To establish a method for related substances determination in timolol maleate by ultra-high performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry（UPLC-Q/Orbitrap HRMS），by which the impurities both in active pharmaceutical ingredients（APIs） and preparations can be recognized and determined.

An ACE Excel3 C₁₈-AR column（150 mm×4.6 mm，3 μm）was used for the separation and a mixture of 0.01 mol·L^-1 ammonium acetate solution with 0.02% formic acid and methanol was employed as the mobile phase by gradient elution，at a flow rate of 0.6 mL·min^-1. The detection wavelength for UV detector was 295 nm，an HESI（heated ESI）ion source was employed in both the positive mode and negative mode. The possible fragmentation patterns prediction was conducted with the help of Mass Frontier 8.0 and Compound Discover 3.3. The related substances could be recognized and determined by means of the forced degradation of the APIs，with the calibration by the correction factors and confirmation by the mass spectrum data from UPLC-Q/Orbitrap HRMS.

The timolol impurity B[3-(tert-butylamino)-2-(4-morpholino-1，2，5-thiadiazol-3-yloxy)propan-1-ol]，timolol impurity D(4-morpholino-1，2，5-thiadiazol-2-ol)，timolol impurity E((S，Z)-4-(｛1-(tert-butylamino)- 3-[(4-morpholino-1，2，5-thiadiazol-3-yl) oxy] propan-2-yl｝oxy)-4-oxobut-2-enoic acid maleate salt) and timolol impurity C[N-(tert-butyl)-2，3-bis (4-morphloline-1，2，5-thiadiazol-3-yloxy) propan-1-amine maleate] were produced from the APIs under selected conditions and separated well in the specified HPLC condition，the limit of quantitation was 0.05 μg·mL^-1 and the limit of detection was 0.015 μg·mL^-1 for HPLC-UV. The contents of individual impurities were between 0.000 4%-0.09% and the results of total impurities were between 0.02%-0.12% for the samples from 4 different manufactures. The probable chemical structures of the 6 unspecified impurities were speculated according to the fragmentation pattern of fragment ions，combined with the fragment information，chemical structure of API and the references.

The system solution can be obtained by the degradation of the API，and be implied in the impurity analysis for the timolol maleate. The results can be used as a reference for the quality control of timolol maletae.