Sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis of Alkaloid Nitrogen Oxides at Three Different Processing Stages of Binglang

Betel nut (Areca nut, AN) products are extensively consumed across East and South Asian countries, with Binglang being the predominant chewable AN product in China. Given the increased risk of carcinogenicity associated with alkaloid nitrogen oxides compared to the primary alkaloids in AN, this study aimed to establish a precise and sensitive method for quantitative analysis of the arecoline N-oxide (ACNO) and arecaidine N-oxide (ADNO) at three different processing stages of Binglang using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The developed method underwent comprehensive validation, evaluating key analytical parameters including selectivity, linearity, sensitivity, accuracy, precision, and stability. Notably, the method demonstrated excellent sensitivity, with the limits of detection (LOD) ranging from 0.029 to 0.135 ng/mL, while the limits of quantification (LOQ) ranging from 0.095 to 0.450 ng/mL. Subsequently, the established analytical technique was applied to analyze 15 Binglang samples at three different processing stages, providing the first evidence of the content and variability of alkaloid nitrogen oxides throughout these stages. ACNO and ADNO were detected in all samples, and their total contents ranged from 4.31 to 32.82 mu g/g. The changes observed across different processing stages indicated that the roasting process at 80 degree celsius resulted in the promotion of alkaloid nitrogen oxides. This analytical approach constitutes a valuable tool for the quality control and risk assessment of Binglang and can be extended to other AN products, thereby assisting in addressing health concerns associated with these products.