Abstract: Nicotinic acid and its derivatives serve as crucial synthetic precursors of "key substances associated with multi-dimensional anti-aging biomarkers". They play significant physiological roles in processes such as anti-aging, skin lightening and reducing the risk of chronic disease. Rice is a major staple food for more than half of the global population. Notably, brown rice contains higher levels of niacin and its related derivatives compared with other foods. Owing to this property, it emerges as a potential candidate for research on the dietary uptake of endogenous nicotinamide adenine dinucleotide (NAD⁺) precursors. This study aims to establish a quantitative method capable of simultaneously determining the content of nicotinic acid and its derivatives in various types of rice, in order to fill the technical gap in the development of functional rice products and in the regulatory field of natural NAD⁺ precursors. The effects of extraction solvents, solvent volumes, and extraction time on the extraction efficiencies of the seven target compounds from brown rice were compared. Additionally, the adsorption materials and purification conditions were optimized. The brown rice samples were ultrasonically extracted with 60% methanol aqueous solution (acidified with 0.1% formic acid) for 20 min and purified using an Oasis HLB solid-phase extraction cartridge. Seven target compounds were separated using gradient elution of acetonitrile and 25 mmol/L ammonium carbonate on an XBridge^TM Amide column (150 mm×2.1 mm, 3.5 μm). Detection was carried out in positive ion and multiple reaction monitoring (MRM) modes. The experimental results showed that the seven nicotinic acid and its derivatives exhibited good linearity, with correlation coefficients (r) all greater than 0.995 0. The limits of detection (LODs) and limits of quantification (LOQs) were 0.001-0.07 mg/kg and 0.004-0.2 mg/kg, respectively. The recoveries of the seven target compounds, spiked at three levels in six rice matrices (indica, japonica, glutinous, red, purple, and black rice), ranged from 78.3% to 109.8%, with relative standard deviations (RSDs, n=6) of 0.4%-6.7%. This method was applied to determine the contents of the seven nicotinic acid and its derivatives in 343 rice samples. The results revealed significant differences in the contents of these compounds among different rice varieties. Nicotinamide riboside (NR) was found to be the most abundant derivative among the tested varieties, while nicotinamide (NAM) was the least prevalent consistently. The method established in this study features simple and efficient extraction, high accuracy, and excellent repeatability, thus enabling reliable qualitative and quantitative analysis of the seven nicotinic acid and its derivatives in various rice varieties.

Key words: liquid chromatography-tandem mass spectrometry (LC-MS/MS), solid-phase extraction column, nicotinic acid and its derivatives, rice