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Original Article

Application of high-performance liquidchromatographyeUV detection to quantificationof clenbuterol in bovine liver samples

Fredy Morales-Trejo a, Salvador Vega-y Leon a, Arturo Escobar-Medina b,Rey Gutierrez-Tolentino a,*aDepartment of Agricultural and Animal Production, Autonomous Metropolitan University-Xochimilco,

Calz. del Hueso 1100, Col. Villa Quietud, Zp 04960, MexicobNational Center for Agricultural Health (Centro Nacional de Sanidad Agropecuaria), Zp 10, San Jose de las Lajas,

La Habana, Cuba

a r t i c l e i n f o

Article history:

Received 26 February 2013

Received in revised form

20 April 2013

Accepted 28 June 2013

Available online 7 October 2013

Keywords:

Bovine liver

Clenbuterol

High performance liquid

chromatography

Mexican food

* Corresponding author. Department of Agricdel Hueso 1100, Colonia Villa Quietud, Zp 04

E-mail address: reygut@correo.xoc.uam.m1021-9498/$ e see front matter Copyright ª 201

http://dx.doi.org/10.1016/j.jfda.2013.09.009

a b s t r a c t

A method for the determination of clenbuterol (CLB) concentration in bovine liver by

reversed-phase high-performance liquid chromatography (HPLC) with UV detection was

developed. The sample was extracted with acetonitrile and isopropanol, followed by HPLC

analysis. A reverse-phase column C18 was used, with a UV detector at 214 nm and 0.05 M

NaH2PO4 (pH 3.0)/acetonitrile (85:15, v/v) as the mobile phase. The accuracy of the

analytical method was estimated by spiking bovine liver samples with three different

concentrations of CLB (5.24 ng/g, 20.98 ng/g, and 41.96 ng/g) and recovery of 111.7%, 82.0%,

and 84.8%, respectively, was obtained. The precision of the method was estimated by the

relative standard deviation, which was < 4.74%. The limit of detection and quantification of

CLB were 0.20 ng/g and 0.42 ng/g of liver sample, respectively, and the retention time was

24.82 minutes. The recent discovery of CLB contamination in Mexican food led to the

specific inspection of a distribution center for this b-agonist, involving the analysis of a

total of 78 bovine liver samples. Of all samples screened, 62% of them had concentrations

above the maximum residue limit of 0.6 ng/g set by the United Nations Food and Agri-

cultural Organization for CLB. The analytical method was found to be rapid, sensitive,

accurate, repeatable, and reproducible, and could be applied to the measurement of CLB

concentration in bovine liver.

Copyright ª 2013, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan

LLC. All rights reserved.

1. Introduction agonist [1]. It is used as a bronchodilator, tocolytic, and

Clenbuterol {4-amino-alpha-[(tert-butylamino)methyl]-3,5-

dichlorobenzyl alcohol hydrochloride, CLB} is a b2-adrenergic

ultural and Animal Produ960, Mexico City, Federalx (R. Gutierrez-Tolentin3, Food and Drug Adminis

mucolytic agent in the management of respiratory disorders

[2,3]. It also possesses physiological effects similar to anabolic

steroids, which promote the growth of muscular tissue and

ction, Autonomous Metropolitan University-Xochimilco, CalzadaDistrict, Mexico.

o).tration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved.

j o u r n a l o f f o o d and d ru g an a l y s i s 2 1 ( 2 0 1 3 ) 4 1 4e4 2 0 415

reduction of body fat, hence it is illegally used as a reparti-

tioning agent inmeat-producing animals [4e6]. The use of CLB

as a growth promoter in cattle destined for human con-

sumption is illegal and implies a threat to public health,

because several human poisonings have been linked to the

consumption of meat products contaminated with CLB resi-

dues [7]. The use of CLB as a growth promoter in cattle is

banned in the European Union [8], Mexico [9e11], and many

other countries. The human effects include increased heart

rate and blood pressure, anxiety, palpitation, and tremor in

skeletal muscle [12]. The Codex Alimentarius Commission

recommends maximum residue limits (MRLs) for cattle of

0.2 ng/g in muscle and fat, 0.6 ng/g in liver and kidney, and

0.05 ng/mL for cattle milk, expressed as parent drug [13].

To analyze residues in animal tissues, it should be noted

that they are highly complex feature matrices; furthermore,

the extraction and detection of substances present in con-

centrations of the order of ng/g (ppb), or even less, should be

performed with high selectivity [14]. For food security, the

analytical method for determining residues of CLB is indis-

pensable. There are various methods for determining CLB,

including immunological techniques such as enzyme-linked

immunosorbent assay [14,15], gas chromatography coupled

with mass spectrometry (GC/MS) [16e18], high-performance

liquid chromatography (HPLC)eUV detection [5,19,20], and

liquid chromatography coupled with tandem mass spec-

trometry (LC/MS/MS) [21] or with mass spectrometry (LC/MS)

[22] . Of the reported methods, immunological techniques are

the least used for quantification, although they are fast qual-

itative screening techniques. GC/MS methods involve tedious

steps for derivatization of CLB [23]. Thus, HPLC was chosen as

the technique for fast and accurate quantification of CLB in

bovine liver samples.

In the present work, a rapid, accurate, and sensitive

method for determining CLB by HPLC with UV absorbance

detection was optimized with reference to published pro-

cedures of the United States Department of Agriculture

(USDA) [12] and Chang et al [5]. A simple and time-saving

method for extraction and filtration for direct analysis was

used in this experiment. The main objective of this study was

to develop the optimal conditions for the HPLCmethod, which

could be applied to the determination of CLB in bovine liver.

This analytical method was then implemented to determine

the CLB levels in samples purchased from a meat distribution

center in Mexico City.

2. Methods

2.1. Reagents

Standard clenbuterol hydrochloride (�95%)was obtained from

SigmaeAldrich (St Louis, MO, USA). Acetonitrile (J.T. Baker,

Phillipsburg, NJ, USA) and isopropanol (Burdick & Jackson,

Muskegon, MI, USA) were of HPLC grade. The other chemicals

and solvents, such as sodium chloride, sodium sulfate, mag-

nesium sulfate, and sodium dihydrogen phosphate, were of

analytical-reagent grade and purchased from J. T. Baker (Eca-

tepec, Mexico). All aqueous solutions were prepared using

Type I ultrapure water (Purelab Flex, Elga LabWater, UK).

2.2. Preparation of stock and standard solutions of CLB

The stock solution of clenbuterol hydrochloride was prepared

by dissolving an appropriate amount of the drug in ultrapure

water to obtain a concentration of w1 mg/mL and stored in

the dark at 4�C for no longer than 2 months. The clenbuterol

hydrochloride concentration was corrected for salt and purity

[12] because CLB is the analyte of interest as opposed to

clenbuterol hydrochloride, therefore, the real concentration in

the stock solution was 839.56 mg/mL. Working standard solu-

tions were prepared each day by gradual dilution with the

mobile phase. Standard curves were plotted using peak areas

versus known concentrations.

2.3. Equipment

The HPLC system was a Hitachi Elite LaChrom chromatogra-

pher (Tokyo, Japan) equipped with an L-2420 UV-Vis detector,

an L-2300 column oven, an L-2200 autosampler, and an L-2130

pump. EZChrom Elite 3.3.2 SP2 software (Agilent Technolo-

gies, CA, USA) was used for data acquisition.

2.4. Chromatographic conditions

Chromatographic separation was executed in a Restek Ultra

C18 analytical column (250 mm � 4.6 mm, internal diameter,

5 mm; Bellefonte, PA, USA). The mobile phase was 50 mM

NaH2PO4 (adjusted to pH 3.0 with orthophosphoric acid/

acetonitrile; 85:15, v/v) and its flow rate was set at 1.0 mL/min.

Prior to use the mobile phase was filtered through a Millipore

0.45-mm filter (EMD Millipore Corporation, MA, USA). The

detection wavelength of the detector was set at 214 nm. The

column temperature was maintained at 25�C.

2.5. Bovine liver

A total of 78 bovine liver samples were collected from a meat

distribution center in Mexico City on five different dates dur-

ing July 2012 to November 2012. Each sampling collected

13e17 samples, which weighed w250 g. Samples were kept

frozen at �20�C until analysis.

2.6. Extraction

For sample extraction, 5.0 � 0.1 g of homogenized liver was

weighed, and 4 mL acetonitrile and 2 mL isopropanol were

added, and then the mixture was ground for 30 seconds. The

mixture was placed into a 50-mL centrifuge tube and 1.2 g

NaCl was added and vortexed for 2 minutes, then 4 g Na2SO4

and 0.5 g MgSO4 were added and vortexed for 2 minutes. The

samples were centrifuged for 15 minutes at 2000g. The total

extract was transferred into a 50-mL round-bottom flask and

rotoevaporated to dryness. The residue was dissolved in

1.0mL ofmobile phase, vortexed for 2minutes, sonicated for 2

minutes, and filtered using a 3-mL syringe and 0.45-mm nylon

filter into a vial. Fifty microliters of this solution was injected

into the HPLC column. Peak identification was made by

comparing the retention times of samples with those of the

standard solutions.

j o u rn a l o f f o o d a nd d r u g an a l y s i s 2 1 ( 2 0 1 3 ) 4 1 4e4 2 0416

Spiked tissues were prepared by adding standard solutions

to the homogenate of control tissue prior to adding salts, fol-

lowed by extraction and analysis at the same time as the

samples.

2.7. Specificity of retention time for CLB

Three replicates from the same sample of liver were prepared,

which were subjected to the extraction process described

above. Two of them were fortified with CLB prior to homoge-

nization, with 2 ng/g and 25 ng/g of liver sample, whereas a

replicate was not fortified (blank).

2.8. Linearity and range for standard curve of CLB

The working solutions were prepared from the stock solution

of CLB so as to contain the drug in the range of 6.5e104.9 ng/

mL. The solutions were injected in triplicate into the HPLC

column, with a constant injection volume of 50 mL and the

chromatograms were recorded. The peak area versus con-

centration datawere treated by least squares linear regression

analysis.

2.9. Limit of detection and limit of quantification

The limit of detection (LOD) and limit of quantification (LOQ)

were determined according to the method based on extrapo-

lation of the calibration curve to zero concentration [24,25] by

injecting a series of spiked samples of known concentrations

near the expected limits (0.66 ng/g, 1.31 ng/g, and 1.97 ng/g) in

triplicate, and the area versus concentration datawere treated

by least squares linear regression analysis.

We used Equations 1 and 2 for the calculation of LOD and

LOQ, respectively.

LOD ¼ ðYbl þ 3SblÞ=b [1]

LOQ ¼ ðYbl þ 10SblÞ=b [2]

Where Ybl is the estimate of the blank response, Sbl is the es-

timate of the SD of the blank, and b is the slope.

2.10. Precision of the method for determining CLB

Precision was evaluated by analyzing three replicates of three

different bovine liver samples on the same day under the

optimal conditions with the same reagents and equipment.

The mean (X) and SD were calculated for each sample

analyzed, besides the “grouped estimator” of the standard

deviation (sg) and the relative standard deviation (RSD) using

Equations 3 and 4 [24].

sg ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi�

1N� k

�Xk

j¼1

Xn

i¼1

�Xi � Xi

�2vuut (3)

RSD ¼ sg$100

X(4)

where N is the total number of determinations, k is the

number of samples, n is the number of determinations on

each sample, Xi is the measured value in i test, and Xi is the

estimator of the population mean m.

2.11. Accuracy of the method for determining CLB

The accuracy of the assay method was evaluated in fortified

bovine liver in triplicate at three known concentration levels

of clenbuterol (5.24 ng/g, 20.98 ng/g and 41.96 ng/g). The re-

covery of the spiked drug was determined by Equation 5:

Recovery ¼ XbX$100 (5)

Where X is the mean value and X is the spiked value.

3. Results and discussion

3.1. Extraction and chromatography

A fast, simple, and efficient extraction procedure was an

essential part of the quantification method in the present

study. The extraction procedure was a modification of that

described by USDA [12] and Chang et al [5] for the screening of

b-agonists in bovine liver by HPLC. However, in the present

study, the totality of extract was dried to increase the signal

of CLB, and the chromatographic conditions were modified.

The HPLC method was modified from previously described

methods in which a UV detector was used [20,26]. The

extraction of b-agonists from meat and other tissues has

usually been accomplished by solid-phase extraction (SPE)

using different reversed-phase adsorbents [2,4], but previous

work (data not shown) in this laboratory using SPE to separate

CLB from bovine liver indicated that it resulted in low

extraction efficiency.

Several HPLC methods that have been published for the

extraction and determination of CLB in liver using large vol-

umes of solvents are expensive and not environmentally

friendly [2e5]. Meanwhile, compared with GC methods that

are generally used for the determination of b-adrenergics, the

major advantages of the present method were a shorter

extraction time and the absence of a derivatization step [4].

Therefore, the procedure described here enables the direct

extraction of CLB without complementary purification steps.

Chang et al [5] reported that to avoid the tardy and tailing

peak, the optimal conditions of the mobile phase for CLB

determination were 50 mM NaH2PO4 (pH 3.0)/acetonitrile

(80:20, v/v) at 212 nm. However, in the present study, a UV

spectral scanning for CLB was tested using 50 mM NaH2PO4

(pH 3.0)/acetonitrile (85:15, v/v). The maximum UV absorp-

tion present at 214 nm was selected for CLB detection in this

study.

3.2. Specificity of retention time for CLB

The specificity test was to guarantee that CLB was reliably

determined, eliminating the possibility of false-positive re-

sults due to the presence of interference of the constituents

of the matrix used in the test or due to the decomposition-

derived elements [24,25]. In Fig. 1, representative chro-

matograms of a CLB standard, a control blank sample, and a

Fig. 1 e Chromatograms obtained from (A) a standard solution of clenbuterol (209.8 ng/mL), (B) a bovine liver (blank), and (C)

a bovine liver sample fortified with 25 ng/g of clenbuterol. High-performance liquid chromatography conditions are

described in the text.

j o u r n a l o f f o o d and d ru g an a l y s i s 2 1 ( 2 0 1 3 ) 4 1 4e4 2 0 417

spiked sample are shown. The retention time was 24.8

minutes.

3.3. Linearity for standard curve of CLB

The calibration plot for the assay of CLB was linear over the

investigated range of 6.5e104.9 ng/mL, with a correlation

coefficient of 0.9997 (p < 0.05), and the equation of the line

for the best fit of the standard calibration curve was

y ¼ 1340.8563x � 1539.7319 (Fig. 2).

3.4. LOD and LOQ

Asociacion Espanola de Farmaceuticos de la Industria [25]

guidelines were followed to calculate the values of LOD and

LOQ for CLB. According to values obtained in the line with

lower concentrations to the calibration curve (0.66 ng/g,

1.31 ng/g, and 1.97 ng/g), equation y¼ 1108.9xþ 119.38 and the

respective SD equation y ¼ �5.4637x þ 34.69 were obtained, of

which the corresponding values were: Ybl ¼ 119.38; Sbl ¼ 34.69;

and b ¼ 1108.9. Therefore, the LOD and LOQ values were

0.20 ng/g and 0.42 ng/g, respectively. These values were

slightly higher than those reported by Chang et al [5]

(LOD ¼ 0.1 ng/g), but below the MRL established by the

United Nations Food and Agricultural Organization for CLB.

Fig. 2 e The calibration curve of clenbuterol dissolved in

mobile phase [0.05 M NaH2PO4 (pH 3.0)/CH3CN (85:15, v/v)].

3.5. Precision of the method for determining CLB

The %RSD value of the grouped estimator for the precision

study was found to be 4.74%, indicating that the method was

sufficiently precise (Table 1).

Oneof the factors thatmay influence the repeatabilityof the

method of analysis is the concentration of the analyte because

the SD of the responses obtained increases with decreasing

analyte concentration. For the analysis of impurities, the As-

sociation of Official Analytical Chemists proposes several limit

values of RSD of themethod according to the concentration of

the analyte, such that when working with levels between

1000 ng/g and 100 ng/g, the acceptable RSD limits are 11% and

15%, respectively [26]. Therefore, the results obtained in the

present study (RSD ¼ 4.74%) met the specifications set.

3.6. Accuracy of the method for determining CLB

The analysis of liver samples treated with CLB was similar to

that for the CLB standard, indicating that this compound was

recovered using our technique. Accuracy was determined by

spiking control bovine liver with three different concentra-

tions and treating and analyzing them as samples. The ac-

curacy was the ratio of the measured (calculated)

concentration to the added concentration. The accuracy was

111.7%, 82.0%, and 84.8% (Table 2). These values are consid-

ered acceptable because the trace analysis does not always

Table 1 e Results of the precision study at optimalconditions of extraction.

Parameter Sample A Sample B Sample C Groupedestimator

Mean 1,004,215 609,656 626,336 746,736

SD 56,308 20,273 13,579 d

sg d d d 35,431

RSD 5.6 3.3 2.2 4.74

RSD ¼ relative standard deviation; SD ¼ standard deviation.

Table 2 e Percentage recovery of bovine liver samplesartificially spiked with clenbuterol.

Amountadded (ng/g)

Value obtained(ng/g)

Recovery (%) RSD (%)

5.24 5.9 � 0.5 111.7 � 8.6 7.7

20.98 17.2 � 1.0 82.0 � 4.8 5.8

41.96 35.6 � 1.0 84.8 � 2.5 2.9

RSD ¼ relative standard deviation.

j o u rn a l o f f o o d a nd d r u g an a l y s i s 2 1 ( 2 0 1 3 ) 4 1 4e4 2 0418

achieve high recoveries and are regarded as usual recovery

values between 60% and 80% [24,26]. Chang et al [5] reported

obtained average recoveries of CLB in beef of 82.6e90.6% with

a coefficient of variation of 3.57%.

High percentage recovery revealed that the proposed

methodwas accurate and could be adopted for routine quality

control analysis.

3.7. Application of the method to quantification of CLBin bovine liver

The method developed was successfully applied to the

quantification of CLB. Seventy-eight bovine liver samples

were collected from a meat distribution center in Mexico City

and treated according to the methodology described above for

the extraction of CLB and analyzed by HPLCeUV. On the

whole, 49 (62.8%) bovine liver samples contained higher levels

of CLB than the MRL set by the Codex Alimentarius Commis-

sion, which is 0.6 ng/g for liver [13]. Meat and bovine viscera

are part of the Mexican diet, thus, there have been intensified

efforts to prevent the distribution and slaughter of animals

that are fed with CLB, through the implementation and

execution of government programs in slaughter houses

[11,27]. These steps have led some livestock producers to

avoid sending animals with drug residues to markets. To

complement these actions, it is necessary to monitor period-

ically places of introduction and marketing of meat products

and to pay special attention to the collection centers of high

concentration of these products in Mexico, through the sam-

pling of meat and viscera. However, according to the results

obtained, it is a reality that illegal use of this substance during

growth remains a common practice by some producers,

especially considering that CLB accumulates in the liver in

Fig. 3 e Samples with residues of clenbuterol by each

sampling. CLB [ clenbuterol.

large amounts (less in muscles). Therefore, the consumption

of animal viscera has a higher risk of food poisoning than

meat.

The request for meat with less fat, especially for con-

sumers in some states of Central Mexico, promotes the use of

CLB on livestock farms, so its inclusion continues. Fig. 3 shows

the trend for each batch tested monthly (Lots 1e5). The pro-

portion of samples with CLB remained constant in each

sampling, indicating consistency regarding the use of this

drug during the entire sampling period (5 months).

The use of CLB for cattle defattening is not allowed inter-

nationally. However, there have been reported cases of

human poisoning by this substance because of its presence in

bovine liver. Fig. 4 shows the levels of CLB in bovine liver

samples obtained in this investigation, and there were resi-

dues with values up to 185.3 ng/g in each sample. This was

similar to that found in Spain in 1990 by Martınez-Navarro

[28], which affected 135 peoplewho suffered the characteristic

symptoms of this contamination; in that case it was found

that CLB residues ranged from 160 ng/g to 291 ng/g in fresh

liver. The people were hospitalized with reversible symptoms

such as increased heart rate, muscle tremors, headache,

nausea, fever, and chills. A similar incident in France with 22

cases, also linked to bovine liver (375e500 ng/g) has been re-

ported [6,29].

Prolonged administration of a growth-promoting dose of

CLB to cattle could lead to residue accumulation in the liver as

an edible tissue, which may induce a pharmacotoxicologic

reaction in consumers. Adverse effects have been reported in

humans after ingestion of CLB at an oral dose of 10 mg/person,

four times daily. The consumption of 100 g of contaminated

liver with CLB at concentrations of 160e500 ng/g may exceed

the level of pharmacological effect [6]. The levels of CLB in

bovine liver marketed in Mexico found in the present research

(3.9e185.3 ng/g), corroborate the imminent risk that exists for

people to consume quantities that could cause severe damage

to their health.

In general, the bovine liver samples analyzed in the present

study presented with CLB concentrations lower than those

reported for cases of food poisoning in different countries:

Spain (160e291 ng/g in veal liver, 1990 [28], 19e5395 ng/g in

veal liver, 1995 [30], and 500 ng/g in veal liver, 1997 [31]); France

(375e500 ng/g in veal liver, 1991 [29]); Italy (800e7400 ng/g in

bovine meat, 1998 [32] and 1140e1480 ng/g in beef, 2000 [33]);

and Portugal (1420 ng/g in liver, 2003 and 2005) [17,34].

In Europe, Canada, and the USA, use of CLB has been

considered illegal since 1988. In Mexico, NOM-061-ZOO-1999

banned its use in the same year, but it continues to be found

in cases of human poisoning. In determining the b-agonist

in Mexico, Ortiz-Borges et al [35] reported that no CLB res-

idue was found in liver of 138 cattle slaughtered in the

municipal slaughter house of Merida. However, in 2008,

Estrada-Montoya et al [36] found CLB residues in beef pur-

chased from markets in Sonora, with values up to 6.12 ng/g.

Little information exists about the levels of CLB in food

poisoning cases reported in Mexico. However, work has

been done to evaluate the presence of CLB in various

matrices by enzyme-linked immunosorbent assay [37,38],

including serum of people affected by eating contaminated

meat [39].

Fig. 4 e Clenbuterol levels in bovine liver samples extracted and analyzed by high-performance liquid chromatography with

UV detection.

j o u r n a l o f f o o d and d ru g an a l y s i s 2 1 ( 2 0 1 3 ) 4 1 4e4 2 0 419

The problem for the human consumer is that the action of

CLB on livestock muscle development comes only with

pharmacological doses at least 10 times higher than the

therapeutic ones [40]. Based on the validation of this method,

it serves as an accurate and reliable method for the identifi-

cation and quantification of CLB, due to the low detection

limit, and good degrees of chromatographic separation, pre-

cision, accuracy, and sensitivity. The simplicity of this

method in the pretreatment of samples allows one to deter-

mine and quantify CLB levels in bovine liver by HPLCeUV. It

also saves reagents used as compared with other extraction

methods.

The levels of CLB found in some liver samples in our study

warn of a possible public health problem in Mexico City.

4. Conclusions

In this paper, we describe a method for measuring CLB in

bovine liver. The method was precise, specific, and accurate

for this b-agonist. The recovery was reproducible (precision of

4.74%) and good (in the accepted range of 82.0e111.7%). As a

better alternative to others methods, the currently described

HPLC method determines CLB by using only a UV detector. At

lower concentrations of CLB, the extraction method produced

better results in extraction efficiency than SPE did. In view of

this, the proposedmethod could be adopted for quality control

and routine analysis. With the proposedmethod, we achieved

simple and rapid detection of CLB in bovine liver samples; part

of the animal where the largest amount of CLB is accumulated

andwhere it remains for the longest time after any treatment.

As a result of recent shocking cases of food contamination

with CLB residues in Mexico, it is important to conduct as-

sessments of the quality of meat and meat products in order

tomagnify the real problem that exists in the country with the

b-agonist.

Acknowledgments

The first author is grateful to the Autonomous Metropolitan

University-Xochimilco for the award of Postdoctoral Fellow-

ship to carry out this research.

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