Authenticity Assessment of (E)-Cinnamic Acid, Vanillin, and Benzoic Acid from Sumatra Benzoin Balsam by Gas Chromatography Combustion/Pyrolysis Isotope Ratio Mass Spectrometry

Authenticity assessment of (E)-cinnamic acid, vanillin, and benzoic acid from various origins (n = 26) was performed using gas chromatography-isotope ratio mass spectrometry coupled with combustion and pyrolysis mode (GC-C/P-IRMS). For that reason, the above three compounds (1–3) from synthetic, natural, and Sumatra benzoin balsam (laboratory prepared, adulterated, and commercial) were investigated. The δ13CV-PDB and δ2HV-SMOW values for compounds 1–3 from synthetic samples (S1–S5) ranging from −26.9 to −31.1‰ and 42 to 83‰, respectively, were clearly different from those of authentic samples (N1–N4, L1–L9) varying from −29.8 to −41.6‰ and −19 to −156‰. In adulteration verification testing, for compounds 1 and 3, both δ13CV-PDB and δ2HV-SMOW data of A1 (5.0% added) and A2 (2.5% added) except A3 (0.5% added) fell into the synthetic region, whereas for compound 2, the δ2HV-SMOW data of adulterated samples (A1–A3) fell into the synthetic region, and even the lowest adulterated sample A3 is no exception. With this conclusion, some commercial Sumatra benzoin balsam samples were identified to be adulterated with synthetic 1 (C1, C3, and C5) and synthetic 2 (C3, C4, and C5) but not with synthetic 3. GC-C/P-IRMS allowed clear-cut differentiation of the synthetic and natural origin of 1, 2, and 3 and definite identification of whether a Sumatra benzoin balsam was adulterated or not.


Introduction
Sumatra benzoin is a natural balsamic resin, exuded from a small tree, Styrax benzoin Dryander, grown extensively in Sumatra and Malaya, mostly cultivated in Vietnam, ailand, and China [1]. Sumatra benzoin balsam was obtained by extraction, filtration, and vacuum distillation of the crude benzoin. It has a sweet, balsamic-cinnamic characteristic odor, which is used extensively as fixative in perfumery, food, and tobacco flavoring [2]. Driven by business interests, Sumatra benzoin balsam was often adulterated with synthetic flavors (E)-cinnamic acid (1), vanillin (2), and benzoic acid (3) (Figure 1) to claim that it was a better grade or "originated from Siam Benzoin" [3].
Current research on benzoin resin and balsam mainly focuses on the analysis of the different volatile and nonvolatile components in various species or different places of origin [4][5][6][7][8], and little information is available about authenticity assessment. Concerning flavor authenticity and traceability, IRMS has been widely used due to the high precision of the method, the requirement for small samples, and the fact that the same technique can be used for almost any type of food or beverage [9][10][11][12][13]. Fink et al. studied hydrogen isotope ratio and carbon isotope ratio of the natural, synthetic, and semi-synthetic methyl cinnamate and showed that different sources of methyl cinnamate, 2 H/ 1 H and 13 C/ 12 C, have different distribution ranges [8].
is result shows that isotope analysis can be used to verify the authenticity of flavor compounds. In this study, we undertook the authenticity study of (E)-cinnamic acid (1), vanillin (2), and benzoic acid (3) from synthetic, natural, and Sumatra benzoin balsam through 13 C/ 12 C and 2 H/ 1 H isotope ratios measured by GC-C/P-IRMS analysis.

GC-C/P-IRMS Conditions.
A Finnigan Delta V Advantage isotope ratio mass spectrometer coupled to an HP 6890°N gas chromatograph via the open-splitof combustion and pyrolysis interface was used. e GC was equipped with an HP-INNOWAX fused silica capillary column (30 m × 0.32 mm × 0.25 μm). e following conditions were employed: for GC: 1-μL solution was injected in splitless mode (250°C); the injector temperature was 250°C; the initial oven temperature was 60°C, held for 1 min, then heated to at a rate of 180°C at 8°C/min, raised to 240°C at a rate of 6°C/ min and held at 240°C for 17 min; the carrier gas was He at a flow rate of 1.5 mL/min. For 13 C/ 12 C: the solutions flow was online combusted into to CO 2 at 960°C in the oxidative reactor (Al 2 O 3 , 0.5 mm×1.5 mm×320 mm) with Cu, Ni, and Pt (each 240 mm×0.125 mm); the water separated by Nafion membrane. For 2H/1H: the effluent from the GC were directed to a high-temperature ceramic tube (Al 2 O 3 , 0.5 mm×320 mm) and pyrolyzed to H 2 at 1440°C. In addition, coupling GC isolink elemental analyzer system to the IRMS was realized for offline control determination of reference samples. Daily system stability checks were performed by measuring reference samples with known 13 C/ 12 C and 2 H/ 1 H ratios. e reference samples were using International Atomic Energy Agency (IAEA, Vienna, Austria) standards, and IAEA-601 used for 13 C/ 12 C and IAEA-601, and VSMOW used for 2 H/ 1 H, respectively. e isotope ratios are expressed in per mil (‰) deviation relative to the V-PDB and VSMOW international standards, and the calculation method is the same as reference [8]. In general, 6-fold determinations were carried out and standard deviations were calculated. e latter were ±0.2 and ±5‰ for δ 13 C V-PDB and δ 2 H V-SMOW determinations, respectively.

Results and Discussion
To check potential isotope discrimination in the course of sample preparation, the three synthetic reference samples under study (S1, 1-3) were subjected to the steps used for the laboratory prepared balsam. e data summarized in Table 1 showed that sample preparation did not affect the isotope values.
e data from treated samples S1a did not differ significantly from those the untreated reference samples S1.

Conclusion
In conclusion, the δ 13 C V-PDB and δ 2 H V-SMOW values for authenticity assessment of (E)-cinnamic acid (1), vanillin (2), and benzoic acid (3) from various origins including synthetic, natural, and Sumatra benzoin balsam (laboratory prepared, commercial, and adulterated) were demonstrated. Despite the limited number of samples, GC-C/P-IRMS allowed clear-cut analytical differentiation of the synthetic and natural origin of 1, 2, and 3 and definite identification of whether a Sumatra benzoin balsam was adulterated or not. Future work will be done to extend the amounts of 1, 2, and 3 from natural plant sources, particularly Siam benzoin, which has antioxidative effect, economic value, and flavoring application [2], until finally build the IRMS database for their authenticity identification.

Data Availability
e reference data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors are affiliated to and funded by China Tobacco Yunnan Industrial Co. Ltd. e authors attest that China Tobacco Yunnan Industrial Co. Ltd. had no influence on design of this study or its outcomes. International Journal of Analytical Chemistry 5