Clenbuterol Hydrochloride

CHAPTER TWO

Clenbuterol Hydrochloride Abdulrahman A. Al-Majed, Nasr Y. Khalil, Ibraheem Khbrani, Hatem A. Abdel-Aziz College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

Contents 1. Description 1.1 Nomenclature 1.2 Formula 1.3 Elemental Analysis 1.4 Appearance 1.5 Uses and Applications 2. Methods of Preparation 3. Physical Characteristics 3.1 Ionization Constant 3.2 Solubility Characteristics 3.3 X-Ray Powder Diffraction Pattern 3.4 Thermal Method of Analysis 3.5 Spectroscopy 3.6 Mass Spectrometry 3.7 NMR Spectrometry 4. Methods of Analysis 4.1 Compendial Method 4.2 Reported Methods of Analysis 4.3 Electrochemical Methods 4.4 Chromatography 5. Stability 6. Pharmacokinetics, Metabolism, and Excretion 6.1 Pharmacokinetics 6.2 Metabolism 6.3 Excretion 7. Pharmacology References

Profiles of Drug Substances, Excipients, and Related Methodology, Volume 42 ISSN 1871-5125 http://dx.doi.org/10.1016/bs.podrm.2017.02.002

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2017 Elsevier Inc. All rights reserved.

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1. DESCRIPTION 1.1 Nomenclature 1.1.1 Systemic Chemical Names 1-(4-Amino-3,5-dichlorophenyl)-2-tert-butylaminoethanol hydrochloride; 4-amino-3,5-dichloro-α-[[(1,1-dimethylethyl)-amino]methyl]benzenemethanol hydrochloride; and 4-amino-α-[(tert-butylamino)methyl]-3,5dichlorobenzyl alcohol hydrochloride [1]. 1.1.2 Nonproprietary Names Clenbuterol hydrochloride [1–3]. 1.1.3 Proprietary Names Broncodil; Clenasma; Contrasmina; Contraspasmin; Monores; Prontovent; Spiropent; Ventolase; Ventipulmin [4].

1.2 Formula 1.2.1 Empirical Formula, Molecular Weight, and CAS Number [4] Empirical Formula

Molecular Weight

CAS Number

C12H18Cl2N2O

277.2

37148-27-9

C12H18Cl2N2O
HCl

313.65

21898-19-1

1.2.2 Structural Formula for Clenbuterol Hydrochloride (Fig. 1) IUPAC Name: 1-(4-Amino-3,5-dichlorophenyl)-2-(tert-butylamino) ethanol hydrochloride (MW ¼ 313.65) [3,4]. OH

H N

CI

•HCI NH2 CI

Fig. 1 Structure of clenbuterol hydrochloride.

Clenbuterol Hydrochloride

93

1.3 Elemental Analysis C: 52.00%, H: 6.55%, Cl: 25.58%, N: 10.11%, O: 5.77% [1].

1.4 Appearance Colorless, microcrystalline powder [4].

1.5 Uses and Applications Clenbuterol hydrochloride is a direct-acting sympathomimetic agent with mainly beta-adrenergic activity and a selective action on β2 receptors (a β2 agonist). It has properties similar to those of salbutamol. It is used as a bronchodilator in the management of reversible airways obstruction, as in asthma and in certain patients with chronic obstructive pulmonary disease. A usual oral dose is 20 μg twice daily. Clenbuterol hydrochloride has also been given by inhalation. In patients with asthma, as-required beta agonist therapy is preferable to regular use. An increased need for, or decreased duration of effect of, clenbuterol indicates deterioration of asthma control and the need for review of therapy. Clenbuterol hydrochloride may be restricted in certain sports and competitors should check with the appropriate sports authorities. Clenbuterol has been used illicitly in animal feeds in an attempt to promote weight gain and to increase muscle to lipid mass. Adverse effects typical of sympathomimetic activity have been attributed to such misuse both in farmers perpetrating such acts and in innocent persons consuming meat products from affected animals. Clenbuterol has been abused by sportsmen for its anabolic effects, although it is doubtful as to whether it enhances performance. Myocardial infarction was described in an otherwise healthy 17-year-old bodybuilder after abuse of clenbuterol. Coronary artery spasm and temporary thrombosis were suggested as possible explanations for this adverse effect [2].

2. METHODS OF PREPARATION By bromination of 4-amino-3,5-dichloroacetophenone (I) with Br2 in CHCl3 to give 4-amino-3,5-dichloro-alpha-bromoacetophenone (II), m.p. 140–145°C, which is condensed with tert-butylamine (III) in CHCl3 to 4-amino-3,5-dichloro-alpha-tert-butylaminoacetophenone hydrochloride (IV), m.p. 252–257°C, this product is finally reduced with NaBH4 in methanol (Scheme 1) [5]. Clenbuterol has also been synthesized from p-aminoacetophenone through benzene ring chlorination using polymer Lewis acid PVC–FeCl3

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O

O

Cl

Cl CH3

H 2N

(III)

(I)

H N

Cl

CH3 CH3 · HCl

H2N Cl

O

H2N—tBu Br

Br2

CH3

H2N Cl

Cl (II)

(IV)

NaBH4

OH Cl

H N

CH3 CH3 CH3

H2N Cl

Scheme 1 Preparation of clenbuterol from 4-amino-3,5-dichloroacetophenone.

as catalyst, α-hydrogen brominating using CuBr2 as brominating agent, amination with H2NCMe3, and reduction with KBH4. The total yield was 40.8%. The improved method is more convenient and effective [6].

3. PHYSICAL CHARACTERISTICS 3.1 Ionization Constant pKa: 5–7 [2].

3.2 Solubility Characteristics Very soluble in water, methanol, and ethanol; slightly soluble in chloroform; and insoluble in benzene [4].

3.3 X-Ray Powder Diffraction Pattern Toro et al. [7] presented a structural characterization of a new form of clenbuterol, the well-known decongestant and bronchodilator which is also used as a performance-enhancing drug. In the PDF-4/Orgs. 2012 database, there are six entries related to this compound: three for its hydrochloride salt calculated using single-crystal data, two for a methanol and a dimethyl sulfoxide solvate of two Cu-clenbuterol complexes, and one experimental unindexed pattern. In this contribution the powder diffraction pattern and the crystal structure are determined using single-crystal X-ray diffraction techniques of clenbuterol hemihydrate, C12H18Cl2N2O
0.5H2O.

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3.4 Thermal Method of Analysis 3.4.1 Melting Behavior Clenbuterol melts at 174–175.5°C [4] (173–175°C). 3.4.2 Differential Scanning Calorimetry Signoretti et al. [8] used differential scanning calorimetry (DSC) to predict the physicochemical compatibility between clenbuterol and some mucolytic drugs. Mixtures of clenbuterol and ambroxol, bromhexine, tiopronin, sobrerol, eprazinone, and carbocysteine were examined. Using this method, only eprazinone and carbocysteine were found to be compatible with clenbuterol. Gopferich et al. [9] measured the solubility of clenbuterol in thin films of surfactant-free Eudragit NE30D. Light microscopy and DSC were supplemented by a technique based on the measurement of the rate of drug release from the films. The clenbuterol crystals had the form of fractals which could be shown by a computer simulation of diffusion-controlled aggregation.

3.5 Spectroscopy 3.5.1 Ultraviolet Spectroscopy Fig. 2 shows the ultraviolet absorption spectrum of clenbuterol hydrochloride (20 μg/mL, in methanol) that was obtained using a Shimadzu 2.6

Absorbance

2.2 1.8 1.4 1 0.6 0.2 –0.2 200

220

240

260

280

300

320

340

360

380

400

Wavelength(nm)

Fig. 2 Ultra violet absorption spectrum of clenbuterol hydrochloride (20 μg/mL) in methanol.

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Fig. 3 Infrared absorption spectrum of clenbuterol hydrochloride (KBr pellet).

Spectrophotometer (Model UV-1800) and showed three maxima at 210, 247, and 302 nm. 3.5.2 Infrared Spectroscopy The infrared absorption spectrum of clenbuterol hydrochloride was recorded using KBr disk sampling in a Shimadzu FTIR 8400S spectrometer. The broad absorption bands of the OH, NH, and NH2 groups appeared in the region 3350–3250 cm1. The absorption band of aromatic CH appeared at 3129 cm1, while the band of CH aliphatic appeared at 2972 cm1 (see Fig. 3).

3.6 Mass Spectrometry The mass spectrum of clenbuterol hydrochloride (C12H18Cl2N2O
HCl, FW ¼ 313.65) was obtained using an Agilent 6320 Ion trap mass spectrometer (Agilent Technologies, USA) equipped with an electrospray ionization interface (ESI). A connector was used instead of column. The mobile phase was composed of a mixture of solvents A and B (50:50), where A is HPLC (high-performance liquid chromatography) grade water and B is acetonitrile. The stock sample solution was prepared by weighing the solid substance to 1 mg/mL in DMSO and diluted with the mobile phase. The test solution was prepared by diluting the stock solutions with the mobile phase depending on the ions intensities. The flow rate was 0.4 mL/min,

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⫻102 +Scan (0.428 min) CL2.d 1.4

277.0

1.3 1.2 1.1

202.9

1 0.9 0.8 0.7

259.1

0.6 0.5 0.4 0.3 0.2 0.1 0 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300

Counts vs mass-to-charge (m/z)

Fig. 4 Mass spectrum of clenbuterol hydrochloride.

and the run time was 5 min. MS parameters were optimized. The scan was ultra-scan mode. MS2 scans were performed in the mass range of 150–300 m/z. The ESI was operated in the positive mode. The source temperature was set to 350°C, the nebulizer gas pressure was 55.00 psi, and the dry gas flow rate was 12.00 L/min. Fig. 4 shows the peak of molecular ion of clenbuterol at m/z ¼ 277.0 [C12H18Cl2N2O].

3.7 NMR Spectrometry 3.7.1 1H NMR Spectrometry The 1H NMR spectrum of clenbuterol hydrochloride was scanned in DMSO-d6 on a Bruker NMR spectrometer operating at 500 MHz. Chemical shifts are expressed in δ-values (ppm) relative to TMS as an internal standard. Coupling constants (J) are expressed in Hz (Table 1 and Fig. 5A and B). 3.7.2 13C NMR Spectrometry The 13C NMR spectrum of clenbuterol hydrochloride was obtained in DMSO-d6 on a Bruker NMR spectrometer operating at 125 MHz. Chemical shifts are expressed in δ-values (ppm) relative to TMS as an internal standard (Table 2 and Fig. 6).

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Table 1 1H NMR of Clenbuterol Hydrochloride (DMSO-d6) 13 OH Cl

3

1

4 H2N 7

H N 11 9 10

2 8

12b 12c

6 5 Cl

12a

HCl 14

Clenbuterol hydrochloride

Signal

Location (δ)

Shape

Integration

Correspondences

1

1.31

s

9H

–NH–C–(CH3)3 (Hs12a–c)

2

2.89–2.94

m

1H

–CH2–NH–C–(CH3)3 (Ha9)

3

2.99–3.01

m

1H

–CH2–NH–C–(CH3)3 (Hb9)

4

4.89

m

1H

–CH–OH (H8)

5

5.51

s

2H

–NH2 (Hs7)

6

6.17

br. s

1H

–OH (H13)

7

7.29

s

2H

ArH (H2 + H6)

8

8.5

br. s

1H

–NH (H10)

9

9.3

br. s

1H

HCl (H14)

The underlining and italics indicate that the NMR recorded is the proton NMR.

4. METHODS OF ANALYSIS 4.1 Compendial Method 4.1.1 British Pharmacopeia [10] Identification A. Infrared absorption spectrophotometry. Comparison with clenbuterol hydrochloride CRS; the spectrum of the sample must be equivalent to that of the Standard. B. Thin-layer chromatography 1. Test solution: Dissolve 10 mg of the substance to be examined in 10 mL of methanol R. 2. Reference solution: Dissolve 10 mg of clenbuterol hydrochloride CRS in 10 mL of methanol R. • Plate: TLC silica gel F254 plate R. • Mobile phase: ammonia R, anhydrous ethanol R, toluene R (0.15:10:15, v/v/v).

99

6.5

5.5

1.310

5.0

0

–1

–3 ppm

4.0

3.5

2.511

3.380

4.5

–2

9.07

1.01

1

3.013 2.989 2.938 2.918 2.894

3.013 2.989 2.938 2.918 2.894 2.511

3.380

1.06 2.03

2

4.891

4.891

2.02

5.505

6

3

3.0

2.5 1.01

7.0

4

2.03

7.5

5

1.06

8.0

6

1.00

8.5

2.01

9.0

1.05

0.84

9.5

7

6.173

7.294

B

8

0.95

9

2.02

10

2.01

12 11

0.95

14 13

0.84

15

1.05

16

1.00

7.294

6.173

A

5.505

Clenbuterol Hydrochloride

ppm

Fig. 5 (A) 1H NMR spectrum of clenbuterol hydrochloride (3.0 to 16.0 ppm). (B) 1H NMR spectrum of clenbuterol hydrochloride (2.5–9.5 ppm).

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Table 2 Signal

13

C NMR of Clenbuterol Hydrochloride (DMSO-d6) Location (δ) Correspondences

1

25.49

–NH–C–(CH3)3 (3 C12a–c)

2

48.67

–CH2–NH–C–(CH3)3 (C9)

3

58.76

–CH2–NH–C–(CH3)3 (C11)

4

68.03

–CH–(OH)– (C8)

5

118.32

C2 + C6

6

126.24

C3 + C5

7

131.12

C1

8

141.02

C4

The underlining and italics indicate that the NMR recorded is the proton NMR

Fig. 6

13

C NMR spectrum of clenbuterol hydrochloride.

• • • •

Application: 10 μL. Development is performed over a path of 10 cm. Dry in air. Detection: Spray with a 10 g/L solution of sodium nitrite R in 1 M hydrochloric acid and dip after 10 min in a 4 g/L solution of naphthylethylenediamine dihydrochloride R in methanol R. Allow to dry in air.

Clenbuterol Hydrochloride

101

• Results: The principal spot in the chromatogram obtained with the test solution is similar in position, color, and size to the principal spot in the chromatogram obtained with the reference solution. 4.1.1.1 Tests

Solution S Dissolve 0.5 g in 10 mL of carbon dioxide-free water R. Appearance of the solution Solution S is not more opalescent than reference suspension II (2.2.1) and not more intensely colored than reference solution Y6 (2.2.2, Method II). pH 5.0–7.0 for solution S. Optical rotation • 0.10 degree to +0.10 degree. • Dissolve 0.30 g in water R and dilute to 10.0 mL with the same solvent. Filter if necessary. Related substances Liquid chromatography • Test solution: Disperse 100.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 mL with the mobile phase. • Reference solution (a): Dilute 0.1 mL of the test solution to 100.0 mL with water R. • Reference solution (b): Dissolve 5 mg of clenbuterol impurity B CRS in 10 mL of the mobile phase, add 2.5 mL of the test solution and dilute to 25.0 mL with the mobile phase. • Column: Size: L ¼ 0.125 m, Ø ¼ 4 mm Stationary phase: End-capped octadecylsilyl silica gel for chromatography R (5 μm) Temperature: 40°C. • Mobile phase: Mix 200 volumes of acetonitrile R, 200 volumes of methanol R, and 600 volumes of a solution prepared as follows: dissolve 3.0 g of sodium decanesulfonate R and 5.0 g of potassium dihydrogen phosphate R in 900 mL of water R, adjust to pH 3.0 with dilute phosphoric acid R, and dilute to 1000 mL with water R. • Flow rate: 0.5 mL/min. • Detection: UV detector at 215 nm. • Injection: 5 μL. • Run time: 1.5 times the retention time of clenbuterol.

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• •

Retention time of clenbuterol: about 29 min. System suitability: Reference solution (b): Resolution: minimum of 4.0 between the peaks due to impurity B and clenbuterol. • Limits: Impurities A, B, C, D, E, F: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1%) Any other impurity: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1%). • Total: not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2%). • Disregard limit: 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05%). Water Maximum 1.0%, determined on 0.500 g. Sulfated ash Maximum 0.1%, determined on 1.0 g. 4.1.1.2 Assay

Dissolve 0.250 g in 50 mL of ethanol (96%) R and add 5.0 mL of 0.01 M hydrochloric acid. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). Read the volume added between the two points of inflexion. 1 mL of 0.1 M sodium hydroxide is equivalent to 31.37 mg of C12H19Cl3N2O. 4.1.1.3 Impurities

Specified impurities: A, B, C, D, E, F. O R2 R1 H2N R2

A: R1 ¼ H, R2 ¼ Cl: 4-amino-3,5-dichlorobenzaldehyde B: R1 ¼ CH2–NH–C(CH3)3, R2 ¼ Cl: 1-(4-amino-3,5-dichlorophenyl)2-[(1,1-dimethylethyl)amino]ethanone

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C: R1 ¼ CH3, R2 ¼ Cl: 1-(4-amino-3,5-dichlorophenyl)ethanone D: R1 ¼ CH3, R2 ¼ H: 1-(4-aminophenyl)ethanone E: R1 ¼ CH2Br, R2 ¼ Cl: 1-(4-amino-3,5-dichlorophenyl)-2bromoethanone F: Bromoclenbuterol: (1RS)-1-(4-amino-3-bromo-5-chlorophenyl)-2[(1,1-dimethylethyl)amino]ethanol OH CH

C1

CH2

NHBu-t

H2N Br

4.2 Reported Methods of Analysis 4.2.1 Immunochemical Methods Yamamoto and Iwata [11] established a sensitive double antibody and heterologous enzyme immunoassay (EIA) for the quantitation of clenbuterol. Specific antiserum to this agent was raised in rabbits by immunization with diazotized clenbuterol and human serum albumin conjugate. For competitive reactions, antibody was incubated with a mixture of diazotized clenbuterol analog (NA 1141) labeled with beta-D-galactosidase and unlabeled standard or sample clenbuterol. The antibody-bound enzyme hapten was separated from free hapten by antirabbit IgG immobilized to a polystyrene ball. Activity of the enzyme on the solid phase was fluorometrically determined. The assay system made it possible to ascertain values as low as 0.5 pg/tube of clenbuterol. By use of this assay method, the time course of plasma levels of clenbuterol was examined after a single oral administration (20 μg) to three healthy volunteers. It was shown that the maximum level was achieved after 2–3 h with approximately 10 ng clenbuterol/dL of plasma. Degand et al. [12] developed an EIA based on the competition between clenbuterol and CLB-horse radish peroxidase. This test was capable of detecting CLB residues present in urine samples in the region of 0.5 ng/mL. Haasnoot et al. [13] described the development of a clenbuterol (CLB) EIA, by using the cross-reactivity of the antibody with structurally similar compounds to CLB; they were able to detect at least four other β-agonist compounds which had been linked to illegal use (cimbuterol, brombuterol, mapenterol, and mabuterol).

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Chen et al. [14] presented a novel method to analyze clenbuterol based on a competitive microfluidic immunoassay scheme with a micro-ELISA system and obtained a limit of detection (LOD) that is less than 0.1 ng/mL with a quantitative working range of 0.1–27 ng/mL. The approach was envisaged to be a promising method for efficient onsite clenbuterol control with good sensitivity and portability. Hajrulai-Musliu et al. [15] introduced ELISA and GC–MS (gas chromatography/mass spectrometry) methods to monitor the presence of clenbuterol residues in bovine meat muscle collected from the veterinarian inspectors, using screening and confirmatory methods. From 55 bovine meat samples analyzed by the ELISA test, 1 (1.8%) of presented clenbuterol residues was 1.19 μg/kg, 7 (12.7%) from 0.5 to 1.0 μg/kg, 13 (23.64%) from 0.1 to 0.5 μg/kg, and 34 (61.82%) were below the LOD of 0.1 μg/kg. In this study, from 21 meat samples analyzed by GC–MS, only one sample was confirmed positive for clenbuterol. The obtained results indicated that clenbuterol was still illegally used as a bovine growth promoter. Haughey et al. [16] developed a rapid and sensitive biosensor assay to detect CLB residues in bovine urine. The method involved a simple extraction procedure using tert-butyl methyl ether followed by analysis on the biosensor with results obtained against a buffer calibration curve. The assay allowed up to 88 samples to be analyzed per working day. Haughey and Baxter [17] performed a study to develop an assay capable of detecting CLB residues present in bovine urine using an optical biosensor assay. Following the validation of this test, the results obtained in detecting CLB residues in samples taken from treated animals were compared with those found with the traditional immunoassay (EIA and RIA) and GC/MS methods routinely used in testing laboratories. 4.2.2 Spectrophotometric Methods 4.2.2.1 Ultraviolet Spectrometry

Biosca et al. [18] developed an ultrasensitive absorption procedure for the determination of clenbuterol in pharmaceutical preparations. Clenbuterol was diazotized with nitrite and coupled with 1-(naphthyl)ethylenediamine, and the absorbance of the azo dye formed was measured by both spectrophotometry and ultrasensitive thermal lens spectrometry (TLS). The TLS limit of detection was 1.5 ppb, 14-fold lower than with a Hewlett–Packard diode array spectrophotometer. Thus, the TLS procedure can be advantageously applied to quality control of clenbuterol at the individual dose level

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and in small samples. Repeatability as relative standard deviation was 1.5% (50 ppb, n ¼ 6). 4.2.2.2 Mid-Infrared Spectroscopy

Meza-Ma´rquez et al. [19] reported a mid-infrared spectroscopic (FT-Mid IR) method coupled with a multivariate analysis to predict clenbuterol in beef meat, liver, and kidney. A soft independent modeling of class analogy (SIMCA) model was also developed to discriminate between pure (beef meat, liver, and kidney) and spiked with clenbuterol samples (beef meat clenbuterol, liver-clenbuterol, and kidney-clenbuterol). The best models to predict clenbuterol concentrations were obtained using the partial least squares algorithm with an R2 > 0.9 and SEC and standard error of prediction <0.296 and 0.324, respectively. The SIMCA model used to discriminate pure and spiked with clenbuterol samples showed 100% correct classification rate. Method detection limit was 2 μg/kg. FT-Mid IR coupled with chemometrics could be a simple and rapid screening tool for monitoring clenbuterol in beef meat, liver, and kidney implicated in food poisoning. This method could be used for screening purposes. 4.2.2.3 Colorimetery

Zhang et al. [20] developed a highly sensitive method for the detection of trace amount of clenbuterol based on gold nanoparticles (AuNPs) in the presence of melamine. Hydrogen-bonding interaction between clenbuterol and melamine resulted in the aggregation of AuNPs and a consequent color change of AuNPs from wine red to blue. The concentration of clenbuterol could be determined with naked eye or a UV–vis spectrometer. Results showed that the absorption ratio (A(670)/A(520)) was liner with the logarithm of clenbuterol concentration in the range of 2.8  1010 to 2.8  107 M and 2.8  107 to 1.4  106 M with linear coefficients of 0.996 and 0.993, respectively. The detection limit was 2.8  1011 M (S/N ¼ 3), which was much lower than most existing methods. The coexisting substances including DL-epinephrine, phenylalanine, tryptophan, alanine, uric acid, glycine, glycerol, glucose, MgCl2, CaCl2, and NaCl did not affect the determination of clenbuterol. The proposed method could be successfully applied to the determination of clenbuterol in human urine. Kang et al. [21] demonstrated a simple visual and rapid colorimetric sensor for detection of clenbuterol (CLB) based on AuNPs modified with cysteamine (CA) and characterized by transmission electron microscopy,

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dynamic light scattering, and UV–vis. The solution color from red to blue gray with increasing clenbuterol concentration resulted from the aggregation of AuNPs. The detection limit of clenbuterol is 50 nM by naked eyes. The selectivity of CA-AuNP detection system for clenbuterol is excellent compared with other interferents in food. This sensor has been successfully applied to detect clenbuterol in real blood sample.

4.3 Electrochemical Methods 4.3.1 Voltammetric Method Chai and Liu [22] developed a novel immunosensor for determination of clenbuterol by using horseradish peroxidase and monoclonal antibody against clenbuterol which was covalently immobilized into the cellulose acetate membrane which was used as one-touch immunochips. After 20 min of competitive reaction of clenbuterol in samples against glucose oxidase-conjugated clenbuterol (CLB-GOD) with anti-clenbuterol antibody immobilized on the membrane, the one-touch transducer was fixed on the Nafion-ferrocene-modified glass-carbon electrode (GCE, working electrode) by “O” ring. The increased current changes (ΔI) were recorded for the quantitative determination of clenbuterol by derivative cyclic voltammetry. Experimental results show that a very effective system for electron transmission had been established using dual-enzyme system and Nafion-ferrocene-modified GCE. Quantitative analysis of clenbuterol can be easily performed on this kind of immunosensor, showing the advantage of time-saving and high sensitivity. Moane et al. [23] developed a method for the determination of clenbuterol in bovine urine using differential-pulse voltammetry (DPV), based on the electrochemical behavior of clenbuterol at a Nafion-modified carbon paste electrode (CPE). Clenbuterol is irreversibly oxidized at high positive potentials, its irreversibility being due to a chemical follow-up reaction, which results in a product showing quasi-reversible electrochemical behavior at much lower potentials. It is the oxidation peak of this product, arising in acidic media at 0.42 V, which was analyzed using DPV, again following the accumulation of clenbuterol at the Nafion-modified CPE. Electrode renewal was achieved by holding the potential at 0.6 V for 120 s in 0.1 M NaOH. The determination of clenbuterol in the presence of interfering compounds present in bovine urine samples was then carried out after a two-step clean-up of the urine involving liquid–liquid extraction (LLE) followed by a mixed-mode solid-phase extraction (SPE) procedure. This allowed clenbuterol to

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be detected down to a level of 1.02  109 mol/L in bovine urine extracts. 4.3.2 Amperometric and Photoelectric Methods Zhang et al. [24] used a nanocomposite consisting of zinc sulfide quantum dots and polyaniline (ZnSQD@PANI) placed on a gold electrode along with antibody against clenbuterol to give an amperometric immunosensor for clenbuterol. Compared to the use of pristine PANI, the electrode modified with the ZnSQD@PANI nanocomposite adsorbs clenbuterol antibody much better and therefore exhibits higher sensitivity to clenbuterol. The biosensor, when operated at a working potential of 0.21 V (vs Ag/AgCl), displays a detection limit as low as 5.5 pg/mL and works over the 0.01–10 ng/mL concentration range. Related species such as salbutamol and ractopamine, urine components such as urea and uric acid, and the ions Ca(II), Na(I), and K(I) do not interfere. Liang and Yaxin [25] described an invention which relates to a detection device for rapid quantitative detection of clenbuterol. The detection device for rapid quantitative detection of clenbuterol comprises a reagent card, transfer means, and information reading means; the reagent card includes a protective film, base plate, and in turn connected sample absorbent pad, colloidal gold pad, nitrocellulose membrane, and absorbent pads provided on the base plate; the protective film is provided on one side surface of the nitrocellulose membrane; the delivery device comprises a plurality of reagent card slot provided on its surface; and said information reading means includes a light source, photoelectric sensors, the optical information anal. module, processor module, and display module. The present invention also relates to a detection method for rapid quantitative detection of clenbuterol. Detecting device and detection method for rapid quantitative detection of clenbuterol provided by the present invention can effectively prevent the color influence of air environmental factors on reagent card detection line, with advantages of good detecting effect and accurate quantitative test results. 4.3.3 Potentiometric Methods Liang et al. [26] described a polymeric membrane ion-selective electrode for determination of clenbuterol based on a molecularly imprinted polymer as an ionophore for molecular recognition, which can be synthesized by precipitation polymerization using clenbuterol hydrochloride as the template molecule. Under optimized conditions, the proposed membrane electrode

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exhibits Nernstian response to the protonated clenbuterol over the concentration range of 1.0  107–1.0  104 M with a slope of 55.7 mV/dec, and a detection limit of 7.0  108 M. The MIP-based sensor shows excellent selectivity, rapid response time, and satisfactory long-term stability. The potentiometric sensor has been successfully applied to the determination of clenbuterol in pig urine samples with recoveries between 98% and 107% and an analysis time of less than 3 min. Feng et al. [27] reported a method for determination of clenbuterol by using ethanolamine and oleylamine as reduction, and dicaprylyl ether and 1,2-hexadecanediol as medium; PtNiFe (8–13 nm) nanoflower particles. By utilizing a graphite rod as a carbon source and a quench method to get the carbon nanosheets, the clenbuterol electrochemical sensor was prepared by absorbing PtNiFe nanoflower into carbon nanosheets to modified carbon glassy electrode for the detection of clenbuterol residual in the metabolite of animal. The effects of some experiment conditions such as buffer, enrichment potential, and time on the electrochemical behavior of this sensor were investigated. The liner range was 1.9  107–4.9  102 mol/L, and the detection limit is 6.0  108 mol/L. This sensor has good selectivity, high sensitivity, good reproducibility, and stability. Guo et al. [28] developed a potentiometric method for the determination of trace clenbuterol. A multiwalled carbon nanotube-Nafion composite was used to modify the GCE. The modified electrode showed high sensitivity and good selectivity for clenbuterol detection. It offered a linear range of 1.0  109–1.0  106 mol/L with a detection limit of 5.0  1010 mol/L.

4.4 Chromatography 4.4.1 Thin-Layer Chromatography Presa et al. [29] developed a simple method for semiquantitative clenbuterol residue analysis in urine. After an SPE clean-up step and washings with hexane at low pH, the resulting aqueous phase was alkalinized and extracted with tert-butyl-methyl ether. The dry residue was resuspended in ethyl acetate and applied on the high-performance thin-layer chromatography (HPTLC) plate. Visualization of clenbuterol was performed by means of the transformation in this azo-compound. The detection limit is about 1 mg/L and the recoveries in urine were between 80% and 100%, values obtained by means of visual appreciation of the spots. Huang et al. [30] developed a thin-layer chromatography scanning determination of clenbuterol (CLB) residue in swine liver using EtOAc– MeOH–HOAc (8:1:0.8) as the mobile phase, and on the silica gel GF plate,

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the Rf value of CLB was 0.8 L. The calibration curve was linear in the range of 0.004–0.050 μg. The average recovery was 86.5%–94.9% and the RSD of CLB was 5.0%–5.8% (n ¼ 6). 4.4.2 High-Performance Liquid Chromatography Guan et al. [31] developed a liquid chromatographic method with electrospray for quantification and confirmation of the presence of CLB in equine plasma, urine, and tissue samples. The method involved LLE, separation by liquid chromatography (LC) on a short cyano column, and pseudo-multiple reaction monitoring (pseudo-MRM) by electrospray ionization quadruple time-of-flight tandem mass spectrometry (ESIQTOF-MS/MS). At very low concentrations (picograms of CLB/mL), LLE produced better extraction efficiency and calibration curves than SPE. The operating parameters for electrospray QTOF and yield of the product ion in MRM were optimized to enhance sensitivity for the detection and quantification of CLB. The quantification range of the method was 0.013–10 ng of CLB/mL of plasma, 0.05–20 ng/0.1 mL of urine, and 0.025–10 ng/g of tissue. The detection limit of the method was 13 pg/mL of plasma, 50 pg/0.1 mL of urine, and 25 pg/g of tissue. The method was successfully applied to the analysis of CLB in plasma, urine, and various tissue samples, and in pharmacokinetic (PK) studies of CLB in the horse. CLB was quantified for 96 h in plasma and 288 h in urine postadministration of CLB (1.6 μg/kg, twice daily for 7 days). This method is useful for the detection and quantification of very low concentrations of CLB in urine, plasma, and tissue samples. Bazylak and Nagels [32] developed an HPLC method with potentiometric detection using poly(vinyl chloride) (PVC)-based liquid membrane electrode coatings for determination of a series of 18 beta-adrenoceptor binding drugs (5 agonists and 13 antagonists) in cation exchange-HPLC and RP-HPLC systems. Incorporation of the lipophilic cation-exchanger tetrakis(p-chlorophenyl)borate (TCPB) alone or in combination with trioctylated α-cyclodextrin into the polymeric liquid membrane yielded very sensitive responses for racemic forms of bufuralol, propranolol, carazolol, clenbuterol, mabuterol, cimaterol, bisoprolol, oxprenolol, alprenolol, tertatolol, and bevantolol, especially in the cation exchange-HPLC system applying acetonitrile–40 mM phosphoric acid (15:85, v/v, pH 2.35) as the mobile phase. In both applied orthogonal HPLC modes, using TCPB-containing electrodes (no addition of neutral macrocyclic ionophores) gives more than fivefold improvement in LOD down to

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107 M for mabuterol, bufuralol, alprenolol, and tertatolol in comparison with UV detection. These results suggest that potentiometric detection, especially in RP-HPLC employing hybrid polymer-silica packing, can be considered as the promising alternative in the high-throughput drug abuse or doping control procedures of investigated beta-adrenergic agonists and beta-adrenolytics in humans and animals. The quantitative structure–potentiometric response relationships were developed for a set of 18 beta-adrenergic drugs and a set of PVC-based electrodes using TCPB alone or in admixture with trioctylated α-cyclodextrin, dibenzo-18crown-6, or calix arene hexaethylacetate ester. A multiple linear regression model based on computationally derived set of molecular descriptors was used to predict detection limits of beta-blocking agents and betaadrenergic agonists from their molecular structure in the developed potentiometric detectors. Principal component analysis of the data, considering determined potentiometric detection limits, revealed that it can be used to establish a reliable pharmacological classification of compounds with beta-adrenoceptor activity, especially for the differentiation of cardioselective and noncardioselective beta-1 antagonists. Polettini et al. [33] have studied two approaches: (i) a multiresidue method for the clean-up and separation of eight different β-agonists (isoprenaline, cimaterol, terbutaline, salbutamol, fenoterol, ractopamine, clenbuterol, and mabuterol) and (ii) a single-residue method focussed at the detection of clenbuterol residues in samples of urine. Both approaches provided efficient procedures to process urine samples automatically with coupled-column LC. Wen et al. [34] prepared a hydroxylated poly(glycidyl methacrylate-coethylene dimethacrylate) (GMA-co-EDMA) monolithic capillary for polymer monolith microextraction. Coupled to HPLC with UV detection, this extraction medium was successfully applied to establish a simple and fast method for the analysis of CLB in urine. To obtain optimum extraction performance, the effects of pH value and ionic strength of the sample matrix on extraction efficiency were investigated. The linearity of the method was evaluated over a concentration range of 10–2000 ng/mL, and the correlation coefficient (R2 value) was 0.9985. The detection limit and the quantification limit were 2.3 and 7.7 ng/mL, respectively. Good reproducibility of the method was obtained, yielding the intra- and interday RSDs less than 5.1% and 9.1%, respectively. Moreover, the hydroxylated poly(GMA-coEDMA) monolithic capillary exhibited good preparation reproducibility and long-term extraction life. When applied to the determination of

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CLB in urine samples, an effective removal of interfering compounds was achieved and recoveries were in the range of 87.6%–106%. The determination of CLB from one real sample including pretreatment, extraction, and analysis could be finished within 30 min. Su et al. [35] described a normal phase chromatographic method for rapid detection of clenbuterol in meat. The experimental results showed that the standard deviation was 0.049, the average recovery rate was about 92.49%, and the correlation coefficient (as R2) reached 0.9997. It is proved that the method has a good linear relationship, and is accurate with short analysis time. It meets the requirement for rapid detection of clenbuterol in meat. Zhang and Zhang [36] developed a method for the enantioseparation of clenbuterol by HPLC. The enantiomeric resolution of clenbuterol was achieved on two Pirkle-type chiral stationary phases (α-Burke—2 and Pirkle—1J). Factors affecting the enantioresolution, such as the type and concentration of buffer solution, the type and content of organic modifier, and temperature, were investigated. The optimal separation conditions were as follows: dichloromethane–ethanol (19:1, v/v) containing 5 mM ammonium acetate as the mobile phase, at a flow rate of 2.0 mL/ min, column temperature of 20°C. Under the above conditions, the enantiomers of clenbuterol were successfully separated on α-Burke—2 and Pirkle—1J chiral stationary phases with the resolution factors of 1.85 and 0.64, respectively. The effective π–π donor–acceptor interaction and hydrogen bonding interaction between the two Pirkle-type chiral stationary phases and clenbuterol were expected to be responsible for the chiral recognition. The method is simple and rapid for the quality control and stereoselective PK study of clenbuterol. Gigosos et al. [37] introduced a liquid chromatographic method of analysis of clenbuterol residues in food products of animal origin (cow liver). The paper reports a method employing diphasic dialysis extraction for the rapid confirmation of clenbuterol in cow liver by RP-HPLC with diode array detection. The most suitable means of homogenizing the liver is in the barium hydroxide–barium chloride buffer; the optimum extraction solvent is tert-butyl methyl ether at an extraction temperature of 37°C; stirring should be applied at 150 RPM for 4 h. They claim that their method improves clenbuterol recovery values to 98.9%. Biondi and coworkers [38] explained a precolumn derivatization method for the analysis of clenbuterol by HPLC with UV detection. After preliminary studies, N,N-dimethylaniline was chosen as reagent against clenbuterol diazonium salt to form a diazo compound. The final derivative

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was separated on a reversed-phase column using a phosphate buffer– acetonitrile mixture as eluent. The reproducibility was increased using a compound homologous to clenbuterol as the internal standard. The method was applied to the analysis of clenbuterol in calf urine. Degroodt et al. [39] developed a method for the analysis of cimaterol and clenbuterol residues in liver, with detection limits of 0.25 and 0.5 μg/kg, respectively. The recovery varied from 55% to 60%. After extraction, a clean-up procedure with Baker-spec C-18 columns was performed. The two chemical compounds of interest were eluted with methanol. Cimaterol and clenbuterol were quantitatively determined by HPLC using an RP-Select B (5 μm) column and a postcolumn reaction procedure. The positive results were confirmed by HPTLC as this technique reaches the same level of sensitivity as the HPLC method. Furusawa [40] described a reserved-phase HPLC method for detecting clenbuterol (CLB) and ractopamine (RP) using an isocratic solvent-free mobile phase. The separations were performed using a Kaseisorb® LC C1-300-5 (100  4.6 mm, 5 μm) with 0.05 M octanesulfonic acid mobile phase and a photodiode array detector. The total run time was less than 5 min. The system suitability was well within the international acceptance criteria. The detection limits were 0.68 ng/mL for CLB and 1.24 ng/mL for RP, respectively. A method for simultaneous detecting CLB and RP was developed and may be further applied to the quantification in foods. 4.4.3 Liquid Chromatography–Mass Spectroscopy Garcia and coworkers [41] described a method which they claimed to be a sensitive method using LC/ESI-MSn on a quadrupole linear ion trap mass analyzer for the detection of nine β2 agonists (cimaterol, clenbuterol, fenoterol, formoterol, mabuterol, terbutaline, ractopamine, salbutamol, and salmeterol) in horse urine. The method consists of SPE on CSDAU cartridges before analysis by LC/ESI-MSn. The efficiency of extraction combined with the sensitivity and the selectivity of MSn allowed the detection of these compounds at pg/mL levels. Administration studies of fenoterol and formoterol are reported and show their possible detection after inhalation. The method is applicable for screening and confirmatory analysis. Pleadin et al. [42] developed liquid chromatographic-electrospray ionization tandem mass spectrometry method for determination of clenbuterol residues in liver tissue as a regulatory matrix for the control of clenbuterol abuse as an anabolic. To investigate the level of clenbuterol residues during the withdrawal period, male food-producing pigs were exposed to

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subchronic repeat oral administration of a clenbuterol growth-promoting dose for 28 days. The analytical procedure showed acceptable validation results for all liver spiked samples analyzed and proved to be useful as a quantification and confirmation method in supporting regulatory enforcement programs of clenbuterol misuse monitoring. The highest level of clenbuterol in the liver of treated animals was recorded on day 0 of treatment cessation (21.58  14.29 ng/g), followed by 6.59  3.11 ng/g on day 3, 0.83  0.27 ng/g on day 7, and 0.44  0.08 ng/g on day 14 of withdrawal. At the end of the study period (day 35), the concentration of clenbuterol was less than LOD of the method (<0.1 ng/g).

4.4.4 Gas Chromatography–Mass Spectroscopy Henze et al. [43] developed a new method for a comprehensive screening and confirmation of beta-2 agonists in human urine based on gas chromatography–low-resolution mass spectrometry (GC–MS) using electron impact ionization (EI). After hydrolysis of the conjugates with beta glucuronidase/arylsulfatase, a derivatization step with formaldehyde converts fenoterol, orciprenaline, reproterol, and terbutaline to one derivative (a tetrahydroisoquinoline), while the other beta-2 agonists remain unchanged. Liquid–liquid extraction and trimethylsilylation steps follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behavior. The detection limit of these four beta-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/mL of urine. The other beta-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented. Damasceno et al. [44] described a GC–MS procedure for the detection of different beta-agonists in urine samples based on two consecutive derivatization steps. The derivatization procedure is based on the consecutive formation of cyclic methylboronate derivatives followed by a second derivatization step with MSTFA (N-methyl(trimethylsilyl)trifluoroacetamide) on the same extract, forming TMS derivatives. Injections in the GC–MS system may be carried out after each one of the derivatization steps, obtaining enough information for unambiguous identification. Limits of detection for the two derivatization steps ranged from 0.5 to 5 ng/mL. This procedure was tested with the beta-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol, alpha-hydroxy-salmeterol, and terbutaline.

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Polettini et al. [45] developed a GC-MS method for determination of CLB in bovine urine that uses a double washing of the acidified sample with tert-butyl methyl ether and an SPE with Bond-Elut Certify® columns, derivatization was performed with trimethylboroxine. The extract was then analyzed by gas chromatography/electron impact mass spectrometry. The advantages of the methylboronate derivative with respect to other derivatives are discussed in terms of chromatographic properties and, above all, of specificity of the electron impact mass spectrum. Van Eenoo et al. [46] developed a GC–MS method for the detection of clenbuterol in urine. Alkaline liquid–liquid extraction was followed by derivatization to a cyclic methyl boronate derivative and analysis on a Finnigan MAT GCQ instrument. Method validation showed good linearity in the range 0.1–2.0 ng/mL, excellent repeatability, and specificity. The limit of quantitative detection of the method was 0.1 ng/mL. Different instrumental parameters of the ion trap mass spectrometer were changed to increase the number of diagnostic ions for the cyclic methyl boronate derivative of clenbuterol. The influence of these changes and their applicability within the requirements and the criteria for mass spectrometry set by the responsible regulatory bodies are discussed. Clenbuterol was administered via nebulization to five standard bred mares (0.4 μg/kg body weight). Analysis of the urine samples resulted in the detection of clenbuterol, as early as 2 h postadministration and for up to 36 h posttreatment. Generally, maximum urinary concentrations of 1.2 ng/mL were reached after 6–9 h. Li and coworkers [47] described a GC–MS method for the determination of residual amount of clenbuterol in pork. The pork sample was extracted under alkaline conditions with ethyl acetate and back-extracted with HCl solution as described in the Chinese standard: NY/T 468-2006. The solution obtained was adjusted to pH 4.5 and purified by passing through an SCX SPE column. After adding 40 μL of 1.6 mg/L metoprolol solution as an internal standard, the sample solution was evaporated to dryness using a nitrogen flow at 60°C. 200 μL of toluene and 100 μL of bistrifluoroacetamide (BSTFA) were then added to the residue and derivatized for 1 h in an oven at 80°C. After cooling, 200 μL of toluene was added, and the derivatives in the solution were separated on a DB-5MS capillary column. MS determination was performed under SIM mode, and quantification was carried out by the internal standard method. The monitoring ions of m/z 86 (as quantitative ion) and of m/z 262, 243, and 187 (as qualitative ions) were selected by analytical methods. The

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linearity range for clenbuterol was between 16 and 512 μg/L, and the detection limit found was 1.0 μg/kg. On the basis of a blank sample, a test for recovery was made by the standard addition method, giving values of recovery in the range of 79.7%–86.6%, with RSD values (n ¼ 6) in the range of 4.8%–6.7%. Keskin et al. [48] developed a GC–MS analysis of clenbuterol from urine. Timolol was used as an internal standard. Extraction and derivatization procedures of clenbuterol from urine were developed. Clenbuterol was extracted by using diethylether/tert-butanol (4:1, v/v) and pH 12 K2CO3/KHCO3 (3:2, w/w) buffer. MSTFA/NH4I (1 mL/10 mg) mixture was used for derivatization of clenbuterol. Selected ions of clenbuterol-bis-TMS were m/z: 405, 337, 336, 335, 300, and 227. Extraction yield and minimum detection limit of clenbuterol from urine were identified. Extraction yield was 94.30% and minimum detection was found 0.02 ng/mL urine. It has been concluded that the GC–MS method is sensitive, accurate, precise, and reproducible for analyzing of clenbuterol from urine. Batjoens and coauthors [49] introduced a gas chromatographic-tandem mass spectrometric analysis of clenbuterol residues in feces. They claimed that “…in full scan MS it is impossible to fulfil the EU criteria of four diagnostic ions with one single ionization mode. Some alternative possibilities are: (1) the use of two different ionization modes, (2) the use of different derivatization methods or (3) the use of tandem MS. Each derivatization or ionization mode on its own did not give a sufficient number of ions. By combining these different possibilities we were able to obtain four ions, fulfilling the EU criteria.” Sniegocki and others [50] claimed a sensitive and specific GC–MS method for the determination of clenbuterol in urine and liver samples. Their method includes SPE for clean-up of biological samples, derivatization, and analysis of the clenbuterol derivatives in the selected ion monitoring mode. During the studies, six derivatization procedures have been tested, of which BSTFA + 1% TMCS gave the best results. This procedure was validated. The LOD was established at 0.5 ng/g in the liver and 0.5 ng/mL in urine, and the recoveries were better than 80%. 4.4.5 Capillary Electrophoresis Shi et al. [51] developed a capillary electrophoresis (CE) method, using field-amplified sample injection, for separation and determination of some β2-agonists, such as cimaterol, clenbuterol, and salbutamol. The optimum

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conditions for this system had been investigated in detail. The precision of the migration time, peak height, and accuracy were determined in both intraday (n ¼ 5) and interday (n ¼ 15) assays. Under the optimum conditions, the detection limits (defined as peak S/N ¼ 3) of this method were found to be lower than 2.0 ng/mL for all of these three β2-agonists, which were much lower than that of the conventional electromigration injection method, and the enhancement factors were greatly improved to be 30–40-fold. Such a lower detection limit allows this method to be suitable for determination of above-mentioned β2-agonists in urine samples. The mean recoveries in urine were higher than 96.2%, 95.6%, and 95.3% for cimaterol, clenbuterol, and salbutamol, respectively, with relative standard deviations less than 3.5%. Sirichai et al. [52] reported a CE method (with UV detection) for the simultaneous and short-time analysis of clenbuterol, salbutamol, procaterol, and fenoterol. Optimized conditions were found to be a 10 mmol/L borate buffer (pH 10.0), a separation voltage of 19 kV, and a separation temperature of 32°C. UV detection was used at an analyzing wavelength of 205 nm. Under the optimized conditions, analyses of the four analytes in pharmaceutical and human urine samples were carried out in approximately 1 min. Interference by the sample matrix was not observed. The limits of detection (defined at S/N of 3:1) were found between 0.5 and 2.0 mg/L for the analytes. The linearity of the detector response was within the range from 2.0 to 30 mg/L with a correlation coefficient exceeding 0.996. Zhou et al. [53] reported a CE method (with UV detection) for separating seven beta-adrenergic blocking agents (atenolol, celiprolol, clorprenaline, fenoterol, metoprolol, propranolol, terbutaline) and clenbuterol. In order to simultaneously separate all analytes, a tromethamine phosphate solution was used that contained titanium dioxide nanoparticles such as BGEs. The effects of important factors, such as concentration of TiO2 NPs, optimum pH, run buffer concentration, and separation voltage, were investigated so as to achieve the best CE separation. The eight analytes could be well separated applying a separation voltage of 15 kV in 75 mM Tris– H3PO4 buffer at a pH of 2.40, containing 6.0  106 g/mL TiO2 nanoparticles. Under these optimal conditions, the RSD values for peak areas and for migration times were less than 2.7% and 2.3%, respectively. The detection limits were 0.1 μg/mL for celiprolol, 0.1 μg/mL for propranolol, 0.2 μg/mL for fenoterol, 1.0 μg/mL for atenolol, 1.0 μg/mL for clenbuterol, 1.0 μg/mL for clorprenaline, 1.0 μg/mL for metoprolol, and 1.0 μg/mL for terbutaline. The method was successfully applied for the rapid

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CE determination of antihypertensive beta-blocking compounds (atenolol, metoprolol, terbutaline, and propranolol) in pharmaceutical tablets. Toussaint et al. [54] developed an on-line coupling of capillary zone electrophoresis with mass spectrometry (CZE-MS) for the separation of enantiomers that were hampered by the presence in the separation buffer of nonvolatile chiral selectors such as cyclodextrins. This problem was overcome by use of a partial filling technique where only a part of the capillary is filled with the separation buffer containing chiral selectors. Since the electroosmotic flow is almost completely suppressed at acidic pH, and since dimethyl-β-cyclodextrin is neutral, no free cyclodextrin would reach the MS detector when using a partially filled capillary. By this method, clenbuterol enantiomers were successfully resolved and separated from salbutamol (the internal standard) in aqueous solution and in plasma samples. An SPE was used for the preparation of plasma samples before analysis. Kim et al. [55] investigated five racemic β2-agonists enantiomers by CE employing a hydroxypropyl-β-cyclodextrin (HP-β-CD). The effects of the concentration of HP-β-CD added to the background electrolyte and of the pH of the buffer on the effective mobility and resolution of the studied compounds were examined. Very good resolution was achieved for terbutaline and clenbuterol; salbutamol and bambuterol were able to be partially resolved. Enantioselectivity and resolution were influenced by the concentration of HP-β-CD, the buffer composition, and solution pH. Zhou et al. [56] established a competitive immunoassay for detecting clenbuterol in urine by CE with laser-induced fluorescence. The clenbuterol was conjugated with bovine serum albumin, and then the derivative was labeled with fluorescein isothiocyanate (FITC) to compete for antibody with free clenbuterol in the sample. Under the optimal conditions, free and bound FITC-labeled clenbuterol was separated within 8 min with the relative standard deviations 0.72% for migration time and 2.8% for peak area. The detection limit was 0.7 ng/mL.

5. STABILITY Signoretti et al. [57] used DSC to study the physicochemical compatibility between this drug and various excipients commonly used in manufacturing of tablets in order to improve the formulation of clenbuterol. Using this method, clenbuterol was found to be compatible with talc, stearic acid, magnesium stearate, and titanium dioxide. An incompatibility was

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demonstrated with maize starch, pregelatinized starch, sodium starch glycolate, polyvinylpyrrolidone, Avicel PH101, and lactose. Martin et al. [58] studied the heat stability of clenbuterol. The drug was shown to be stable in boiling water at 100°C. However, in cooking oil at 260°C, losses were observed, indicating a half-life of about 5 min. The effect of a range of cooking processes (boiling, roasting, frying, and microwaving) on clenbuterol residues in fortified and incurred tissue was studied. No net change in the amount of clenbuterol was observed in any of the cooking processes investigated, except for deep frying using extreme conditions. There was little observed migration from the tissue into the surrounding liquid or meat juices. Clenbuterol residues were found not to be evenly distributed in the incurred raw tissue used for the investigation. The findings of this investigation show that the data obtained from the measurements of raw tissue are applicable for use in consumer exposure estimates and dietary intake calculations.

6. PHARMACOKINETICS, METABOLISM, AND EXCRETION 6.1 Pharmacokinetics Yamamoto et al. [59] reported the pharmacokinetics of clenbuterol therapeutic doses (20, 40, and 80 μg/man) by the oral administration of clenbuterol hydrochloride to healthy volunteers, where the unmetabolized drug in plasma and urine was determined by EIA. The plasma levels of clenbuterol reached maximum values of 0.1, 0.2, and 0.35 ng/mL, respectively, in a dose-dependent manner within 2.5 h, which lasted for over 6 h after administration. The half-life of clenbuterol in plasma was estimated to be about 35 h. When the drug was orally administered repeatedly to men twice a day, the plasma level reached a plateau within 4 days after the initial administration. At that time, the plasma levels of the unchanged form were 0.2–0.3 and 0.5–0.6 ng/mL at doses of 20 and 40 μg/man, respectively. The bound ratio of the drug to plasma protein was estimated to be 89%–98% at a single administration of 80 μg of the drug. The cumulative urinary excretion of unchanged compound corresponded to about 20% of the administered dose as measured at 72 h following a single oral administration. When clenbuterol hydrochloride was orally administered to rats at a dose of 2 μg/kg, the plasma level reached its maximum at about 1 h after administration. In rabbits, the plasma concentrations reached the maximum value of about 0.2 and 0.8 ng/mL within 2 h following

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administration of clenbuterol hydrochloride at doses of 0.5 and 2 μg/kg, respectively. The half-life of clenbuterol in plasma was about 30 h in rats and about 9 h in rabbits.

6.2 Metabolism Zalko et al. [60] reported that the metabolic fate of clenbuterol remains unknown in the main target species (i.e., bovine), and that only limited data have been published concerning its biotransformation in laboratory animals. A metabolic study has been carried out in the rat using 3H- and 14C-labeled clenbuterol. Urine appeared to be the major excretion pathway. Using a soft technique for urine preparation, extraction, and purification, as well as adequate analytical tools in order to preserve labile metabolites, N-oxidation products of the parental drug on the primary amine function were identified for the first time. Clenbuterol hydroxylamine was the major compound, but 4-nitroclenbuterol was also detected. The metabolic pathway leading to the formation of clenbuterol hydroxylamine prevails at high dosages. Clenbuterol hydroxylamine (but not 4-nitroclenbuterol) was also formed extensively when the drug was incubated with rat liver microsomal fractions in aerobic conditions. It is concluded that oxido-reduction during urine preparation have previously impaired the identification of this toxicologically important clenbuterol metabolic route.

6.3 Excretion Excretion is mainly via the kidneys for intravenous administration and biotransformation for oral administration. No accumulation has been observed [2]: (1) Renal clearance (rate): 38–60 mL/min [61]. (2) Renal excretion: 30% [61]. (3) At steady-state with clenbuterol in oral doses of 20 μg twice daily, approximately 30% of the dose is recovered unchanged in the urine, suggesting some degree of hepatic metabolism [61]. When administered at the recommended dose of 0.8 mg/kg per 12 h, clenbuterol reaches plasma levels that should relax airway smooth muscle. However, the measurable degree of bronchodilation achieved may not always be clinically obvious even in horses with heaves. After a 10-day treatment (0.8 mg/kg per 12 h), the half-life of elimination from the plasma is about 10 h and plasma levels are below the pharmacological threshold

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within 3 days. Urinary elimination is prolonged and irregular so that control of clenbuterol usage in racehorses should be based on blood rather than urine levels [62].

7. PHARMACOLOGY The most important action of clenbuterol and other β2-agonists in the lung is relaxation of airway smooth muscle. For this reason, such drugs are widely used for relief of bronchospasm in human asthma and similar diseases in animals. When these drugs bind to β2-adrenoceptors, they activate adenylyl cyclase, which leads to an increase in the intracellular concentration of the second messenger cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA). In the tracheobronchial tree and in the uterus, β2-agonists, cAMP, and PKA inhibit smooth muscle contraction by opening K+ channels and by downregulation of myosin light-chain kinase activity [62].

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