Solving a meaty problem
6 Dec 2011 by Evoluted New Media
ß-agonists can be used illegally to beef-up our meat – but how do we catch those who are doing it? Here we discover that LC/MS is on the front line of keeping our meat honest Beta-agonists (ß-agonists) are synthetically manufactured compounds which have a stimulatory effect on ß-adrenergic receptors and are used in human and veterinary medicine as bronchodilatory agents. Since the mid 1970s these compounds have also been used for their ability to significantly induce weight gain and increase growth in animals bred for consumption. One of the most extensively used ß-agonists is Clenbuterol, a non-steroidal anabolic and metabolism accelerator which is illegally used to increase the leanness and protein content of food-producing animals. Residues of ß-agonists with high oral bioavailability, long plasma half-life and slow rates of elimination, represent a significant health risk to consumers. Acute toxic effects such as heart palpitations, muscle tremors, tetany and severe migraines can be a result of high ß-agonists content in animals1.
The use of ß-agonists in stock farming is widely prohibited, both within the European Union and by other worldwide regulatory agencies. However, current monitoring programs have shown that ß-agonists are still illegally used to increase weight gain efficiency in animals. Modified structures of these compounds and analog development are continuously being introduced in routine practice. As a result, it is essential to have rapid screening methods and stringent detection practices to monitor levels of ß-agonists in samples of animal origin, including urine, plasma and tissues.
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Institutions such as The Chemical and Veterinary Analytical Institute Münsterland-Emscher-Lippe (CVUA-MEL) work to analyse food and feed samples in order to ensure public health complies with EU legislative requirements. The CVUA-MEL is the central official laboratory for food and feed control in the Münster district of Germany. In order to provide an accurate and efficient service, the organisation increasingly requires the development of new liquid chromatography analytical methodologies to monitor and detect levels of ß-agonists in food-producing animals. The combination of high resolution Orbitrap mass spectrometry (Exactive LC-MS) has enabled CVUA-MEL to achieve accurate and streamlined detection and analysis of priority ß-agonists in animals through the automated screening of urine samples.
Council Directive 96/22/EC2 prohibits the use of ß-agonists and other substances that contain a hormonal or thyrostatic action in stock farming. The Directive covers substances that have an anabolising action such as ß-agonists and are used illegally in livestock rearing in order to stimulate the growth and yield of animals. According to the legislation, administering medicinal products based on ß-agonists to animals may be authorised only for well-defined therapeutic purposes. Meat or products derived from animals to which ß-agonists have been administered in accordance with the dispensatory provisions of this Directive may not be placed on the market for human consumption, unless the animals in question have been treated with legal veterinary medicinal products and the withdrawal period stipulated was observed before the animals were slaughtered. Additionally, the Directive identifies the need for maximum withdrawal periods to be set for veterinary medicinal products containing hormonal substances or ß-agonists.
The Council Directive 96/23/EC3 requirement to monitor certain substances and residues in live animals and animal products specifies that EU Member States should draft a national residue monitoring plan for substances having anabolic effect, unauthorised substances and veterinary drugs and contaminants, including ß-agonists. The Directive establishes the frequencies and level of sampling and the groups of substances to be controlled for each food commodity.
[caption id="attachment_25546" align="alignleft" width="300" caption="Table 1: The list of analysed beta agonists"]
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Commission Decision 2002/657/EC4 implements Council Directive 96/23/EC and details criteria and procedures to be used for the validation of analytical methods to ensure the quality and comparability of analytical results generated by official laboratories. In addition, the Decision establishes common criteria for the interpretation of test results and introduces a procedure to progressively enforce minimum required performance limits (MRPL) for analytical methods employed to detect substances for which no permitted limit has been established. This is particularly important for substances not authorised for use or specifically prohibited in the EU, such as ß-agonists.
To ensure regulatory compliance and safeguard consumer health, a stipulated number of samples must be taken and analysed in order to detect illegal drugs administered in animals and to ensure that withdrawal periods are being upheld by farmers.
Molecularly imprinted polymer (MIP) solid phase extraction (SPE) column clean-up has been traditionally used for sample preparation during determination of ß-agonists in samples of animal origin. However, this technique is associated with a number of shortcomings that limit its efficiency. MIP synthesis is difficult to control due to uncertainties relating to polymer architecture, chemical environment diversity and site specificity. This is a particularly crucial limitation given that different MIPs must be synthesised for each new analyte or class of analytes. Factors such as the gel effect during cross linking contribute to the heterogeneity of imprinted matrices, whereas the restricted mobilities and high local viscosities noted in the latter stages of MIP synthesis can lead to residual reactive groups in the final material.
One of the detection techniques being used at CVUA-MEL is ultra performance liquid chromatography (UPLC) coupled with Time-of-Flight (TOF) MS. The major drawback of this technique is the occurrence of matrix effects, which can reduce or enhance substantially the response signal, thereby delivering unreliable results. Additionally, processing and reviewing TOF screening data can be a complex and time-consuming task, requiring positive peaks to be first identified and then quantified. A further shortcoming of the technique is that the transfer from qualitative to quantitative processes is often performed manually, thereby introducing a higher probability of errors and placing a greater burden on analysts.
[caption id="attachment_25547" align="alignright" width="300" caption="Table 2: Method performance characteristics."]
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A new method has been introduced, combining high resolution Orbitrap LC-MS technology and TurboFlow technology, offering significant gains in simplicity, speed and sensitivity for high-throughput food safety laboratories (Figure 1).
A rapid and automated method for screening of priority ß-agonists in urine samples was developed by CVUA-MEL. Liquid chromatography mass spectrometry analyses were performed using the benchtop Orbitrap single stage Exactive LC-MS. The LC-MS instrument was coupled to a TurboFlow system Aria TLX-1 consisting of loading and eluting pumps, a switching valve module and an autosampler. All LC-MS measurements were performed at resolving power settings of 50,000 FWHM, while separation of analytes was carried out on a Hypersil GOLD Phenyl column 4.6 x 100mm x 5µm.
The optimisation of the TurboFlow method was performed using a standard solution containing salbutamol, clenbuterol and mabuterol at 100µg.L-1 levels. These compounds were selected for optimisation due to their physico-chemical properties and strong dependency on the pH of the mobile phases. During method development specific TurboFlow parameters were optimised, including column selection whereby evaluation of different types of columns was undertaken with respect to the retention of analytes. The composition and pH of the loading mobile phase as well as the pH, flow rate and time of the elution mobile phase were also optimised. Additionally, it was possible to optimise the initial gradient composition to effectively trap and focus all compounds of interest.
The optimised TLX method was then applied to the analysis of spiked and unspiked urine samples and standard solutions. The urine was spiked at different concentration levels, centrifuged for 10 minutes and diluted with water 1:1; pH was adjusted to 6 with 0.1M HCl. Prepared urine samples were directly injected into the TLX/Exactive system. Calibration was performed using urine matrix matched standards at concentration levels of 0.25; 0.5; 1; 5; 10; 15; 20 and 25µg/L. The typical chromatogram of the standard solution at 0.25µg/L and spiked urine sample at concentration level of 1µg.L-1.
Table 1 shows the group of priority ß-agonists analysed with this method. With the use of this method, all analysed compounds could be successfully detected at low µg.L-1 concentration levels in urine. In neat standard, all compounds are detected easily at 0.25µg/L. Also, in a few cases coelutions occurred (salbutamol, terbutalin, pirbuterol) but the peak could be distinguished from the matrix.
Table 2 summarises detailed performance characteristics of the method evaluated by analyses of urine samples spiked at 1µg.L-1. For estimation of precision and accuracy the spiked samples were analysed in six replicates. Carryover sample (blank urine) was analysed immediately after the spiked samples. All calibrations were carried out using matrix matched standard solutions to eliminate matrix effects during the ionisation step.
Conventional methods have previously limited the efficiency of detecting toxic compounds in urine samples of farm animals. Using the combination of high-resolution Orbitrap LC/MS technology and TurboFlow clean-up technology, the CVUA-MEL is now able to use a streamlined, automated and accurate process for the screening of urine samples, which optimises the institution’s ability to monitor and detect ß-agonists that have been illegally administered to increase weight gain in animals.
References:
- Botsoglou, N.A., Fletouris D.J., Drug Residues in Food. Pharmacology, Food Safety and Analysis, Marcel Dekker: New York, 2001
- COUNCIL DIRECTIVE 96/22/EC of 29 April 1996, concerning the prohibition on the use in stockfarming of certain substances having a hormonal or thyrostatic action and of beta-agonists, and repealing Directives 81/602/EEC, 88/146/EEC and 88/299/EEC, http://eur-lex.europa.eu/LexUriSer/LexUriServ.do?uri=CONSLEG:1996L0022:20081218:EN:PDF
- COUNCIL DIRECTIVE 96/23/EC of 29 April 1996, on measures to monitor certain substances and residues thereof in live animals and animal products and repealing Directives81/602/EEC, 85/358/EEC and 86/469/EEC and Decisions 89/187/EEC and 91/664/EEC, http://ec.europa.eu/food/food/chemicalsafety/residues/council_directive_96_23ec.pdf
- COMMISSION DECISION (2002/657/EC) of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results
