High-pH mobile phase in reversed-phase liquid chromatography-tandem mass spectrometry to improve the separation efficiency of aminoglycoside isomers

In chromatography, the use of extreme conditions can often lead to unique separation selectivity. In this study, a highly basic mobile phase (pH > 11), which is not typically employed for reversed-phase liquid chromatography (RPLC), was utilized in RPLC-tandem mass spectrometry (MS/MS) to achieve effective separation between electrically neutral bases of aminoglycosides (AGs). A mixture of AGs was simultaneously analyzed using 500 mmol L−1 ammonia aqueous solution (pH 11.8) as the mobile phase. A total of 11 AGs, including 2 stereoisomers of neomycin (B and C) and 5 structurally similar components of gentamicin (C1, C1a, C2, C2a, and C2b), were completely separated for the first time. The high separation performance for AGs was mainly due to two factors: First, slight differences in hydrophobicity among the AGs were significantly enhanced at a high pH by the complete acid dissociation of amines. Second, the high pH of the mobile phase minimized any electrostatic interactions between the AGs and residual silanol groups in the stationary phase, resulting in extremely sharp peaks for the AGs. The sensitivity of spectinomycin decreased by more than 20% when using the highly basic mobile phase (pH 11.8) due to its degradation, therefore, a mixture of 10 AGs was analyzed with 250 mmol L−1 ammonia aqueous solution (pH 11.5) with less degradation as the optimum condition. The developed analytical method could be used to determine the concentrations of trace AGs in milk with high accuracy and precision. Thus, RPLC-MS/MS using a high-pH mobile phase has great potential for the efficient separation of basic compounds containing amino sugars such as AGs.