Development and Validation of a Rapid Detection Method for Boar Taint by means of Solid Phase Microextraction and a Person-Portable GC-MS
K. Verplanken1, J. Wauters1, J. Van Durme2, D. Claus3, J. Vercammen3,S. De Saeger4, and L. Vanhaecke1
1 Ghent University, Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, B-9820 Merelbeke, Belgium
2Research Group Molecular Odor Chemistry, Department of Microbial and Molecular Systems (M2S), Research Cluster Food and Biotechnology, KU Leuven Campus Ghent, Technology Campus, B-9000 Ghent, Belgium
3 IS-X, Avenue Jean-Etienne Lenoir 2, B-1348 Louvain-La-Neuve, Belgium
4 Laboratory of Food Analysis, Ghent University, Faculty of Pharmaceutical Sciences, B-9000 Ghent, Belgium
Introduction
During the past decades, the awareness on animal welfare has significantly increased. Indeed, in 2010 the ban on surgical castration of male piglets, starting January 2018, was announced in a European Treaty. One viable alternative for surgical castration of pigs is the production of entire males. However, the main setback in the raise of entire males is the possible occurrence of boar taint, i.e. an off-odour caused by the release of androstenone (AEON), skatole (SK) and indole (IND) when meat or fat of boars is heated(1). As a consequence, boar taint provokes negative consumer reactions(2), which can cause severe economic losses in pig husbandry. Since currently, apart from castration, no methods for the complete elimination of boar taint are available, there is an urgent need for rapid detection methods at the slaughter line.In this study, headspace solid phase microextraction (HS-SPME) coupled to GC-MS was used in the development and optimization of a candidate method for fast and accurate at-line detection of the boar taint compounds.
Material and Methods
Development and optimization of the HS-SPME-GC-MS method initially occurred on a bench top Hewlett Packard 6890 GC coupled to a Hewlett Packard 5973 quadrupole MS (Agilent Technologies, Palo Alto, CA). Fast sample extraction was achieved by applying the soldering iron as a heating device prior to SPME. This process was optimized by evaluating the performance of different SPME fibres (PDMS 100 µm, DVB/CAR/PDMS 50/30 µm, CAR/PDMS 75 µm, PA 85 µm) for the extraction of AEON, SK and IND from adipose tissue. Because of the complex nature of extraction of the boar taint compounds from adipose tissue, further optimization occurred by means of experimental designs. A D-optimal design was applied to identify influential extraction variables such as extraction time, extraction temperature, sample size, headspace volume and desorption time. Variables that had a significant influence on the extraction protocol were included in a central composite face-centred (CCF) design for further optimization.
After optimization, the HS-SPME-GC-MS method was validated according to the criteria of the European Commission by evaluating specificity, selectivity, linearity, trueness and precision. The limits of detection (LOD) and quantification (LOQ) were calculated according the ISO 17025 standard.
To prove the applicability of the newly developed HS-SPME-GC-MS method on boar samples, boar taint positive carcasses were selected at the slaughter line by means of the soldering iron method as optimized by Bekaert et al.(3)To confirm the presence of the boar taint compounds in neck fat of the selected carcasses, each sample was analysed with a validated in-house UHPLC-HR-MS method. After confirmation of the presence of the boar taint compounds, the samples were analysed by the newly developed HS-SPME-GC-MS method.
Finally, the validated HS-SPME-GC-MS method was transferred to a portable GC-MS instrument (Torion Technologies, PerkinElmer, UT) employable at the slaughter line, and its performance characteristics were evaluated.
Results and Discussion
SPME fibre comparison showed that the use of the DVB/CAR/PDMS 50/30 µm fibre resulted in better extraction efficiency and repeatability at short extraction times and was therefore selected for the rapid determination of the boar taint compounds. After fibre selection, a D-optimal design was executed, which identified extraction temperature, extraction time, desorption time and sample size as influential parameters. Further optimization of the latter in a CCD design showed that optimal extraction of the boar taint compounds from adipose tissue was achieved at an extraction temperature of 400 °C, extraction time of 40 sec, desorption time of 150 sec and sample size of 2 g.
Validation proved good specificity and selectivity of the method since no interfering peaks were observed at the specific retention times of the boar taint compounds and selected ion monitoring was applied. Linearity of the method was proven in a range of 0 to 5000 µg kg-1 (R2 > 0.99) and no lack of fit was observed (95% confidence interval; F-test, p > 0.05). Recovery values were within the permitted range (-20% - +10%) and good precision was observed: repeatability (RSD% < 15%) and within-laboratory reproducibility (RSD% < 20%). Finally, LOD values were determinedat 50 and 10 µg kg-1 and LOQ values at 70 and 30 µg kg-1 for AEON and the indolic compounds, respectively. The latter values are far below the odour threshold, 500, 200 and 100 µgkg-1for AEON, SK and IND, respectively as set in adipose tissue. Furthermore, analysis of boar samples proved the applicability of the HS-SPME-GC-MS method and precise quantification.
Finally, the performance characteristics of the method on a portable GC-MS instrument, employable at the slaughter line, were evaluated. Analysis of fortified blank samples showed that sensitivity of the portable GC-MS instrument was insufficient for the detection of the boar taint compounds at threshold levels using the current extraction protocol (HS-SPME). To improve sensitivity on the portable GC-MS instrument, the extraction protocol should be adjusted (e.g. derivatisation, trapping etc.); however, taking into account the practical obstacles for on-site analysis.
Conclusion
In this study, a candidate HS-SPME-GC-MS method for the fast and accurate at-line detection of the boar taint compounds was developed and validated. Fast extraction (40 sec) of the boar taint compounds from adipose tissue was achieved by singing the fat with a soldering iron and SPME. Validation of the HS-SPME-GC-MS and analysis of boar samples proved the quality and applicability of the method on boar samples. However, sensitivity of the portable GC-MS instrument for this application was questioned and adaptations to the method should be made before allowing fast and sensitive screening of pig carcasses at the slaughter line.
Acknowledgements
K. Verplanken is supported by the Agency for Innovation by Science and Technology in Flanders.
References
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