# Construction of IsoVoc Database for the Authentication of Natural Flavours
## 3 Results and Discussion
### Authenticity Assessment of Commercial Samples  
Based on isotopic mass balance using \(\delta^{13}\)C values of individual compounds, estimating the average amount of synthetic compound in the sample is possible, as shown by [PERSON] et al. [16]. Here we adapted the method to work for any distribution, not necessarily a normal one, where \(\delta^{13}\)C distribution is described by the min, max, and median value. Thus, the \(\delta^{13}\)C value of a mixture (_mix_) of two compounds, both natural (_nat_) and synthetic (_syn_), can be expressed with the following relationship:  
\[\delta^{13}\mathrm{C}_{mix}=(1-x)*\delta^{13}\mathrm{C}_{nat}+x*\delta^{13} \mathrm{C}_{syn}, \tag{2}\]  
where fraction \(x\) corresponds to the added synthetic compound in the mixture, and in the case where \(x=0.5\), both components are present in equal amounts. Assuming an extensive dataset of samples that is representative of the population, we can use \(\delta^{13}\mathrm{C}_{nat,max}\)_and_\(\delta^{13}\mathrm{C}_{nat,min}\) as the range of \(\delta^{13}\)C values in natural compounds (with a median value M) to estimate the lowest value \(x\) that can be detected for compounds in those regions where natural and synthetic samples are separated. When the range of synthetic \(\delta^{13}\)C values is higher than the natural range, we use the following expression:  
\[x=\frac{\delta^{13}\mathrm{C}_{nat\ max}-\delta^{13}\mathrm{C}_{nat}}{\delta^ {13}\mathrm{C}_{syn}-\delta^{13}\mathrm{C}_{nat}}. \tag{3}\]  
For example, we chose benzaldehyde with a median \(\delta^{13}\mathrm{C}_{nat}\) value of \(-31.3\%\) and max value \(\delta^{13}\mathrm{C}_{nat\ max}\) of \(-28.0\%\). If the median \(\delta^{13}\mathrm{C}_{syn}\) value is \(-25.7\%\), then \(x=0.59\), meaning that if a mixed sample contains more than \(59\%\) of a synthetic fraction, it is likely (with \(50\%\) chance) that aduliteration will be suspected since the shift in the \(\delta^{13}\)C values will be significant (i.e., out of range of natural compounds). If the calculation is made using the minimum measured values of \(\delta^{13}\mathrm{C}_{nat}=\delta^{13}\mathrm{C}_{nat\ min}\) and \(\delta^{13}\mathrm{C}_{syn}=\delta^{13}\mathrm{C}_{syn\ min}\), then \(x=0.79\), meaning that if a mixed sample contains more than \(79\%\) of a synthetic fraction, the measured \(\delta^{13}\)C value will fall out of natural sample range. If the range of synthetic \(\delta^{13}\)C values is lower than natural ones, the corresponding fraction \(x\) of a synthetic sample is calculated by replacing the maximum values of the measured ranges with the minimum values and vice versa. The detectable fractions of synthetic compounds for different VOCs are presented in Table 2.  
The necessary synthetic fraction of selected aroma compounds lies between \(5\%\) and \(83\%\) for a \(50\%\) detection threshold and between \(19\%\) and \(100\%\) for a \(100\%\) detection threshold. Therefore, the method is the most sensitive for detecting aduliteration with \(\beta\)-ionone, acetic acid, \(\alpha\)-ionone, and \(1\)-butanol and less sensitive at detecting aduliteration of ethyl \(2\)-methyl butyrate and hexanal. However, evaluating their usefulness will require a study on how adding different amounts of synthetic aroma compounds to a sample with a weak intrinsic aroma can affect the odour. Finally, we assessed the authenticity of 33 commercial fruit flavourings, including natural banana, blueberry, peach, grape, pear, apple, strawberry, kiwi, raspberry, blackberry, plum, and sour cherry aroma. A shift in the \(\delta^{13}\)C values towards the synthetic range for 2-heptanone, benzaldehyde, butyl acetate, ethyl butyrate, hexanal, hexanoic acid, isoamyl acetate, methyl acetate, methyl hexanoate, and propyl acetate suggest the possible falsification of specific fruit VOCs despite being labelled as natural fruit extracts (Figure 5). Results imply that the authenticity of flavoured products on the market can be questioned, and extensive testing is necessary.