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b -Agonist Residues in Food, Analysis by LC

Nikolaos A. Botsoglou

Aristotle University, Thessaloniki, Greece

INTRODUCTION

animals for growth-promoting purposes has been pro-

hibited by regulatory agencies in Europe, Asia, and the

Americas. Clenbuterol, in particular, has been banned by

the FDA for any animal application in the United States,

whereas tishighly ikelytobebannedevenfor

therapeutic use in the United States in the near future.

However, veterinary use of some b-agonists, such as

clenbuterol, cimaterol, and ractopamine, is still licensed in

several parts of the world for therapeutic purposes.

b-Agonists are synthetically produced compounds that, in

addition to their regular therapeutic role in veterinary

medicine as bronchodilatory and tocolytic agents, can

promote live weight gain in food-producing animals. They

are also referred to as repartitioning agents because their

effect on carcass composition is to increase the deposition

of protein while reducing fat accumulation. For use in

lean-meat production, doses of 5 to 15 times greater than

the recommended therapeutic dose would be required,

together with a more prolonged period of in-feed

administration, which is often quite near to slaughter to

obviate the elimination problem. Such use would result in

significant residue levels in edible tissues of treated

animals, which might in turn exert adverse effects in the

cardiovascular and central nervous systems of the

consumers. [1]

There are a number of well-documented cases where

consumption of liver and meat from animals that have

been illegally treated with these compounds, particularly

clenbuterol, has resulted in massive human intoxifica-

tion. [1] In Spain, a foodborne clenbuterol poisoning

outbreak occurred in 1989–1990, affecting 135 persons.

Consumption of liver containing clenbuterol in the range

160–291 ppb was identified as the common point in the 43

families affected, while symptoms were observed in 97%

of all family members who consumed liver. In 1992,

another outbreak occurred in Spain, affecting this time

232 persons. Clinical signs of poisoning in more than half

of the patients included muscle tremors and tachycardia,

frequently accompanied by nervousness, headaches, and

myalgia. Clenbuterol levels in the urine of the patients

were found to range from 11 to 486 ppb. In addition, an

incident of food poisoning by residues of clenbuterol in

veal liver occurred in the fall of 1990 in the cities of

Roanne and Clermont-Ferrand, France. Twenty-two

persons from eight families were affected. Apart from

the mentioned cases, two farmers in Ireland were also

reported to have died while preparing clenbuterol for

feeding to livestock.

Although, without exception, these incidents have all

been caused by the toxicity of clenbuterol, the entire

group of b-agonists are now treated with great suspicion

by regulatory authorities, and use of all b-agonists in farm

MONITORING

Monitoring programs have shown that b-agonists have

been used illegally in parts of Europe and United States

by some livestock producers. [1] In addition, newly

developed analogues, often with modified structural

properties, are continuously introduced in the illegal

practice of application of growth-promoting b-agonists

in cattle raising. As a result, specific knowledge of

the target residues appropriate to surveillance is very

limited for many of the b-agonists that have potential

black market use. [2] Hence, continuous improvement

of detection methods is necessary to keep pace with

the rapid development of these new, heretofore unknown

b-agonists. Both gas and liquid chromatographic meth-

ods can be used for the determination of b-agonist

residues in biological samples. However, LC methods are

receiving wider acceptance because gas chromatographic

methods are generally complicated by the necessity of

derivatization of the polar hydroxyl and amino functional

groups of b-agonists. In this article, an overview of the

analytical methodology for the determination of b-agonist

in food is provided.

ANALYSIS OF b -AGONISTS BY LC

Included in this group of drugs are certain synthetical-

ly produced phenethanolamines such as bambuterol,

bromobuterol, carbuterol, cimaterol, clenbuterol, dobut-

amine, fenoterol, isoproterenol, mabuterol, mapenterol,

metaproterenol, pirbuterol, ractopamine, reproterol, rimi-

terol, ritodrine, salbutamol, salmeterol, terbutaline, and

Encyclopedia of Chromatography

DOI: 10.1081/E-ECHR 120028860

ORDER REPRINTS

b-Agonist Residues in Food, Analysis by LC

tulobuterol. These drugs fall into two major categories,

i.e., substituted anilines, including clenbuterol, and

substituted phenols, including salbutamol. This distinc-

tion is important because most methods for drugs in the

former category depend on pH adjustment to partition

the analytes between organic and aqueous phases. The

pH dependence is not valid, however, for drugs within

the latter category, because phenolic compounds are

charged under all practical pH conditions.

ether/n-butanol as extraction solvents. [5,7,8] The organic

extracts are then either concentrated to dryness, or repar-

titioned with dilute acid to facilitate back extraction of the

analytes into the acidic solution. A literature survey shows

that liquid–liquid partitioning cleanup resulted in good

recoveries of substituted anilines such as clenbuterol, [7,8]

but it was less effective for more polar compounds such

as salbutamol. [5] Diphasic dialysis can also be used for

purification of the primary sample extract. This procedure

was only applied in the determination of clenbuterol re-

sidues in liver using tert-butylmethyl ether as the ex-

traction solvent. [6]

EXTRACTION PROCEDURES

b-Agonists are relatively polar compounds that are

soluble in methanol and ethanol, slightly soluble in

chloroform, and almost insoluble in benzene. When

analyzing liquid samples for residues of b-agonists,

deconjugation of bound residues, using 2-glucuronidase/

sulfatase enzyme hydrolysis prior to sample extraction,

is often recommended. [3,4] Semisolid samples, such as

liver and muscle, require usually more intensive sample

pretreatment for tissue breakup. The most popular ap-

proach is sample homogenization in dilute acids such

as hydrochloric or perchloric acid or aqueous buffer. [3–6]

In general, dilute acids allow high extraction yields

for all categories of b-agonists, because the aromatic

moiety of these analytes is uncharged under acidic con-

ditions, whereas their aliphatic amino group is positively

ionized. Following centrifugation of the extract, the

supernatant may be further treated with b-glucuronidase/

sulfatase or subtilisin A to allow hydrolysis of the con-

jugated residues.

SOLID-PHASE EXTRACTION

Solid-phase extraction is, generally, better suited to the

multiresidue analysis of b-agonists. This procedure has

become the method of choice for the determination of

b-agonists in biological matrices because it is not labor

and material intensive. It is particularly advantageous

because it allows better extraction of the more hy-

drophilic b-agonists, including salbutamol. b-Agonists

are better suited to reversed-phase solid-phase extraction

due, in part, to their relatively non-polar aliphatic moiety,

which can interact with the hydrophobic octadecyl- and

octyl-based sorbents of the cartridge. [9–11] By adjusting

the pH of the sample extracts at values greater than 10,

optimum retention of the analytes can be achieved.

Adsorption solid-phase extraction, using a neutral

alumina sorbent, has also been recommended for

improved cleanup of liver homogenates. [5] Ion-exchange

solid-phase extraction is another cleanup procedure that

has been successfully used in the purification of liver and

tissue homogenates. [12] Because multiresidue solid-phase

extraction procedures covering b-agonists of different

types generally present analytical problems, mixed-phase

solid-phase extraction sorbents, which contained a

mixture of reversed-phase and ion-exchange material,

were also used to improve the retention of the more polar

compounds. Toward this goal, several different sorbents

were designed, and procedures that utilized both in-

teraction mechanisms have been described. [5,9,13]

CLEANUP PROCEDURES

The primary sample extract is subsequently subjected to

cleanup using several different approaches, including

conventional liquid–liquid partitioning, diphasic dialysis,

solid-phase extraction, and immunoaffinity chromatogra-

phy cleanup. In some instances, more than one of these

procedures is applied in combination to achieve better

extract purification.

IMMUNOAFFINITY CHROMATOGRAPHY

LIQUID–LIQUID PARTITION

Liquid–liquid partitioning cleanup is generally performed

at alkaline conditions using ethyl acetate, ethyl acetate/

tert-butanol mixture, diethyl ether, or tert-butylmethyl

Owing to its high specificity and sample cleanup

efficiency, immunoaffinity chromatography has also

received widespread acceptance for the determination of

b-agonists in biological matrices. [3,4,12,14]

The potential

ORDER REPRINTS

b-Agonist Residues in Food, Analysis by LC

of online immunoaffinity extraction for the multiresidue

determination of b-agonists in bovine urine was recently

demonstrated, using an automated column switching

system. [14]

CONCLUSION

This literature overview shows that a wide range of

efficient extraction, cleanup, separation, and detection

procedures is available for the determination of b-agonists

in food. However, continuous improvement of detection

methods is necessary to keep pace with the ongoing

introduction of new unknown b-agonists that have poten-

tial black market use, in the illegal practice.

SEPARATION PROCEDURES

Following extraction and cleanup, b-agonist residues are

analyzed by liquid chromatography. Gas chromatographic

separation of b-agonists is generally complicated by the

necessity of derivatization of their polar hydroxyl and

amino functional groups. LC reversed-phase columns are

commonly used for the separation of the various b-agonist

residues due to their hydrophobic interaction with the C 18

sorbent. Efficient reversed-phase ion-pair separation of

b-agonists has also been reported, using sodium dodecyl

sulfate as the pairing counterion. [15]

REFERENCES

1. Botsoglou, N.A.; Fletouris, D.J. Drug Residues in Food.

Pharmacology, Food Safety, and Analysis; Marcel Dekker:

New York, 2001.

2. Kuiper, H.A.; Noordam, M.Y.; Van Dooren-Flipsen,

M.M.H.; Schilt, R.; Roos, A.H. Illegal use of beta-

adrenergic agonists—European Community. J. Anim. Sci.

1998, 76, 195 – 207.

3. Van Ginkel, L.A.; Stephany, R.W.; Van Rossum, H.J.

Development and validation of a multiresidue method for

beta-agonists in biological samples and animal feed.

J. AOAC Int. 1992, 75, 554 – 560.

4. Visser, T.; Vredenbregt, M.J.; De Jong, A.P.J.M.; Van

Ginkel, L.A.; Van Rossum, H.J.; Stephany, R.W. Cryo-

trapping gas-chromatography Fourier-transform infrared

spectrometry—A new technique to confirm the presence of

beta-agonists in animal material. Anal. Chim. Acta 1993,

275, 205 – 214.

5. Leyssens, L.; Driessen, C.; Jacobs, A.; Czech, J.; Raus, J.

Determination of beta-2-receptor agonists in bovine urine

and liver by gas-chromatography tandem mass-spectrom-

etry. J. Chromatogr. 1991, 564, 515 – 527.

6. Gonzalez, P.; Fente, C.A.; Franco, C.; Vazquez, B.;

Quinto, E.; Cepeda, A. Determination of residues of the

beta-agonist clenbuterol in liver of medicated farm-animals

by gas-chromatography mass-spectrometry using diphasic

dialysis as an extraction procedure. J. Chromatogr. 1997,

693, 321 – 326.

7. Wilson, R.T.; Groneck, J.M.; Holland, K.P.; Henry, A.C.

Determination of clenbuterol in cattle, sheep, and swine

tissues by electron ionization gas-chromatography mass-

spectrometry. J. AOAC Int. 1994, 77, 917 – 924.

8. Lin, L.A.; Tomlinson, J.A.; Satzger, R.D. Detection of

clenbuterol in bovine retinal tissue by high performance

liquid-chromatography with electrochemical detection.

J. Chromatogr. 1997, 762, 275 – 280.

9. Elliott, C.T.; Thompson, C.S.; Arts, C.J.M.; Crooks,

S.R.H.; Van Baak, M.J.; Verheij, E.R.; Baxter, G.A.

Screening and confirmatory determination of ractopamine

residues in calves treated with growth-promoting doses of

the beta-agonist. Analyst 1998, 123, 1103 – 1107.

10. Van Rhijn, J.A.; Heskamp, H.H.; Essers, M.L.; Van de

Wetering, H.J.; Kleijnen, H.C.H.; Roos, A.H. Possibilities

for confirmatory analysis of some beta-agonists using 2

DETECTION PROCEDURES

Following LC separation, detection is often performed in

the ultraviolet region at wavelengths of 245 or 260 nm.

However, poor sensitivity and interference from coex-

tractives may appear at these low detection wavelengths

unless sample extracts are extensively cleaned up and

concentrated. This problem may be overcome by post-

column derivatization of the aromatic amino group of

the b-agonist molecules to the corresponding diazo dyes

through a Bratton-Marshall reaction, and subsequent de-

tection at 494 nm. [15] Although spectrophotometric de-

tection is generally acceptable, electrochemical detection

appears more appropriate for the analysis of b-agonists

due to the presence on the aromatic part of their molecule

of oxidizable hydroxyl and amino groups. This method

of detection has been applied in the determination of

clenbuterol residues in bovine retinal tissue with sufficient

sensitivity for this tissue. [8]

CONFIRMATION PROCEDURES

Confirmatory analysis of suspected liquid chromatograph-

ic peaks can be accomplished by coupling liquid chro-

matography with mass spectrometry. Ion spray LC-MS-

MS has been used to monitor five b-agonists in bovine

urine, [14] whereas atmospheric-pressure chemical ioniza-

tion LC-MS-MS has been used for the identification of

ractopamine residues in bovine urine. [9]

ORDER REPRINTS

b-Agonist Residues in Food, Analysis by LC

different derivatives simultaneously. J. Chromatogr. 1995,

665, 395 – 398.

11. Gaillard, Y.; Balland, A.; Doucet, F.; Pepin, G. Detection

of illegal clenbuterol use in calves using hair analysis.

J. Chromatogr. 1997, 703, 85 – 95.

12. Lawrence, J.F.; Menard, C. Determination of clenbuterol

in beef-liver and muscle-tissue using immunoaffinity

chromatographic cleanup and liquid-chromatography with

ultraviolet absorbency detection. J. Chromatogr. 1997,

696, 291 – 297.

13. Ramos, F.; Santos, C.; Silva, A.; Da Silveira, M.I.N.

Beta(2)-adrenergic agonist residues—Simultaneous meth-

ylboronic and butylboronic derivatization for confirmatory

analysis by gas-chromatography mass-spectrometry. J.

Chromatogr. 1998, 716, 366 – 370.

14. Cai, J.; Henion, J. Quantitative multi-residue determination

of beta-agonists in bovine urine using online immunoaf-

finity extraction coupled-column packed capillary liquid-

chromatography tandem mass-spectrometry. J. Chroma-

togr. 1997, 691, 357 – 370.

15. Courtheyn, D.; Desaever, C.; Verhe, R. High-performance

liquid-chromatographic determination of clenbuterol and

cimaterol using postcolumn derivatization. J. Chromatogr.

1991, 564, 537 – 549.

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