Development of a Candidate Reference Method for the Simultaneous Quantitation of the Boar Taint Compounds Androstenone, 3α-Androstenol, 3β-Androstenol, Skatole, and Indole in Pig Fat by Means of Stable Isotope Dilution Analysis–Headspace Solid-Phase Microextraction–Gas Chromatography/Mass Spectrometry

Abstract

The steroidal pig pheromones androstenone (5α-androst-16-en-3-one), 3α-androstenol (5α-androst-16-en-3α-ol), and 3β-androstenol (5α-androst-16-en-3β-ol) as well as the heterocyclic aromatic amines skatole and indole, originating from microbial degradation of tryptophan in the intestine of pigs, are frequently recognized as the major compounds responsible for boar taint. A new procedure, applying stable isotope dilution analysis (SIDA) and headspace solid-phase microextraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS) for the simultaneous quantitation of these boar taint compounds in pig fat was developed and validated. The deuterated compounds androstenone-d(3), 3β-androstenol-d(3), skatole-d(3), and indole-d(6) were synthesized and successfully employed as internal standards for SIDA. The new procedure is characterized by a fast, simple, and economic sample preparation: methanolic extraction of the melted fat followed by a freezing and an evaporation step allows for extraction and enrichment of all five analytes. Additional time-consuming cleanup steps were not necessary, as HS-SPME sampling overcomes fat-associated injector and column contamination. The method has been validated by determining intra- and interday precision and accuracy as well as the limit of detection (LOD) and limit of quantitation (LOQ). Additionally, a cross-validation for androstenone, skatole, and indole was carried out comparing the results of 25 back fat samples obtained simultaneously by the new SIDA-HS-SPME-GC/MS procedure with those obtained in separate GC/MS and high-performance liquid chromatography fluorescence detection (HPLC-FD) measurements. The cross-validation revealed comparable results and confirms the feasibility of the new SIDA-HS-SPME-GC/MS procedure.

Analysis of taints and off-flavours

“…Direct solvent extraction can be used for analysis of food taints, for example, for the determination of indole and skatole in meat from male pigs. Although, more recently, headspace solid-phase microextraction (HS-SPME) with stable isotope dilution has been employed (Fischer et al, 2011). Soxhlet extraction, pressurised liquid extraction and microwave extraction have all been used for determination of trichloroanisole in corks.…”

Preliminary study of FMO1, FMO5, CYP21, ESR1, PLIN2 and SULT2A1 as candidate gene for compounds related to boar taint

“…Fat samples were collected from the neck and stored at −20°C until used for boar taint compound measurement including androstenone, skatole and indole. For the quantification of androstenone, skatole and indole an in-house gas-chromatography/mass spectrometry (GC-MS) method was applied as described previously (Fischer et al, 2011). The details of the phenotypes are listed in Table 1.…”

Quantitative determination of the boar taint compounds androstenone, skatole, indole, 3α-androstenol and 3β-androstenol in wild boars (Sus scrofa) reveals extremely low levels of the tryptophan-related degradation products

“…The obtained methanolic supernatant, containing the analytes of interest, was then transferred into a headspace vial and evaporated to dryness by compressed air. Finally the headspace vial was sealed and passed to the automated HS-SPME-GC/MS measurement (Fischer et al, 2011).…”

“…As there are a lack of data concerning both major and minor boar taint compounds in the adipose tissue of adult male wild boars, the aim of the present work was to determine these compounds in a set of 23 animals, shot during hunting season in 2011/12. Quantitation of ANON, SK, 3a-OL, 3b-OL and IND was performed by applying a recently developed stable isotope dilution analysis-headspace solid-phase microextractiongas chromatography/mass spectrometry (SIDA-HS-SPME-GC/MS) procedure (Fischer et al, 2011).…”