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

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

Development of Oligonucleotide Analysis Methodology with RP-HPLC Column
Designed for Separation of Highly Polar Compounds
Noriko Shoji, Chie Yokoyama, and Naohiro Kuriyama
YMC Co., Ltd., Ishikawa, Japan
3.
Introduction
Introduction
Results and Discussion
Figure 4: Influence of salt concentration on separation of d(pT)2-20
5
7
2
4
6
3
8
10
11
9
Hydrosphere C18
(A000922A)
5
6,7
1
2
4
10
8
9
3
11
YMC-Pack Pro C18
(A001018A)
5
1. 5'-UTP
2. 5'-UDP
3. 5'-CMP
4. 5'-UMP
5. 5'-dCMP
6. 5'-GMP
7. 5'-ATP
8. 5'-IMP
9. 5'-ADP
10. 5'-TMP
11. 5'-AMP
3
4
7,6
10
8
11
9
Competitor WX
(A001031A)
0
5
10
15
Column
Flow rate
Temperature
Detection
Eluent
a) 10-13% ACN (0-12min)
Slope 0.25 %/min
T2
(F050216D)
(F050203A)
(05012000.D)
Rs(20mer/19mer)=1.53
(F050216A)
Rs(3mer/2mer)=5.21
(05012002.D)
b) 10-13% ACN (0-15min)
Slope 0.20 %/min
(F050127A)
0
(05012003.D)
c) 10-13% ACN (0-17min)
Slope 0.18 %/min
Rs(3mer/2mer)=5.64
d) 10-13% ACN (0-20min)
Slope 0.15 %/min
(05012005.D)
Rs(20mer/19mer)=1.89
Rs(3mer/2mer)=4.18
(05021809.D)
4
6
8
10
12
14
16
18
e) 11-14% ACN (0-20min)
Slope 0.15 %/min
Competitor WX
C18
C18
: 150×4.6mmI.D., 5µm : 1.0 mL/min
: 30℃
: UV at 260 nm
: 100mM KH2PO4-K2HPO4 (pH5.5)
18
min
0
3, 5
120
5
5
120
Figure 3 : Comparison of d(pT)2-20 separation among TEAA,
acetate and phosphate buffers
125
15.5
yes
: Hydrosphere C18 (3µm) 50×4.6 mmI.D.
: 1.0 mL/min
: 35 ℃
: UV at 269 nm
: 5µL (25pmol/component)
: Three kinds of gradient systems are used in buffer concentration
range of 10-100mM at pH6.0
Eluent 1 : triethylammonium acetate (TEAA) buffer/acetonitrile
Eluent 2 : ammonium acetate buffer/acetonitrile
Eluent 3 : potassium phosphate buffer/acetonitrile
Gradient conditions are shown in the figures
20
25
10mM
(F050209B)
min
0
5
10mer
80
20mer
60
120
2mer
5mer
10mer
20mer
80
60
40
60
80
100
20
25
30
35
20mM
10mM
min
: A) 10-100mM potassium phosphate buffer (pH6.0)
: B) Eluent A / acetonitrile (80/20)
Gradient : 45-52%B (0-42min)
Salt concentration vs. Retention
140
100
15
30mM
Eluent
Salt concentration vs. Retention
5mer
20
10
50mM
120
2mer
100
5mer
10mer
80
20mer
60
40
0
120
20
40
60
80
100
120
0
Salt concentration of acetate buffer (mM)
20
40
60
80
100
120
Salt concentration of phosphate buffer (mM)
In Figure 4, the influence of salt concentration on separation and resolution of d(pT)2-20 is evaluated among the TEAA, ammonium acetate and potassium
phosphate buffers. Regardless of salt type, the retention decreased and the peak broadened with decreasing salt concentration, except for the case of d(pT)2. The
retention was unexpectedly influenced by salt concentration of the acetate or phosphate buffer, which has no ion-paring interaction, but this phenomenon might be
associated with the charge status of oligonucleotides.
In case of the TEAA buffer, the favorable separation was maintained even at a low salt concentration, such as 10mM. The mobile phase consisting of TEAA and
acetonitrile has been used commonly in RP-HPLC separation of oligonucleotides, but a high salt concentration is usually necessary to improve the poor retention
and peak shape. Hydrosphere C18 can be used at a lower TEAA concentration than ordinary C18 phases.
Figure 5: Comparison of d(pT)2-20 separation among
Hydrosphere C18 and two ordinary C18 phases
a)
T10
T2
T20
Hydrosphere C18
(J050209A)
b)
T2
10mM TEAA
T10
T20
YMC-Pack Pro C18 10mM TEAA
T2-20
c)
(J050209B)
Competitor WX
10mM TEAA
(J050214A)
Competitor WX
100mM TEAA
T10
T2
d)
T20
5
10
Column
Flow rate
Temperature
Detection
Injection
Eluent
Gradient
Eluent 2 : acetate buffer
15
20
25
min
30
: 150×4.6mmI.D., 5µm : 1.0 mL/min
: 35℃
: UV at 269 nm
: 5mL (25pmol/component)
: A) 10mM TEAA, pH6.0 (a-c) or 100mM TEAA, pH6.0 (d)
B) Eluent A / acetonitrile (80/20)
: 55-61%B (0-30min)
In Figure 5, the chromatographic performance was compared among
Hydrosphere C18 and two ordinary C18 phases under the same condition which
was found suitable to Hydrosphere C18 in Figure 4, i.e. use of the 10mM TEAA
buffer. The ordinary C18 phases could not achieve acceptable retention and
resolution with the 10mM TEAA buffer. Even in case of Competitor WX used at
a salt concentration of 100mM, the separation was less favorable as compared
with Hydrosphere C18 used at 10mM.
Figure 6 shows the analysis results of a crude synthetic 21mer
oligonucleotide obtained with Hydrosphere C18 and Competitor WX. Even
when the loading amount was increased from 50pmol to 500pmol, many
impurities could be separated from the target product on Hydrosphere C18 with
the resolution capacity maintained high as distinct from Competitor WX.
Furthermore, a volatile buffer of a low concentration, like 10mM TEAA, is easily
removed from the collected fractions by lyophilization or vaccum centrifugation
as an additional advantage. Hydrosphere C18 may be suitable for purification
of synthetic oligonucleotides.
4.
Conclusions
(F050218A)
Figure 6: Analysis of a crude synthetic 21mer oligonucleotide
yes
yes
15
(F050209C)
140
Salt concentration of TEAA buffer (mM)
Eluent 1 : TEAA buffer
16.0
10
20mM
: A) 10-100mM ammonium acetate buffer (pH6.0)
: B) Eluent A / acetonitrile (80/20)
Gradient : 45-53%B (0-30min)
40
(F050126E)
12.0
5
(F050209A)
Eluent
20 min
HPLC conditions for separation of d(pT)2-20 in Figures 2-4
Column
Flow rate
Temperature
Detection
Injection
Eluent
16
(F050217A)
2mer
0
: A) 100mM TEAA (pH6.0)
: B) Eluent A / acetonitrile (80/20)
Gradient : a) 50-65%B (0-12min)
b) 50-65%B (0-15min)
c) 50-65%B (0-17min)
d) 50-65%B (0-20min)
e) 55-70%B (0-20min)
Eluent 3 : phosphate buffer
(F050210B)
YMC-Pack Pro C18
14
100
Eluent
Stationary Particle size Pore size Carbon content Endcapping
phase
(µm)
(Å)
(%)
C18
12
10mM
120
min
Table 1: Specifications of stationary phases used in this study
Hydrosphere C18
10
(F050216C)
100mM
(F050208B)
30mM
(F050216B)
20mM
(F050207D)
50mM
30mM
Salt concentration vs. Retention
140
Rs(20mer/19mer)=1.77
2
8
100mM
(F050215E)
: A) 10-100mM TEAA (pH6.0)
: B) Eluent A / acetonitrile (80/20)
Gradient : 50-65%B (0-20min)
Rs(3mer/2mer)=5.31
0
6
50mM
Eluent
Experiments
Brand name
4
Rs(20mer/19mer)=1.64
0
2.
2
(F050214G)
k' at each mM / k' at 100mM (%)
T10
Rs(3mer/2mer)=5.17
100mM
(F050126E)
T20
(J050210E)
1
2
Rs(20mer/19mer)=1.36
The influences of gradient conditions on separation of polydeoxythymidylic
acids, d(pT)2-20, are shown in Figure 2. The retention and resolution were
greatly influenced by both gradient slope and initial composition of mobile
phase. As shown in Chromatograms a-d, a slight change in slope from 0.25%
ACN/min to 0.15% ACN/min resulted in enhanced retention and improved
resolution especially in case of longer oligonucleotides, and it enabled baseline
separation of d(pT)2-20 even by one-nucleotide difference. Furthermore, even
with the same slope, only a 1% increase in initial acetonitrile concentration
could shorten each run time maintaining the favorable separation, as shown in
Chromatograms d and e. It seems therefore important to choose the
appropriate gradient slope and initial composition of mobile phase carefully for
achieving better separation of oligonucleotides.
Figure 1 : Comparison of nucleotide separation among
Hydrosphere C18 and two ordinary C18 phases
1
Figure 2 : Influences of gradient conditions on separation of
d(pT)2-20
k' at each mM / k' at 100mM (%)
Synthetic oligonucleotides have been used extensively as primers of DNA
sequencing or PCR, hybridization probes, and antisense drugs, and
separation methodology using reversed-phase HPLC has been widely applied
also to analysis and purification of these oligonucleotides. Since it is difficult
to retain and separate highly polar compounds like short oligonucleotides on
the ordinary reversed-phase columns, an ion-pairing buffer containing
triethylammonium acetate (TEAA) at a high concentration, e.g., 100-200mM,
has been commonly used to improve the poor retention and resolution.
However, in case of a buffer containing TEAA at a concentration higher than
50mM, the signal intensity decreases in electrospray ionization mass
spectrometry (ESI-MS), which is one of the most important analytical
methodologies for oligonucleotides. 1 Moreover, the high concentration of
buffer has an adverse effect on column lifetime. In addition, peak tailing is
often caused by basic functional groups residing in the oligonucleotide
molecules.
We have 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 in case of basic compounds, and it can be used with a 100%
aqueous mobile phase.
Figure 1 compares nucleotide separation among
Hydrosphere C18 and two C18 phases shown in Table 1. Hydrosphere C18
shows enhanced retention and improved resolution of highly polar compounds
like nucleotides.
We are presenting here some example cases of analyzing 2-20mer Polyd(pT) oligonucleotides by means of Hydrosphere C18 under different mobile
phase conditions. We also compare the retention and elution achieved with
Hydrosphere C18 and those achieved with other C18 columns.
Eluent 3 : phosphate buffer (pH 6.0)
Eluent 2 : acetate buffer (pH 6.0)
Eluent 1 : TEAA buffer (pH 6.0)
k' at each mM / k' at 100mM (%)
1.
0
2
4
6
8
10
12
14
16
18
min
Sample : Primer of DNA sequencing
5'-CCCGTGTTCCTTGCCACAGAC -3'
Eluent A : 1) 100mM TEAA (pH6.0)
: 2) 100mM ammonium acetate buffer (pH6.0)
: 3) 100mM potassium phosphate buffer (pH6.0)
Eluent B : Eluent A / acetonitrile (80/20)
Gradient : 50-65%B (0-20min)
Figure 3 compares d(pT)2-20 separation among the TEAA, ammonium
acetate and potassium phosphate buffers under the same gradient condition.
The retention achieved with the TEAA buffer was stronger than that achieved
with the acetate or phosphate buffer. This indicates that the retention/elution
mechanism in using a mobile phase containing triethylamine is mainly based
on the ion-paring interaction between the highly hydrophilic phosphodiester
groups of oligonucleotides and the somewhat lipophilic triethylammonium ions
in the buffer, and the ion-paring interaction produces some interaction between
the hydrophilic oligonucleotides and the lipophilic sorbent on the stationary
phase, resulting in stronger retention of oligonucleotides.
Competitor WX
Hydrosphere C18
21mer
61.6% purity*
The ordinary C18 phases could not achieve any acceptable separation at
a relatively low concentration of TEAA, e.g., 10mM. However,
Hydrosphere C18 showed strong retention and good resolution of
oligonucleotides under the same conditions.
impurities
21mer
71.7% purity*
impurities
(J050216E)
(J050216A)
0
2
4
6
8
10
12
14
16
Column
Flow rate
Temperature
Detection
Injection
Eluent
Gradient
18
500 pmol
50 pmol
min
(J050217C)
(J050217B)
0
2
: 150×4.6mmI.D., 5µm : 1.0 mL/min
: 35℃
: UV at 260 nm
: 5mL (10nmol/mL or 100nmol/mL)
: A) 10mM TEAA, pH6.0
B) Eluent A / acetonitrile (80/20)
: 55-65%B (0-20min)
4
6
8
10
12
14
16
Oligonucleotide separation was compared under various mobile phase
conditions using Hydrosphere C18. The retention and resolution of
oligonucleotides were greatly influenced by gradient condition, salt type,
and salt concentration.
18
500 pmol
50 pmol
Hydrosphere C18 may be applicable to analysis and purification of
various types of oligonucleotides.
min
Reference
* Purity of 21mer oligonucleotides was determined
from peak area at UV 260nm.
1) Alex Apffel, John A. Chakel, Steven Fischer, Kay Lichtenwalter, William s. Hancock,
Anal. Chem. 1997, 69, 1320-1325