Development and Validation of an LC-MS/MS Assay for Quantitative Analysis of Nusinersen in Human CSF and Plasma

Nusinersen is the first antisense oligonucleotide (ASO) drug approved for the treatment of spinal muscular atrophy (SMA) in China; however, its pharmacokinetics (PK) in Chinese SMA patients remains unknown. The objective of this study was to develop and validate a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methodology for quantifying nusinersen in human plasma and cerebrospinal fluid (CSF) samples. The samples were prepared by protein precipitation, and then the gradient was eluted on a column of Acquity UPLC Xbridge C18 by using acetonitrile with 0.5% triethylamine (TEA) and 0.5% hexafluoroisopropanol (HFIP) and water with 0.5% TEA and 0.5% HFIP as the mobile phase. Detection was performed on a QTRAP6500⁺ tandem mass spectrometer in the negative ion multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The optimized method was successfully qualified for the nusinersen in human plasma and CSF samples over the range of 5.00 to 1000 ng/mL and 2.00 to 400 ng/mL, respectively. Importantly, our work is the first report of quantification of nusinersen in human plasma and CSF by LC-MS/MS methodology. The developed methodology is reliable and will be applied to PK study of nusinersen in Chinese SMA patients.