Isolation, structural characterization and quantification of impurities in bupivacaine

Bupivacaine was found to be unstable during the accelerated storage condition(40 ℃ and 75% relative humidity), and two degradation impurities with the same protonated molecular ion were observed by high performance liquid chromatography-mass spectrometry (LC-MS). A semipreparative method was used to separate and purify the two impurities, and their structures were elucidated via comprehensive HR-MSMS and NMR spectroscopy analyses. Their stereo structures were characterized through single crystal X-ray diffraction. Meanwhile, an LC-MS method was developed and validated to quantify the two degradation impurities of bupivacaine. Chromatographic separation was performed on a C18 reversed-phase column (4.6 × 150 mm, 5 µm) using an equivalent elution with water and methanol. The limits of quantitation for the two degradation impurities (named RS1 and RS2) were 0.89 and 0.65 ng, respectively, and the average recoveries were in the range of 90∼108% and relative standard deviations were less than 5.0%. The proposed LC-MS method can be used to control the quality of bupivacaine and its formulations. DATA AVAILABILITY: Data will be made available on request.