Characterize Your Oligos With Confidence
App Note / Case Study
Last Updated: September 14, 2023
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Published: May 22, 2023
Credit: iStock
Oligo synthesis can result in the formation of impurities. Hence, final products must be characterized to ensure they meet quality assurance and control standards.
While LC/MS methods offer efficient solutions for oligo characterization, ion-pairing reagents can suppress ionization in positive mode polarity and decrease system performance. Therefore, most methods require a dedicated negative mode polarity system.
Discover an effective method for LC separation and high-resolution mass data collection that is free from ion-pairing reagents.
Download this app note to learn how you can:
- Achieve precise and accurate QC without the need for extensive system maintenance and cleaning
- Separate a wide variety of sample types in only 15 minutes
- Achieve excellent retention time reproducibility and faster system re-equilibration
Application Note
BioPharma
Authors
Peter Rye and Cody Schwarzer
Agilent Technologies, Inc.
Introduction
The synthesis of RNA and DNA oligonucleotides (oligos) is an iterative process that,
despite highly optimized chemistry, results in impurities that must be characterized
for research and quality assurance and quality control (QA/QC) efforts. While many
techniques exist for analyzing complex oligo samples, LC/MS offers both efficient
chromatographic separation and identification capabilities through high‑resolution
accurate mass information.
IP-RP chromatography has traditionally been used for LC/MS analysis of oligos for
sensitivity and exceptional chromatographic resolution.1–7 However, these methods
often require a dedicated negative mode polarity system, since the memory effect
of the ion-pairing reagents can suppress ionization in positive mode and decrease
performance in that polarity. For mixed-use systems where positive mode data
collection is required, the use of positively charged ion-pairing reagents is often
not acceptable.
MS1 Oligonucleotide Characterization
Using LC/Q-TOF with
HILIC Chromatography
2
In this application note, LC separation and MS1 mass
identification of a variety of oligos without the use of
ion‑pairing reagents is demonstrated. The LC separation
allows subsequent positive mode use with little to no
flushing or hardware changes. This HILIC-based method
uses an Agilent InfintyLab Poroshell 120 HILIC-Z column
and MS-friendly ammonium acetate-based mobile phases.
The samples were analyzed on an Agilent 1290 Infinity II LC
system and a 6545XT AdvanceBio quadrupole time-of-flight
mass spectrometer (LC/Q-TOF).
Experimental
Instrumentation
– Agilent 1290 Infinity II LC including:
– Agilent 1290 Infinity II high-speed pump (G7120A)
– Agilent 1290 Infinity II multisampler (G7167B) with the
optional sample cooler (G7167-60005)
– Agilent 1290 Infinity II multicolumn thermostat
(G7116B)
– Agilent InfintyLab Poroshell 120 HILIC-Z column,
2.1 × 100 mm, 1.9 µm (part number 685675-924)
– Agilent 6545XT AdvanceBio LC/Q-TOF equipped with an
Agilent dual spray Jet Stream source
Materials
Oligos used in this study (Table 1) were purchased through
Integrated DNA Technologies (Coralville, Iowa) and purified
by standard desalting. InfinityLab Ultrapure LC/MS solvents
were provided by Agilent Technologies (Santa Clara, CA) and
LC/MS-grade ammonium acetate, ammonium hydroxide,
and acetic acid were purchased through Sigma-Aldrich
(St. Louis, MO).
Sample preparation
Oligo samples were resuspended to 100 µM in water and
stored at –80 °C. For analysis, oligo aliquots were diluted to
5 to 10 µM (experiment dependent) in polypropylene vials
using mobile phase A (see Table 2) and stored in the chilled
autosampler for up to 2 days.
LC/MS analysis
The LC/MS methods used in this study are described
in Table 2. Mobile phases were prepared by first adding
the desired amount of ammonium acetate to the water
component and then adding acetonitrile to the final
composition. These solutions were prepared just before
acquisition and were used for up to 2 days.
Name Length
Approximate Average
Molecular Weight (Da) Sequence
PRL20 20 6,108 CTAGTTACTTGCTCAGCGGA
PRL40 40 12,278 CTAGTTACTTGCTCAGCGGACTAGTTACTTGCTCAGCGGA
PRL60 60 18,448 CTAGTTACTTGCTCAGCGGACTAGTTACTTGCTCAGCGGACTAGTTACTTGCTCAGCGGA
PRL80 80 24,617 CTAGTTACTTGCTCAGCGGACTAGTTACTTGCTCAGCGGACTAGTTACTTGCTCAGCGGACTAGTTACTTGCTCAGCGGA
PRL100 100 30,787 CTAGTTACTTGCTCAGCGGACTAGTTACTTGCTCAGCGGACTAGTTACTTGCTCAGCGGACTAGTTACTTGCTCAGCGGACTAGTTACTTGCTCAGCGGA
PR1 20 6,148 AGAGTTTGATCCTGGCTCAG
PR2 20 6,103 GGCCACGCGTCGACTAGTAC
PR3 20 6,007, 6,031, 6,047 TTTTTTTTTTTTTTTTTTTV
PR4 38 11,564, 11,588, 11,604 GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTTV
PR5 24 7,289 CGCCAGGGTTTTCCCAGTCACGAC
PR6 20 6,204 CTTGGGTGGAGAGGCTATTC
PR7 21 6,102 /5Phos/TTTTTTTTTTTTTTTTTTTT
PR8 18 5,505 CTAGTTATTGCTCAGCGG
PR9 23 7,145 CCGGGAGCTGCATGTGTCAGAGG
Aptamer 28 9,116 /52FC/mGmGrArA/i2FU//i2FC/mAmG/i2FU/mGmAmA/i2FU/mG/i2FC//
i2FU//i2FU/mA/i2FU/mA/i2FC/mA/i2FU//i2FC//i2FC/mG/3InvdT/
siRNA sense 18 8,111 mGmCrAmGrUmGrUrUrArArUmArUmCrGmCrUmUrUrGrUrGrAmAG
siRNA antisense 18 8,633 rCrUmUrCrArCrArAmArGmCrGmArUrArUrUrArArCrArCmUrGmCmCmU
Code Description
A 2'-deoxyribose adenine
C 2'-deoxyribose cytosine
G 2'-deoxyribose guanine
T 2'-deoxyribose thymine
mA 2'-O-methyl A
mC 2'-O-methyl C
mG 2'-O-methyl G
mU 2'-O-methyl U
rA Ribose adenine
rC Ribose cytosine
rG Ribose guanosine
rU Ribose uradine
V Mixed C, A, and G
/3InvdT/ 3' Inverted T
/32MOErG/ 3' Methoxyethoxy G
/5Phos/ 5' Phosphate
/52FC/ 5' 2’-Fluoro C
/52MOErT/ 5' 2-Methoxyethoxy T
/i2FC/ Internal 2'-fluoro C
/i2FU/ Internal 2'-fluoro U
Table 1. Oligos used in this study and their associated code notations. All sequences are written in the 5' to 3' orientation.
3
The gradient consisted of a 1 minute hold at 15% mobile
phase B (MPB), then ramping to 40% MPB at 10 minutes, then
increasing to 60% at 13 minutes. The gradient was returned
to starting conditions at 14 minutes and held for 1 minute.
The column was equilibrated for a total of 10 minutes
(1 minute in the gradient plus 9 minutes of post time) before
the next injection. This 15 minute gradient proved suitable
for chromatographic resolution of our oligo ladder (20 to
100-mer range) but could easily be modified based on
separation needs.
HILIC separations were performed on a 1290 Infinity II LC
system. The column used was an InfinityLab Poroshell 120
HILIC-Z. Mass spectrometric detection was performed on a
6545XT AdvanceBio LC/Q-TOF with a dual spray Jet Stream
source. Results were analyzed using Agilent MassHunter
BioConfirm software 12.0 and Qualitative Analysis
software 11.0.
Results and discussion
Several factors were considered for the preliminary
chromatographic method including the ionic strength of the
mobile phases as well as the HILIC column choice. Previous
work has established that the concentration of ammonium
acetate (AA) used in HILIC-based mobile phases can affect
oligo retention, peak shape, and MS signal.8
Review of
these findings led us to conclude that 15 mM AA provides a
favorable balance of multiple LC/MS performance criteria.
Our own experiments corroborated these results (data not
shown) and 15 mM AA, in both mobile phase A (MPA) and
B (MPB), was chosen for subsequent studies in the future.
The InfinityLab Poroshell 120 HILIC-Z material, which uses a
novel zwitterionic phase, was chosen because of its excellent
tolerance to a wide pH range beyond what traditional silica
Agilent 1290 Infinity II LC Conditions
Column InfinityLab Poroshell 120 HILIC-Z, 2.1 × 100 mm, 1.9 µm
(p/n 685675-924)
Column Temperature 30 ºC
Injection Volume 2 to 5 µL
Autosampler
Temperature 4 °C
Needle Wash Methanol/water 50/50
Mobile Phase A) 70% acetonitrile : 30% water + 15 mM ammonium acetate
B) 30% acetonitrile : 70% water + 15 mM ammonium acetate
Flow Rate 0.4 mL/min
Gradient Program
Time (min) B (%)
1.00 15
11.00 40
13.00 60
14.00 15
15.00 15
Stop Time 15.00 min
Post Time 9.00 min
6545 XT AdvanceBio LC/Q-TOF Conditions
Ion Polarity Dual AJS Negative
Data Storage Both (centroid and profile)
Gas Temperature 350 ºC
Drying Gas Flow 13 L/min
Nebulizer Gas 35 psi
Sheath Gas Temperature 400 ºC
Sheath Gas Flow 12 L/min
Capillary Voltage 4,500 V
Nozzle Voltage 2,000 V
Fragmentor 180 V
Skimmer 65 V
Oct 1 RF Vpp 750 V
Mass Range 400 to 3,200 m/z
Acquisition Rate 1 spectra/sec
Table 2. LC/MS methods used in this study.
4
particles normally provide. This wide range was considered
advantageous because recent literature9
indicates that
mobile phases with elevated pH (e.g., pH 9) promote oligos
of different chemical makeup to have similar charge state
distributions and subsequent column separation. In support
of these findings, mobile phases at pH 9 yielded better MS
sensitivity and chromatographic performance than those
at pH 7 for a number of oligos. Ultimately, even though the
column could tolerate these high pHs, neutral pH mobile
phases were chosen for subsequent studies. The experiments
showed that the results at pH 7 were more than acceptable
and easier to reproduce.
Chromatographic resolution and spectral deconvolution
of oligo ladder species
To evaluate the ability of the optimized method to separate
oligos over a wide mass range and provide high-quality
data for impurities analysis, an oligo ladder standard was
injected. The oligo standard consisted of a 20, 40, 60, 80, and
100‑mer DNA (25 pmol/each on column). Baseline separation
of the first four components was achieved, and the 80 and
100‑mer exhibited slight overlap (see Figure 1). This overlap
proved not to be problematic for intact mass determination
or impurities assessment of any oligo. The unique extraction
and deconvolution of all five species was easily accomplished
by automatic peak spectrum background subtraction, defined
by the average of spectra at peak start and end. As expected,
low-abundance depurination and depyrimidination impurities
were easily identified across each oligo in the series. Figure 2
shows the deconvoluted results for the 20 and 100‑mer,
showing intact mass determinations and a multitude of
low‑abundance impurities in these samples. A deeper dive
into impurities assignments was subsequently conducted and
is described in more detail in the section titled “Identification
of low abundance impurities".
0
1
2
3
4
5
6
7
8
Acquisition time (min)
2 3 4 5 6 7 8 9 10
20-mer
40-mer
60-mer
80-mer 100-mer
×105 Counts
Figure 1. Extracted ion chromatogram overlay for a 20, 40, 60, 80,
100‑mer ladder.
0
1
2
3
4
5
6
7
8 30,787
30,137
30,000 31,000 32,000
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
A B 6,108
Deconvoluted mass (amu)
5,000 6,000 7,000
×104
×106
Counts
Deconvoluted mass (amu)
Counts
Figure 2. Spectral deconvolution of a 20-mer (PRL20, left) and 100-mer
(PRL100, right), showing the applicability of the LC/MS method for intact
mass and impurity determinations for a wide distribution of oligo lengths.
MS data from oligos of different size
While the LC/MS method was able to chromatographically
resolve a wide size range of oligos, a decreasing response
was observed with increasing analyte length. Figure 3 shows
how the deconvoluted peak height decreases significantly
with oligo size, beyond what is expected based on comparing
the number of molecules on column.
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
5,000 10,000 15,000 20,000 25,000 30,000
20-mer
40-mer
60-mer 80-mer 100-mer
Deconvoluted
(overlaid)
×106
Deconvoluted mass (amu)
Counts
Figure 3. Overlay of deconvoluted spectra from a 20, 40, 60, 80, and 100‑mer
DNA strand.
Inspection of the mass to charge (m/z) spectra from the
HILIC method (Figure 4A) shed some light on this trend,
especially when compared to m/z data collected on the
same samples using IP-RP conditions (Figure 4B). The m/z
data from the IP-RP conditions show a characteristic wide
distribution of charge states for each oligo that include both
relatively high and low charged species (at low and high
m/z, respectively). Comparison of the most predominant ion
intensity for each sample shows a difference of 4-fold (54
for the 100‑mer to 25
for the 60‑mer). The results indicate
relatively little difference in desolvating these samples under
IP-RP conditions.
5
In contrast, the m/z spectra from the HILIC method display
a narrow distribution of charge states that are shifted
heavily towards the lower charge states (higher m/z). These
observations are discussed in depth in the following section.
Also, comparison of the most predominant ion intensity for
each sample shows a difference of greater than a 10‑fold
(33
for the 100‑mer to 44
for the 20‑mer). These results
indicate that larger oligos are harder to desolvate under these
conditions. The combination of low ion number and dwindling
intensity for the larger oligo ions helps explain the significantly
lower deconvoluted heights for the larger oligos. These
behaviors may challenge the utility of this specific method on
longer samples. Separately, the right shifting of the ion m/z
range for oligos of increasing size highlights the importance
of an extended m/z range on the mass spectrometer.
Evaluation of secondary structure with the HILIC method
Interestingly, the m/z results from the HILIC method are
consistent with what is often observed for biomolecules
under native conditions. These results show a narrower
charge state distribution focused around lower charged
species (as compared to denaturing conditions). Based on
these observations, the HILIC LC/MS data for three samples
with significantly different potential for forming higher-order
structures were compared. The comparison included a
poly dT oligo (PR7, with low potential for self-annealing), an
oligo containing a variety of building blocks (PR1, with some
potential for self-annealing), and an siRNA duplex composed
of complementary 18‑mers (siRNA, with high potential for
self‑annealing). Several data features were compared for each
of these samples.
First, since chromatographic peak widths can be influenced
by molecules with structural diversity, the total ion
chromatograms were compared. As can be seen in Figure 5,
the peak widths increase from PR7 (top left) to the siRNA
(top right). Interestingly, even though PR1 is one building
block shorter than PR7, its peak width is larger because the
mixed base composition allows for higher-order structures
(e.g., hairpins and homodimers). This composition produces
a structural mixture that spreads on column. Next, it was not
a surprise that the siRNA peak is broad and nonsymmetrical
because it is composed of an array of single and double
stranded formations.
Second, the m/z spectra for the three samples were
compared. The spectrum for the oligo sample with low
potential for higher-order structures (PR7, middle left)
displayed a wide charge state distribution composed of
highly charged ions. In contrast, the spectra for PR1 and
siRNA, which can form higher-order structures, shows narrow
charge state distributions of lesser relative charge. These
observations are consistent with higher-order structures
being preserved and have been described elsewhere on linear
versus hairpin forming strands.10
–4
–6
–8
–9
–10
20-mer
40-mer
60-mer
80-mer
100-mer
20-mer
40-mer
60-mer
80-mer
100-mer
×103
×103
×103
×104
×104 ×104
×104
×105
×105
×105
Counts
Counts
Mass-to-charge (m/z) Mass-to-charge (m/z)
0
0
0
0
1
1.0
2
0
1
0
1
0
1
2
5.0
0
5
0
5
1,400 1,800 2,200 2,600 3,000 3,400 3,800
0
2
2
2.5
0.5
4
800 1,200 1,600 2,000 2,400 2,800
Figure 4. Raw m/z spectra for 20, 40, 60, 80, and 100-mers, run under HILIC conditions (left) and IP-RP conditions (right).
6
Third, the deconvoluted spectra for the three samples were
compared. The predominant peaks for PR7 and PR1 matched
expectations (6,102 and 6,148 Da, respectively). The most
striking result was observed for the siRNA (bottom right)
in which the main peak matched the mass of the duplex
(16,744 Da). Results for this sample also showed peaks
for the individual sense and antisense strands at 8,111 and
8,633 Da, respectively. However, the height of the peak at
16,744 Da was approximately 3-fold higher than those for the
individual strands, consistent with higher-order structures
being preserved throughout the entire analysis.
Column equilibration and retention time reproducibility
For HILIC methods, inadequate stability of peak retention
times and areas can result from inadequate re-equilibration or
poor column properties.11,12 Moreover, some have described
HILIC re-equilibration times as exceptionally long.13 It has
been hypothesized that the InfinityLab Poroshell 120 HILIC-Z
column would afford relatively fast re-equilibration time
because the particles are superficially porous, constructed
of a solid silica core and a porous outer layer. This design
Mass-to-charge (m/z)
0
0.2
0.4
0.6
0.8
1.0
Counts
1,400 1,800 2,200 2,600 3,000
m/z spectrum
for siRNA
×104
0
0.2
0.4
0.6
0.8
1.0
400 800 1,200 1,600 2,000 2,400 2,800
Mass-to-charge (m/z)
m/z spectrum for PR1
–4
–5
Counts
×105
0
0.5
1.0
1.5
2.0
2.5
Mass-to-charge (m/z)
600 1,000 1,400 1,800 2,200 2,600 3,000
m/z spectrum for PR7
–4
–5
–6
–7
–8
–9 –3
Counts
×105
Acquisition time (min)
2
3
4
5
6
Counts
3 4 5 6 7 8 9 10
Chromatogram
for siRNA
×105
Acquisition time (min)
0
1
2
3
4
5
6
3 4 5 6 7 8 9 10
Chromatogram for PR1
Counts
×105
0
1
2
3
4
Acquisition time (min)
3 4 5 6 7 8 9 10
Chromatogram for PR7
Counts
×106
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Counts (%)
Deconvoluted mass (amu)
8,000 10,000 12,000 14,000 16,000
Deconvoluted spectrum for siRNA
Sense
strand
Antisense
strand
siRNA
duplex
×102
0
0.2
0.4
0.6
0.8
1.0
5,000 6,000 7,000 8,000 9,000
Deconvoluted mass (amu)
Deconvoluted spectrum for PR1
Counts
×106
0
1.0
2.0
3.0
4.0
Deconvoluted mass (amu)
5,000 6,000 7,000 8,000 9,000 10,00011,000
Deconvoluted spectrum for PR7
Counts
×107
Figure 5. Comparison of LC/MS data for samples (organized by column) with different potentials for higher-order structures.
shortens the diffusion path in/out of the particle and enables
higher resolution, faster resolution, and faster re-equilibration.
To evaluate the retention time stability that could be achieved
with a relatively short re-equilibration time, 10 replicate
injections (20 pmol on column) of PR1 were conducted. The
injections were run with just 10 minutes of initial condition
time between each. The method resulted in excellent peak
retention time stability with an RT RSD of <0.1% (Figure 6).
In addition, shorter equilibration times were possible after two
initial preconditioning runs, which likely allowed a dynamic
equilibrium to be achieved. The effect can be observed in
Figure 6 where the baseline is initially elevated but rapidly
decreases. These preconditioning runs avoid the need for a
full equilibration of the column, which can take a long time
depending on column size and flow rate.12 By including these
initial preconditioning injections, it was found that that further
decreasing the equilibration time to a total of 5 minutes only
marginally increased the RSD of the RT over five injections to
<0.2% (data not shown). The peak area reproducibility for both
the standard and short equilibration times was calculated to a
<2% RSD following the two preconditioning runs.
7
For more information regarding HILIC method development
and optimization, please refer to technical overview
5991-9271EN: Hydrophilic Interaction Chromatography
Method Development and Troubleshooting.
Identification of low abundance impurities
A common technique for the identification and relative
quantification of product-related impurities takes advantage
of the high dynamic range offered by modern mass
spectrometers, such as the 6545XT AdvanceBio LC/Q-TOF.
This system displays up to five orders of in-spectra dynamic
range. To evaluate the ability of the method to detect low
abundance impurities in the presence of a highly abundant
target, PR7 was injected (10 µM, 50 pmol on column).
The resulting data were processed in Agilent MassHunter
BioConfirm software 12.0 using both targeted and untargeted
methods. The untargeted method was optimized to match
and label truncations, depyrimidations, and loss of the
terminal phosphate group from a single deconvolution result
(from a single chromatographic peak). The deconvolution
results are shown in Figure 7. The full-length product (FLP)
was determined at 6,102 Da and multiple low-abundance
impurities were identified. The impurities included products
with loss of 3' T (–304 mu, 0.52% of FLP), loss of the
5' phosphate (–81 mu, 0.28% of FLP), and the gas phase
depurination of T (–126 mu, 0.26% of FLP). The targeted
method was optimized to interrogate depyrimidations, loss
of the terminal phosphate, and all possible truncations using
Find-by-Formula. In this case, 49 impurities were found in the
m/z data, some of which had abundances well under 0.1%
of the FLP (data not shown). These studies illustrate how
even low-level impurities or degradation products can be
characterized and quantified relative to the full-length product,
even when they coelute.
0
1
2
3
4
5
6
7
8
9
{3-Trunc-L} {/Depur-T Gas/} {/5Phos mod/}
5,780 5,800 5,820 5,840 5,860 5,880 5,900 5,920 5,940 5,960 5,980 6,000 6,020 6,040 6,060 6,080 6,100
0
1
2
3
4
5
6
7
8
9 {3-Trunc-L}
{/5Phos mod/} {/Depur-T Gas/}
5,780 5,800 5,820 5,840 5,860 5,880 5,900 5,920 5,940 5,960 5,980 6,000 6,020 6,040 6,060 6,080 6,100
×10 Zoom 4
×106
Counts
Deconvoluted mass (amu)
Counts
Deconvoluted mass (amu)
Figure 7. Identification of low-abundance impurities.
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Acquisition time (min)
4.5 5.0 5.5 6.0 6.5 7.0
Preconditioning
injections
×102
Counts (%) Figure 6. Retention time stability for repeat injections following two initial
preconditioning injections.
8
Evaluating the method on sample mixtures
Often, chromatographic separation of different oligo species
is not possible because of insufficient chemical difference
or method throughput needs. In these situations, resolution,
and independent measurement of these species by the mass
spectrometer is critical. To evaluate the ability of the method
to mass-resolve multiple components without the need for
chromatographic separation, two samples containing variable
bases (V, indicative of a mixture of C, A, and G) at their 3' end
were injected. In both cases, chromatographic separation
of the mixtures was not observed (data not shown). First,
a 20-mer poly dT strand containing a 3' V (oligo PR3) was
analyzed. Deconvolution results (Figure 8A) show peaks at
6,007, 6,031, and 6,047 Da that perfectly match expectations,
based on sequences ending in C, A, and G respectively. A
second, a 38‑mer oligo containing a 3' V (oligo PR4) was
analyzed. Like the first sample, deconvolution results for
PR4 (Figure 8B) show three peaks, at 11,564, 11,588, and
11,604 Da, which match calculations. In both cases, the
relative abundances/heights match expectations, confirming
the ability of the method to provide meaningful information
about oligo mixtures without chromatographic separation of
the individual components.
0
1
2
3
4
11,500 11,600 11,700
×105
0
1
2
3
4
5
5,950 6,050 6,150
×106
Deconvoluted mass (amu)
Counts
Deconvoluted mass (amu)
Counts
A B
Figure 8 . Deconvolution results for 20-mer (A) and 38-mer (B)
oligo mixtures.
Evaluating method performance on oligos of different
sizes and chemistries
Many oligos studied by LC/MS, for confirmation of Target
Plus Impurities, are constructed from chemically modified
building blocks. Subsequently, the versatility of LC/MS
methods for oligos of different size and chemical makeup
is of interest. To evaluate the HILIC method in this regard,
a variety of 18 to 34‑mer oligo samples were analyzed
individually for their LC/MS behavior. Overlaid deconvolution
results for eight disparate samples (Figure 9) showed
high‑quality data across a simple to a complex sample
group. Results show data for a relatively simple 18‑mer
sample (PR8, 5 µM, 25 pmol on column) to the more complex
aptamer with multiple modified bases (50 µM, 100 pmol
on column) through an siRNA duplex (50 µM, 100 pmol on
column) where the duplex state of two 18‑mer strands was
maintained throughout. These results provide confidence in
the ability of the method to perform well across a wide range
of chemistries on 20 to 40‑mer samples.
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
6,000 8,000 10,000 12,000 14,000 16,000 PR8 PR2 PR6PR5PR9 Aptamer PRL40
siRNA
×102 Counts (%)
Deconvoluted mass (amu)
Figure 9. Overlaid deconvolution spectra from oligos of different sizes
and chemistries.
www.agilent.com
DE32357668
This information is subject to change without notice.
© Agilent Technologies, Inc. 2023
Printed in the USA, January 31, 2023
5994-5631EN
Conclusion
– The analysis of oligos via HILIC chromatography allows
for effective separation and high-resolution mass data
collection without contaminating the instrumentation with
ion-pairing reagents.
– The MS-friendly, ammonium acetate-based additive
necessitated little system maintenance and source
cleaning, allowing fast and easy LC/MS switching between
negative and positive mode applications.
– The 15-minute LC/MS method described here provided
chromatographic separation of a wide range of oligo
samples including 20 to 100-mers, variable base mixtures,
aptamers, and siRNA duplexes.
– The HILIC-Z column enabled excellent retention time
stability with short re-equilibration times.
– The HILIC method was able to preserve higher-order oligo
structures – in one case maintaining an siRNA duplex
throughout the analysis.
– The native-like conditions observed did result in
narrow charge state distributions (relative to IP-RP)
and significantly lower signal intensity for the longer
synthetic oligos.
– The HILIC method performed well for impurity analyses,
enabling the determination of modifications over a wide
dynamic range.
References
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