Identification of impurities in polymyxin B and colistin bulk sample using liquid chromatography coupled to mass spectrometry.
The European Pharmacopoeia (Ph. Eur.) describes liquid chromatography-ultraviolet (LC-UV) methods using C(18) stationary phases for the analysis of polymyxin B and colistin. Several unknown impurities were detected in commercial samples of those polypeptide complexes. However, the Ph. Eur. does not specify any related substances for polymyxin B and colistin. Since both methods use non-volatile buffers, the mobile phases were incompatible with mass spectrometry (MS). For the identification of related substances in bulk samples by LC/MS, volatile mobile phase systems were developed. However, the LC/MS methods (with volatile additives) showed inferior chromatographic separation compared to the LC-UV method (with non-volatile additives). Moreover, previously identified impurities by LC/MS could not be assigned in LC-UV methods as the separation in both systems was different. In this study, known impurities were traced in the LC-UV methods and new impurities present in polymyxin B and colistin bulk samples were characterized. To achieve this, each peak from the non-volatile system was collected separately and reinjected into an LC system with a volatile mobile phase coupled to MS. This way, collected impurity peaks were rechromatographed on a reversed phase column in order to separate the analyte from the buffer salts. Using this method, out of 39 peaks, five novel related substances were characterized in a polymyxin B bulk sample. Fourteen impurities, which were already reported in the literature were traced as good as possible in the LC-UV method. In the case of colistin, a total of 36 peaks were investigated, among which four new compounds. Additionally, 30 known impurities were traced in the LC-UV method.
Katholieke Universiteit Leuven, Faculteit Farmaceutische Wetenschappen, Leuven, Belgium.
Larissa
L
Van den Bossche
Ann
A
Van Schepdael
Shruti
S
Chopra
Jos
J
Hoogmartens
Erwin
E
Adams
D000900:Anti-Bacterial Agents / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D003091:Colistin / Q000737:chemistry*; D004340:Drug Contamination; D015394:Molecular Structure; D004364:Pharmaceutical Preparations / Q000737:chemistry; D011112:Polymyxin B / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D000900:Anti-Bacterial Agents; D004364:Pharmaceutical Preparations; D011112:Polymyxin B; D003091:Colistin