Abstract OBJECTIVE To introduce the developing procedure of reference standards of nitrosamines by taking 1-methyl-4-nitrosopiperazine(MNP) as example and discuss the problems needing attention when using nuclear magnetic resonance(NMR) quantitative method. METHODS The structure of the material was confirmed by mass spectrometry, infrared spectrum and nuclear magnetic methods. In terms of quantitative assessment, the purity of the material was analyzed by HPLC, and the water was investigated. The content of MNP was determined by mass balance method, the content of the material was verified by NMR quantitative method. RESULTS MNP was consistent with its structure. In the quantitative NMR investigation, it was found that the use of different internal standards had a great impact on the calibration results. CONCLUSION Since MNP is basic, it is easy to reduce the final determination result by using an acidic internal standard in NMR quantitative analysis. Therefore, it is suggested that an appropriate internal standard should be selected for this kind of sample in the process of NMR quantitative determination.