andShuangyang Ding11Department of Veterinary Pharmacology and Toxicology, College ofVeterinary Medicine, China Agricultural University, Beijing, China2College of Animal Science, Henan Institute of Science and Technology,Xinxiang, Henan, China3Faculty of Veterinary Medicine, Ludwig-Maximilians-Universitat Munchen,Oberschleißheim, Germany

A salbutamol-bovine serum albumin and a salbutamol-ovalbumin coating antigen were

synthesized, and six New Zealand white rabbits were treated with the immunogen to obtain

polyclonal antibodies to develop a rapid, sensitive, and indirect chemiluminescent enzyme

immunoassay (ciCLEIA) for the analysis of swine and bovine urine. The prepared

antibodies showed high cross-reactivities with clenbuterol (139.6%) and brombuterol

(225%). Under the optimized conditions, the linear dynamic range and the limit of

detection (LOD) for salbutamol were from 0.007 to 0.17lgL�1 and 0.003lgL�1, with

a correlation coefficient of 0.9965 and a half maximum inhibition concentration of

0.028lgL�1. Recoveries for salbutamol, clenbuterol, and brombuterol were from 78.8%

to 119.0% with intra-assay and inter-assay coefficients of variation less than 13.9% and

19.7%, respectively. The reported ciCLEIA was about 10-fold more sensitive for salbutamol

and 20-fold more sensitive for clenbuterol compared to conventional methods. This study

showed that ciCLEIA was a reliable, convenient, and sensitive method for the simultaneous

determination of salbutamol, clenbuterol, and brombuterol in swine and bovine urine.

Keywords: Brombuterol; Chemiluminescent enzyme immunoassay; Clenbuterol; Polyclonal antibody;

Salbutamol; Urine

Received 16 December 2013; accepted 15 April 2014.

Address correspondence to Shuangyang Ding, Department of Veterinary Pharmacology and

Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.

E-mail: dingsy@cau.edu.cn

Color versions of one or more of the figures in the article can be found online at www.tandfonline.

com/lanl.

Analytical Letters, 47: 2761–2773, 2014

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2014.917422

2761

INTRODUCTON

Salbutamol or albuterol is a b2-agonist pharmaceutical with functions similarto clenbuterol for the treatment of respiratory diseases and is also a tocolytic agentin human and veterinary medicine. Thus, these compounds have been widely usedas growth promoters and fattening agents in livestock (Warriss et al. 1990; Garssenet al. 1995). Nowadays, b2-agonists are used illegally as ‘‘lean meat agents’’ forfood-producing animals and may cause food-safety accidents. In the 1990s, severaloutbreaks of food poisoning were reported due to the consumption of meatcontaminated with b2-agonist residues (Vanoosthuyze, Arts, and Van Peteghem1997; Meng et al. 2010). In 2011, some meat products were contaminated by ‘‘leanmeat agents’’ (Li et al. 2013). Hence the use of b2-agonists as growth promotors isforbidden in the European community (Council Directive 96=23=EC, 1996), the Uni-ted States, and China (Ministry of Agriculture of China 235th Bulletin, 2002). TheWorld Anti-Doping Agency has also restricted the use of salbutamol (WADA 2013).

A variety of analytical methods such as gas chromatography (Caban et al. 2011;Eenoo and Delbeke 2002; Forsdahl and Gmeiner 2004), capillary electrophoresis-mass spectrometry (Anurukvorakun et al. 2010; Hung et al. 2010), and high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS–MS)(Ventura et al. 2009; Garcia et al. 2011) have been applied for the determination ofsalbutamol. However, these instrumental methods require time-consuming samplepreparation and sophisticated technicians. Thus, they are usually used as precisequantitative and confirmatory methods, and are not suitable for field analysis or rapidscreening. Immunoassay techniques are widely used due to high sensitivity, goodspecificity, simplicity, and high-throughput field screening. Immunoassay methodsbased on enzyme-linked immunoassays (ELISA) (Eenoo and Delbeke 2002),immunochromatographic assays (Khamta et al. 2009), electrochemiluminescentimmunosensor assays (Li et al. 2013), electrochemical immunosensors (Liu et al.2011), hapten microarrays (Zuo et al. 2010), and chemiluminence (Barnett,Hindson, and Lewis 1999; Sanchez and Rocha 2008) have shown great promise inthe determination of b2-agonists in animal urine and blood.

In recent years, mixtures of low concentrations of several substances(‘‘cocktails’’) are extensively used. To circumvent this problem, more sensitiveanalytical methods to detect such abuse are mandatory. Compared to conventionalcolorimetric detection, chemiluminescent ELISA (ciCLEIA) nay improve thesensitivity of the immunoassay by 1 to 3 orders of magnitude, and thus it is a suitableapproach for screening samples. The purpose of this study was to developa sensitive ciCLEIA for simultaneous determination of salbutamol, clenbuterol,and brombuterol in swine and bovine urine.

MATERIALS AND METHODS

Chemicals and Reagents

Salbutamol hemisulfate salt (99%), clenbuterol hydrochloride (98.5%),ractopamine hydrochloride (98%), cimaterol hydrochloride (98%), terbutaline(99%), and brombuterol hydrochloride (99%) were obtained from Dr. Ehrenstorfer(Germany); phenyl ethanolamine a (99%) and clorprenaline hydrochloride (99%)

2762 Y. WU ET AL.

were from Shanghai Anpel Scientific Instrument (Shanghai, China). Fenoterolhydrobromide (100 ng mL�1) and zilpaterol hydrochloride (98%) were purchasedfrom European Pharmacopoeia Reference Standard (Council of Europe, Strasbourg,France). Stock concentrations were 1m gmL�1, prepared in methanol, and storedat �20�C; working standards were prepared from the stock by serial dilution inassay buffer.

A series of buffers such as coating buffer, blocking buffer, washing solution,0.2M sodium phosphate buffer (PB, pH 7.2), phosphate-buffered saline (PBS, pH7.4), assay buffer, and substrate buffer (pH 5.0) were prepared according to theliterature (Tao et al. 2012; Tao et al. 2013). A pH 6.0 buffer was prepared from0.1M 2-(N-morpholino) ethanesulfonic acid buffer and 0.5molL�1 NaCl. Salbutamolhemisulfate salt, succinic anhydride, bovine serum albumin (BSA), ovalbumin (OVA),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 1,4-dioxane, and2-(N-morpholino) ethanesulfonic acid were purchased from Pierce (Rockford, IL,USA). Isobutyl chloroformate, dicyclohexylcarbodiimide, tributylamine (>98%),triethylanine (>99.5%), dimethylformamide, and Freund’s complete and incompleteadjuvant were purchased from Sigma-Aldrich (St. Louis, MO, USA). Goat-anti-rabbit immunoglobulins labeled horseradish peroxidase (goat anti-rabbit IgG-HRP conjugate) was provided by the Jackson ImmunoResearch Laboratories (WestGrove, PA, USA). Isobutyl alcohol was purchased from Sinopharm ChemicalReagent (Shanghai, China). Deionized water was prepared on a Milli-Q system(Millipore, MA, USA). All other chemicals and organic solvents were of reagentgrade and were from Beijing Chemical (Beijing, China). Chemiluminescencesubstrate solution (SuperSignal) was purchased from Pierce (Rockford, IL, USA).ELISA-kits were obtained from Beijing WDWK Biotech (Beijing, China).

Apparatus

Immunoassay absorbance was read with a Sunrise microtiter plate reader(Tecan, Groedig, Austria). Ultraviolet-visible spectra were recorded using aUV-3000 spectrophotometer (Shimadzu, Japan). Centrifugation was carried outwith a Mikro 22R centrifuge from Hettich Laborapparate (Tuttlingen, Germany).Chemiluminescence was measured with a Veritas Microplate Luminometer (TurnerBioSystems, Sunny Vale, CA, USA). Transparent 96-well microtiter ELISAplates and white opaque high binding plates were used for chemiluminescentmeasurements (Costar Cambridge, USA). An LB-W-24 bath-typed nitrogeninstrument (Organomation Associates, Jnc., USA) and FTS-EI585-Q freeze dryer(Stone Ridge, NY, USA) were used.

Production of Polyclonal Antibodies

Immunogen salbutamol-BSA and coating antigen salbutamol-OVA weresynthesized by the mixed anhydride and 1-ethyl-3-(3-dimethylaminopropyl) carbo-diimide hydrochloride methods (Degand and Bernes-Duyckaerts 1993). The mono-succinyl derivative of salbutamol (salbutamol succinate) was prepared by mixingthe salbutamol hapten with succinic anhydride, linked to BSA or OVA and an

SENSITIVE CHEMILUMINESCENCE ENZYME IMMUNOASSAY 2763

immunogen or a coating antigen conjugation. Detailed procedures are in thefollowing sections.

Salbutamol hemisulfate salt (80mg, 0.33mmol) was dissolved in methanol(10mL) using a magnetic stirrer and 36mg (0.36mmol) of succinic anhydride wasadded. After 36 h at room temperature, the reaction was complete; the productwas dried in vacuo at 50�C for 6 h, and the salbutamol succinate was stored at�20�C until use. The synthetic route is shown as Figure 1.

Salbutamol succinate (40mg, 0.12mmol) was dissolved in a 28.3 mL mixture(1, 4-dioxane: triethylamine: distilled water, 25:3:0.3, v=v=v) using a magnetic stirrer.While stirring, 30 mL (0.23mmol) of isobutyl chloroformate was added dropwise.After 30min at room temperature, the reaction was divided to two equal portions.An ice-cold protein solution of 80mg (0.0012mmol) BSA in 25mL in distilled waterwas added dropwise to one portion. The reaction mixture was stirred for 2 h at roomtemperature followed by overnight at 4�C. Subsequently, dialysis against PBS wascarried out and stirred for 5 days. During dialysis, the PBS was replaced with freshsolution every 24 h to remove the uncoupled free hapten. The dialyzed solution was

Figure 1. Synthesis schemes of (a) monosuccinyl derivative of salbutamol-salbutamol succinate,

(b) salbutamol immunogen, and (c) salbutamol coating antigen.

2764 Y. WU ET AL.

characterized by spectrophotometry and used as the immunogen. Salbutamol, BSA,and dialyzed solution were measured at wavelengths between 220 nm and 400 nm todetermine whether the linking was successful (Figure 2).

Coating antigen was synthesized by the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride method. Ovalbumin (1mg) was dissolved in 0.1mL2-(N-morpholino) ethanesulfonic acid buffer; the monosuccinyl derivative ofsalbutamol (2mg) dissolved in 0.1mL 2-(N-Morpholino) ethanesulfonic acid bufferwas added; 400mL (4mg) of freshly prepared 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added; and the mixture reacted at room tempera-ture for 4 h and was maintained at 4�C overnight. Dialysis against 1, 4-dioxane,2-(N-morpholino) ethanesulfonic acid solution was carried out for 5 days. Thesynthesis scheme for salbutamol immunogen and coating antigen are shown in Figure 1.

Six New Zealand white rabbits were immunized with salbutamol-BSA at 5 sitesby hypodermic injection. Primary immunizations were composed of 500 mL ofPBS containing 1mg of immunogen emulsified in 500 mL of Freund’s completeadjuvant. Subsequent immunizations were of the same volume with completeadjuvant replaced by Freund’s incomplete adjuvant at 3–4 week intervals. After fivebooster injections, anti-salbutamol antisera were produced. The sera werecollected and purified by saturated (NH4)2SO4. The supplement was characterizedby spectrophotometry, followed by addition of an equal volume of glycerol, andstorage at �20�C until tested.

Figure 2. Ultraviolet spectrum of the immunogen of salbutamol.

SENSITIVE CHEMILUMINESCENCE ENZYME IMMUNOASSAY 2765

Sample Extraction

Bovine and swine urine were collected from the local slaughter house andbovine farm at Beijing and Guangzhou, China. The technique described by Courtheynet al. (1994) was used with small modifications in sample preparation. A volume of2mL of urine were added into a 50-mL polystyrene centrifuge tube for extraction,mixed vigorously for 2min, and centrifuged at 10,000 g for 10min. The supernatantwas transferred into a new tube, 1M NaOH were added dropwise to adjust the pHto 9.8, mixed with 6mL isobutyl alcohol, and centrifuged at 8,000 g for 5min. Theorganic layer was transferred into a fresh tube and evaporated to dryness undera gentle steam of nitrogen at 50�C. To evaluate matrix effects on the immunoassay,the extracts of blanks were diluted with assay buffer and prepared to developmatrix-matched inhibition curves. The residue was dissolved in 2mL of assay buffer.

Optimal Concentrations of Antibodies and Coating Antigens

White opaque high binding plates were coated overnight at 4�C with 100 mLof the salbutamol-OVA with different dilution factors (10,000, 20,000, 40,000,and 80,000) dissolved in coating buffer. The plates were washed with 260 mL=wellwashing solution five times, blocked with 150 mL=well of blocking buffer, and incu-bated at 37�C for 1 h. 50 mL=well of standard in assay buffer, followed by 50 mL=wellof polyclonal antibody of salbutamol at different dilutions (5,000, 10,000, 20,000,and 40,000) in 0.2M sodium phosphate solution (pH 7.2) were added. The com-petitive reaction occurred for 30min at room temperature. The 100 mL of goatanti-rabbit IgG-HRP conjugate (1,000- or 5,000-fold dilution) were added to eachwell, and the reaction took place for 30min at room temperature. After washing fivetimes, 100mL freshly prepared substrate mixture of SuperSignal substrate solution wasadded in darkness and the intensity of emission light was measured immediately at425 nm using a chemiluminescence reader. The ELISA procedures were performedas previously describe, but the reaction occurred 15min after addition of 100mL=wellsubstrate buffer, and absorbance was measured within 10min at 450 nm using a micro-titer plate reader after addition of 50mL=well 2.0M H2SO4 to stop the reaction.

Data Analysis

A four parameter logistic equation was used to fit the ELISA and CLEIA data.Standards and samples were made in triplicate and mean chemiluminescenceintensities were processed. Standard curves were obtained by plotting B =B0 againstthe logarithm of analyte concentration and fitted to the four parameter logisticequation using Origin Pro 8.0 software (Version, Microcal, USA). The equation was:

y ¼ f A�Dð Þ=½1þ x=Cð ÞB�g þD ð1Þ

where A and D are the asymptotic maximum (chemiluminescence intensity in theabsence of analyte, RLUmax) and the minimum asymptotic (background signal),respectively; B is the curve slope at the inflection point; and C is the x value at theinflection point (corresponding to the analyte concentration that reduces RLUmax

to 50%, corresponding that the value of B =B0 is 0.5).

2766 Y. WU ET AL.

Cross-Reactivity Study

Nine drugs structurally related and unrelated to salbutamol were analyzed bythe competitive indirect chemiluminescent enzyme immunoassay (ciCLEIA) method.The cross-reactivity (CR) values were calculated with the following equation:

CR% ¼ IC50 of salbutamol nMð Þ=IC50 of salbutamol analogues nMð Þ � 100% ð2Þ

RESULTS AND DISCUSSION

Haptens and Immunogens

Haptens are molecules of low molecular weight, which are not naturallyantigenic. When haptens were conjugated to proteins for an immunogen, antibodiesthat specifically bind free hapten with high affinity were generated. As smallmolecules, salbutamol (molecular weight¼ 239.31 Da) or albuterol sulfate (mole-cular weight¼ 548.64) must be conjugated to carrier proteins in order to makethem immunogenic or to improve their immunogenicity. While designing happensfor antibody production, it is preferable to preserve characteristic groups or fractionsof the target molecular and maximize exposure of haptenic determinants to theimmune system (Goodrow and Hammock 1998). Conjugation of salbutamol toBSA was verified by ELISA (data not shown) and absorbance spectral analysis.The absorbance spectra indicates that effective coupling of salbutamol and BSAwas achieved (Figure 2). According to the absorbance additivity principle, thecoupling ratio of hapten to protein was approximately 21:1. Immunogenicity maybe reduced due to the space shielding effects with high binding ratios of haptensto carrier proteins. Low ratios of haptens to proteins may reduce the specific immuneinduction to the hapten. A coupling ratio ranging from 8:1 to 25:1 was consideredappropriate. In this study, the coupling ratio of hapten to protein was estimatedabout 21:1, which was in the correct proportion.

Optimization of CLEIA Conditions

Optimal concentrations of antibody and coating antigen were determined bycheckerboard analysis. The performance of CLEIA competitive immunoassayusually relies on assay conditions, such as the concentration of coating antigen orantibody, incubation time, the ionic strength, and the pH of the coreactant. Severalexperimental factors influencing the CLEIA performance were studied. TheRLUmax=IC50 ratio has been shown to be a convenient and useful parameter toestimate the effect of a certain factor on the CLEIA performance; the highest ratioindicating highest sensitivity (Mercader and Montoya 1999). RLUmax reflectsthe antibody recognition of the competitive coating antigen in the absence ofanalyte, whereas IC50, is an expression of the assay sensitivity, reflecting the antibodyaffinity for the target analyte. To optimize the experimental conditions, checker-board analysis was used. The optimized results are summarized in Table 1. It wasfound that the concentration of coating antigen and primary antibody were the mostimportant factors to the stability, sensitivity, and the signal-to-noise ratio of

SENSITIVE CHEMILUMINESCENCE ENZYME IMMUNOASSAY 2767

the system. The dilution of the goat anti-rabbit IgG-HRP conjugate showed greatinfluence on the intensity and detection range.

Sensitivity, Stability, and Specificity of the ciCLEIA System

Under the optimized conditions, the equation for standard calibrationcurve was y ¼ f(1.0149�0.0531)=[1þ (x=0.0285)^0.9341]g þ0.0531 with a correlationcoefficient (R2) of 0.9965 and an IC50 value of 0.028 mgL�1 (0.117 n M). The lineardynamic range and the limit of detection (LOD) were from 0.007 to 0.17 mgL�1 and0.003 mgL�1, respectively. The developed ciCLEIA was compared with ELISA forthe determination of salbutamol; the former was approximately 10 timesmore sensitive (Figure 3). Compared with reported ELISA methods for salbutamol

Table 1. Analytical characteristics of the ciCLEIA procedure for salbutamol determination

Characteristic Results

ciCLEIA parameters

Coating antigen 1:40000

Dilution of polyclonal antibody 1:10000

Dilution of goat anti-rabbit IgG-HRP conjugate 1:5000

Reaction time (min) 30

Reaction temperature (�C) RT

Analytical parameters of salbutamol standard curve

LC50 (mgL�1) 0.028

LOD (mgL�1) 0.003

20–80% inhibition (mgL�1) 0.007–0.17

Figure 3. Calibration curve by CLEIA (.) for salbuterol under optimized conditions and the standard

curve (&) obtained by the developed ELISA.

2768 Y. WU ET AL.

(Lei et al. 2008; Zhang et al. 2013; Meng et al. 2010; Pleadin et al. 2013), anelectrochemical immunosensor for salbutamol (Liu et al. 2011), and reported CLEIAmethods for clenbuterol (Xu et al. 2005), the sensitivity of the developed ciCLEIAmethod was improved by 20-fold.

When salbutamol, clenbuterol, and brombuterol were spiked in swine orbovine urine at concentrations of 0.02, 0.05, or 0.1 mgL�1, recoveries ranged from78.8% to 119.0%. The intra-assay and inter-assay coefficients of variation (CV) were<13.9% and <19.7% (Table 2), respectively, which are in accordance with therequired accuracy in trace analysis (CV< 20%), indicating acceptable precision.

The polyclonal antibody of salbutamol, which exhibits 139.6% and 225%cross-reactivity with clenbuterol and brombuterol (Table 3), was used in the developedciCLEIA for the determination of salbutamol, clenbuterol, and brombuterol inurine.

Matrix Effects

Although blood sampling showed advantages of immediate availability andreliability (Pleadin et al. 2013; Pleadin et al. 2010), urinalysis was quick andeasy-operative for determination of b2-agonist residue. Salbutamol is excreted inurine as a mixture of the unchanged drug and its conjugate metabolite, mainly asthe sulfate; therefore, the urine matrix was investigated.

Table 2. Intra- and inter-assay recoveries and precision of fortified urine

Analyte sample

Level

(ngL�1)

Intra-assaya Inter-assayb

Measured Recovery CVc Measured Recovery CV

n (ngL�1) (%) (%) n (ngL�1) (%) (%)

Salbutamol 20 6 23.8� 1.9 119.0 8.0 3 20.4� 2.8 102.0 13.7

swine 50 6 57.5� 6.6 115.0 13.9 3 54.0� 9.5 108.0 17.6

urine 100 6 95.2� 7.5 95.2 7.9 3 89.2� 8.2 89.2 9.1

20 6 20.8� 2.39 104.0 11.5 3 22.8� 4.5 114.0 19.7

bovine 50 6 54.0� 6.5 108.0 12.0 3 46.5� 4.6 93.0 9.9

urine 100 6 96.1� 8.0 96.1 8.3 3 86.4� 9.6 86.4 11.1

Clenbuterol 20 6 23.0� 3.2 115.0 13.9 3 21.6� 3.8 108.0 17.6

swine 50 6 47.6� 3.8 95.2 7.9 3 45.0� 4.0 90.0 8.8

urine 100 6 108.2� 10.0 108.2 9.2 3 102.4� 14.0 103.0 13.6

20 6 21.6� 2.6 108.0 12.0 3 18.9� 1.8 94.5 9.5

bovine 50 6 48.2� 4.0 96.4 8.3 3 43.4� 4.8 86.8 11.1

urine 100 6 104.0� 12.4 104.0 11.9 3 114.4� 15.6 114.4 13.6

Brombuterol 20 6 20.8� 2.6 104.0 12.5 3 18.7� 2.0 93.7 11.5

swine 50 6 42.1� 3.2 84.2 7.6 3 44.3� 5.6 88.6 12.6

urine 100 6 87.7� 9.8 87.7 11.2 3 78.8� 9.1 78.8 11.5

20 6 18.5� 2.1 92.5 11.2 3 17.2� 1.5 86.2 8.6

bovine 50 6 47.4� 5.9 94.8 12.4 3 51.2� 5.3 102.4 10.3

urine 100 6 100.7� 8.4 100.7 8.3 3 102.5� 12.2 102.5 11.9

aIntra-assay variation was determined by six replicates on a single day.bInter-assay variation was determined by three replicates on three different days.cCoefficient of variation.

SENSITIVE CHEMILUMINESCENCE ENZYME IMMUNOASSAY 2769

Matrix interferences are a common and challenging problem when applyingimmunoassays to real samples. False positives may result, inhibiting enzyme activityand antibody binding. The simplest way to overcome this interference is to preparesamples and standards in the same medium. When determining the matrixeffects, interferences are quantified by comparing a standard inhibition curve witha standard curve generated in urine known to be free of salbutamol. The two curvesfor salbutamol are superposable, indicating that the effects of the matrix were notsignificant (Figure 4), allowing the samples to be analyzed using the standard curveinstead of the matrix curve. These results showed that the developed technology forsample pretreatment was feasible.

Table 3. Cross reactivity of salbutamol and its analogsa

Compound IC50 (nM) Cross reactivity (%)

salbutamol 0.117 100.0

clenbuterol 0.084 139.6

terbutaline 0.457 25.6

cimaterol 2.417 4.8

zilpaterol 9.567 1.2

brombuterol 0.052 225.0

clorprenaline >100 <0.1

phenolethanolamine >1000 NDb

ractopamine >1000 ND

fenoterol >1000 ND

aIC50 value was the competitor concentrations by indirect competitive ELISA.

Data was obtained from three separate experiments run on three different days.bNot determined.

Figure 4. The standard inhibition curve by ciCLEIA for salbutamol in (&) assay buffer and (.) urine.

2770 Y. WU ET AL.

CONCLUSIONS

Based on the polyclonal antibody of salbutamol, a rapid, sensitive ciCLEIA forsalbutamol, clenbuterol, and brombuterol were developed for analysis of swine andbovine urine. Under the optimized condition, the ciCLEIA for salbutamol was moresensitive than the ELISA in this study. Compared with previously reported CLEIAfor clenbuterol, the sensitivity of the developed ciCLEIA was improved 20-fold. Thenovel ciCLEIA was a reliable, convenient, and sensitive method for simultaneousscreening of salbutamol, clenbuterol, and brombuterol residue in swine and bovineurine. Further research will consider the determination of these compounds inhuman urine.

FUNDING

This work was supported by the Special Fund for Agro-Scientific Research inthe Public Interest (No. 201203069-2) and the Fund of Modern Agriculture IndustrySystem Innovation in Beijing City Team.

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