(T1030-06-39) Optimal Mobile Phase and Antisense Oligonucleotide Modifications for the Internal Standard in Bioanalysis of Therapeutic Oligonucleotides by Ion-Pair LC-MS/MS

Purpose: Recently, LC-MS/MS has been widely utilized for bioanalysis of therapeutic oligonucleotides due to its advantages, such as selectivity and wide quantitative range. However, oligonucleotides are detected as negative multivalent ions which can form an adduct with other elements such as sodium and potassium, thus complex MS spectra make the process to select target ions for quantitation and internal standards (IS) difficult. In addition, when the analyte and IS are antisense oligonucleotides (ASOs) having a common product-ion, the separation in the LC needs to be improved to ensure selectivity. In this work, we investigated the optimal combination of alkyl-amines as ion-pair reagent and fluorinated alcohols in mobile phase, and the optimal phosphorothioate (PS) ASO modification for the selection of IS to achieve high sensitivity and resolution.

Methods: ASOs were separated using a Nexera X2 UHPLC with an ACQUITY Premier BEH C18 column, and detected using a Sciex API5000 tandem mass spectrometer with selected reaction monitoring (SRM) in negative mode. By a ternary flow system with three pumps, mobile phase A (100 % Milli-Q water), mobile phase B (75%MetOH and 25%ACN), and mobile phase C (1 mol/L fluorinated alcohol and 150 mmol/L alkyl-amine in methanol) were mixed and introduced. Alkyl-amines used were N,N-diisopropylethylamine (DIPEA), octylamine (OA), diisopropylamine (DIPA), hexylamine (HA), tributylamine (TBA), and N,N-dimethylcyclohexylamine (DMCHA). Fluorinated alcohols were hexafluoroisopropanol (HFIP), trifluoroethanol (TFE), and pentafluoropropanol (PFP). Various modified synthetic ASOs were used as test compounds (analyte and IS). The analyte sequence (ASO-1) was A(L)^G(L)^T(L)^a^c^t^a^t^a^g^c^a^t^5(L)^T(L)^G(L) (16mer)¹⁾. The (L) and (^) letters indicate locked nucleic acid (LNA) and phosphorothioate, respectively. The upper and lower cases show RNA, and DNA, respectively, and 5(L) means LNA_5-Methylcytosine.The IS candidates were set as follows.
ASO-2: ASO with the same modification as ASO-1 but with a different sequence
PS ASO-1: ASO-1 with PS modification only
2'-OMe ASO-1: ASO-1 with 2'-O-methyl (2'-OMe) modification instead of LNA
MOE ASO-1: ASO-1 with 2'-O-methoxyethyl (MOE) modification instead of LNA
ASO-3: The 5' terminal G(L) was deleted from ASO-1

Results: Firstly, the sensitivity of ASOs was verified in various combinations of alkyl-amines and fluorinated alcohols, and it was found that the combination of DIPEA, DIPA, TBA or HA as an ion-pair reagent and HFIP or TFE as a fluorinated alcohol has improved sensitivity for the analyte and PS ASO-1 (Figure 1). Subsequently, mobile phase conditions were optimized for the separation of ASO-1 and PS ASO-1. The results indicated superior resolution when used in a combination of DIPEA or DIPA as the ion-pair reagent and HFIP as the fluorinated alcohol (Figure 2). Finally, the separation of IS candidates with the combination of mobile phases selected from the above results were examined and the results indicated that MOE ASO-1 and PS ASO-1 had superior resolution with ASO-1 (Figure 3).

Conclusion: To achieve high sensitivity and high resolution, the most suitable analytical method is to use HFIP as fluorinated alcohol and DIPEA or DIPA as ion-pair reagent in mobile phases. Furthermore, when the target for quantitation is LNA ASO, the MOE or PS ASO is considered to be suitable as IS judging from the separation in the LC.

References: 1) Paymaan Jafar-Nejad, et al. The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration. Nucleic Acids Res. 2021 Jan 25;49(2):657-673.

Figure 1. Sensitivity of ASO-1 and PS ASO-1 in combination of alkyl-amines and fluorinated. Alcohols
Product ions were unified to 94.9 for all transitions from molecular ions. In addition, 8- to 11-valent precursor ions were monitored.

Figure 2. Separation of ASO-1 and PS ASO-1 (one of the IS candidates) in each mobile phase combination. Resolution was calculated by the half-width method (below). Resolution = (𝟏.𝟏𝟖×|𝒕𝑹𝟐−𝒕𝑹𝟏|) / (W𝟎.𝟓𝒉𝟏+W𝟎.𝟓𝒉𝟐) 𝒕𝑹1 ASO-1 retention time, 𝒕𝑹𝟐: IS retention time, W𝟎.𝟓𝒉𝟏: ASO-1 half-width, W𝟎.𝟓𝒉𝟐: IS half-width

Figure 3. Separation of ASO-1 and IS candidates in optimal mobile phase. Resolution was calculated by the half-width method (below). Resolution = (𝟏.𝟏𝟖×|𝒕𝑹𝟐−𝒕𝑹𝟏|) / (W𝟎.𝟓𝒉𝟏+W𝟎.𝟓𝒉𝟐)
𝒕𝑹1: ASO-1 retention time, 𝒕𝑹𝟐: IS retention time, W𝟎.𝟓𝒉𝟏: ASO-1 half-width, W𝟎.𝟓𝒉𝟐: IS half-width