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Validation of radioimmunoassay screening methods

for -agonists in bovine liver according to Commission
Decision 2002/657/EC
a a a a b
R.H.M.M. Granja , A.M. Montes Nio , F. Rabone , R.E. Montes Nio , A. Cannavan &
a
A. Gonzalez Salerno
a
Microbioticos Laboratories , Campinas, Brazil
b
Agrochemicals Unit, FAO/IAEA Agriculture & Biotechnology Laboratory, IAEA Laboratories ,
Seibersdorf, Austria
Published online: 02 Dec 2008.

To cite this article: R.H.M.M. Granja , A.M. Montes Nio , F. Rabone , R.E. Montes Nio , A. Cannavan & A. Gonzalez Salerno
(2008) Validation of radioimmunoassay screening methods for -agonists in bovine liver according to Commission Decision
2002/657/EC, Food Additives & Contaminants: Part A, 25:12, 1475-1481, DOI: 10.1080/02652030802308464

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 Food Additives and Contaminants
Vol. 25, No. 12, December 2008, 14751481

Validation of radioimmunoassay screening methods for b-agonists in bovine liver

according to Commission Decision 2002/657/EC
R.H.M.M. Granjaa*, A.M. Montes Ninoa, F. Rabonea, R.E. Montes Ninoa, A. Cannavanb and
A. Gonzalez Salernoa
a
Microbioticos Laboratories, Campinas, Brazil; bAgrochemicals Unit, FAO/IAEA Agriculture & Biotechnology Laboratory,
IAEA Laboratories, Seibersdorf, Austria
(Received 14 December 2007; final version received 25 June 2008)

Validation studies were carried out on a multi-residue screening method for anilinic type -agonists (clenbuterol,
mabuterol, brombuterol, cimaterol, cimbuterol, mapenterol, clenpenterol) and a method for the phenolic type
Downloaded by [Northeastern University] at 17:51 14 November 2014

-agonist, salbutamol, in bovine liver. The validation was performed according to the European Union
Commission Decision 2002/657/EC (European Commission 2002), which establishes criteria and procedures for
the determination of parameters such as the detection capability (CC), specificity, stability of standard solutions
and stability of the analyte in matrix. CC values for the eight target compounds were between 0.25 and
0.5 mg kg1. The stability of standard solutions and analytes in matrix and the specificity of the antibody were
characterized. The methods are applicable for qualitative screening of -agonists for regulatory programmes
according to European Union performance requirements, or as a semi-quantitative research tool for known
target compounds.
Keywords: -agonists; radioimmunoassay; residues; validation

Introduction the slowest depletion of residues. These organs are,

-Agonists are frequently used in farm animals for the
treatment of pulmonary diseases and bronchospasm.
Lipolysis and increased protein synthesis are well-
known side-effects and, therefore, these substances can
also be misused as growth promoters (Buttery and
Dawson 1990). However, because of the potential
harmful effects on human health of residues of these
drugs in food, the European Union has banned their
use in animal production systems through Council
Directive 96/22/EC (European Commission 1996).
Clenbuterol and mabuterol are sympathomimetic
bronchodilating agents belonging to the family of
2-agonists. These compounds are used in veterinary
and human medicine for the treatment of chronic and
obstructive pulmonary diseases (Murai et al. 1984;
Nazzal 1985). Clenbuterol is probably the most well
known of the aniline type 2-agonists, and is the most
lipophylic. When administrated orally in livestock, it
can be absorbed by liver, lungs, kidneys and secretor
organs, such as pancreas and supra-renal glands
(Miller et al. 1988; Sauer et al. 1995; Smith and
Paulson 1997; The European Agency for the
Evaluation of Medical Products (EMEA) 2000).
Tissue concentrations of clenbuterol decrease rapidly,
with liver, eyeball (retina), hair and feathers showing
therefore, suitable for detection of the drug during or
after the withdrawal periods in the animal (Malucelli
et al. 1994; Sauer and Anderson 1994). On the other
hand, the metabolism and pharmacokinetic character-
istics of mabuterol are largely unknown in livestock,
but reliable data from rodent and human studies
showed that mabuterol had a long-lasting effect after
oral administration and was extensively absorbed
along the entire small intestine of rats (Guentert
et al. 1984; Yuge et al. 1984). Both clenbuterol and
mabuterol have been abused to improve by producers
carcass quality and other members of the -agonist
group of drugs may be used in the same way.
Because of the diversity of the substances illegally
used in meat production, the presence of these
compounds at trace levels and the complexity of the
biological matrices analysed, there is a continuous need
for development of a multiresidue strategy of analysis,
involving a quick and easy sample pretreatment,
followed by a specific and sensitive determination of
several residues within the same run. A number of
techniques have been developed or applied to screen
for -agonists. A surface plasma resonance (SPR)
biosensor assay was described by Traynor et al. (2003).
The assay was able to detect mabuterol down to

*Corresponding author. Email: rodrigo@microbioticos.com

ISSN 0265203X print/ISSN 14645122 online

2008 Taylor & Francis
DOI: 10.1080/02652030802308464
http://www.informaworld.com
 1476 R.H.M.M. Granja et al.
0.02 mg kg1, clenbuterol at 0.11 mg kg1 and salbuta-
mol at 0.19 mg kg1, and a range of other -agonists at
concentrations below 1.5 mg kg1. An enzyme-linked
immunosorbent assay (ELISA) has been described
for -agonists in hair (Haasnoot et al. 1998), permit-
ting on-farm testing, with detection limits for
clenbuterol, bromobuterol, mapenterol and mabuterol
of 1.01.5 mg kg1 for white and 3.04.0 mg kg1 for
black hair, though the assay did not detect cimbuterol,
salbutamol or terbutaline. Other ELISA screening
methods have been described for single analytes such
as clenbuterol (Posyniak et al. 2003), ractopamine
(Elliott et al. 1998a) and zilpaterol (Shelver and Smith
2004). These methods each have drawbacks, especially
for use in developing countries. Biosensor (SPR)
instruments and chips/reagents are expensive and kits
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are not currently commercially available for many
applications. The robustness of the technology is,
as yet, largely unproved in developing country situa-
tions. Methods based on ELISA have been shown to
be frequently prone to poor performance in developing
countries, probably due to degradation of reagents
(especially the enzyme-conjugate) under less than ideal
conditions during transport and storage (International
Atomic Energy Agency (IAEA) 2003; Cannavan and
Elliott 2004). Radioimmunoassay (RIA) is a mature,
robust and reliable technology capable of meeting the
requirements in terms of sensitivity, specificity and
method performance to achieve the objective of
a multiresidue screening method (Delahaut et al.
1991; Collins et al. 1994). Brazil routinely carries out
immunological tests for the detection of -agonists
such as clenbuterol and salbutamol, which are prohib-
ited for use as growth promoters, in official monitoring
programmes. Since it is possible that other members of
this family of drugs may also be used as growth
promoters, there is a demand for validated screening
methods for other -agonistic compounds.
The aim of this study was to develop a general
strategy for the screening of liver samples of bovine
origin for -agonists, using RIA tests. Liver is an
excellent matrix for the detection of residues of
-agonists, since it is considered a site of accumulation
of these drugs and good recovery of the analytes can be
attained from this matrix. Hence, analysis of liver
samples taken at slaughter provides a good way to
track the illegal use of -agonists in animal production.
In the present work, we initially developed an
analytical method for monitoring of mabuterol resi-
dues in bovine liver samples and subsequently extended
the method validation, according to Decision 2002/
657/EC (European Commission 2002) and ISO/IEC
17025: 2005 (ISO/IEC 2005) guidelines to other anilinic
type -agonists, including clenbuterol, brombuterol,
cimaterol, cimbuterol, mapenterol and clenpenterol.
A separate extraction procedure was also developed to
permit the analysis of the phenolic type -agonist
salbutamol, which, because of its different chemical
properties, was not satisfactorily extracted by the
sample preparation procedure for the anilinic com-
pounds. The methods were characterized with respect
to parameters such as antibody cross-reactivity, detec-
tion capability (CC), stability of standard solutions,
and stability of the analyte in matrix. To facilitate the
use of the method as a semi-quantitative research tool,
the decision limit (CC) was also estimated. Typical
measurement uncertainty values were estimated in
order to comply with ISO 17025 requirements.
Materials and methods
Materials and reagents
All the solvents used were analytical grade from Merck
(Darmstadt, Germany) or Carlo Erba (Milan, Italy).
Other chemicals used were phosphate buffer (pH 6.8),
phosphate buffer PBSgelatin (pH 7.4), acetate buffer
(pH 5.2) and Trisma base buffer (pH 9.1). Water was
purified by a Milli-Q Gradient System A-10 (Millipore,
Billerica, MA, USA). Solid-phase extraction (SPE,
Bond Elut C-18 cartridge) (the average size of particles
was 4760 mm) was supplied by Varian (Palo Alta, CA,
USA). -Glucuronidase from Helix pomatia (pH 5.3)
and salbutamol standard were purchased from
Sigma Aldrich (Saint Louis, MO, USA). Cimaterol,
brombuterol, cimbuterol, clenproperol, and mapen-
terol standards were provided by Witega, GmbH
(Germany). Tritium-labelled clenbuterol marker
(3H-clenbuterol) was supplied by Laboratorie
dHormonologie (Belgium) and a polyclonal antibody
to range -agonists was supplied by the Chemical
Surveillance Department, Agri-Food and Biosciences
Institute (Belfast, UK).
Apparatus
The apparatus used included a refrigerated centrifuge
(Revan Cycle CR), liquid scintillation counter
(Beckman LS 6000 TA), sample evaporation system
(TecVap TE-0194), nitrogen evaporation system,
SPE vacuum manifolds (Baker) and an incubator
(Olidef CZ).
Extraction of liver samples anilinic b-agonists
Liver (0.5 g) was weighed into a 15 ml centrifuge tube.
The analyte extraction was performed by homogeniza-
tion with 5 ml of a phosphate buffer (pH 6.8)methanol
mixture (95:5). Subsequently a liquidliquid extraction
with 2 ml of methanol, 3 ml of chloroform was
performed, followed by centrifugation (1560g, 4 C,
10 min). The aqueous supernatant was transferred to
another tube, sodium hydroxide (0.7 ml) was added,
and the analytes were extracted with diethyl ether (5 ml).

The upper (ether) layer was evaporated to dryness
at 37 C under a gentle steam of nitrogen. After this
simple extraction/clean-up procedure, the anilinic type
-agonists were quantified by RIA.
Extraction of liver samples salbutamol
(phenolic b-agonist)
A total of 1.0 g of liver was weighed into a 15 ml falcon
tube, and the sample was hydrolysed by incubation
(2 h, 37 C) with acetate buffer and -glucuronidase
(200 U). The sample was then ultrasonicated in an
ultrasonic bath for 15 min and centrifuged (1560g, 4 C,
10 min). The supernatant was transferred to a 15 ml
falcon tube and 5.5 ml of Trisma base buffer was
added. The sample was applied to a C18 SPE cartridge
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preconditioned with Trisma base buffer, methanol and
water. The column was rinsed with a water/acetonitrile
mixture, dried and eluted with methanolphosphate
buffer (99.5 : 0.5 ml). The organic phase was evapo-
rated at 60 C under a stream of nitrogen and
salbutamol was then determined by RIA.
Radioimmunoassay
Aliquots (0.1 ml) of clenbuterol standard solutions in
phosphate buffer (PBSgelatin, pH 7.4; 0.01 M with
0.01% sodium azide) were added to RIA tubes giving
levels of 0, 7.8, 15.6, 31.3, 62.5 125, 250, 500 and
1000 pg for preparation of a calibration curve. Tubes
to quantify non-specific binding were also prepared.
Aliquots (0.3 ml) of liver extracts were added to RIA
tubes. An aliquot (0.1 ml) of tritiated clenbuterol
solution (3500 cpm) was added to each tube.
Antiserum (0.1 ml) was added to all tubes except
those used to quantify non-specific binding. The tubes
were vortex mixed and incubated overnight (4 C).
Dextran-coated charcoal mixture (0.5%, 0.5 ml) was
added and the tubes were incubated for 10 min at 4 C.
The tubes were then centrifuged (1560g, 10 min, 4 C).
The supernatant (0.5 ml) was transferred into scintilla-
tion vials and the level of radioactivity in each vial was
measured on a scintillation counter. The concentration
of -agonist was calculation by comparison with the
calibration curve.
Method validation
For the method validation, blank liver matrix was
obtained by pooling 20 samples which had presented
analytical signals equivalent to the 100% binding
concentration signal (B0) of the RIA method (no
analyte signal). As a final verification, this pooled
matrix was analysed again by RIA and it was certified
that the signal was close to the B0 signal, providing
evidence that there was no -agonist present.
Food Additives and Contaminants 1477
The stability of the standard solutions was
checked at zero, 3, 6 and 9 months after preparation
by comparison of standard calibration curves (AOAC
International 1999). The stability of the analyte in
matrix was determined by spiking six blank samples at
four times the CC value obtained and analysing them
after storage in a refrigerator for 3 months.
Although not required for qualitative methods, the
decision limit, CC, was estimated by analysing 20
blank Brazilian bovine liver samples. The mean of the
signal plus three times the standard deviation of the
blank population was taken as CC. The CC value
was determined by fortifying 20 blank Brazilian bovine
liver samples with low levels of each -agonist
(0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.5 mg kg1) in separate
experiments. The CC value was determined as the
lowest content of -agonist detected in the samples
with an acceptable false negative rate of 5%.
The uncertainty of the method was estimated using
the average of the sum of the squares of the relative
standard deviations (RSD) obtained from the
measured values of spiked samples (AOAC
International 2004). As a quality control measure for
routine application of the method, a reagent blank,
two negative tissue control samples and two positive
control samples (negative tissue spiked with a standard
of known concentration 1 mg of -agonist per kg of
matrix) were included with every batch of samples for
analysis.
The antibody cross-reactivity with a range of
-agonists was determined and its specificity for the
-agonist group of compounds was evaluated by cross-
reactivity experiments with a range of alternative
compounds that might be used as growth promoters.
Routine quality control of the method performance
was carried out by plotting Shewhart charts for various
method performance parameters, including antibody
percentage binding, percentage of non-specific binding,
slope of the calibration curve, and recovery and
precision data from spiked tissue blanks.
Method uncertainty
For the calculation of the method uncertainty
associated with semi-quantitative applications of the
method, a top-down approach was applied. It was
considered that the uncertainty contributions arising
from all the possible sources of both type A and type B
errors associated with the analytical procedure, such as
weighing, sample preparation and preparation of
solutions, were included in the standard deviations
of replicate analyses performed during method valida-
tion. This method can be applied to procedures where
no trends are observed (AOAC International 2004).
The uncertainty estimations were carried out for all
the -agonists included in the scope of the method.
 1478 R.H.M.M. Granja et al.

For the estimation of the method uncertainty, samples concentration of the analyte(s) without knowledge of
were spiked at 0.5, 1.0 and 2.0 mg kg1, or level I, II and which compound is present. However, the method
III, respectively. The combined uncertainty (CU) of the could be used in a semi-quantitative manner where the
method was expressed as: target analyte is known, for example in research
applications.
CU Seven analytes considered in the present work
q
required the same sample pretreatment and clean-up
RSD level I2 RSD level II2 RSD level III2 =3
stages before the RIA test. The exception was
salbutamol. The extraction procedure is not effective
for this compound because it is not an aniline type but
a phenolic type -agonist which exists in tissues in
Results and discussion glucuronide form. Although there are possibilities for
The cross-reactivity of the -agonist antibody used in the analysis of such compounds without deconjugation
(Elliott et al. 1998b), normally a deconjugation step is
the procedure is shown in Table 1. Useful cross-
required before clean-up and analysis. Also, the
reactivity was exhibited for the eight compounds of
addition of sodium hydroxide solution, which is
interest: salbutamol, cimbuterol, clenbuterol, brombu-
Downloaded by [Northeastern University] at 17:51 14 November 2014

included in the clean-up for the anilinic -agonists,

terol, clenpenterol, mabuterol, mapenterol and cima-
causes salbutamol to remain in an ionic state, reducing
terol. The antibody could, therefore, be used in an
the efficiency of the final ethyl ether extraction. It was
immunoassay protocol for the monitoring of these
necessary, therefore, to develop a separate extraction
analytes. Since the cross-reactivity is less than 100%
and clean-up procedure for salbutamol. This was
for some of the compounds, the method can be applied
achieved by including an enzymatic hydrolysis step
for routine monitoring of unknown samples only as using -glucuronidase and employing a solid-phase
a qualitative method to detect the presence of one or extraction clean-up procedure, as described above.
more -agonists. It is impossible to quantify the This new method produced satisfactory results when
submitted to the validation criteria in Decision 2002/
657/EC (European Commission 2002). The sample
Table 1. Cross-reaction of the antibody used on preparation method can also be used for other
the experiment. phenolic type -agonists (data not presented).
The experiments to determine the stability of
Percentage standard solutions prepared in methanol were carried
Standards cross-reaction
out by constructing calibration curves with the same
Salbutamol (free base) 100 standards immediately after preparation, then subse-
Salbutamol (sulfate salt) 100 quently after 3, 6 and 9 months. The solutions were
Cimbuterol 100 stored in a refrigerator during this period. A seven-
Clenbuterol 100
point calibration curve for each analyte was prepared
Brombuterol 80
Clenpenterol 62 by dilution of appropriate stock standard solutions in
Mabuterol 60 PBS, pH 7.4. Analysis of the data at each time point
Carbuterol 40 using the Students t-test confirmed that the standard
Mapenterol 30 solutions for all analytes studied were stable for at least
Terbutaline 30
9 months (Figure 1).
Cimaterol 15
Isoproteranol 2.3 Table 2 shows data for CC, CC, and uncertainty
Pirbuterol 3 estimates for each of the -agonists. The most critical
Epinephrine (adrenaline) 50.01 parameter to be estimated for a screening method is
Dexamethasone 50.01 CC. The CC values for this method were between
Methytestosterone 50.01
0.25 and 0.5 mg kg1 for all the -agonists.
Zeranol 50.01
Diethylstilbestrol 50.01 The stability of the analytes in matrix was
Nortestosterone 50.01 monitored, according to Decision 2002/657/EC
Medroxyprogesterone acetate 50.01 (European Commission 2002), by spiking six blank
Trenbolone 50.01 samples at four times the CC value obtained. Samples
Ethynylestradiol 50.01
were analysed initially, then 3 months after the spiking
Progesterona 50.01
Testosterona 50.01 procedure, corresponding to more than ten times the
17--Oestradiol 50.01 average storage time of a sample in the laboratory
Arterenol (noradrenaline) 50.001 before analysis. The results for each analyte, presented
DL-4-hydroxy-3,3-metoxy 50.001 as counts per minute (cpm) when measured using the
mandelic acid
competitive RIA format, are presented in Table 3.
DL-3,4-dihydroxymandelic acid 50.001
The analytical signals varied by less than 1% for
 Food Additives and Contaminants 1479

each -agonist, demonstrating the stability of the
compounds in the liver matrix.
For quality control of the method in routine use,
Shewhart control charts on analyte recovery and
calibration curve parameters were plotted.
The parameters checked included antibody percentage
binding, percentage of non-specific binding, slope of
the calibration curve, and recovery and precision data
from spiked tissue blanks. The criteria used for the
acceptability of such data were: (1) no more than seven
consecutive points above or below the main line; (2) no
more than seven consecutive points ascending; (3) no
more than seven consecutive points on descending; and
Downloaded by [Northeastern University] at 17:51 14 November 2014
(4) no more than one point above or below the control
limits. Examples for one analyte, mabuterol, are shown
in Figure 2, the quality control of antibody percentage
binding, and Figure 3, recovery data. The success of
such control is an indicator of the robustness of the
method.
The main purpose of this method was as
a screening tool to identify negative samples or provide
strong evidence of the presence of one or more of the
analytes in a sample, indicating the need for a
confirmatory analysis. Additional characterization of
the method was performed to permit its application
as a semi-quantitative assay for research purposes.

(a) 3

3 Months 6 Months 9 Months

tcrit 2.26
2

1
tstat

2
tcrit - 2.26

3
Mabuterol Cimaterol Mapenterol Salbutamol

(b) 3

3 Months 6 Months 9 Months

tcrit 2.13
2

1
tstat

2 tcrit - 2.13

3
Brombuterol Cimbuterol Clenproperol

Figure 1. Stability of standard solution at 3, 6 and 9 months: (a) mabuterol, cimaterol, mapenterol and sabutamol; and
(b) brombuterol, cimbuterol and clenproperol.
 1480 R.H.M.M. Granja et al.

Table 2. Decision limit (CC), detection capability (CC) Quality control on % of Union data - Mabuterol

Deviation from mean

and uncertainty in the -agonist studied. 3.00
2.00
1.00
CC CC 0.00
Analyte (mg kg1) (mg kg1) Uncertainty (%) 1.00 1 4 7 10 13 16 19 22 25 28 31 34 37 40
2.00
Clenbuterol 0.10 0.25 7.92 3.00
Mabuterol 0.10 0.25 4.58 Batch no
Cimaterol 0.10 0.5 6.56
Figure 2. Quality control chart of antibody percentage
Brombuterol 0.03 0.3 8.77
binding for mabuterol.
Cimbuterol 0.03 0.25 6.56
Clenproperol 0.03 0.35 5.10
Mapenterol 0.10 0.5 5.39
Salbutamol 0.10 0.5 7.68 Quality control information of spike recovery data

Deviation from mean

4.00
3.00
2.00
1.00
Table 3. Stability of the analytes studied in liver matrix
0.00
considering two analytical occasions. 1.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39
Downloaded by [Northeastern University] at 17:51 14 November 2014

2.00
Average Signal Batch no
Analytes blank signal Signal (3 months) Figure 3. Quality control chart of recovery data for
mabuterol.
Clenbuterol 1456 259 265
Mabuterol 1443 371 326
Cimaterol 1216 252 297
Brombuterol 1410 267 323
Cimbuterol 1506 283 266 requirements of the European Union for screening
Clenproperol 1501 337 370 methods. The ongoing quality control data, including
Mapenterol 1225 234 251 Shewhart charts, are valuable, demonstrating that the
Salbutamol 1291 246 278 method is under control and robust when used for
routine analysis. The method can be applied to help
ensure a reliable monitoring program for -agonists
The trueness of the method was estimated for each according to the criteria of one important Brazilian
analyte by comparison of the values obtained beef export market destination.
from spiked samples against the response curve.
However, these values are not reported here, since the
method is intended primarily as a screening method. Acknowledgements
Commission Decision 2002/657/EC (European The authors thank the International Atomic Energy Agency
(IAEA) for financial support of the project (Research
Commission 2002) states that for screening methods, Contract No. 11880/RBF).
even quantitative ones, it is not necessary to calculate
the trueness of the procedure. The precision of the
method has not been reported for the same reason. References
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