IMPROVING MS SENSITIVITY THROUGH THE
REDUCTION OF METAL SALT ADDUCTS IN IP-RPLC/MS
OLIGONUCLEOTIDE ANALYSES
Robert Birdsall, Martin Gilar, Brooke Koshel, Joe Fredette, Ananya Dubey, and Ying Qing Yu
Waters Corporation. Milford MA
INTRODUCTION
METHODS
Due to the negatively charged phosphodiester backbone, chargebased separations such as ion-pairing reversed phase
chromatography (IP-RPLC) have become a popular choice for the
analysis and characterization of oligonucleotides.
Efforts to minimize alkali metal salt adduct formation were taken prior to data
acquisition. As a potential point source of metal salt ions, solvent glassware
and sample vials were replaced with plastic alternatives constructed from
polyethylene and polypropylene, respectively.
When adsorbed onto hydrophobic bonded phases, IP agents such as
n-alkyl amines, provide a means to separate oligonucleotides with
high separation efficiency based on charge interaction of the
phosphodiester backbone. However, as a charge-based separation,
positively charged cations of alkali metal salts such as sodium (Na+)
and potassium (K+), are electrostatically attracted to the negatively
charged polyanionic backbone of oligonucleotides which can
interfere with the separation mechanism and impact assay
performance.
Mobile phase:
A: H2O, 15mM TEA:400 mM HFIP, pH 8.0
B: MeOH, 15mM TEA:400 mM HFIP
C: H2O, 0.2 % FA
High pH Reconditioning Gradient
D: MeOH
Time
(min)
Flow
(mL/min)
%A
%B
%C %D
0.00
0.200
82.0
18.0
0.0
0.0
4.00
0.200
80.0
20.0
0.0
0.0
4.01
0.200
50.0
50.0
0.0
0.0
6.01
0.200
50.0
50.0
0.0
0.0
6.02
0.200
82.0
18.0
0.0
0.0
10.00
0.200
82.0
18.0
0.0
0.0
Column manager
Auto-sampler
High pH
Reconditioning
Tunable UV Detector
Quaternary
solvent manager
A common practice to address this known phenomenon is to
periodically take the system off-line and clean using mobile phase
additives such as EDTA or CDTA which act as cation scavengers to
reduce trace alkali metal salts concentrations. While this strategy can
be effective, it is less than ideal in that it requires system downtime
and resources.
ACQUITY QDa Detector
Figure 1. An ACQUITY H-Class Bio chromatography system configured with
a single quadrupole mass spectrometer (ACQUITY QDa Detector) configured
post TUV detection was used to monitor separation performance and adduct
formation.
Time
(min)
ACQUITY® QDa Settings:
Sample rate: 2 points/sec
Mass range: 410 – 1250 m/z
Cone voltage: 20 V
Capillary voltage: 0.8 kV
Probe Temperature: 600 °C
LC Conditions:
Flow Cell: 5mm Ti
Absorption Wavelength: 260 nm
Injection:5 uL at 10 pmol/uL
Column: OST BEH C18, 2.1x50 mm
Column Temperature: 60 °C
By understanding the formation of alkali metal salts in oligonucleotide
analyses, we have developed a mitigation strategy that maintains
consistent chromatographic performance with minimal impact on
productivity.
Low pH Reconditioning Gradient
Low pH
Reconditioning
Flow
(mL/min)
%A
%B
%C %D
0.00
0.200
82.0
18.0
0.0
0.0
4.00
0.200
80.0
20.0
0.0
0.0
4.01
0.200
0.0
0.0
50.0
50.0
6.01
0.200
0.0
0.0
50.0
50.0
6.02
0.200
82.0
18.0
0.0
0.0
10.00
0.200
82.0
18.0
0.0
0.0
RESULTS
Alkali metal salts can severely disrupt the ion-pairing equilibrium and interfere with the charge-based ion-pairing retention mechanism
RT = 2.47
0.25
Deconvoluted Mass
6
5x10
6
1.0x10
M
6
4x10
5
8.0x10
Injection #1
6
5
0.15
0.10
6.0x10
5
4.0x10
adducts
5
2.0x10
0.05
0
1
2
3
4
5
6
1x10
745
750
755
760
765
5x10
~50 % loss in
spectral
abundance
5
8.0x10
6
4x10
5
peak
fronting
Intensity
RT drift
0.10
6.0x10
Intensity
Intensity
0.15
M
adducts
5
4.0x10
5
M
6
2x10
1x10
0.0
0.00
0
1
2
3
4
Na+/K+
Na+/2K+
2Na+/2K+
0
740
5
K+
Na+
6
2.0x10
0.05
63 %
adducts
6
3x10
Retention time (min)
745
750
755
760
765
770
m/z
775
780
6650 6700 6750 6800 6850
m/z
Mass (Da)
Mass
(Da)
2.55
2.50
time
60
2.45
2.40
40
2.35
20
2.30
2.25
0
10
20
6800 6850
6
6
adducts
0
770
6650 775
6700 780
6750
Mass (Da)
1.0x10
RT = 2.27
0.20
Na+
m/z
Injection #48
0.25
6%
adducts
6
2x10
2.60
80
0
740
Retention time
A.U.
3x10
0.0
0.00
Figure 2. Study design. Using The high-pH
reconditioning method an 8-hr time study
was conducted incorporating a full 48-well
sample plate, a ssRNA sample
(5′-UCGUCAAGCGAUUACAAGGTT-3′) with
a MW of 6,693.1 Da was prepared in
MS-grade water and transferred to
polypropylene vials across the 48 wells with
a control loaded in every 7th vial position.
Intensity
Intensity
Intensity
A.U.
AU
0.20
M
retention time
100
30
40
Retention time (min)
MS Spectra
Spectral Abundance (%)
spectral abundance
Chromatography
50
Injection #
Figure 3. Impact of alkali metal salts in oligonucleotide analyses. Two selected
chromatograms representing the initial injection (blue trace) and end injection (red
trace) of the time study are shown. As shown in the chromatograms a shift in retention
time and significant peak deterioration were observed over the course of the 8-hr time
study. MS data acquired simultaneously using the ACQUITY QDa indicated the
accumulation of trace alkali metal salts resulted in a significant increase in the relative
amount of adduct ions (6% to 63%) and over a 50% drop in spectral abundance of the
parent peak, M. The shift in retention time and peak deterioration observed correlates
with the degree of alkali metal adducts as shown in the bar plot. These observations
demonstrate that salts, when present at elevated concentrations, can severely disrupt
the ion-pairing equilibrium and interfere with the charge-based ion-pairing retention
mechanism leading to decreased chromatographic performance and MS sensitivity.
Non-specific adsorption sites located throughout the LC fluidic path perpetuate adduct formation in oligonucleotide analyses
detector
100.0
100.0
[M]
mobile
phase
metal
surfaces
sample
impurities
metal
surfaces
metal
surfaces
metal
surfaces
waste
Figure 4. impurity source study. Impurities
found in samples ,solvents, make-up water,
IP agents, and buffer components as well
glass and metal surfaces throughout the
LC system can act as point source
contributions to adduct formation. As one of
the largest volume contributors the mobile
phase was investigated as possible
contaminant source. To achieve this, the
10 minute method previously used for the
time study was modified to include a
conditioning step at the end of the gradient
to systematically increase the number of
CVs the fluidic path is exposed to at initial
mobile phase conditions on a clean
system.
Relative Intensity (%)
90.0
100.0
[M]
120 CV
100.0
[M]
90.0
90.0
260 CV
100.0
[M]
80.0
80.0
80.0
80.0
80.0
70.0
70.0
70.0
70.0
70.0
60.0
60.0
60.0
60.0
50.0
50.0
50.0
50.0
40.0
40.0
40.0
40.0
30.0
30.0
30.0
30.0
20.0
20.0
20.0
20.0
+Na
10.0
+Na
10.0
0.0
10.0
6720
6760
6800
Mass (Da)
6680
6720
6760
6800
Mass (Da)
50.0
30.0
+K
6720
6760
6800
+Na/+K
+2K
20.0
+Na/+K
10.0
6680
Mass (Da)
6720
6760
6800
Mass (Da)
experiment
mobile phase study
80
Initial time-trial study
60
40
20
0
0.0
0.0
6680
+K
+Na
40.0
10.0
0.0
0.0
6680
+Na
60.0
+Na
C)
Neutral Peak [M]
100
[M]
90.0
90.0
B)
Mass (Da)
experiment
mobile phase study
40
Initial time-trial study
30
20
10
0
10
6680 6720 6760 6800
260 CV
50
adducts
column
50 CV
Spectral abundance (%)
injector
20 CV
A
pump
0 CV
Spectral abundance (%)
A)
20
50
120
2
3
4
Column volume
260
5
[M]
1
Na
K
Na/K
2
3
4
Adduct species
K/K
5
Figure 5. Mobile phase purity. A) Adduct formation was observed to increase with CVs flowed through the system. B) A comparison of spectral abundance of the neutral peak
[M] in the current mobile phase experiment (green bar) exhibited nearly identical trending behavior as the initial time study experiment (red-bar). C) Closer inspection of the
260CV data set indicates almost identical distribution profiles of observed adduct species between the experimental data sets corroborating the notion that trace impurities in
the mobile phase is a contributing factor to adduct formation. These observations combined with the fact the LC system could routinely be brought back to baseline
performance with minimal adducts using a low pH cleaning protocol indicated non-specific adsorption sites located throughout the LC fluidic path perpetuate adduct formation
in oligonucleotide analyses performed with mobile phases with pH >7.
Low pH recondition,
High pH recondition,
100
retention time (mean of 6)
retention time (mean of 6)
2.55
80
2.50
60
2.45
2.40
40
2.35
20
2.30
2.25
0
0
10
20
CONCLUSION
2.60
30
40
50
Injection #
book 11 (avg)
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Retention time (min)
Figure 6. Adduct mitigation strategy. The non-specific
adsorption observed in this study, which exhibits
behavior analogous to a cation exchange surface,
requires exposure to solutions containing low pH
(acidic) to displace adsorbed alkali ions. the study
was repeated incorporating a 1 minute low pH column
regeneration step in the method after the separation
gradient using 0.1% FA to displace non-specifically
adsorbed metal salt cations. The low-pH regeneration
method (blue bar) was sufficient in mitigating alkali
metal adducts resulting in high spectral abundance
(>92%) and retention time stability (mean 2.44 min,
RSD 0.57%) of the target oligonucleotide (M) when
compared to the initial time study (red bar).
Spectral Abundance (%)
A short low pH reconditioning step was observed to effectively displace trace metal salts adsorbed to surfaces in the fluidic path

Trace alkali metal impurities interfere with charge-based ionpairing retention mechanism

Accumulation of alkali metal adducts reduce MS sensitivity
and increase spectral complexity

An efficient “low-pH” mitigation strategy can be built into the
chromatographic method avoiding system downtime and
yielding greater consistency in results
References
1)
Birdsall, et al. Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase
chromatography/mass spectrometry analysis. Rapid Commun. Mass Spectrom.,
2016, vol. 30 (14), p. 1667.
©2017 Waters Corporation