A validated multi-matrix platform for metabolomic fingerprinting of human urine, feces and plasma using ultra-high performance liquid-chromatography coupled to hybrid orbitrap high-resolution mass spectrometry.

In recent years, metabolomics has surfaced as an innovative research strategy in human metabolism, whereby selection of the biological matrix and its inherent metabolome is of crucial importance. However, focusing on a single matrix may imply that relevant molecules of complementary physiological pathways, covered by other matrices, are missed. To address this problem, this study presents a unique multi-matrix platform for polar metabolic fingerprinting of feces, plasma and urine, applying ultra-high performance liquid-chromatography coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry, that is able to achieve a significantly higher coverage of the system's metabolome and reveal more significant results and interesting correlations in comparison with single-matrix analyses. All three fingerprinting approaches were proven 'fit-for-purpose' through extensive validation in which a number of endogenous metabolites were measured in representative quality control samples. For targeted and untargeted validation of all three matrices, excellent linearity (coefficients of determination R2 ≥ 0.99 or 0.90 respectively), recovery and precision (coefficients of variance ≤ 15% or 30% respectively) were observed. The potential of the platform was demonstrated by subjecting fecal, urine and plasma samples (collected within one day) from ten healthy volunteers to metabolic fingerprinting, yielding respectively 9 672, 9 647, and 6122 components. Orthogonal partial least-squares discriminant analysis provided similar results for feces and plasma to discriminate according to gender (p-value, R2(X), R2(Y) and Q2(Y)), suggesting feces as an excellent alternative biofluid to plasma. Moreover, combining the different matrices improved the model's predictivity, indicating the superiority of multi-matrix platforms for research purposes in biomarker detection or pathway elucidation and in the selection of the most optimal matrix for future clinical purposes.

Sensory evaluation of boar meat products by trained experts.

Rearing entire male pigs, one of the alternatives for surgical castration, entails the possible occurrence of boar taint. This study aimed at the investigation of the acceptability of meat from entire male pigs in 8 different meat products (cutlets, bacon, blade loin, tenderloin, dry fermented sausage, cooked ham, dry-cured ham and minced meat) by trained assessors. Generally, the sensory evaluation of meat samples was affected the most in the androstenone (AEON) group, indicating that AEON is the most offensive boar taint compound for sensitive assessors. Differences between the meat products showed the highest potential for processing tainted meat in cold meat products, which was most likely due to the serving temperature on the one hand and production-related influences on the other. However, more insights regarding reducing and masking effects of production-related factors on boar taint are necessary.

Sensory evaluation of boar-taint-containing minced meat, dry-cured ham and dry fermented sausage by a trained expert panel and consumers.

One of the main issues related to entire male pigs is the occurrence of boar taint, an off-odour, which compromises meat consumability. In this study, odour thresholds (indole: 24-65µgkg-1, skatole: 44-89µgkg-1, androstenone: 121-342µgkg-1) for the boar taint compounds were estimated in minced meat, dry fermented sausage and dry-cured ham. Afterwards, sensory evaluation of these products containing 10% tainted meat (minced meat and dry fermented sausage) or moderate boar taint compound levels (dry-cured ham) occurred. The beneficial effect of diluting tainted meat was demonstrated, as no significant difference in consumability was observed between gilts and 10% tainted meat by experts as well as consumers. Also dry-curing proved a promising technique for masking boar taint and preventing consumer dissatisfaction. The obtained results demonstrate the applicability of the estimated thresholds in meat as a tool for identifying masking and reducing strategies on the perception of boar taint.

Rapid evaporative ionization mass spectrometry for high-throughput screening in food analysis: The case of boar taint.

Boar taint is a contemporary off-odor present in meat of uncastrated male pigs. As European Member States intend to abandon surgical castration of pigs by 2018, this off-odor has gained a lot of research interest. In this study, rapid evaporative ionization mass spectrometry (REIMS) was explored for the rapid detection of boar taint in neck fat. Untargeted screening of samples (n=150) enabled discrimination between sow, tainted and untainted boars. The obtained OPLS-DA models showed excellent classification accuracy, i.e. 99% and 100% for sow and boar samples or solely boar samples, respectively. Furthermore, the obtained models demonstrated excellent validation characteristics (R2(Y)=0.872-0.969; Q2(Y)=0.756-0.917), which were confirmed by CV-ANOVA (p<0.001) and permutation testing. In conclusion, in this work for the first time highly accurate and high-throughput (<10s) classification of tainted and untainted boar samples was achieved, rendering REIMS a promising technique for predictive modelling in food safety and quality applications.

Rapid method for the simultaneous detection of boar taint compounds by means of solid phase microextraction coupled to gas chromatography/mass spectrometry.

Because of animal welfare issues, the voluntary ban on surgical castration of male piglets, starting January 2018 was announced in a European Treaty. One viable alternative is the fattening of entire male pigs. However, this can cause negative consumer reactions due to the occurrence of boar taint and possibly lead to severe economic losses in pig husbandry. 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 detection of the boar taint compounds. Remarkably fast extraction (45s) of the boar taint compounds from adipose tissue was achieved by singeing the fat with a soldering iron while released volatiles were extracted in-situ using HS-SPME. The obtained method showed good performance characteristics after validation according to CD 2002/657/EC and ISO/IEC 17025 guidelines. Moreover, cross-validation with an in-house UHPLC-HR-Orbitrap-MS method showed good agreement between an in-laboratory method and the new candidate method for the fast extraction and detection of skatole and androstenone, which emphasizes the accuracy of this new SPME-GC-MS method. Threshold detection of the boar taint compounds on a portable GC-MS could not be achieved. However, despite the lack of sensitivity obtained on the latter instrument, a very fast method with run-to-run time of 3.5min for the detection of the boar taint compounds was developed.

Boar taint compound levels in back fat versus meat products: Do they correlate?

Surgical castration of male pigs will soon be abandoned, turning a major advantage of this practice, the elimination of boar taint, into the biggest challenge for pig industry when raising intact male pigs becomes common practice. To map the (economical) consequences in relation to boar-taint consumer acceptance, as well as offer a processing strategy for tainted carcasses to stockholders, the current study investigated not only back fat boar taint levels, but additionally generated information on the levels of boar taint compounds recovered after the production of commercially relevant meat products using UHPLC-HRMS laboratory analysis. Our results demonstrate that levels of androstenone, skatole and indole in back fat and meat products tend to correlate strongly, particularly in fatty meat products (generally r>0.80). Concentration values in the edible (lean) meat fraction were significantly lower compared to back fat and fat sampled from fresh or processed meat (p<0.05).

Development and validation of a UHPLC-HR-Orbitrap-MS method for the simultaneous determination of androstenone, skatole and indole in porcine meat and meat products.

Boar taint is an off-odour that entails negative consumer reactions. In this study two extraction and UHPLC-HRMS analysis methods, valuable for evaluation of consumer acceptance towards boar meat, were developed for quantification of indole, skatole, and androstenone in different meat products. Sample pretreatment consisted of extraction with methanol and a homogenising step (cooked ham, minced meat, tenderloin, bacon, cutlets, blade loin, uncooked ham) or a melting step (salami sausage and liver paste). Both methods were validated according to CD 2002/657/EC and ISO 17025 guidelines. Good performance characteristics were obtained. Good linearity (R(2) ⩾ 0.99) and no lack of fit was observed (95% confidence interval; F-test, p > 0.05). Also good recovery (89-110%) and satisfactory precision: repeatability (RSD ⩽ 14.9%) and within-laboratory reproducibility (RSD ⩽ 17.2%) were obtained. Analysis of cooked ham and salami sausage samples proved the applicability of both methods for routine analysis.