High-resolution tandem mass spectrometry (HRMS2) with electrospray ionization is frequently applied to study polar organic molecules such as micropollutants. Fragmentation provides structural information to confirm structures of known compounds or propose structures of unknown compounds. Similarity of HRMS2 spectra between structurally related compounds has been suggested to facilitate identification of unknown compounds. To test this hypothesis, the similarity of reference standard HRMS2 spectra was calculated for 243 pairs of micropollutants and their structurally related transformation products (TPs); for comparison, spectral similarity was also calculated for 219 pairs of unrelated compounds. Spectra were measured on Orbitrap and QTOF mass spectrometers and similarity was calculated with the dot product. The influence of different factors on spectral similarity [e.g., normalized collision energy (NCE), merging fragments from all NCEs, and shifting fragments by the mass difference of the pair] was considered. Spectral similarity increased at higher NCEs and highest similarity scores for related pairs were obtained with merged spectra including measured fragments and shifted fragments. Removal of the monoisotopic peak was critical to reduce false positives. Using a spectral similarity score threshold of 0.52, 40% of related pairs and 0% of unrelated pairs were above this value. Structural similarity was estimated with the Tanimoto coefficient and pairs with higher structural similarity generally had higher spectral similarity. Pairs where one or both compounds contained heteroatoms such as sulfur often resulted in dissimilar spectra. This work demonstrates that HRMS2 spectral similarity may indicate structural similarity and that spectral similarity can be used in the future to screen complex samples for related compounds such as micropollutants and TPs, assisting in the prioritization of non-target compounds.

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具有电喷雾电离的高分辨率串联质谱（HRMS2）通常用于研究极性有机分子，例如微污染物。片段化提供结构信息以确认已知化合物的结构或提出未知化合物的结构。已建议结构相关化合物之间的HRMS2光谱相似，以方便鉴定未知化合物。为了验证这一假设，计算了243对微量污染物及其结构相关转化产物（TPs）的参考标准HRMS2光谱的相似度；为了进行比较，还计算了219对无关化合物的光谱相似性。在Orbitrap和QTOF质谱仪上测量光谱，并计算点积的相似性。考虑了不同因素对光谱相似性的影响[例如，归一化碰撞能量（NCE），来自所有NCE的合并片段以及因片段对的质量差而移动的片段]。较高的NCE时，光谱相似度增加，相关对的最高相似度得分是通过合并的光谱（包括测得的片段和移位的片段）获得的。去除单一同位素峰对于减少假阳性现象至关重要。使用0.52的光谱相似性评分阈值，40％的相关对和0％的不相关对都高于该值。用Tanimoto系数估计结构相似性，具有较高结构相似性的对通常具有较高的光谱相似性。其中一种或两种化合物都包含杂原子（例如硫）的对通常会导致光谱互不相同。

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