A liquid chromatography–tandem mass spectrometry (LC–MS-MS) method to determine clenbuterol-like beta-agonist residues in
hair was developed and validated. Hair samples were extracted with 0.1 mol/L hydrochloric acid and 0.2 mol/L ammonium acetate
buffer (pH 5.0) and cleaned up using a solid-phase extraction cartridge (60 mg/3 mL). Linearity was assessed in the 0.1–50
ng/L (r = 0.9951–0.9996) range for 18 betaagonists in hair samples. The analytical recoveries were acceptable; between 72.76%
and 122.22%, with intraday and interday precisions of relative standard deviation (RSD) between 1.6% and 17%, and the limits
of detection were all lower than 0.5 ng/L. The proposed LC–MS–MS method has high sensitivity and strong specificity with a
relatively easy procedure for sample preparation. Thus, it can be used to detect beta-agonist residues in hair, providing
a basis for regulating food safety.
Beta-adrenoceptor agonists (β-agonists) are used to treat bronchiectasis and to increase pulmonary ventilation. Clenbuterol
is the best known of the aniline-like β-agonists. When the drug dosage exceeds the treatment amount by 5–10-fold, it can cause
a substantial increase in the muscle mass of animals such as cows, lambs, pigs and poultry. Because the drug has stable chemical
characteristics and cannot be destroyed by general heating, it remains in a significant residual amount in edible animal tissue.
For example, clenbuterol is stable in 100 °C water and has a half life of 5 min in 260 °C oil (1). When a person eats the
animal tissue (for example liver, kidney, lung, or meat), toxicity symptoms may develop (2). Thus, the public representatives
of the European Economic Community in July 1997 approved a law to ban the use of β-agonists in all animals destined for human
consumption (3). All β-agonists are currently banned from use in edible animals in China, the United States and the European
There have been few studies on the development of a standard method of assaying nondestructive living samples from animal
hair. For this reason, we investigated the development of an assay using hair for the detection of β-agonists.
Materials and Methods
Reagents and Equipment: Methanol, ethyl acetate, acetonitrile and formic acid were all high performance liquid chromatography (HPLC)–grade (Merck,
Darmstadt, Germany). Sodium dihydrogen phosphate, hydrochloric acid, and ammonium hydroxide solution were of analyticalreagent
grade. Water was produced by a Milli-Q system (Millipore Corp., Molsheim, France).
A clenbuterol hydrochloride reference substance was purchased from the National Institute for the Control of Pharmaceutical
and Biological Products in China, which was equivalent to 88.38% clenbuterol. Terbutaline sulfate, salbutamol, fenoterol hydrobromide
and salmeterol were all in accordance with the European Pharmacopoeia; ractopamine hydrochloride, brombuterol hydrochloride, cimaterol, clenpenterol hydrochloride, mabuterol hydrochloride, tulobuterol
hydrochloride, mapenterol hydrochloride, cimbuterol and bambuterol hydrochloride were all supplied by WITEGA Laboratorien
Berlin-Adlershof GmbH (Berlin, Germany); ritodrine hydrochloride, isoxsuprine hydrochloride, and penbutolol hydrochloride
were all in accordance with the United States Pharmacopeia (USP); D9-clenbuterol and labetalol hydrochloride were all supplied by Sigma (Carlsbad, California, USA).
Hair samples were obtained, both from pigs from the Shanghai hoggery and from people in the outskirts of Shanghai; the hair
was processed, washed and measured separately. Hair that tested drug-negative was regarded as a blank hair sample.
The equipment included an Agilent liquid chromatograph connected to a triple-quadrupole mass spectrometry (MS) detector with
electrospray ionization (ESI) sources (Agilent Technologies Corp., Santa Clara, California, USA); another Agilent liquid chromatography
system, including a binary pump, degasser, column oven, and autosampler (Agilent Technologies, Waldbronn, Germany); an ultrasonic
instrument (Shanghai Kudos Ultrasonic Instrument Corp., Shanghai, China); ultracentrifugation (Thermo Electron Corp., Osterode,
Germany); a constant temperature incubator (Shanghai Medical Constant Temperature Equipment Factory, Shanghai, China); a vortex
mixer (IKA Corp., Staufen, Germany); a nitrogen blower (Organomation Associates Inc., Berlin, Massachusetts, USA); an electronic
balance (Beijng Sartorius Balances Co., Beijing, China); a solid-phase extraction (SPE) manifold (Waters, Milford, Massachusetts,
USA); a PCX SPE column (60 mg/3 mL) (Tianjin Agela Technologies Corp., Tianjin, China); an MCX SPE column; and the Milli-Q
water purification system (Millipore Corp., Molsheim, France).