Development of SPE Method for the Extraction of Phosphorothioate Oligonucleotides from Serum Samples

Abstract

Aim: Comprehensive development of a method for SPE extraction of antisense phosphorothioate oligonucleotide and its metabolites and their determination with the use of UHPLC. Results: Polymer-based adsorbent and high percentage of methanol in elution solvent provided high recoveries compared with silica-based octadecyl cartridge. As to the type and concentration of ion pair reagent and organic solvent, the mixture of 5 mM of N,N-dimethylbutylamine/150 mM of 1,1,1,3,3,3-hexafluoroisopropanol and methanol was selected. Relatively high recoveries in the range of 79.2–81.2% with the SDs of 3.4–6.2% were obtained for the oligonucleotide and its metabolites extracted from human serum. Conclusion: The developed method may be successfully applied for routine analysis of antisense oligonucleotides in serum since it is relatively easy, quick and reliable.

Development of the Method for Nusinersen and Its Metabolites Identification in the Serum Samples of Children Treated with Spinraza for Spinal Muscular Atrophy

“…In the next step, the LLE extracts were further purified and concentrated using other methods, namely SPE, MEPS, and MNPs. The extraction procedures were previously developed in our laboratory and used to extract OGNs from enriched serum samples [ 26 , 27 , 28 ]. In this study, we wanted to compare the efficiency of the developed methods in the study of real samples.…”

“…We also used three other procedures for additional sample purification, which we applied directly to extracts after LLE using phenol/chloroform/isoamyl alcohol (before extraction with chloroform). These procedures were developed previously in our laboratory for the extraction of antisense OGNs from plasma samples [ 26 , 27 , 28 ].…”

Oligonucleotides Isolation and Separation—A Review on Adsorbent Selection

“…C18 adsorbent can be used in SPE for OGN extraction, mainly for phosphorothioate and single and double-stranded DNA. It was used to extract these analytes from mammals’ plasma (including humans) [ 16 , 55 , 61 , 66 , 101 ]. The application of C18 for the extraction of ASOs (e.g., phosphorothioate) provided recoveries approximately 80% [ 61 , 66 , 101 ].…”

“…It was used to extract these analytes from mammals’ plasma (including humans) [ 16 , 55 , 61 , 66 , 101 ]. The application of C18 for the extraction of ASOs (e.g., phosphorothioate) provided recoveries approximately 80% [ 61 , 66 , 101 ]. However, the application of modified silica for the extraction can cause analyte losses.…”

“…It is often used in the SPE extraction of phosphorothioate, 2′-fluoro or 2′-O-methyl OGNs and their metabolites from different biological samples (human, monkey, mouse, and rat plasma, rat urine, mouse liver, and kidney homogenate), as well as samples from in vitro incubation with enzymes (3′ exonucleases), human liver microsomes, and primary rat hepatocytes [ 11 , 13 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 66 , 68 , 101 ]. Typically, the OGN recovery values for Oasis HLB columns ranged between 60–80% [ 11 , 13 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 66 , 101 ]. Moreover, purification of crude, unmodified DNA OGNs with Oasis HLB gave 90–95% purity with a recovery of the target compound of 60–96% [ 11 ].…”