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 KuriyamaYMC Co., Ltd., Ishikawa, Japan

LC-MS analyses conditions in Fig. 3 and 4

Results and DiscussionFigure 2: Comparison of d(pT)2-20 separation under different ion-pairing buffers and organic solvents

IntroductionIntroduction1.

Experimental2.

Conclusions4.

3. Results and Discussion

Figure 3: Influences of mobile phase conditions on intensity of ESI-MS

Figure 4: LC-MS analysis of synthetic 27-30 mer oligonucleotides

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 ESI-MS. The separation and intensity with Competitor WX were less favorable as compared with Hydrosphere C18 in identical conditions.

Column : 50×2.0mmI.D., 3 µm　Flow rate : 0.2 ml/minTemperature: 35℃Detection : UV at 269 nm and ESI negative-modeInjection : 1 µl (10 pmol/component)Mobile phase: A) 10 mM DBAA (pH 6.0) B) Mobile phase A/acetonitrile (50/50)Gradient : 58-62 %B (0-20 min), 62 %B (20-25 min)

Figure 1: Comparison of d(pT)2-20 separation among Hydrosphere C18 and two ordinary C18 phases

(J050214A)min0 5 10 15 20 25 30

(J050210E)

(J050209A) Hydrosphere C18 10 mM TEAA

YMC-Pack Pro C1810 mM TEAA

(J050209B) Competitor WX 10 mM TEAA

Competitor WX 100 mM TEAA

Column : 150×4.6 mmI.D., 5 µm　Flow rate : 1.0 ml/minTemperature : 35℃Detection : UV at 269 nmInjection : 5 µl (25 pmol/component)Mobile phase : A) 10 mM TEAA, pH 6.0 (a-c) or 100 mM TEAA, pH 6.0 (d), B) Mobile phase A/acetonitrile (80/20)Gradient : 55-61 %B (0-30 min)

a)

c)

d)

T2 T10T20

T2-20

T10T20

b)T20

T10T2

T2

HPLC conditions for separation of d(pT)2-20 in Fig. 2 Column : Hydrosphere C18, 50×4.6 mmI.D., 3 µm Flow rate : 1.0 ml/minTemperature : 35℃Detection : UV at 269 nmInjection : 5 µl (25 pmol/component)Mobile phase : 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

Column : 50×2.0 mmI.D., 3 µmFlow rate : 0.2 ml/minColumn temperature : 35℃Injection : 5 µl (25 pmol/component) (Fig. 3) 1 µl (10 pmol/component) (Fig. 4)Mobile phase : Mobile phase conditions are shown in the figuresIonization mode : ESI negative-mode Capillary : 2.50 kVCone : 35 V (Fig. 3) 140 V (Fig. 4)Source temperature : 100℃Desolvation temperature: 350℃

10 mM TEAA10.0-13.0 % ACN(0-20 min)

(F050126E)

5 mM DBAA15.0-37.5 % ACN(0-20 min)

(F050419A)

10 mM DBAA15.0-37.5 % ACN(0-20 min)

(F050415A)

5 mM DBAA33.6-56.0 % MeOH(0-20 min)

(F050426A)

10 mM DBAA33.6-56.0 % MeOH(0-20 min)(F050423A)

(F050228B)

10 mM TEAA22.0-25.0 % MeOH(0-25 min)

0 5 10 15 min

a)

b)

c)

e)

f)

d)

Chromatogram a Mobile phase : A) 10 mM TEAA buffer (pH 6.0) B) Mobile phase A/acetonitrile (80/20) Gradient : 50-65 %B (0-20 min)

Chromatogram b and c Mobile phase : A) 5 or 10 mM DBAA buffer (pH 6.0) B) Mobile phase A/acetonitrile (50/50) Gradient : 30-75 %B (0-20 min)

(F060120C-03)0

100

% T2 T20T1010 mM TEAA

a)

Scan ES- TIC7.50e7

10 mM TEAA

d)

(F060120B-03)0

100

% T2 T10

10 mM DBAA

5 mM DBAA

10 mM DBAA

5 mM DBAA

400 mM HFIP-TEA

buffer/acetonitrile

(F060119D-02)0

100

%

b)

c)

0

100

%

(F060118B-02)

T2 T20T10

T2

T10T20

0 5 10 15 min

0

100

% T2T20

T10

(F060119A-02)

(F060118A-02)0

100

%

0

100

%

(F060125D-01)

0 5 10 15 20 min

Scan ES- TIC7.50e7

buffer/methanol

e)

f)

g)

T2T20T10

T2 T20

T10

raw mass spectratotal ion chromatograms (TIC)

545.3

0

100

%

F060118A-02 22 (1.583)

1.51e6992.7

744.0

1489.4

0

100

%

F060118A-02 158 (9.069)

1.86e61504.7

1203.6

600 1000 1400 1800 m/z0

100

%

F060118A-02 258 (14.575)

2.07e6

545.2

0

100

%

F060125D-01 26 (1.740)

1.33e61488.4

992.7

744.2

0

100

%

F060125D-01 250 (13.317)

1.04e61504.4

1002.7859.61203.6

600 1000 1400 1800 m/z0

100

%

F060125D-01 335(17.710)

4.03e5T2 T10 T20

Sample : Primer of DNA sequencing 5'-CCGCTCGAGCTAAAAAAAGCCTGTGTTACC-3' (30 mer)

Hydrosphere C18 Competitor WX

0 5 10 15 20 min

UV

TIC

0 5 10 15 min

UV

TIC

30 mer2928

27

27-30 mer

References1) Alex Apffel, John A. Chakel, Steven Fischer, Kay Lichtenwalter, William s. Hancock, Anal. Chem. 1997, 69, 1320-13252) Noriko Shoji, Chie Yokoyama, Naohiro Kuriyama, Pittcon 2005, 760-12P

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.

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.

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 reversed-phase 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.

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 .

min0 5 10 15 20

(F060214B-03)(F060213C-04)

Chromatogram d Mobile phase : A) 10 mM TEAA buffer (pH 6.0) B) Mobile phase A/methanol (50/50) Gradient : 44-50 %B (0-25 min)

Chromatogram e and f Mobile phase : A) 5 or 10 mM DBAA buffer (pH 6.0) B) Mobile phase A/methanol (20/80) Gradient : 42-70 %B (0-20 min)

Mobile phase and gradient condition

Mobile phase : A) 400 mM HFIP-TEA buffer (pH 7.0) B) methanolGradient : 7-35 %B (0-20 min)

T2

T2

T2

T10

T10

T10

T20

T20

T20

T2 T10T20

T2

T10 T20

T2

T10 T20

M4-

M5-M6-M7-

Scan ES -

M-M2-

M3-

M4-

T2Scan ES -

T20M4-

M5-

T10

M2-

M3-

M4-

M-

Other Mobile phase conditions are the same as those of corresponding chromatograms in Fig. 2

Mobile phase and gradient condition of chromatogram g

600 1000 1400 1800 m/z600 1000 1400 1800 m/z

600 1000 1400 1800 m/z 600 1000 1400 1800 m/z

1002.7M6-

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.

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.

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.