(M1030-06-39) Robust Quantification Method Development for Antisense Oligonucleotide by LC-MS/MS

Purpose: LC-MS/MS method is frequently selected for bioanalysis of antisense oligonucleotides (ASOs) because of the high selectivity, even though there are some problems caused by LC condition in the development of quantification method for oligonucleotides using LC-MS/MS, such as carry-over due to metal adsorption and the decrease of internal standard (IS) sensitivity caused by degradation of ion-pair reagent. Thus, we intend to optimize the conditions of the analytical method. In order to develop an optimal quantification method, we conducted experiment, focusing on the carry-over and decrease of IS sensitivity.

Methods: 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 (PS) respectively. The upper and lower case show RNA, and DNA, respectively, and 5(L) means LNA_5-methylcytosine. Moreover, the IS was PS ASO-1. Detection of ASOs was conducted on a Sciex Triple Quad™ 7500 mass spectrometer with selected reaction monitoring (SRM) in negative mode. A Nexera X3 ultra-high-performance liquid chromatograph (UHPLC) (Shimadzu) with an ACQUITY Premier BEH C18 column with VanGuard FIT 100 x 2.1 mm Waters) was used to separate ASOs. We examined two LC configurations (Figure 1) as follows. In configuration Figure 1a), a binary flow system with two pumps was set up with mobile phase A (100 mM hexafluoroisopropanol (HFIP), 15 mM N,N -Diisopropylethylamine (DIPEA) in 10% methanol) and B (100 mM HFIP, 15 mM DIPEA in 75% methanol and 25% acetonitrile).The initial condition had a total flow rate of mobile phase at 0.30 mL/min. After holding the initial condition of 2.5% B for 0.2 min, the separation gradient was ramped up to 30% B within 8.0 min. After the separation gradient, the wash step was held at 90% B from 8.01 to 11 min. After the wash, the mobile phase composition was returned to the initial condition for re-equilibration for 4 min, then finishing the complete method at 15 min. The column temperature was maintained at 60 °C. In configuration Figure 1b), a ternary flow system with three pumps was set up, with mobile phase A (100% Milli-Q water), B (75% methanol and 25% acetonitrile), and C (1 M HFIP and 150 mM DIPEA in methanol). The initial condition had flow rates of combined mobile phase A/B at 0.27 mL/min and mobile phase C at 0.03 mL/min, delivering a total flow rate of 0.3 mL/min. After holding the initial condition of 2.5% B for 0.2 min, the separation gradient was ramped up to 30% B within 8.0 min. After the separation gradient, the wash step was held at 90% B from 8.01 to 11 min. In this step, 3 or 4 injections (Figure 2a) of injection reagent which were 5 mM EDTA, blank, 5% ammonia, and 2′-O-methoxyethyl (MOE)-containing blank, for carry-over prevention were conducted. And in the same step, multiple gradient wash cycles were included. After the wash, the mobile phase composition was returned to the initial condition for re-equilibration for 4 min, then finishing the complete method at 15 min. The column temperature was maintained at 60 °C.

Results: As a carry-over strategy, 5 mM EDTA, blank, 5% ammonia, and MOE-containing blank were injected 3 or 4 times (Figure 2a). EDTA and ammonia were used as stripping reagents, and blank and MOE were used as stripping and blocking reagents. In the case of non-injection during the wash, 21.6% area peak compared with LLOQ was observed as carry-over (Figure 2b). Compared with this, in the case of injection of stripping reagents was effective against carry-over and blocking reagent was able to act remarkably against carry-over. Additionally, we found 3 times-injection during the wash were enough for elimination of carry-over. To prevent a decrease of IS sensitivity, the flow system was changed from binary to tertiary (Figure 1a and 1b) which led to stable sensitivity results during 3 day’s analysis (Figure 3).

Conclusion: We could solve some problems for the robust ASOs’ analysis by LC-MS/MS. The results indicate that preventing degradation of ion-pair reagents due to the micellization of alkylamines²⁾ is achievable, and that 3 times-injection of MOE-containing blank protected the flow line in the UHPLC system and could solve carry-over problems. By introducing the ion-pair reagent from the water-free mobile phase, it was possible to avoid a decrease in sensitivity of the IS during the measurement in blank samples.

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; 49(2):657-673
2) Li N, et al. Alkylamine ion-pairing reagents and the chromatographic separation of oligonucleotides. J. Chromatogr A. 2018; 1580, 110-119

Figure 1. Configuration of binary flow system (a) and ternary flow system (b)

Figure 2. Gradient program for carry-over prevention (a) and the comparison of carry-over between injection reagents (b)

Figure 3. The plot of IS intensity during 3day’s analysis