Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

Application Note

Analysis of Oligonucleotide Impurities on the 
BioAccord System with ACQUITY Premier

Catalin E. Doneanu,

 Chris Knowles, Jonathan Fox, Emma Harry, Ying Qing Yu, Joseph Fredette, Weibin Chen

Waters Corporation

Abstract

This application note demonstrates an automated, compliance-ready LC-MS workflow for purity analysis and 

intact mass confirmation of extensively modified oligonucleotides and their impurities.

Benefits

A category of products incorporating the MaxPeak High Performance Surfaces (HPS), including the ACQUITY 

Premier UPLC BSM System and the ACQUITY Premier OST Columns, provide critical advantages for 

oligonucleotide impurity analysis

■

An automated, compliance-ready workflow is shown to provide good mass accuracy (better than 15 ppm) for 

intact mass confirmation of modified oligonucleotides and their impurities analyzed with an ion-pairing 

reversed-phase (IP-RP) LC-MS assay

■

In addition, this automated, compliance-ready workflow provides purity information for all the sample 

components, down to 0.2% abundance levels

■

Introduction

Oligonucleotide therapeutics have emerged in recent years as a powerful alternative to small molecule and 

protein therapeutics.1,2 Manufacturing and quality control of oligonucleotide therapeutics requires highly 

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

selective and sensitive LC-MS methods. One of the well-accepted mass spectrometry-based methods for 

oligonucleotide analysis is ion-pairing reversed-phase chromatography (IP-RP) separation followed by ESI-MS 

detection in negative ion mode. Using this method, an automated workflow for oligonucleotide analysis 

employing the BioAccord System – operating under compliance-ready waters_connect data acquisition and 

processing software – was recently described.3,4 A fully integrated LC-MS system comprised of an ACQUITY 

Premier UPLC BSM System, a Tunable Ultraviolet (TUV) Detector, and an ESI-Tof ACQUITY RDa Mass Detector 

(system shown in Figure 1) was used in this application note for the analysis of low-level oligonucleotide 

impurities. This is a challenging analysis because oligonucleotides contain many negatively charged phosphate 

groups prone to interact strongly with metal surfaces. To address these challenges, Waters has developed a 

family of new technologies containing a more inert surface specifically designed to address difficult to analyze 

analytes, MaxPeak High Performance Surfaces (HPS).5-10 The ACQUITY Premier UPLC BSM System has this 

technology implemented across the entire fluidic path in order to provide a very effective barrier that significantly 

reduces analyte interactions with all types of metal surfaces. Along with the ACQUITY Premier 

OST Columns introduced in 2020, the entire UPLC System was recently tested for bioanalysis related LC-MS 

applications of oligonucleotides.9 Here we investigated the capabilities of this UPLC system for intact mass 

confirmation of oligonucleotides and their associated impurities. All datasets were acquired in full scan MS mode 

on the BioAccord System with ACQUITY Premier and processed in waters_connect using the BayesSpray mass 

spectral charge deconvolution algorithm to produce accurate intact mass measurements for each compound.

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

Figure 1. Components of the BioAccord LC-MS System with ACQUITY Premier.

Experimental

Reagents and Sample Preparation

Triethylamine (TEA, 99.5% purity, catalogue number 65897-50ML) and methanol (LC-MS grade, catalogue 

number 34966-1L) were obtained from Honeywell (Charlotte, NC), while 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, 

99% purity, catalogue number 105228-100G) was purchased from Sigma Aldrich (St Louis, MO). HPLC grade 

deionized (DI) water was purified using a MilliQ system (Millipore, Bedford, MA). Mobile phases were prepared 

fresh and used on the same day. A 21-mer heavily modified oligonucleotide, containing a 2’-OMe modification on 

19 of its nucleosides, having the sequence GUA ACC AAG AGU AUU CCA UTT and the elemental composition C

229H306N76O143P20 was purchased from ATDBio (Southhampton, UK). Stock solutions were prepared in DI water 

at a concentration of 1 µM (or 2.34 µg/mL), from which a 10 µL volume was injected, which corresponds to 

loading 10 picomoles of the 21-mer oligonucleotide on-column. 

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

LC Conditions

LC-MS system:

BioAccord System with ACQUITY Premier

Columns:

1) ACQUITY Premier OST Column 1.7 µm, 130 Å, 2.1 

x 100 mm, (p/n: 186009485)

2) Conventional ACQUITY OST Column 1.7 µm, 130 

Å, 2.1 x 100 mm, (p/n: 186003950)

3) ACQUITY Premier OST Column 1.7 µm, 130 Å, 

2.1 x 50 mm, (p/n: 186009484)

Column temperature:

60 °C

Flow rate:

300 µL/min

Mobile phases:

Solvent A: 40 mM HFIP (hexafluoroisopropanol), 7 

mM TEA (triethylamine) in DI water

Solvent B: 20 mM HFIP (hexafluoroisopropanol), 

3.5 mM TEA (triethylamine) in 50% methanol

Sample temperature:

6 °C

Sample vial:

Certified Clear Glass Vials (p/n: 186000327C)

Injection volume:

10 µL

Wash solvents:

Purge solvent: 50% MeOH

Sample Manager wash solvent: 50% MeOH

Seal wash: 20% acetonitrile in DI water

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

Gradient

MS Conditions

Ionization mode:

ESI(-)

Capillary voltage:

0.8 kV

Cone voltage:

40 V

Source temperature:

120 °C

Desolvation temperature:

400 °C

Desolvation gas (N2) pressure:

6.5 bar

Tof mass range:

400–5000

Acquisition rate:

2 Hz

Lock-mass:

waters_connect Lockmass solution (p/n: 

186009298)

Data acquisition software:

waters_connect

Data processing software:

waters_connect

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

Results and Discussion

A 21-mer oligonucleotide containing a variety of low-level oligonucleotide impurities was separated on two C18 

columns, a regular 2.1 x 100 mm OST column (p/n: 186003950 <

https://www.waters.com/nextgen/us/en/shop/columns/186003950-acquity-uplc-oligonucleotide-beh-c18-

column-130a-17--m-21-mm-x-1.html> ) and a recently introduced ACQUITY Premier 2.1 x 100 mm OST Column 

(p/n: 186009485 <https://www.waters.com/nextgen/us/en/shop/columns/186009485-acquity-premier-

oligonucleotide-c18-column-130a-17--m-21-x-100-m.html> ). For the 21-nt oligomer, a significant portion of the 

molecule (19 nucleosides) contained modified nucleobases (as illustrated in the sequence listed in the 

experimental section and in Figure 2). The 2’-OMe modification was attached to three guanosines (G) labeled in 

blue and seven adenosines (A) labeled in green in the oligonucleotide sequence. Besides the attachment of the 

same 2’-OMe functional group to uridines and cytidines, the nucleobases of these two nucleosides were further 

modified by the attachment of a 5-Methyl group to produce five 2’-OMe 5-Me uridines (U, labeled in purple) and 

four 2’-OMe 5-Me cytidines (C, labeled in red). The only nucleosides left unmodified are the two deoxythymidines 

(TT) at the 3’-end of the 21-mer. All the oligonucleotide modifications can be summarized in the following 

sequence: OMEG OME5mU OMEA OMEA OME5mC OME5mC OMEA OMEA OMEG OMEA OMEG OME5mU 

OMEA OME5mU OME5mU OME5mC OME5mC OMEA OME5mU dT dT, which uses a specially designed 

nomenclature. When analyzed on the ACQUITY Premier OST Column, the separation of the 21-nt oligonucleotide 

reveals a rather complex impurity profile as shown in Figure 2A. Fourteen oligonucleotide impurities were 

separated and detected by both TUV and MS detectors. Analysis of the same sample on a conventional column 

(with stainless-steel casing), having the same dimensions and packed with the same stationary phase (C18 1.7 µm 

particles, 130 Å pores), resolved only half (seven) of the same impurities, completely missing a significant portion 

of the early eluting impurities, as indicated in Figure 2B. A recent publication7 indicated that untreated column 

frits are mainly responsible for analyte adsorption on metal surfaces. The results presented in Figure 2B can be 

explained by considering the adsorption effects of oligonucleotides on the inlet and outlet frit of a conventional 

column. When the sample is loaded at the inlet of the column, the major oligonucleotide component can be used 

to passivate the inlet frit, such that minor oligonucleotide impurities are not adsorbed to this frit. However, after 

undergoing the IP-RP separations, because most of the oligonucleotide impurities elute before the major 

component, there is a great possibility that the outlet frit would retain some of these impurities until it gets fully 

passivated. It is very likely that in the example shown in Figure 2B, the first seven early eluting impurities were 

not detected because they were adsorbed to the outlet frit.

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

 Figure 2. TUV chromatograms 

showing the separation of oligonucleotide impurities from a 21-nt sample: (A) UV chromatogram recorded on an 

ACQUITY Premier OST Column; (B) UV chromatogram recorded on an extensively conditioned conventional 

column. The red traces from each figure correspond to the blanks preceding each injection; (3) Three replicates 

injections performed on the ACQUITY Premier OST Column. For better clarity, the red and blue traces are offset 

by +0.2 min from the previous trace. 

The ACQUITY Premier OST Column belongs to a family of columns packed with sub 2 µm particles, featuring the 

MaxPeak High Performance Surfaces (HPS) Technology.8-10 Oligonucleotides contain a negatively charged 

phosphate backbone known to interact with metal surfaces (like stainless-steel, titanium, or MP35N—a Ni-Co 

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

alloy) typically found in the fluidic path of the UPLC system. These interactions are often responsible for 

oligonucleotide losses, poor chromatographic peak shapes, or poor data reproducibility. The MaxPeak HPS 

Technology implemented along the UPLC fluidic path and the OST Column significantly reduced these 

unwanted interactions, as demonstrated by the number and % abundance (related to UV peak areas) of 

impurities detected. The conventional OST Column showed much more modest results in terms of impurity 

recovery, even after extensive passivation. The separations performed on the ACQUITY Premier Column are 

highly reproducible, as shown in Figure 2C where three replicate injections are overlaid. The ESI-MS spectrum of 

the major oligonucleotide component is presented in Figure 3A. The lowest abundance impurity (0.18% 

according to the UV data, see Table 1), confidently identified based on its ESI-MS spectrum, is an 11-mer 

oligonucleotide missing all 10 nucleosides from the 5’-end of the molecule. The ESI-MS spectrum of this impurity 

showing two major charge states (doubly and triply charged ions) is displayed in Figure 3B in the bottom panel. 

There was no signal detected for this impurity in UV (see Figure 2B) and the ESI-MS spectrum recorded at the 

expected elution time (top panel of Figure 3B) indicates that, very likely, this impurity was trapped (irreversibly 

adsorbed) by metal surfaces inside the regular column (outlet column frit very likely), as the 

same ACQUITY Premier UPLC System was used in both experiments. The full list of oligonucleotide impurities 

detected in the 21-mer analyzed here is displayed in Table 1, along with their sequences, elemental compositions, 

accurate average masses, and percent abundances calculated using the UV peak areas. The data presented in 

Table 1 was used for setting up an automated processing using the BayesSpray charge deconvolution algorithm 

from waters_connect and Figure 4 illustrates the processing results obtained. All fourteen oligonucleotide 

impurities were detected across a relatively wide dynamic range (~500 fold) with deconvoluted mass accuracies 

under 15 ppm.

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

Figure 3. Ion pairing reversed-phase (IP-RP) ESI-MS spectra recorded for: (A) the most abundant sample 

component, the 21-mer heavily modified oligonucleotide; (B) the least abundant sample component, an 11-mer 

oligonucleotide impurity (see Table 1 for its sequence), present at 0.18% according to the UV peak area 

measurement. All the oligonucleotide modifications are summarized using a specially designed nomenclature as 

shown in Figure 3A.

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

Table 1. Fourteen oligonucleotide impurities identified in a 21-mer extensively modified oligonucleotide.

Figure 4. Screenshot showing the waters_connect processing results obtained after BayesSpray charge 

deconvolution of the ESI-MS spectra recorded for the 21-mer major component and fourteen of its 

oligonucleotide impurities. The mass accuracy error for measuring the accurate average masses was better than 

15 ppm for all sample components. The row corresponding to the main component is highlighted in blue and 

indicates a purity of 83.03%, while the abundance of the lowest abundant species (an 11-mer oligo) was 0.18% 

(highlighted by a red circle).

While it is important to be able to detect low-level oligonucleotide impurities with both UV and MS detectors, it is 

equally important to measure their abundances accurately. In other words, it is critical that these impurities 

produce a linear response in UV over the same dynamic range (~500 fold). To test the linearity of the UV assay, 

the 21-mer oligonucleotide was diluted and analyzed on a shorter ACQUITY Premier Column (2.1 x 50 mm, p/n: 

186009484 <https://www.waters.com/nextgen/us/en/shop/columns/186009484-acquity-premier-

oligonucleotide-c18-column-130a-17--m-21-x-50-mm.html> ) using faster (15-min) LC-MS runs. The UV 

chromatogram recorded for lowest detected concentration of 0.5 nM, corresponding to a 1:2000 dilution of the 

stock sample (1 µM), is shown in Figure 5A along with the preceding blank injection. Four replicate injections of 

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

the same solution (0.5 nM or 1.17 ng/mL) have peak area RSDs under 15%, demonstrating that low-level 

oligonucleotides can be analyzed reproducibly on the ACQUITY Premier Columns as highlighted in Figure 5B. A 

calibration plot, showing the UV response (peak area) for a wide range of concentrations of the 21-nt oligomer 

(eight concentrations in the range of 0.5 nM to 1000 nM), is presented in Figure 6. Taken together, the data 

presented in Figures 5A, B, and Figure 6 clearly proves that the 21-mer oligonucleotide displays a linear 

chromatographic behavior over a wide dynamic range (2000 fold) and has very good column recoveries even at 

the lowest concentration - 0.5 nM, which corresponds to 5 femtomoles (or ~12 picograms)  of oligonucleotide 

loaded on-column. Finally, the carryover of the assay was evaluated by the injection of a Solvent A blank (10 µL) 

right after the last replicate of the highest oligonucleotide concentration analyzed (1 µM). The result is displayed 

in Figure 5C and it emphasizes the inertness of the ACQUITY Premier UPLC System and Column: there is no 

detectable signal for the 21-mer oligonucleotide following the injection of a large amount (10 picomoles) on-

column.

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

 Figure 5. Testing the inertness 

of the ACQUITY Premier UPLC System and ACQUITY Premier OST Column using the 21-nt oligomer: (A) UV 

chromatogram of the lowest detectable concentration (0.5 nM, or 5 femtomoles of the 21-mer oligonucleotide 

loaded on-column) compared against the preceding blank injection; (B) replicate UV chromatograms obtained 

for the lowest detectable concentration (0.5 nM), indicating that the UV peak areas had RSDs below 15%; (C) 

carryover evaluation, showing the UV chromatogram of a blank injection (red trace) following the injection of the 

highest sample concentration (1000 nM or 10 picomole oligonucleotide loaded on-column). There is no 

detectable signal from the 21-mer in the blank, suggesting that the UPLC system and column do not retain any 

analyte through non-specific adsorption to the various coated metal surfaces found in the fluidic flow-path.

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

Figure 6. Calibration curve of the 21-mer oligonucleotide showing linearity over three orders of 

magnitude. Peak areas obtained from the TUV detector were plotted against a wide range of 

oligonucleotide concentrations, including 0.5, 1, 5, 10, 20, 100, 200, and 1000 nM. Excellent signal 

linearity was obtained for this assay, indicating that very low oligonucleotide concentrations can be 

recovered completely from a very inert LC system that does not interact in any way with the analyte. 

The inset shows the calibration curve in the 0.5–10 nM concentration range.

In conclusion, the BioAccord System with ACQUITY Premier is capable of measuring and accurately detecting 

oligonucleotide impurities down to 0.2% and also provides intact mass confirmation capabilities via an 

automated, compliance-ready workflow using the waters_connect Software.

Conclusion

A category of products incorporating the MaxPeak High Performance Surfaces (HPS) – including the 

ACQUITY Premier UPLC BSM System and the ACQUITY Premier OST Columns – provide critical advantages 

for oligonucleotide impurity analysis

■

Improved oligonucleotide analysis in terms of low detection limit and chromatographic reproducibility is 

demonstrated using the BioAccord LC-MS System with ACQUITY Premier operated under compliant-ready 

■

13

Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

software

The oligonucleotide impurity analysis workflow provides mass confirmation for oligonucleotide impurities as 

well as their relative abundance. The results from our study indicate that the LC-MS platform provides good 

mass accuracy (better than 15 ppm) for intact mass confirmation of modified oligonucleotides and their 

impurities, while the LC-UV information allows for the measurements of all the sample components, down to 

0.2% relative abundance levels

■

References

Sharma VK, Watts JK Oligonucleotide Therapeutics: Chemistry, Delivery and Clinical Progress, Future Med 

Chem, 2015, 7(16), 2221–2242.

1. 

Roberts TK, Langer R, Wood MJA Advances in Oligonucleotide Drug Delivery, Nat Reviews, 2020, 19, 673–694.

2. 

Catalin E. Doneanu, Jonathan Fox, Emma Harry, Chris Knowles, Ying Qing Yu, Joseph Fredette, Weibin Chen. 

An Automated Compliance-Ready LC-MS Workflow for Intact Mass Confirmation and Purity Analysis of 

Oligonucleotides, 2020, Waters Application Note, 720006820EN.

3. 

Catalin E. Doneanu, Jonathan Fox, Emma Harry, Chris Knowles, Ying Qing Yu, Joseph Fredette, Weibin Chen. 

Intact Mass Confirmation Analysis on the BioAccord LC-MS System for a Variety of Extensively Modified 

Oligonucleotides, 2020, Waters Application Note, 720007028EN.

4. 

M. Lauber, T. H. Walter, M. Gilar, M. DeLano, C. Boissel, K. Smith, R. Birdsall, P. Rainville, J. Belanger, and K. 

Wyndham. Low Adsorption HPLC Columns Based on MaxPeak High Performances Surfaces, 2020, Waters 

White Paper, 720006930EN <https://www.waters.com/webassets/cms/library/docs/720006930en.pdf> .

5. 

DeLano M, Walter TH, Lauber MA, Gilar M, Jung MC, Nguyen J, Boissel C, Patel AV, Bates-Harrison A, 

Wyndham K Using Hybrid Organic-Inorganic Surface Technology to Mitigate Analyte Interactions with Metal 

Surfaces in UHPLC, Anal Chem, 2021, 93, 5773–5781.

6. 

Gilar M, DeLano M, Gritti F Mitigation of Analyte Loss on Metal Surfaces in liquid chromatography, J Chrom A, 

2021, 1650, 5773–5781.

7. 

Amit Patel, Jennifer Simeone, Mathew Delano, Jason Dyke, Susan C. Rzewuski, Moon Chul Jung, Stephen J. 

Shiner. Waters Premier Standards to Investigate the Inertness of Chromatographic Surfaces, 2021, Waters 

Application Note, 720007105EN.

8. 

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier

Kathryn Brennan, Mary Trudeau, Paul D. Rainville. Utilization of the ACQUITY Premier System and Column for 

Improved Oligonucleotide Bioanalytical Chromatographic Performance, 2021, Waters Application Note, 

720007119EN.

9. 

Brooke M. Koshel, Robert E. Birdsall, Ying Qing Yu. Improving Recovery and Quantitation of Oligonucleotide 

Impurities Using the ACQUITY Premier with MaxPeak HPS Technology, 2021, Waters Application Note, 

720007238EN.

10. 

Featured Products

BioAccord LC-MS System with ACQUITY Premier <https://www.waters.com/waters/nav.htm?cid=135087537

>

■

ACQUITY Premier System <https://www.waters.com/waters/nav.htm?cid=135077739>

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ACQUITY UPLC Tunable UV Detector <https://www.waters.com/514228>

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ACQUITY RDa Detector <https://www.waters.com/waters/nav.htm?cid=135077027>

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waters_connect <https://www.waters.com/waters/nav.htm?cid=135040165>

■

720007301, July 2021

© 2021 Waters Corporation. All Rights Reserved.

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Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier