Abstract:A method for determining β-nicotinamide mononucleotide and its analogues in milk powder was established based on high performance liquid chromatography（HPLC）. The sample was dissolved in deionized water，and 45 mL acetic acid was added for protein precipitation. The extract was then passed through a C18 solid-phase extraction cartridge. Gradient elution was carried out in the Venusil HILIC column with ultrapure water，acetonitrile，and 0.1% trifluoroacetic acid and the compounds were detected by a photo-diode array detector. The results showed that the correlation coefficients（R2）of β-nicotinamide mononucleotide and its analogues ranged from 0.999 1 to 0.999 7 within the linear range. The method detection limits（MDLs）and method quantification limits（MQLs）were 0.030 8-0.635 0 mg/kg and 0.102-2.120 mg/kg，respectively. The recovery of the compounds varied within the range of 80.4%-101.7%，and the intra-day precision（RSD）（n=6）of the established method was within the range of 1.22%-8.20%.This method is not only simple and cost-effective but also enjoys excellent precision，recovery，and reproducibility.Therefore，it is suitable for determining β-nicotinamide mononucleotide and its analogues in milk powder.