Instrument : Shimadzu Nexera / LCMS-8030 PlusColumn : Shodex HILICpak VN-50 2D (2.0 mm I.D. x 150 mm) Eluent : (A) 50 mM HCOONH4 aq. / (B) CH3CN

Linear gradient (High pressure)Flow rate : 0.2 mL/minDetector : PDA (190-350 nm) & ESI-MS SIM(-)Column temp. : 40℃ Injection vol. : 1 μL

260 nm

191213 14

15

16 17

11

79

1086

5

423

20mer

18

0 5 10 15 20 min

・Packing material： Poly vinyl alcohol particle with diol group・Housing： PEEK ・Usable pH： 2～13・Usable temp.： 4～60℃・Usable solvent： H2O, CH3CN, CH3OH (mixable at any ratio)・Hydrophilic substance can be retained without derivatization or ion pair agent

・Suitable for HILIC analysis of oligosaccharides and oligonucleotides

LC/MS analysis of oligonucleotides using new polymer-based HILIC column having diol group

Introduction

Experimental

Nucleic acid drug development and quality control areexpected to drastically increase within the pharmaceuticalsector. LC/MS measurements are a highly sensitive andhighly selective analytical tool required for quality control.Conventional ion pair reverse phase mode has been widelyused for the analysis of oligonucleotides. However, the ionpair agent tends to remain in the instrument.

In this study, a novel column ShodexTM HILICpakTM VN-502D column was applied for LC/MS measurement ofoligonucleotides. A method was developed to separateoligonucleotides under a gradient condition of volatile saltsolution and acetonitrile without using ion pairing agent and toconduct highly sensitive analysis.

Conclusions

Shodex® HILICpak® VN-50 series

ShodexTM HILICpakTM VN-50 2D, a newly developed polymer-based diol-type HILIC column, was used with LC/UV/ESI-MS to elute oligonucleotides in order ofpolymerization degree. It was confirmed that highly selective analysis was possible. The eluent was a gradient of 50 mM ammonium formate aqueous solutionand acetonitrile, a condition that does not require the addition of an ion pair reagent in reverse phase mode or addition of a high concentration salt in ionexchange mode. This condition seems to have superiority in terms of simplification of desalting process even for preparative applications. Alkaline washing isalso possible. The VN-50 series is a useful column for the development and quality control of nucleic acid medicine.

LC/MS of Oligo DNA 20mer

Wide pH range

not using ion pair agent and HFIP

Results and Discussion

PVAOH

OH

20mer

0 5 10 15 min

1533.3(-)

Synthetic oligodeoxyribonucleotides(salt-free grade)(1) 20mer 5’-ATACCGATTAAGCGAAGTTT-3’(2) 20mer 5’-ATACCAATTAAACAAAATTT-3’(3) 62mer 5’-CATGAGAAGTATGACAACAGCCCCACACCGG

CTGTTGTCATACTTCTCATGGTTCTTCGGAA-3’* All samples contain trace amounts of imperfect oligo-DNAs as impurity

LC/MS of Oligo DNA 62mer

expanded260 nm

0 5 10 15 20 25 min

Condition of (A=50mM / B%=62%56%) is suitable

260 nm

0 5 10 15 20 25 min

Sample 12.2 mg/mL(in H2O)

B%=57%(constant)

B%=60%(0min)→50%(10-15min)→60%(15.01-20min)

B%=60%(0min)→55%(10-15min)→60%(15.01-20min)

B%=62%(0min)→56%(10-20min)→62%(20.01-25min)

B%=62%(0min)→57%(10-25min)→62%(25.01-30min)

B%=62%(0min)→57%(10-25min)→62%(25.01-30min)

B%=62%(0min)→57%(10-25min)→62%(25.01-30min)

A=100mM

A=50mM

A=20mM

* B%=60%(0min)→55%(10-15min)→60%(15.01-20min)

* B%=62%(0 min)→56%(10-20 min)→62%(20.01-25 min)

2mer (TT)

4mer (GTTT)

3mer (TTT)

5mer (AGTTT)

6mer (AAGTTT)

7mer (GAAGTTT)

8mer (CGAAGTTT)

9mer (GCGAAGTTT)

10mer (AGCGAAGTTT)

11mer (AAGCGAAGTTT)

12mer (TAAGCGAAGTTT)

13mer (TTAAGCGAAGTTT)

14mer (ATTAAGCGAAGTTT)

15mer (GATTAAGCGAAGTTT)

16mer (CGATTAAGCGAAGTTT)

17mer (CCGATTAAGCGAAGTTT)

18mer (ACCGATTAAGCGAAGTTT)

19mer (TACCGATTAAGCGAAGTTT)

20mer (ATACCGATTAAGCGAAGTTT)

545.1(-)

849.2(-)

1178.2(-)

745.2(-)

901.7(-)

1066.2(-)

1210.7(-)

1375.3(-)

1531.8(-)

1688.0(-)

1226.6(-)

1327.9(-)

1432.3(-)

1542.0(-)

1638.3(-)

1734.7(-)

1839.0(-)

1940.4(-)

1533.3(-)

0 5 10 15 20 min

試料1

試料2

2mer (TT)

4mer (ATTT)

3mer (TTT)

5mer (AATTT)

6mer (AAATTT)

7mer (AAAATTT)

8mer (CAAAATTT)

9mer (ACAAAATTT)

10mer (AACAAAATTT)

11mer (AAACAAAATTT)

12mer (TAAACAAAATTT)

13mer (TTAAACAAAATTT)

14mer (ATTAAACAAAATTT)

15mer (AATTAAACAAAATTT)

16mer (CAATTAAACAAAATTT)

17mer (CCAATTAAACAAAATTT)

18mer (ACCAATTAAACAAAATTT)

19mer (TACCAATTAAACAAAATTT)

545.1(-)

849.2(-)

1162.2(-)

737.2(-)

893.7(-)

1050.2(-)

1194.8(-)

1351.3(-)

1507.8(-)

1664.4(-)

1210.6(-)

1311.9(-)

1416.3(-)

1520.7(-)

1617.0(-)

1713.4(-)

1817.7(-)

1919.1(-)

0 5 10 15 20 min1517.3(-) 20mer (ATACCAATTAAACAAAATTT)

Sample 1

2-4mer : [M-H]-5-11mer : [M-2H]2-12-19mer : [M-3H]3-20mer : [M-4H]4-

260nm

2021+22

23

24+25

26

26+27+2829+30

31

10 11

12+1314

15+1617+18+19

1723.7(-)

563.1(-)

892.2(-)1221.3(-)754.7(-)

906.7(-)

1203.2(-)

1058.7(-)

1355.3(-)

1507.3(-)

1671.8(-)

1836.4(-)

1988.4(-)

1429.6(-)

1526.0(-)

1627.3(-)

1825.0(-)

1926.4(-)

1516.8(-)1595.1(-)

1749.3(-)

1671.1(-)

1821.6(-)

1897.6(-)

1979.9(-)

1705.3(-)

1644.5(-)

1771.1(-)

1832.0(-)

1889.8(-)

0 5 10 15 20 min1955.6(-)

2mer (AA)

10mer (TTCTTCGGAA)

20mer (CTTCTCATGGTTCTTCGGAA)

31mer (CTGTTGTCATACTTCTCATGGTTCTTCGGAA)

62mer

* B%=60%(0 min)→50%(10-20 min)→60%(20.01-25 min)

2-4mer : [M-H]-5-13mer : [M-2H]2-14-19mer : [M-3H]3-20-26mer : [M-4H]4-27-32mer : [M-5H]5-33-39mer : [M-6H]6-40-45mer : [M-7H]7-46-52mer : [M-8H]8-53-58mer : [M-9H]9-59-62mer : [M-10H]10-

・Oligo DNAs are eluted in order of degree of polymerization・As oligo DNA contains G, the retention becomes stronger Hydrogen bonding may also be involved in separation

・Good linearity・Capable of high selective quantification

Separable up to 31 mer

Sample 122 μg/mL(in H2O)

Sample 1, 21 mg/mL(in H2O) each

Product name Plate number(TP/Column)Functional

group

Particlesize(μm)

Pore size(Å)

Column size(mm)

I.D. × Length

HILICpak VN-50 4D ≥ 10,000 Diol 5 100 4.6 × 150

HILICpak VN-50G 4A (guard column) Diol 5 100 4.6 × 10

HILICpak VN-50 2D ≥ 3,500 Diol 5 100 2.0 × 150

HILICpak VN-50G 2A (guard column) Diol 5 100 2.0 × 10

Sample 32.8 mg/mL(in H2O)

100 500 1000 1500 m/z

1533.4[M-4H]4-

2-4mer : [M-H]-5-11mer : [M-2H]2-12-19mer : [M-3H]3-20mer : [M-4H]4-

Sample 2

5’-ATACCGATTAAGCGAAGTTT-3’

Mass Intensity6137.073 29.986138.073 74.736139.073 100.006140.073 94.486141.073 70.306142.073 43.676143.073 23.486144.073 11.206145.073 4.826146.073 1.906147.073 0.696148.073 0.246149.073 0.086150.073 0.026151.073 0.01

Isotopic Abundances for C197H247O117N76P19

detected as multivalent negative ion

6135.00 6140.00 6145.00 6150.000.0

20.0

40.0

60.0

80.0

100.0

ShodexCAPTURE THE ESSENCE

TMJunji Sasuga2, Yuzuru Kokido2, Hirobumi Aoki2, Eiji Kagawa2, Leah Block1, and Ronald Benson11Showa Denko America, Inc., 420 Lexington Avenue, Suite 2335A, New York, NY 10170, USA

2Showa Denko K.K., 5-1 Ohgimachi Kawasaki-ku, Kawasaki, 210-0867, JapanContact us at support@shodexHPLC.com For more information, visit www.shodexHPLC.com

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