Cholesterol Stationary Phase in the Separation and Identification of siRNA Impurities by Two-Dimensional Liquid Chromatography-Mass Spectrometry
Abstract
:1. Introduction
2. Results and Discussion
2.1. Retention and Separation Studies for CHOL and AP Columns
2.2. Impurities Analysis with the Use of Q-TOF-MS
2.2.1. The Chemistry behind the Origin of Impurities
2.2.2. Antisense Strand of Patisiran
2.2.3. Sense Strand of Patisiran
2.3. Closely Related Impurities in the Main Peak of Sense and Antisense Strands
2.4. Selective Comprehensive (High-Resolution Sampling) sRP × HILIC-2DLC for Patisiran
3. Materials and Methods
3.1. Materials
3.2. Oligonucleotides
3.3. Instrumentation
3.4. LC-MS Conditions
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Stationary Phase | k | |||
---|---|---|---|---|
pH of 20 mM ammonium formate | ||||
4.5 | 5.5 | 6.3 | 7.5 | |
CHOL | - | 16.62 ± 0.21 | 15.23 ± 0.16 | 13.09 ± 0.09 |
AP | - | 13.22 ± 0.30 | 9.93 ± 0.02 | 7.15 ± 0.02 |
Concentration of ammonium formate | ||||
10 mM | 20 mM | 40 mM | ||
CHOL | 10.44 ± 0.11 | 15.23 ± 0.27 | 13.96 ± 0.20 | |
AP | 8.08 ± 0.18 | 9.93 ± 0.13 | 9.12 ± 0.08 | |
Type of salt | ||||
20 mM ammonium formate | 20 mM ammonium acetate | |||
CHOL | 15.23 ± 0.14 | 10.98 ± 0.25 | ||
AP | 9.93 ± 0.09 | 7.72 ± 0.13 | ||
Type of organic solvent | ||||
MeOH | ACN | |||
CHOL | 15.23 ± 0.29 | 14.17 ± 0.22 | ||
AP | 9.93 ± 0.018 | 8.18 ± 0.21 |
Ion (m/z) | EIC Peak Area | |
---|---|---|
pH 6.3 | pH 7.5 | |
Sense strand | ||
865.173 | 236,618 ± 1302 | 81,850 ± 591 |
1235.711 | 151,654 ± 973 | 68,514 ± 462 |
1155.188 | 66,958 ± 664 | 51,524 ± 349 |
1489.9089 | 87,257 ± 471 | 32,500 ± 588 |
1690.023 | 501,090 ± 1364 | 259,873 ± 1147 |
Antisense strand | ||
pH 6.3 | pH 7.5 | |
850.172 | 198,963 ± 1197 | 71,599 ± 759 |
902.147 | 201,337 ± 1372 | 98,004 ± 1024 |
1074.673 | 73,951± 1005 | 33,820 ± 1153 |
1247.197 | 89,620 ± 826 | 38,391 ± 944 |
1663.732 | 296,281 ± 1277 | 139,009 ± 1039 |
m/z Values for the Most Abundant Ion | Charge | Impurity | Impurity Sequence | Deconvoluted Mass (Da) | Retention Time CHOL pH 6.3 (min) | Retention Time CHOL pH 7.5 (min) |
---|---|---|---|---|---|---|
ANTISENSE STRAND | ||||||
652.082 | −1 | 3′ N-19as | 5′ AU 3′—PO | 653.082 | 8.46 | 5.06 |
979.118 | −1 | 3′ N-18as | 5′ AUG 3′—cyc | 980.118 | 10.76 | 8.68 |
850.170 | −1 | 5′ N-18as | 5′ CdTdT 3′—OH | 851.170 | 10.73 | 9.06 |
545.129 | −1 | 5′ N-19as | 5′ dTdT 3′—OH | 546.129 | 11.15 | 9.94 |
749.128 | −2 | 5′ N-16as | 5′ mUACdTdT 3′—OH | 1500.257 | 13.95 | 11.64 |
835.110 | −2 | 3′ N-16as | 5′ AUGGA 3′—PO | 1672.221 | 14.03 | 9.33 |
902.141 | −2 | 5′ N-15as | 5′ UmUACdTdT 3′—OH | 1806.282 | 14.06 | 11.56 |
1074.665 | −2 | 5′ N-14as | 5′ GCmUACdTdT 3′—OH | 2151.330 | 15.62 | 13.01 |
999.632 | −2 | 3′ N-15as | 5′ AUGGAA 3′—PO | 2001.264 | 16.11 | 10.73 |
1035.139 | −3 | 5′ N-11as | 5′ UUGGUmUACdTdT 3′—OH | 3108.418 | 16.35 | 13.6 |
831.122 | −3 | 5′ N-13as | 5′ GGUmUACdTdT 3′—OH | 2496.367 | 16.8 | 14.23 |
1136.820 | −3 | 5′ N-10as | 5′ CUUGGUmUACdTdT 3′—OH | 3413.459 | 17.04 | 14 |
1043.630 | −4 | 3′ N-8as | 5′ AUGGAAmUACUCUU 3′—PO | 4178.519 | 17.28 | 12.52 |
967.3734 | −4 | 3′ N-9as | 5′ AUGGAAmUAUUUU 3′—PO | 3873.496 | 17.28 | 12.51 |
928.867 | −4 | 5′ N-9as | 5′ UCUUGGUmUACdTdT 3′-OH | 3719.470 | 17.46 | 14.12 |
1005.132 | −4 | 5′ N-8as | 5′ CUCUUGGUmUACdTdT 3′—OH | 4024.528 | 17.46 | 13.78 |
882.455 | −3 | 3′ N-13as | 5′ AUGGAAmUA 3′—PO | 2650.364 | 17.66 | 12.56 |
1187.822 | −3 | 3′ N-10as | 5′ AUGGAAmUACUC 3′—PO | 3566.466 | 17.94 | 12.66 |
1167.402 | −4 | 5′ N-6as | 5′ mUACUCUUGGUmUACdTdT 3′—OH | 4673.609 | 18.21 | 14.16 |
984.135 | −3 | 3′ N-12as | 5′ AUGGAAmUAC 3′—PO | 2955.406 | 18.41 | 12.83 |
1129.892 | −4 | 3′ N-7as | 5′ AUGGAAmUACUCUUG 3′—PO | 4523.570 | 18.7 | 13.13 |
1249.665 | −4 | 5′ N-5as | 5′ AmUACUCUUGGCmUACdTdT 3′—OH | 5002.661 | 18.95 | 14.23 |
1216.152 | −4 | 3′ N-6as | 5′ AUGGAAmUACUCUUGG 3′—PO | 4868.606 | 19.04 | 13.55 |
1580.954 | −4 | 5′ N-1as | 5′ UGGAAmUACUCUUGGUmUACdTdT 3′—OH | 6327.814 | 19.16 | 15.28 |
1418.703 | −4 | 5′ N-3as | 5′ GAAmUACUCUUGGUmUACdTdT 3′—OH | 5676.783 | 19.29 | 15.46 |
1504.957 | −4 | 5′ N-2as | 5′ GGAAmUACUCUUGGUmUACdTdT 3′—OH | 6021.828 | 19.33 | 15.47 |
1531.187 | −4 | 3′ N-2as | 5′ AUGGAAmUACUCUUGGUmUAC 3′—PO | 6128.748 | 19.54 | 15.14 |
1454.931 | −4 | 3′ N-3as | 5′ AUGGAAmUACUCUUGGUmUA 3′—PO | 5823.723 | 19.74 | 14.92 |
1663.732 | −4 | Antisense | 5′ AUGGAAmUACUCUUGGUmUACdTdT 3′—OH | 6656.866 | 20.03 | 15.68 |
SENSE STRAND | ||||||
1011.145 | −1 | 3′ N-18s | 5′ GmUA 3′—PO | 1012.130 | 11.77 | 7.49 |
865.169 | −1 | 5′ N-18s | 5′ mUdTdT 3′—OH | 866.169 | 13.58 | 11.94 |
1340.212 | −1 | 3′ N-17s | 5′GmUAA 3′—PO | 1341.212 | 14.49 | 9.42 |
915.667 | −2 | 5′ N-15s | 5′ mCmCAmUdTdT 3′—OH | 1833.337 | 15.12 | 12.44 |
756.134 | −2 | 5′ N-16s | 5′ mCAmUdTdT 3′—OH | 1514.268 | 15.18 | 12.94 |
1075.689 | −2 | 5′ N-14 OHs | 5′ mUmCmCAmUdTdT 3′—OH | 2153.353 | 16.18 | 13.22 |
1115.676 | −2 | 5′ N-14 POs | 5′ mUmCmCAmUdTdT 3′—PO | 2233.352 | 16.67 | 16.62 |
1235.714 | −2 | 5′ N-13s | 5′ mUmUmCmCAmUdTdT 3′—OH | 2473.396 | 17.21 | 14.12 |
1153.181 | −2 | 3′ N-14s | 5′ GmUAAmCmCA 3′—PO | 2308.362 | 17.92 | 15.7 |
1395.741 | −2 | 5′ N-12 (A-mU)s | 5′ mUmUmUmCmCAmUdTdT 3′—OH | 2793.442 | 18.26 | 14.9 |
933.150 | −3 | 5′ N-12s | 5′ AmUmUmCmCAmUdTdT 3′—OH | 2802.450 | 18.65 | 15.33 |
1039.831 | −3 | 5′ N-11s | 5′ mUAmUmUmCmCAmUdTdT 3′—OH | 3122.492 | 18.79 | 15.48 |
1317.703 | −2 | 3′ N-13s | 5′ GmUAAmCmCAA 3′—Np | 2637.407 | 18.87 | 12.17 |
1154.857 | −3 | 5′ N-10s | 5′ GmUAmUmUmCmCAmUdTdT 3′—OH | 3467.530 | 19.14 | 15.7 |
1264.544 | −3 | 5′ N-9s | 5′ AGmUAmUmUmCmCAmUdTdT 3′—OH | 3796.590 | 20.31 | 16.95 |
1489.237 | −3 | 5′ N-7s | 5′ AGAGmUAmUmUmCmCAmUdTdT 3′—OH | 4470.688 | 20.4 | 16.63 |
1598.928 | −3 | 5′ N-6s | 5′ AAGAGmUAmUmUmCmCAmUdTdT 3′—OH | 4799.718 | 20.58 | 16.96 |
1103.173 | −3 | 3′ N-11s | 5′ GmUAAmCmCAAGA 3′—PO | 3311.495 | 20.62 | 13.39 |
1217.849 | −3 | 3′ N-10s | 5′ GmUAAmCmCAAGAG 3′—PO | 3656.547 | 20.69 | 13.59 |
1358.973 | −4 | 5′ N-4s | 5′ mCmCAAGAGmUAmUmUmCmCAmUdTdT 3′—OH | 5437.841 | 20.68 | 16.58 |
1603.513 | −4 | 5′ N-1s | 5′ mUAAmCmCAAGAGmUAmUmUmCmCAmUdTdT 3′—OH | 6416.030 | 21.08 | 17.07 |
1690.023 | −5 | Sense (guide strand) | 5′ GmUAAmCmCAAGAGmUAmUmUmCmCAmUdTdT 3′—OH | 6761.087 | 21.8 | 17.17 |
1441.236 | −4 | 5′ N-3s | 5′ AmCmCAAGAGmUAmUmUmCmCAmUdTdT 3′—OH | 5766.896 | 22.71 | 17.03 |
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Studzińska, S.; Li, F.; Szumski, M.; Buszewski, B.; Lämmerhofer, M. Cholesterol Stationary Phase in the Separation and Identification of siRNA Impurities by Two-Dimensional Liquid Chromatography-Mass Spectrometry. Int. J. Mol. Sci. 2022, 23, 14960. https://doi.org/10.3390/ijms232314960
Studzińska S, Li F, Szumski M, Buszewski B, Lämmerhofer M. Cholesterol Stationary Phase in the Separation and Identification of siRNA Impurities by Two-Dimensional Liquid Chromatography-Mass Spectrometry. International Journal of Molecular Sciences. 2022; 23(23):14960. https://doi.org/10.3390/ijms232314960
Chicago/Turabian StyleStudzińska, Sylwia, Feiyang Li, Michał Szumski, Bogusław Buszewski, and Michael Lämmerhofer. 2022. "Cholesterol Stationary Phase in the Separation and Identification of siRNA Impurities by Two-Dimensional Liquid Chromatography-Mass Spectrometry" International Journal of Molecular Sciences 23, no. 23: 14960. https://doi.org/10.3390/ijms232314960