Fig. 3(a) shows the mean of the Training Set beef and horse spectra from Lab 1. To aid in annotation, these were compared with a high-field JAK inhibitor 600 MHz 1H NMR spectrum of a single randomly chosen horse sample from Lab 2 (Fig.

3(b); peaks annotated based on Vinaixa et al. (Vinaixa, Rodriguez, Rull, Beltran, Blade, Brezmes, et al., 2010)), and with spectra from the series of triglyceride mixtures prepared at Lab 2 (Fig. 3(c)). The horse spectrum in Fig. 3(a) is qualitatively very similar to the spectra of mixtures with a C18:3 constituent (Fig 3.(c)), consistent with the presence of an appreciable C18:3 component in the extracts from horse meat. Comparison with the high-field spectrum in Fig. 3(b) helps interpretation. Linolenic acid C18:3 ω-3 (α-linolenic acid) contains a double Fluorouracil in vivo bond close to the terminal CH3 that is known to cause a shift to higher ppm values (from 0.87 to 0.97, high-field NMR values) (Alonso-Salces, Holland, & Guillou, 2011). We found peaks at both 0.87 and 0.97 ppm in the high-field horse meat spectrum (Fig. 3(b)) and in the low-field spectra of both horse and C18:3 containing mixtures (Fig. 3 (a) and (c)). Note that the outer lines of the two triplets in panel (b) derive from a coupling constant value in Hz that is independent

of field strength, which is why in ppm the triplet outer lines appear at different values for 600 MHz (b) and 60 MHz (c) spectra. This also results in the third peak of the α-linolenic acid triplet appearing Carnitine palmitoyltransferase II at 0.84 ppm in the 60 MHz spectra and being obscured by a terminal CH3 peak at 0.78 ppm. In contrast, the beef spectrum more closely resembles that of the C18:0 + C18:1 mixture. This is consistent with beef having essentially no C18:3 content. Therefore, linolenic acid, previously identified as a marker for horse meat versus beef, has an NMR signature in the form of a shifted terminal CH3 peak combined with a bis-allylic peak. Note however that in the C18:3 ω-6 (γ-linolenic acid) isomer, the relevant double bond is further away from the CH3 terminal so does not give rise to the same shift. Therefore, for C18:3 ω-6 (γ-linolenic

acid) the CH3 peak is at 0.866 ppm, indistinguishable from those for saturated, oleic and linoleic acids. In other words, the NMR shifted-CH3 marker is not related to total linolenic acid, but specifically to the α-linolenic acid content. The high-field data also helps to identify two peaks visible in the mean horse spectra, but absent in the beef extracts and triglyceride mixtures. These are at 0.67 and 1.00 ppm, and are due to cholesterol (Vinaixa et al., 2010). Such cholesterol peaks appear in some, but not all, of the individual horse spectra and are most apparent in those extracts with the lowest overall triglyceride concentration. This is a consequence of the inflating effect of normalizing by the glyceride peak area.