Addressing New Challenges in the Field of Growth Promoters in Biological Samples

The safety of our food supply can no longer be taken for granted. As the world changes and populations continue to grow, so will the responsibility of organizations to meet the demand for safe food supplies.

One route of human exposure to veterinary substances is through the food chain as a result of malpractice or illegal activities. Some substances of concern include corticosteroid hormones, ß-agonists, and recombinant bovine somatotropin (rbST).

Fifty years after the discovery of natural corticosteroid hormones, many synthetic derivatives of these molecules are available today. In human and veterinary medicine, their legal use is strictly regulated. This includes withdrawal periods between treatment and animal slaughtering, as well as maximum residue limits (MRLs) in edible biological matrices for some compounds. Some of these substances have also been used as growth promoters in cattle, but these practices are banned in Europe.

For many years, various analytical methods have been proposed for the identification of corticosteroid residues in edible tissues or urine samples. Most of these methods are based on liquid chromatography, coupled with multidimensional mass spectrometry with triple quadrupole or ion trap mass analyzers. These are highly efficient for analyzing urine, milk, muscle, or hair samples. Conversely, liver is a special case and remains more problematic due to the extreme complexity of this matrix. As a result, the identification and quantification of corticosteroids in liver with respect to the MRL fixed at the European level (2 µg/kg for dexamethasone and betamethasone, and 10 µg/kg for methylprednisolone and prednisolone) are still a significant analytical challenge. Recent discussions within the Joint FAO/WHO Expert Committee on Food Additives (JECFA) have underlined the lack of appropriate quantitative, efficient methods for this matrix, and a harmonized international MRL for dexamethasone fixed at 2 µg/kg for cattle, pig, and horse liver samples has been proposed.1

In parallel to their regulated use for therapeutic purposes, ß-agonists are potentially misused as growth-promoting agents in food-producing animals.2 As a result, efficient measurement methods based on mass spectrometry have been dedicated to these substances for many years. These methods rely on the direct measurement of drugs in a targeted mode, which only allows for the detection of a restricted number of compounds. Problems arise because there are many possible structures for ß-agonists, which exhibit activity at the ß2-adrenoreceptor level. Therefore, a range of compounds, either of known chemical structure but not yet included in the methods, or of unknown chemical structure, are missed during routine screening and confirmatory analysis. In addition, it has been reported that there are some cases in which “cocktails” of very low amounts of several active substances have been used. These factors make it a challenge to expose illegal practices. The possibility of extended multianalyte monitoring with high sensitivity, as well as the capability of combined acquisition modes for structural elucidation of unknown compounds and/or unknown screening analyses, are examples of new challenges for the last generation of instruments in this field.

Recombinant bovine somatotropin, also known as growth hormone, is used in some countries as a general growth promoter in pigs and cattle, but also in lactating cows to increase milk production.3–5 Different regulations exist regarding its use, but the lack of analytical methods for its detection makes it difficult to apply these regulations. It turns out to be an international issue in terms of animal doping, as well as food safety. Indeed, residues of rbST can be present in food produced by animals treated with this hormone. In order to detect residues of rbST in biological matrices, the analysis is targeted at the tryptic N-terminal peptide of the protein, specific to the difference between the endogenous and recombinant forms. The N-terminal amino acid alanine present in the endogenous form is replaced by a methionine in the recombinant one. This article describes use of the Waters® Xevo™ TQ MS (Waters Corp., Milford, MA) to address some of the analytical challenges previously described when analyzing growth promoters in biological samples.

Experiment setup

Following the extraction and purification of corticosteroids in liver and ß-agonists in urine,6,7 the sample was introduced into an ACQUITY UPLC® fitted with an ACQUITY® BEH C18, 1.7-µm, 2.1 × 50 mm column (Waters Corp.). Mobile phase A was 0.1% formic acid in water (growth hormone) or 0.1% acetic acid in water (corticosteroids and ß-agonists). Mobile phase B was acetonitrile with the same additive as mobile phase A. The gradient used was 0%B for 0.6 min, to 100%B at 4 min, held at 100%B until 5.0 min, and then reequilibrated to the initial starting conditions. The run time for the analysis was 6 min. The injection volume was 2.0 µL in each instance, with the flow rate set at 0.8 mL/min.

Analyte detection was via a Xevo TQ MS system using electrospray ionization (ESI) in positive mode for the growth hormone and ß-agonists, and negative mode for the corticosteroids. The MS capillary voltage was set at 1.50 kV and the cone voltage at 20.00 V with a source temperature of 150 °C and desolvation temperature of 550 °C. The desolvation gas flow was 1000 L/hr, and the collision gas flow was 0.15 mL/min.

Method parameters for both the ß-agonists and recombinant bovine growth hormone were automatically generated using IntelliStart ™ software (Waters Corp.), which included a series of automated tools to streamline Xevo TQ operation and work flow.

All corticosteroids and ß-agonist reference compounds used in the experiment were provided by Sigma (St. Louis, MO). The peptide used as a standard for the growth hormone application (= Nterm rbST) exhibits the following amino acid sequence: MFPAMSLSGLFANAVLR. This peptide was synthesized from MilleGen (Labege, France).

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