Lc ms water

Acetic acid (≥95.0%), formic acid (≥95.0%), ammonia (25%), LC-MS water and Acetonitrile (ACN, LC-MS grade) were obtained from Merck (Darmstadt, Germany). Ammonium acetate (≥99.0%) was supplied by Sigma-Aldrich (St. Louis, MO, USA).
A mixture of 54 metabolites was used to evaluate the HILIC stationary phases behavior. Table 1 shows the metabolites contained in the analyzed mixture. All standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nucleosides and amino acids were from two mix solutions provided by Sigma-Aldrich (St. Louis, MO, USA). A summary of the polarity of the analyzed metabolites is shown in Table S1 in the supplementary information.
Standard stock solutions (1000 μg mL⁻¹) of metabolite mixture were prepared by dissolving an appropriate amount of each metabolite in water and stored at −20 °C until their use. Working standard solutions (20 μg mL⁻¹) were prepared by diluting the stock solution in ACN:H2O (1:1).

Nusinersen is the sodium salt of an 18-mer 2’-O-methyl-phosphorothioate oligoribonucleotide dissolved in sodium dihydrogen phosphate dihydrate, disodium phosphate, sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium hydroxide, hydrochloric acid, and water (Spinraza™). The full sequence of the ASO is 3’-mU-mC-A-mC-mU-mU-mU-mC-A-mU-A-A-mU-G-mC-mU-G-G-5′, while the molecular mass equals 7110 g/mol.
Mobile phases were prepared using high purity solvents such as methanol, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), as well as N,N-dimethylbutylamine (DMBA), N,N-diisopropylethylamine (DIPEA), dipropylamine (DPA) and LC-MS water (Merck KGaA, Darmstadt, Germany). The mixture of phenol/chloroform/isoamyl alcohol (25:24:1) (VWR International, Gdańsk, Poland), acetonitrile, chloroform, 10 mM ammonium acetate (pH 4.5 and 9.0), acetic acid, ammonia solution 25% (Sigma-Aldrich, Gillingham, Dorset, UK) was also used during sample preparation step.