Development and validation of an HILIC/MS/MS method for determination of nusinersen in rabbit plasma

A hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC/MS/MS) method was developed and validated for the quantitative analysis of the fully phosphorothioate modified oligonucleotide nusinersen. HILIC/MS/MS method is more robust and compatible with mass spectrometry than ion pair reversed-phase liquid chromatography-tandem mass spectrometry (IP-RP-LC/MS/MS). Various types and concentrations of additives and different pH of mobile phase affected the mass spectrometry response, chromatographic peak shape and retention of nusinersen. The optimized extraction method of nusinersen employs hydrophilic-lipophilic balance solid phase extraction, with a recovery of up to 80 %. Chromatographic quantification was performed using a gradient system on an amide column and the mobile phase consisted of ammonium acetate, acetonitrile and water in a certain proportion. The fully phosphorothioate modified nusinersen can obtain a high mass spectrometry response by providing greater peak symmetry and high ionization efficiency in a high-pH mobile phase. Moreover, the significant carry over interference was observed at the pH 6.3 of the mobile phase. Adjusting the pH value up to 10, and the carry over interference disappeared. The lower limit of quantitation of this developed HILIC/MS/MS assay was 30.0 ng/mL and the method was systematic methodology validated. This HILIC/MS/MS method provides an attractive and robust alternative for the quantitative analysis of nusinersen and was applied in the pharmacokinetic study of nusinersen in rabbits. •Adjusting the pH of the mobile phase to a high value of 10, the ionization efficiency of phosphorothioate increased significantly, high mass spectrometry response of nusinersen was obtained, and the significant carry over interference disappeared.•A perfect chromatographic behavior of nusinersen was exhibited under the pH value of mobile phase was 10 by detailed investigation and optimization of the type, concentration of additives in the mobile phase.•The hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC/MS/MS) method was developed and validated, the lower limit of quantitation of method was 30.0 ng/mL.•The developed HILIC/MS/MS method successfully applied to the fully phosphorothioate oligonucleotide nusinersen and met the requirement for pharmacokinetic study of nusinersen in rabbits.