Impurity profile analysis of cefprozil using 2D-LC-HR-MS/MS
Objective To analyze the impurity profile of cefprozil API using 2D-LC-HR-MS/MS.Methods The sample chromatogram was acquired by one-dimensional chromatography conditions to confirm the peak positions of each impurity.The YMC Hydrosphere column(250 mm × 4.6 mm,5 μm)was used,with 11.5 g·L-1 ammonium dihydrogen phosphate(pH adjusted to 4.4 with phosphoric acid)as solvent A,a mixture of acetonitrile and solvent A(50:50)as solvent B,and gradient elution;column temperature was 40℃;flow rate was 1.0 mL·min-1;detection wavelength was 230 nm;injection volume was 4 μL.The impurity profile was analyzed by 2D-LC desalting and high-resolution tandem mass spectrometry(HR-MS/MS)after cutting into the mass spectrometer.Based on the results,the structures and formation mechanisms of the impurities were inferred.The Waters BEH C18 column(50 mm × 2.1 mm,1.7 μm)was used,with 0.01％formic acid aqueous solution as solvent A and acetonitrile as solvent B.The peaks were cut and the A phase was gradually increased from 98％to 1％starting from the cut.The column temperature was 40° C,the flow rate was 0.3 mL·min-1,and the mass spectrometer used was the Waters Xevo G2-XS QTof MS system with an ESI ion source,a capillary voltage of 3 kV,a vaporizer temperature of 450 ℃,and a scanning range of m/z 100-2 000.Results Nine impurity peaks were identified in the cefprozil sample,of which five were known impurities.Peak 3 was impurity B(cefhydroxime),peak 5 was impurity D,peak 6 was impurity F,peak 7 was impurity G,and peak 9 was impurity I.Preliminary structural hypotheses were made for the three unknown impurities and the possible generation routes were discussed.Peak 2 has a molecular formula of C18H19N3O6S,which was one oxygen atom more than cefprozil.It was analyzed as an oxidation impurity of cefprozil.Peak 4 has a molecular formula of C16H15N3O6S,which was one oxygen atom and two hydrogen atoms less than impurity B.It was judged that it was the sulfur atom of the reaction product between 7-ACA lactam and HPGM(hydroxyphenylglycine methyl ester)continuing to undergo oxidation.Peak 8 has a molecular formula of C8H9NO2S,which is part of the molecular structure of cefprozil.Peak 1 needs further study.Conclusion This method effectively solved the problem of the chromatographic system with non-volatile phosphate in the mobile phase of cefprozil flowing phase and the incompatibility of chromatography-mass spectrometry rapid identification of impurities.It can simply and quickly determine the qualitative analysis and impurity spectrum of cefprozil-related substances.
2D-LC-HR-MS/MScefprozilrelated substancesimpurity profilequality control
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