Vanillin is the main component of vanilla flavor and is naturally produced from an orchid. However, due to the high cost and time-intensive nature of cultivating natural vanilla pods, most of the vanillin is mainly artificially manufactured. Existing methodologies, such as isotope ratio mass spectrometry (IRMS) and site-specific natural isotopic fractionation by nuclear magnetic resonance (SNIF-NMR), are employed to differentiate natural vanillin from other sources based on carbon and hydrogen isotope measurements. Nevertheless, these methods have limitations, as the carbon isotopic ratio can be counterfeited by adding commercially available enriched vanillin. For this research, we purified 1 mg of vanillin from pods from various geographical and botanical sources. We developed a novel method for analyzing ¹³C/¹²C and ¹⁸O/¹⁶O isotopic ratios of vanillin using direct injection analysis coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). This innovative approach enables the examination of bulk vanillin carbon and oxygen isotopic ratios, as well as specific molecular fragments. By analyzing a characteristic vanillin fragment that provides site-specific ¹⁸O/¹⁶O isotopic ratio data, we achieved superior clustering and discrimination of samples based on their botanical source and geographical origin. Our proposed method holds significant potential for vanillin authentication and can be performed using a mere 20 μg of pure vanillin in just 10 min of analysis time. Subsequent research should focus on acquiring additional vanillin samples from diverse botanical, geographical, and biosynthetic origins while exploring various isotopic ratios to further enhance the reproducibility and reliability of this methodology.

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