Abstract: Clarithromycin has significant antibacterial effect against Gram positive bacteria, and has been widely used as an important clinical antibiotic at present. As a derivative of microbial secondary metabolites, however, clarithromycin products have a large number of impurities, and some trace impurities also need further structural analysis. In this study, the mass spectrometric fragmentation reactions of clarithromycin K and its impurities of KL and KO were systematically studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and quantum calculation. Quantum calculation showed that the amino N atom of deoxyaminoglycosides (the No.5 side chain unit) is the most preferred protonation site in the structure of clarithromycin. Upon collisional activation, the protonated molecules undergo a serie of proton migration and fragmentation reactions on side chain groups as the followings: 1) The ionizing proton transfers to the O5a atom in the No.5 side chain group and triggers the loss of macrolides to form an oxonium ion of the dehydrided deoxyaminoglycoside (K5a-P, m/z 158.1). 2) The ionizing proton transfers to the O5b atom in the No.5 side chain group, which triggers the ring-opeaning reaction of the deoxyaminoglycosides group to give a characteristic fragment ion of N-(3-hydroxybutylidene)-N-methylmethanaminium (K5c-P, m/z 116.1). 3) Migration of the ionizing proton to the O6 atom results in the loss of methanol. 4) Migration of the ionizing proton to the O3a atom results in the formation of K3a, which undergoes migration of the pyranose unit due to the nucleophilic attack of the O5d atom of the No.5 side chain unit, and the subsequent loss of macrolides to form an oxonium ion at m/z 316.2 (K3a-1P). 5) K3a can also undergo nucleophilic reaction due to the nucleophilic attack of the O3c atom of the para hydroxyl, and subsequent fragmentation to produce the characteristic ion K3a-2P by losing C8H12O3, and further cleavage to give the characteristic ion K3a-2P-P via the loss of methanol. The impurities of KL and KO have high similarity to clarithromycin in structure, which only differ in the No.9 side chain unit. As expected, they have very similar tandem mass spectra, in which there are several characteristic neutral losses of CH₄O, C₈H₁₂O₃ and (C₈H₁₂O₃+CH₄O) in the high mass region, and abundant characteristic fragment ions at m/z 316, m/z 158 and m/z 116 in the low mass region. This work summarizes the MS fragmentation law of clarithromycin and its analogue, provides a reference for the structure derivation of the other impurities of clarithromycin and it metabolites.