Noriko Shoji, Chie Yokoyama and Naohiro Kuriyama YMC Co., Ltd

Noriko Shoji, Chie Yokoyama and Naohiro Kuriyama YMC Co., Ltd

Development of LC-MS method for Oligonucleotide Analysis
with RP-HPLC Column Designed for Separation of Highly Polar Compounds
Noriko Shoji, Chie Yokoyama and Naohiro Kuriyama
YMC Co., Ltd., Ishikawa, Japan
1.
3.
Introduction
Introduction
Reversed-phase HPLC has been widely applied to analysis and purification of
synthetic oligonucleotides, such as primers of DNA sequencing or PCR,
hybridization probes, and antisense drugs. Because it is difficult to retain and
separate highly polar compounds like short oligonucleotides on ordinary reversedphase columns, an ion-pairing buffer containing triethylammonium acetate (TEAA)
at a high concentration, e.g., 100-200 mM, has been commonly used to improve
poor retention and resolution. However, for a buffer containing TEAA at a
concentration higher than 50 mM, the signal intensity decreases in electrospray
ionization mass spectrometry (ESI-MS), which is one of the most important
analytical methodology for oligonucleotides. Though it has been reported that the
mobile phase of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)-TEA buffer/methanol
gives an advantage in MS sensitivity, it is necessary to add HFIP, which is not so
commonly used for RP-HPLC, with a relatively high concentration such as 400 mM
to achieve good LC/MS analysis.1
We have developed a silica-based C18-bonded packing material named
Hydrosphere C18, which has been specially designed for separation of highly polar
compounds. It provides strong retention of polar compounds and an excellent
peak shape even for basic compounds, and it can be used with a 100% aqueous
mobile phase.
In our previous study,2 we applied Hydrosphere C18 to separation of short
oligonucleotides up to 20 mer under various mobile phase conditions. As shown in
Fig. 1, Hydrosphere C18 showed enhanced retention and improved resolution at a
low concentration of TEAA buffer, such as 10 mM. Ordinary C18 phases could not
achieve any acceptable separation in identical conditions.
This study developed an efficient analytical method for short oligonucleotides
using Hydrosphere C18 with an ion-pairing buffer containing di-n-butylammonium
acetate (DBAA). It provides both good HPLC separation and high-sensitivity in
ESI-MS.
Figure 1: Comparison of d(pT)2-20 separation among
Hydrosphere C18 and two ordinary C18 phases
b)
T10
T2
a)
T20
T2
(J050209A)
Hydrosphere C18
10 mM TEAA
(J050210E)
YMC-Pack Pro C18 10 mM TEAA
(J050209B)
Competitor WX
Results and
and Discussion
Discussion
Results
Figure 4: LC-MS analysis of synthetic 27-30 mer oligonucleotides
Figure 2: Comparison of d(pT)2-20 separation under different ion-pairing buffers and organic solvents
T10
a)
T20
T2
Sample : Primer of DNA sequencing
5'-CCGCTCGAGCTAAAAAAAGCCTGTGTTACC-3' (30 mer)
d)
T10
T2
(F050126E)
T10
T20
10 mM TEAA
10.0-13.0 % ACN
(0-20 min)
(F050228B)
T20
T10
Hydrosphere C18
10 mM TEAA
22.0-25.0 % MeOH
(0-25 min)
27
27-30 mer
T20
b)
28
e)
T2
(F050415A)
T10
10 mM DBAA
15.0-37.5 % ACN
(0-20 min)
10 mM DBAA
33.6-56.0 % MeOH
(0-20 min)
(F050423A)
T10
T20
f)
(F050419A)
10
5
15
0
5 mM DBAA
15.0-37.5 % ACN
(0-20 min)
TIC
TIC
(F060214B-03)
5
10
15
20
0
5
10
15
Column
Flow rate
Temperature
Detection
Injection
Mobile phase
5 mM DBAA
33.6-56.0 % MeOH
(0-20 min)
(F050426A)
min
min
20
Gradient
Mobile phase
Gradient
Chromatogram b and c
UV
Mobile phase
Gradient
min
0
5
10
15
min
T2
Mobile phase and gradient condition
Chromatogram a
UV
(F060213C-04)
T2
0
29 30 mer
T2
T20
c)
Competitor WX
: A) 10 mM TEAA buffer (pH 6.0)
B) Mobile phase A/acetonitrile (80/20)
: 50-65 %B (0-20 min)
Chromatogram d
: A) 5 or 10 mM DBAA buffer (pH 6.0)
B) Mobile phase A/acetonitrile (50/50)
: 30-75 %B (0-20 min)
Chromatogram e and f
Mobile phase
: A) 10 mM TEAA buffer (pH 6.0)
B) Mobile phase A/methanol (50/50)
: 44-50 %B (0-25 min)
Gradient
Mobile phase
: A) 5 or 10 mM DBAA buffer (pH 6.0)
B) Mobile phase A/methanol (20/80)
: 42-70 %B (0-20 min)
Gradient
Figure 2 compares separation of polydeoxythymidylic acid, d(pT)2-20, among four different gradient systems of TEAA buffer/acetonitrile (a), DBAA
buffer/acetonitrile (b, c), TEAA buffer/methanol (d) and DBAA buffer/methanol (e, f) , with the gradient slope which has been optimized at 10 mM buffer
concentration. In case of the DBAA buffer, d(pT)2-20 can be separated favorably with higher initial concentration and higher gradient slope of organic
solvent with both acetonitrile and methanol than in case of the TEAA buffer. This result indicates that the ion-paring interaction between di-n-butylamine
and oligonucleotides is stronger than that between triethylamine and oligonucleotides. Even at a lower concentration of DBAA, such as 5 mM, the
acceptable separation and good peak shape was maintained under the same gradient condition as 10 mM DBAA buffer, as shown in Chromatograms 2c
and 2f.
: 50×2.0mmI.D., 3 µm : 0.2 ml/min
: 35℃
: UV at 269 nm and ESI negative-mode
: 1 µl (10 pmol/component)
: A) 10 mM DBAA (pH 6.0)
B) Mobile phase A/acetonitrile (50/50)
: 58-62 %B (0-20 min), 62 %B (20-25 min)
Figure 4 shows the LC-MS analyses of a mixture of synthetic 27-30 mer
oligonucleotides using Hydrosphere C18 and the ordinary C18 phase, competitor
WX. The mobile phase consisting of the 10mM DBAA buffer and acetonitrile was
suitable for separation of these oligonucleotides. Hydrosphere C18 can achieve
excellent separation by one-nucleotide difference and sufficient intensity in ESIMS. The separation and intensity with Competitor WX were less favorable as
compared with Hydrosphere C18 in identical conditions.
4.
Conclusions
T10
T20
T2-20
c)
d)
T10
T2
10 mM TEAA
total ion chromatograms (TIC)
T20
(J050214A)
0
5
Column
Flow rate
Temperature
Detection
Injection
Mobile phase
Gradient
10
15
20
25
30
: 150×4.6 mmI.D., 5 µm : 1.0 ml/min
: 35℃
: UV at 269 nm
: 5 µl (25 pmol/component)
: A) 10 mM TEAA, pH 6.0 (a-c) or 100 mM TEAA, pH 6.0 (d),
: 55-61 %B (0-30 min)
Competitor WX
100 mM TEAA
min
B) Mobile phase A/acetonitrile (80/20)
buffer/acetonitrile
raw mass spectra
buffer/methanol
Scan ES- TIC
7.50e7
Scan ES- TIC
7.50e7
a) 100
d)
Scan ES -
100
T2
T10
1.51e6
1.86e6
100
%
2.
Experimental
HPLC conditions for separation of d(pT)2-20 in Fig. 2
Column
Flow rate
Temperature
Detection
Injection
Mobile phase
: Hydrosphere C18, 50×4.6 mmI.D., 3 µm
: 1.0 ml/min
: 35℃
: UV at 269 nm
: 5 µl (25 pmol/component)
: Four kinds of gradient systems are used
10 mM TEAA (pH 6.0)/acetonitrile (a)
5 or 10 mM DBAA (pH 6.0)/acetonitrile (b, c)
10 mM TEAA (pH 6.0)/methanol (d)
5 or 10 mM DBAA (pH 6.0)/methanol (e, f)
Gradient conditions are shown in Fig. 2
LC-MS analyses conditions in Fig. 3 and 4
: 50×2.0 mmI.D., 3 µm
: 0.2 ml/min
: 35℃
: 5 µl (25 pmol/component) (Fig. 3)
1 µl (10 pmol/component) (Fig. 4)
Mobile phase
: Mobile phase conditions are shown in the figures
Ionization mode
: ESI negative-mode
Capillary
: 2.50 kV
Cone
: 35 V (Fig. 3)
140 V (Fig. 4)
Source temperature
: 100℃
Desolvation temperature : 350℃
Column
Flow rate
Column temperature
Injection
Oligonucleotide separation was compared under mobile phase conditions
containing two different ion-pairing buffers, TEAA and DBAA, using
Hydrosphere C18. It is possible to reduce the salt concentration and
increase the organic solvent concentration with the DBAA buffer than with
the TEAA buffer.
Figure 3: Influences of mobile phase conditions on intensity of ESI-MS
T2
T10
%
T20
T2
10 mM TEAA
(F060120C-03)
T10
M-
%
T2
%
(F060119D-02)
1489.4
M6-
744.0
T20
T2
10 mM DBAA
%
M4T10
T20
10 mM DBAA
0
600
1000
1400
1002.7
0
0
1800 m/z
600
1000
1400
F060118A-02 22 (1.583)
(F060119A-02)
1800 m/z
600
1000
F060118A-02 158 (9.069)
1800 m/z
1400
F060118A-02 258 (14.575)
0
0
c) 100
f)
100
T10
T10
T20
T2
%
5 mM DBAA
5 mM DBAA
(F060118B-02)
(F060118A-02)
0
0
0
5
10
15
min
g) 100
Scan ES -
T2
T10
T10
1.33e6
T20
T2
%
400 mM HFIP-TEA
100
T20
1.04e6
M-
1488.4
100
545.2
4.03e5
M2M3-
100
0
5
10
15
20
M4-
min
%
%
M41504.4
M7-
M6-
This simple method using Hydrosphere C18 with a low concentration of
DBAA buffer provides a great potential for various chromatography of
oligonucleotides, such as analytical and preparative HPLC or HPLC-MS.
5859.6 1002.7 M
1203.6
992.7
(F060125D-01)
0
There was sufficient intensity of ESI-MS when 5-10 mM DBAA buffer was
used to analyze oligonucleotides with both acetonitrile and methanol. The
intensity obtained with 5 mM DBAA buffer was equal to or greater than
that obtained with 400 mM HFIP-TEA buffer.
T20
T2
%
Hydrosphere C18 showed strong retention and good resolution of short
oligonucleotides at a relatively low concentration of DBAA, e.g. 5-10 mM,
compared to the ordinary C18 phase .
1203.6
M2-
%
e) 100
T10
%
1504.7
100
M5-
0
b) 100
M4-
10 mM TEAA
(F060120B-03)
0
2.07e6
M3992.7
100
545.3
T20
%
744.2
Mobile phase and gradient condition of chromatogram g
Mobile phase
Gradient
: A) 400 mM HFIP-TEA buffer (pH 7.0)
B) methanol
: 7-35 %B (0-20 min)
0
0
600
1000
1400
1800 m/z
F060125D-01 26 (1.740)
600
1000
1400
1800 m/z
F060125D-01 250 (13.317)
0
600
1000
1400
1800 m/z
F060125D-01 335(17.710)
Other Mobile phase conditions are the same as those of corresponding chromatograms in Fig. 2
In Fig. 3, the influences of mobile phase conditions on signal intensity in HPLC-ESI-MS analyses of d(pT)2-20 are compared among five different gradient
systems of TEAA buffer/acetonitrile (a), DBAA buffer/acetonitrile (b, c), TEAA buffer/methanol (d), DBAA buffer/methanol (e, f) and HFIP-TEA
buffer/methanol (g). The intensity of TIC in ESI-MS obtained with the 10 mM DBAA buffer is much superior to that obtained with the 10 mM TEAA buffer with
both acetonitrile and methanol (Chromatograms 3a, 3b, 3d and 3e). Furthermore, the 5 mM decrease of DBAA concentration in mobile phase resulted in
approximately 1.5-3 times increasing of the intensity of the peaks with sufficient column performance (Chromatograms 3b, 3c, 3e and 3f). The intensity
obtained with 5 mM DBAA buffer/acetonitrile or 5 mM DBAA buffer/methanol can be comparable to that obtained with 400 mM HFIP-TEA buffer/methanol.
Also, the multiply-charged ions of d(pT)2-20, [M-nH]n-, were obtained with good intensity in the raw mass spectra.
References
1) Alex Apffel, John A. Chakel, Steven Fischer, Kay Lichtenwalter, William s. Hancock,
Anal. Chem. 1997, 69, 1320-1325
2) Noriko Shoji, Chie Yokoyama, Naohiro Kuriyama, Pittcon 2005, 760-12P