high resolution lc/ms analysis of therapeutic ... · high resolution lc/ms analysis of therapeutic...

High Resolution LC/MS analysis of Therapeutic Oligonucleotides on a New Porous Polymer-Based Reversed Phase Column Shanhua Lin, Julia Baek, Jim Thayer, Hongxia Wang, Jonathan Josephs, Ilze Birznieks, and Xiaodong LiuThermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085

Po

ster No

te 64

732

Shanhua Lin, Julia Baek, Jim Thayer, Hongxia Wang, Jonathan Josephs, Ilze Birznieks and Xiaodong Liu, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94085

RESULTS

Figure1. LC/MS analysis of failure sequences. a) UV and ion current traces. b) Mass spectrum of 21mer at -4 charge state. c) Mass spectrum of n-1 failure sequence at -4 charge state.

In Figure 5 an unmodified ON and the CpG methylated ON are well resolved on the DNAPac RP column. Figure 4b shows the -3 charge state of unmodified CpG ON at m/z 1517.919 and the -3charge state of methylated CpG ON at m/z 1522.593.The mass difference between the methylated and unmodified peaks corresponds to one methyl group.

ABSTRACTTherapeutic models for synthetic oligonucleotides (ONs) include gene silencing (siRNA), cellular immunity (immunostimulatory [is]) RNA, inhibitory binding (aptamers). These applications require thorough and effective characterization and quality control of synthetic oligonucleotides to satisfy regulatory agencies. High performance LC and LC/MS are the preferred tools for these analyses, and are often used for more common oligonucleotide purity assessments. Ion-pair reversed phase LC is the preferred method for coupling to MS for these analyses. We introduce a new polymeric reversed phase column and ion-pair methods for LC and LC/MS ON analysis.The new column is based on a highly stable hydrophobic, polymer resin capable of operation at temperatures up to 100 ºC and pH up to 14. Coupling the column to a Q Exactive Plus Orbitrap mass spectrometer successfully identified overlapping failure sequences, ONs with characteristic methylation (CpG and 2’-O-methylated), phosphorothioate diastereoisomers and 5’-phosphorylated DNAs.

INTRODUCTIONSynthetic ONs with different functionalities including antisense ONs, small interfering RNAs (siRNAs), aptamers and immunostimulatory RNAs (isRNAs) are candidate therapeutic agents due to their specificity, and well-established synthesis and modification technologies. Still characterization is required to satisfy regulatory agencies that efficacy and safety of these therapeutic ONs are established. Such analyses include characterization of modifications to the base, sugar and backbone linkages, as these are commonly employed to decrease in vivodegradation and increase therapeutic efficacy. High performance liquid chromatography (HPLC) and mass spectrometry (MS) comprise valuable tools for the purity assessment and identification of impurities in oligonucleotide samples. Anion-exchange (AEX) chromatography and ion-pair reversed phase chromatography (IP-RP) are the most commonly used techniques for LC/UV or LC/fluorescence analysis of oligonucleotides. However for LC/MS analysis, AEX chromatography is less popular due to the high salt concentration used in their mobile phases which requires a desalting step before MS analysis. Ion-pair reversed phase LC, with volatile mobile phase components, can be directly coupled to MS. Here we introduce a new polymeric reversed phase column and ion-pair methods for LC/MS ON analysis.

MATERIALS AND METHODS

Samples21mer DNA: GATTGTAGGTTCTCTAACGCT

21mer siRNA sense strand 1: AGCUGACCCUGAAGSUUCAUdCdT

21mer siRNA sense strand 2: A-MeOG-C-MeOU-G-MeOA-s-C-MeOC-C-MeOU-G-MeOA-A-MeOG-s-U-MeOU-C-MeOA-U-dCdT

21mer siRNA sense strand 3: AGCUGACCCUGAAGUUCAUSdSdT

15mer DNA: CGGCATCCTTATTGG

CpG methylated 15mer DNA: iMe-dC/GGCATCCTTATTGG

Liquid Chromatography

HPLC experiments were carried out using a Thermo Scientific™ Dionex™ UltiMate™ 3000 BioRS system equipped with:

SR-3000 Solvent Rack (P/N 5035.9200)LPG-3400RS Biocompatible Quaternary Rapid Separation Pump (P/N 5040.0036)WPS-3000TBRS Biocompatible Rapid Separation Thermostatted Autosampler (P/N 5841.0020)TCC-3000RS Rapid Separation Thermostatted Column Compartment (P/N 5730.0000)VWD-3400RS Rapid Separation Variable Wavelength Detector (VWD) equipped with micro flow cell (P/N 5074.0010)Chromatography was controlled by Thermo Scientific™ Dionex™ Chromeleon™Chromatography Data System .

Mass Spectrometry

The Thermo Scientific™ Q Exactive™ Plus hybrid quadrupole-Orbitrap mass spectrometer was used for this study. All data were acquired in negative ion mode.

CONCLUSIONS

•ON product and n-1 failure sequence, were separated on the DNAPac RP column. High resolution orbitrap mass spectrometer revealed loss of each of the four bases.

•siRNA ONs harboring diastereomers of phosphorothioate with our without 2’-O-methyl modifications were separated using high pH mobile phases. In addition, PO impurity was baseline separated from the PS product.

•CpG methylation was successfully identified using DNAPac RP and high resolution mass spectrometer.

REFERENCES

1.Dias, N. et al. Molecular Cancer Therapeutics (2002) 1, 347-355.

2.Resnier, P. et al. Biomaterials (2013) 34, 6429-6443.

3.Jones, P.A. et al. Nature Reviews Genetics (2002) 3, 415-428.

TRADEMARKS/LICENSING© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

High Resolution LC/MS analysis of Therapeutic Oligonucleotides on a New Porous Polymer-Based Reversed Phase Column

Analysis of failure sequencesSynthetic ONs molecules are used as PCR primers, aptamers, as library adaptors for genomic studies and as therapeutic agents.1,2 High purity ONs in these applications are required. Therefore separation and identification of failure sequences and other impurities is critical for the production of ON drugs.

In Figure 1a, a 21mer ON was analyzed using mass spec compatible mobile phases (TEA, HFIP). A small peak in front of the target peak was observed. The MS data confirmed the desired target product. Monoisotopic m/z value at charge state -4 for the 21 mer DNA was 1605.016 with mass accuracy of 1.87 ppm (Figure 1b). The high resolution mass spectrometer revealed loss of each of the four bases in the n-1 peak. The masses of failure sequences with missing Guanine or Adenine or Cytosine or Thymine were detected (Figure 1c).

Analysis of phosphorothioate and 2’-O-methyl modified siRNAsSynthetic siRNAs are important tools for gene function studies and as potential therapeutic agents.2 Nucleic acids are often modified to increase in vivo stability. A common modification in DNA and RNA is incorporation of phosphorothioate (PS) linkages. Another common, but RNA-specific modification is 2’-O-methylation on ribose. The PS linkage introduces a chiral center at phosphorus in addition to the chiral centers in D-ribose of the nucleic acid. Therefore PS modified linkages produce diastereoisomer pairs at each PS linkage.

Figure 2 shows the separation of a sense strand that has one phosphorothioate linkage incorporated at base 14 in the sequence. The two possible diastereoisomers were baseline separated on the DNAPac RP column using high pH mobile phases. At -4 charge state, m/z value of first and the second peaks were 1655.964 and 1655.971 respectively, indicating these molecules to be diastereoisomers rather than failure sequences other impurities.

In Figure 3, sense strand of the siRNA was 2’-O-methylated on alternate bases and contains phosphorothioate linkages at the 6th and 14th bases. The UV trace and the ion current traces show the separation of all four possible phosphorothioate diastereoisomers. The high resolution MS data reveal identical masses for all four peaks confirming these molecules to be isomers.

In Figure 4, the sense strand is modified with two PS linkages at the 19th and the 20th bases. In this case, three of the four possible diastereoisomers were chromatographically resolved. In addition to the diastereoisomer peaks, an impurity which contains a single PS linkage (PO) was detected. The mass difference between peak 4 and all three of the other peaks is 16 Da, corresponding to the mass difference between the oxygen and sulfur.

Mass range Spray voltage Sheath gas Auxiliary gas Capillary temp. S-lens level

m/z 600-4,000 3.5 kV 40 arb. units 15 arb. units 320 ºC 50

In-source CID Microscans AGC target Maximum IT Resolution Probe temp.

0 eV 1 3 ×106 200 ms 70,000 300 ºC

1 2 3 4 5Time (min)

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Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 15 mM TEA, 400 mM HFIP, pH 7.9Mobile phase B: 15 mM TEA, 400 mM HFIP in

Water / Methanol (50:50 v/v)

Gradient:Time (min) %A %B0.0 70 303.0 48 533.1 10 905.0 10 905.1 70 30

11.0 70 30

Temperature: 60 ºCFlow rate: 0.25 mL/minInj. volume: 4 µL Detection: UV (260 nm)

MS (Negative-ion mode)Mass Spec: Q Exactive PlusSample: 21mer DNA

GATTGTAGGTTCTCTAACGCT

100

Mi=1605.0160

[M+Na+K-6H]4-

[M-4H]4-

[M+Na-5H]4-

[M+K-5H]4-

[M+2Na-6H]4-

1600 1605 1610 1615 1620 1625 1630m/z

0

20

40

60

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1615.2522z=4

1611.0105z=4

1620.4968z=4 1624.7399

z=4 1629.9860z=4

Rel

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RT: 4.10-4.16

a

b

c -T

1520 1522 1524 1526 1528 1530 1532 1534 1536 1538 1540 1542 1544 1546 1548 1550m/z

0

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Rel

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1529.5098z=4

1527.2569z=4

z=4

z=4

1523.2571z=4

Mi=1526.7534

Mi=1522.7602

Mi=1529.0094

Mi=1532.2514

1533.2577z=4

1535.0061

1538.9978z=4

1542.7451z=4

1536.7438

1548.4852z=4

1544.4927z=4 1546.4824

z=4

-G

-A-C

Figure 2. LC/MS analysis of phosphorothioate modified siRNA

Figure 4. Separation of PO impurity from PS product. a) UV and ion current traces. b) Mass spectra of peaks at -4 charge state.

2 3 4 5 6 7 8Time (min)

0

0

Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 35 mM TEA, 40 mM HFIP, pH 9.9Mobile phase B: 35 mM TEA, 40 mM HFIP in Water

/ Methanol (75:25 v/v)


13.0 67 33


MS (Negative-ion mode)Mass Spec: Q Exactive PlusSample: 21mer siRNA

A-MeOG-C-MeOU-G-MeOA-s-C-MeOC-C-MeOU-G-MeOA-A-MeOG-s-U-MeOU-C-MeOA-U-dCdT

100

16

Ion current

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(mA

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1

23

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2 3

4

Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 35 mM TEA, 40 mM HFIP, pH 9.9Mobile phase B: 35 mM TEA, 40 mM HFIP in Water /

Methanol (75:25 v/v)


13.0 93 7



AGCUGACCCUGAAGSUUCAUdCdT

1 2 3 4 5 6 70

0

100

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2Column: DNAPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: 15 mM TEA, 400 mM HFIP, pH 7.6Mobile phase B: 15 mM TEA, 400 mM HFIP in



11.0 75 25

Temperature: 60 ºCFlow rate: 0.25Inj. volume: 3 µL Detection: UV (260 nm)Sample: 1) CGGCATCCTTATTGG

2) /iMe-dC/GGCATCCTTATTGG

1 2 3 4 5 6 7Time (min)

0

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40

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1

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a

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0

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40

60

80

1001518.5880

z=3

Rel

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z=3

Peak 1

[M-3H]3-

1522.5926

1510 1515 1520 1525 1530 1535 1540 1545 1550

m/z

0

20

40

60

80

1001523.2603

z=3

1524.2618z=3

z=3

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Peak 2

1510 1515 1520m/z

[M-3H]3-

a

b

N H 2

ONH

N

N H 2

ONH

NMethylation

Cytosine 5-methyl Cytosine

[M-4H]4-

Peak 1: RT 5.93 min

Peak 3: RT 6.23 min[M-4H]4-

Peak 2: RT 6.13 min


[M-4H]4-

1688 1689 1690 1691 1692 1693 1694 1695 1696m/z

0

100

0

100

0

100

0

1001692.2498

z=41692.5007z=4

1691.7489z=4

1692.9972z=41691.5004

z=4 1693.5048z=4

1692.2515z=4

1692.4994z=41691.7504

z=41692.9998z=41691.4985

z=4 1693.7490z=4

1692.2498z=41691.7498

z=4 1692.7501z=4

1693.0035z=41691.5001

z=4 1693.7549z=4

1692.5012z=4

1692.2517z=4 1692.7518

z=41691.7488z=4 1693.0029

z=41691.5002z=4

Rel

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Figure 3. LC/MS analysis of phosphorothioate and 2’-O-methyl modified siRNA. a) UV and ion current traces. b) Mass spectra of peaks at -4 charge state.

[M-4H]4-Peak 4:RT 4.96 min

Peak 2: RT 5.76 min [M-4H]4-

Peak 1: RT 5.57 min


[M-4H]4-

1655 1656 1657 1658 1659 1660 1661 1662 1663m/z

0

100

0

100

0

100

0

1001656.7231

z=41662.2181

z=41661.7166z=4

1656.9744z=4

1662.7181z=41655.9773

z=4

1660.4620z=4

1660.2134z=4

1660.9648z=4

1659.9624z=4

1661.4653z=4

1660.9640z=41660.4652

z=4 1661.2151z=4

1660.2202z=4 1661.7079

z=41659.9653z=4

1660.7145z=41660.2139

z=4

1661.4708z=4

1659.9581z=4

Rel

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2 3 4 5 6 7 8

0

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AGCUGACCCUGAAGUUCAUSdSdT

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3

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3

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Time (min)

Analysis of CpG methylationMethylation of CpG sequences in the promoter regions suppresses the expression of the gene and aberrant methylation has been implicated in the development and progression of cancer.3 Therefore detection of CpG methylation is important for epigenetics studies and cancer research.

Figure 5. LC/MS analysis of CpG methylation. a) UV and ion current traces. b) Mass spectra of peaks at -3 charge state.

Sodium and potassium adducts


RESULTS















Mass Spectrometry


CONCLUSIONS




REFERENCES















0 eV 1 3 ×106 200 ms 70,000 300 ºC

1 2 3 4 5Time (min)

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11.0 70 30




100

Mi=1605.0160

[M+Na+K-6H]4-

[M-4H]4-

[M+Na-5H]4-

[M+K-5H]4-

[M+2Na-6H]4-

1600 1605 1610 1615 1620 1625 1630m/z

0

20

40

60

80

1615.2522z=4

1611.0105z=4

1620.4968z=4 1624.7399

z=4 1629.9860z=4

Rel

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unda

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RT: 4.10-4.16

a

b

c -T

1520 1522 1524 1526 1528 1530 1532 1534 1536 1538 1540 1542 1544 1546 1548 1550m/z

0

20

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60

80

100

Rel

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1529.5098z=4

1527.2569z=4

z=4

z=4

1523.2571z=4

Mi=1526.7534

Mi=1522.7602

Mi=1529.0094

Mi=1532.2514

1533.2577z=4

1535.0061

1538.9978z=4

1542.7451z=4

1536.7438

1548.4852z=4

1544.4927z=4 1546.4824

z=4

-G

-A-C



2 3 4 5 6 7 8Time (min)

0

0




13.0 67 33




100

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Ion current

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1 2 3 4 5 6 70

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11.0 75 25



1 2 3 4 5 6 7Time (min)

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1

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a

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0

20

40

60

80

1001518.5880

z=3

Rel

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z=3

Peak 1

[M-3H]3-

1522.5926

1510 1515 1520 1525 1530 1535 1540 1545 1550

m/z

0

20

40

60

80

1001523.2603

z=3

1524.2618z=3

z=3

Rel

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Peak 2

1510 1515 1520m/z

[M-3H]3-

a

b

N H 2

ONH

N

N H 2

ONH

NMethylation


[M-4H]4-

Peak 1: RT 5.93 min


Peak 2: RT 6.13 min


[M-4H]4-

1688 1689 1690 1691 1692 1693 1694 1695 1696m/z

0

100

0

100

0

100

0

1001692.2498

z=41692.5007z=4

1691.7489z=4

1692.9972z=41691.5004

z=4 1693.5048z=4

1692.2515z=4

1692.4994z=41691.7504

z=41692.9998z=41691.4985

z=4 1693.7490z=4

1692.2498z=41691.7498

z=4 1692.7501z=4

1693.0035z=41691.5001

z=4 1693.7549z=4

1692.5012z=4

1692.2517z=4 1692.7518

z=41691.7488z=4 1693.0029

z=41691.5002z=4

Rel

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Peak 2: RT 5.76 min [M-4H]4-

Peak 1: RT 5.57 min


[M-4H]4-

1655 1656 1657 1658 1659 1660 1661 1662 1663m/z

0

100

0

100

0

100

0

1001656.7231

z=41662.2181

z=41661.7166z=4

1656.9744z=4

1662.7181z=41655.9773

z=4

1660.4620z=4

1660.2134z=4

1660.9648z=4

1659.9624z=4

1661.4653z=4

1660.9640z=41660.4652

z=4 1661.2151z=4

1660.2202z=4 1661.7079

z=41659.9653z=4

1660.7145z=41660.2139

z=4

1661.4708z=4

1659.9581z=4

Rel

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2 High Resolution LC/MS analysis of Therapeutic Oligonucleotides on a New Porous Polymer-Based Reversed Phase Column


RESULTS















Mass Spectrometry


CONCLUSIONS




REFERENCES















0 eV 1 3 ×106 200 ms 70,000 300 ºC

1 2 3 4 5Time (min)

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11.0 70 30




100

Mi=1605.0160

[M+Na+K-6H]4-

[M-4H]4-

[M+Na-5H]4-

[M+K-5H]4-

[M+2Na-6H]4-

1600 1605 1610 1615 1620 1625 1630m/z

0

20

40

60

80

1615.2522z=4

1611.0105z=4

1620.4968z=4 1624.7399

z=4 1629.9860z=4

Rel

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unda

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RT: 4.10-4.16

a

b

c -T

1520 1522 1524 1526 1528 1530 1532 1534 1536 1538 1540 1542 1544 1546 1548 1550m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

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1529.5098z=4

1527.2569z=4

z=4

z=4

1523.2571z=4

Mi=1526.7534

Mi=1522.7602

Mi=1529.0094

Mi=1532.2514

1533.2577z=4

1535.0061

1538.9978z=4

1542.7451z=4

1536.7438

1548.4852z=4

1544.4927z=4 1546.4824

z=4

-G

-A-C



2 3 4 5 6 7 8Time (min)

0

0




13.0 67 33




100

16

Ion current

UV

Rel

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13.0 93 7




1 2 3 4 5 6 70

0

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11.0 75 25



1 2 3 4 5 6 7Time (min)

0

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40

Rel

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1

12

a

b

0

20

40

60

80

1001518.5880

z=3

Rel

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z=3

Peak 1

[M-3H]3-

1522.5926

1510 1515 1520 1525 1530 1535 1540 1545 1550

m/z

0

20

40

60

80

1001523.2603

z=3

1524.2618z=3

z=3

Rel

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Peak 2

1510 1515 1520m/z

[M-3H]3-

a

b

N H 2

ONH

N

N H 2

ONH

NMethylation


[M-4H]4-

Peak 1: RT 5.93 min


Peak 2: RT 6.13 min


[M-4H]4-

1688 1689 1690 1691 1692 1693 1694 1695 1696m/z

0

100

0

100

0

100

0

1001692.2498

z=41692.5007z=4

1691.7489z=4

1692.9972z=41691.5004

z=4 1693.5048z=4

1692.2515z=4

1692.4994z=41691.7504

z=41692.9998z=41691.4985

z=4 1693.7490z=4

1692.2498z=41691.7498

z=4 1692.7501z=4

1693.0035z=41691.5001

z=4 1693.7549z=4

1692.5012z=4

1692.2517z=4 1692.7518

z=41691.7488z=4 1693.0029

z=41691.5002z=4

Rel

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Peak 2: RT 5.76 min [M-4H]4-

Peak 1: RT 5.57 min


[M-4H]4-

1655 1656 1657 1658 1659 1660 1661 1662 1663m/z

0

100

0

100

0

100

0

1001656.7231

z=41662.2181

z=41661.7166z=4

1656.9744z=4

1662.7181z=41655.9773

z=4

1660.4620z=4

1660.2134z=4

1660.9648z=4

1659.9624z=4

1661.4653z=4

1660.9640z=41660.4652

z=4 1661.2151z=4

1660.2202z=4 1661.7079

z=41659.9653z=4

1660.7145z=41660.2139

z=4

1661.4708z=4

1659.9581z=4

Rel

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b

a

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0

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100

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13.0 93 7




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RESULTS















Mass Spectrometry


CONCLUSIONS




REFERENCES















0 eV 1 3 ×106 200 ms 70,000 300 ºC

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n-1

n

n-1




11.0 70 30




100

Mi=1605.0160

[M+Na+K-6H]4-

[M-4H]4-

[M+Na-5H]4-

[M+K-5H]4-

[M+2Na-6H]4-

1600 1605 1610 1615 1620 1625 1630m/z

0

20

40

60

80

1615.2522z=4

1611.0105z=4

1620.4968z=4 1624.7399

z=4 1629.9860z=4

Rel

ativ

e Ab

unda

nce

RT: 4.10-4.16

a

b

c -T

1520 1522 1524 1526 1528 1530 1532 1534 1536 1538 1540 1542 1544 1546 1548 1550m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1529.5098z=4

1527.2569z=4

z=4

z=4

1523.2571z=4

Mi=1526.7534

Mi=1522.7602

Mi=1529.0094

Mi=1532.2514

1533.2577z=4

1535.0061

1538.9978z=4

1542.7451z=4

1536.7438

1548.4852z=4

1544.4927z=4 1546.4824

z=4

-G

-A-C



2 3 4 5 6 7 8Time (min)

0

0




13.0 67 33




100

16

Ion current

UV

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

1

23

4

1

2 3

4




13.0 93 7




1 2 3 4 5 6 70

0

100

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

Ion current

UV40

1

2

12

Time (min)




11.0 75 25



1 2 3 4 5 6 7Time (min)

0

0

100

40

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

1

12

a

b

0

20

40

60

80

1001518.5880

z=3

Rel

ativ

e Ab

unda

nce 1517.9194

z=3

Peak 1

[M-3H]3-

1522.5926

1510 1515 1520 1525 1530 1535 1540 1545 1550

m/z

0

20

40

60

80

1001523.2603

z=3

1524.2618z=3

z=3

Rel

ativ

e Ab

unda

nce

Peak 2

1510 1515 1520m/z

[M-3H]3-

a

b

N H 2

ONH

N

N H 2

ONH

NMethylation


[M-4H]4-

Peak 1: RT 5.93 min


Peak 2: RT 6.13 min


[M-4H]4-

1688 1689 1690 1691 1692 1693 1694 1695 1696m/z

0

100

0

100

0

100

0

1001692.2498

z=41692.5007z=4

1691.7489z=4

1692.9972z=41691.5004

z=4 1693.5048z=4

1692.2515z=4

1692.4994z=41691.7504

z=41692.9998z=41691.4985

z=4 1693.7490z=4

1692.2498z=41691.7498

z=4 1692.7501z=4

1693.0035z=41691.5001

z=4 1693.7549z=4

1692.5012z=4

1692.2517z=4 1692.7518

z=41691.7488z=4 1693.0029

z=41691.5002z=4

Rel

ativ

e A

bund

ance



Peak 2: RT 5.76 min [M-4H]4-

Peak 1: RT 5.57 min


[M-4H]4-

1655 1656 1657 1658 1659 1660 1661 1662 1663m/z

0

100

0

100

0

100

0

1001656.7231

z=41662.2181

z=41661.7166z=4

1656.9744z=4

1662.7181z=41655.9773

z=4

1660.4620z=4

1660.2134z=4

1660.9648z=4

1659.9624z=4

1661.4653z=4

1660.9640z=41660.4652

z=4 1661.2151z=4

1660.2202z=4 1661.7079

z=41659.9653z=4

1660.7145z=41660.2139

z=4

1661.4708z=4

1659.9581z=4

Rel

ativ

e Ab

unda

nce

b

a

2 3 4 5 6 7 8

0

0

100

56

Ion current

UV




13.0 93 7




Rel

ativ

e Ab

unda

nce

Abs

orba

nce

(mA

U)

3

24

1

3

24

Time (min)





RESULTS















Mass Spectrometry


CONCLUSIONS




REFERENCES















0 eV 1 3 ×106 200 ms 70,000 300 ºC

1 2 3 4 5Time (min)

0

240

100

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

Ion current

UV

n

n-1

n

n-1




11.0 70 30




100

Mi=1605.0160

[M+Na+K-6H]4-

[M-4H]4-

[M+Na-5H]4-

[M+K-5H]4-

[M+2Na-6H]4-

1600 1605 1610 1615 1620 1625 1630m/z

0

20

40

60

80

1615.2522z=4

1611.0105z=4

1620.4968z=4 1624.7399

z=4 1629.9860z=4

Rel

ativ

e Ab

unda

nce

RT: 4.10-4.16

a

b

c -T

1520 1522 1524 1526 1528 1530 1532 1534 1536 1538 1540 1542 1544 1546 1548 1550m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1529.5098z=4

1527.2569z=4

z=4

z=4

1523.2571z=4

Mi=1526.7534

Mi=1522.7602

Mi=1529.0094

Mi=1532.2514

1533.2577z=4

1535.0061

1538.9978z=4

1542.7451z=4

1536.7438

1548.4852z=4

1544.4927z=4 1546.4824

z=4

-G

-A-C



2 3 4 5 6 7 8Time (min)

0

0




13.0 67 33




100

16

Ion current

UV

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

1

23

4

1

2 3

4




13.0 93 7




1 2 3 4 5 6 70

0

100

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

Ion current

UV40

1

2

12

Time (min)




11.0 75 25



1 2 3 4 5 6 7Time (min)

0

0

100

40

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

1

12

a

b

0

20

40

60

80

1001518.5880

z=3

Rel

ativ

e Ab

unda

nce 1517.9194

z=3

Peak 1

[M-3H]3-

1522.5926

1510 1515 1520 1525 1530 1535 1540 1545 1550

m/z

0

20

40

60

80

1001523.2603

z=3

1524.2618z=3

z=3

Rel

ativ

e Ab

unda

nce

Peak 2

1510 1515 1520m/z

[M-3H]3-

a

b

N H 2

ONH

N

N H 2

ONH

NMethylation


[M-4H]4-

Peak 1: RT 5.93 min


Peak 2: RT 6.13 min


[M-4H]4-

1688 1689 1690 1691 1692 1693 1694 1695 1696m/z

0

100

0

100

0

100

0

1001692.2498

z=41692.5007z=4

1691.7489z=4

1692.9972z=41691.5004

z=4 1693.5048z=4

1692.2515z=4

1692.4994z=41691.7504

z=41692.9998z=41691.4985

z=4 1693.7490z=4

1692.2498z=41691.7498

z=4 1692.7501z=4

1693.0035z=41691.5001

z=4 1693.7549z=4

1692.5012z=4

1692.2517z=4 1692.7518

z=41691.7488z=4 1693.0029

z=41691.5002z=4

Rel

ativ

e A

bund

ance



Peak 2: RT 5.76 min [M-4H]4-

Peak 1: RT 5.57 min


[M-4H]4-

1655 1656 1657 1658 1659 1660 1661 1662 1663m/z

0

100

0

100

0

100

0

1001656.7231

z=41662.2181

z=41661.7166z=4

1656.9744z=4

1662.7181z=41655.9773

z=4

1660.4620z=4

1660.2134z=4

1660.9648z=4

1659.9624z=4

1661.4653z=4

1660.9640z=41660.4652

z=4 1661.2151z=4

1660.2202z=4 1661.7079

z=41659.9653z=4

1660.7145z=41660.2139

z=4

1661.4708z=4

1659.9581z=4

Rel

ativ

e Ab

unda

nce

b

a

2 3 4 5 6 7 8

0

0

100

56

Ion current

UV




13.0 93 7




Rel

ativ

e Ab

unda

nce

Abs

orba

nce

(mA

U)

3

24

1

3

24

Time (min)





RESULTS















Mass Spectrometry


CONCLUSIONS




REFERENCES















0 eV 1 3 ×106 200 ms 70,000 300 ºC

1 2 3 4 5Time (min)

0

240

100

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

Ion current

UV

n

n-1

n

n-1




11.0 70 30




100

Mi=1605.0160

[M+Na+K-6H]4-

[M-4H]4-

[M+Na-5H]4-

[M+K-5H]4-

[M+2Na-6H]4-

1600 1605 1610 1615 1620 1625 1630m/z

0

20

40

60

80

1615.2522z=4

1611.0105z=4

1620.4968z=4 1624.7399

z=4 1629.9860z=4

Rel

ativ

e Ab

unda

nce

RT: 4.10-4.16

a

b

c -T

1520 1522 1524 1526 1528 1530 1532 1534 1536 1538 1540 1542 1544 1546 1548 1550m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1529.5098z=4

1527.2569z=4

z=4

z=4

1523.2571z=4

Mi=1526.7534

Mi=1522.7602

Mi=1529.0094

Mi=1532.2514

1533.2577z=4

1535.0061

1538.9978z=4

1542.7451z=4

1536.7438

1548.4852z=4

1544.4927z=4 1546.4824

z=4

-G

-A-C



2 3 4 5 6 7 8Time (min)

0

0




13.0 67 33




100

16

Ion current

UV

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

1

23

4

1

2 3

4




13.0 93 7




1 2 3 4 5 6 70

0

100

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

Ion current

UV40

1

2

12

Time (min)




11.0 75 25



1 2 3 4 5 6 7Time (min)

0

0

100

40

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

1

12

a

b

0

20

40

60

80

1001518.5880

z=3

Rel

ativ

e Ab

unda

nce 1517.9194

z=3

Peak 1

[M-3H]3-

1522.5926

1510 1515 1520 1525 1530 1535 1540 1545 1550

m/z

0

20

40

60

80

1001523.2603

z=3

1524.2618z=3

z=3

Rel

ativ

e Ab

unda

nce

Peak 2

1510 1515 1520m/z

[M-3H]3-

a

b

N H 2

ONH

N

N H 2

ONH

NMethylation


[M-4H]4-

Peak 1: RT 5.93 min


Peak 2: RT 6.13 min


[M-4H]4-

1688 1689 1690 1691 1692 1693 1694 1695 1696m/z

0

100

0

100

0

100

0

1001692.2498

z=41692.5007z=4

1691.7489z=4

1692.9972z=41691.5004

z=4 1693.5048z=4

1692.2515z=4

1692.4994z=41691.7504

z=41692.9998z=41691.4985

z=4 1693.7490z=4

1692.2498z=41691.7498

z=4 1692.7501z=4

1693.0035z=41691.5001

z=4 1693.7549z=4

1692.5012z=4

1692.2517z=4 1692.7518

z=41691.7488z=4 1693.0029

z=41691.5002z=4

Rel

ativ

e A

bund

ance



Peak 2: RT 5.76 min [M-4H]4-

Peak 1: RT 5.57 min


[M-4H]4-

1655 1656 1657 1658 1659 1660 1661 1662 1663m/z

0

100

0

100

0

100

0

1001656.7231

z=41662.2181

z=41661.7166z=4

1656.9744z=4

1662.7181z=41655.9773

z=4

1660.4620z=4

1660.2134z=4

1660.9648z=4

1659.9624z=4

1661.4653z=4

1660.9640z=41660.4652

z=4 1661.2151z=4

1660.2202z=4 1661.7079

z=41659.9653z=4

1660.7145z=41660.2139

z=4

1661.4708z=4

1659.9581z=4

Rel

ativ

e Ab

unda

nce

b

a

2 3 4 5 6 7 8

0

0

100

56

Ion current

UV




13.0 93 7




Rel

ativ

e Ab

unda

nce

Abs

orba

nce

(mA

U)

3

24

1

3

24

Time (min)





RESULTS















Mass Spectrometry


CONCLUSIONS




REFERENCES















0 eV 1 3 ×106 200 ms 70,000 300 ºC

1 2 3 4 5Time (min)

0

240

100

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

Ion current

UV

n

n-1

n

n-1




11.0 70 30




100

Mi=1605.0160

[M+Na+K-6H]4-

[M-4H]4-

[M+Na-5H]4-

[M+K-5H]4-

[M+2Na-6H]4-

1600 1605 1610 1615 1620 1625 1630m/z

0

20

40

60

80

1615.2522z=4

1611.0105z=4

1620.4968z=4 1624.7399

z=4 1629.9860z=4

Rel

ativ

e Ab

unda

nce

RT: 4.10-4.16

a

b

c -T

1520 1522 1524 1526 1528 1530 1532 1534 1536 1538 1540 1542 1544 1546 1548 1550m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1529.5098z=4

1527.2569z=4

z=4

z=4

1523.2571z=4

Mi=1526.7534

Mi=1522.7602

Mi=1529.0094

Mi=1532.2514

1533.2577z=4

1535.0061

1538.9978z=4

1542.7451z=4

1536.7438

1548.4852z=4

1544.4927z=4 1546.4824

z=4

-G

-A-C



2 3 4 5 6 7 8Time (min)

0

0




13.0 67 33




100

16

Ion current

UV

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

1

23

4

1

2 3

4




13.0 93 7




1 2 3 4 5 6 70

0

100

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

Ion current

UV40

1

2

12

Time (min)




11.0 75 25



1 2 3 4 5 6 7Time (min)

0

0

100

40

Rel

ativ

e A

bund

ance

Abs

orba

nce

(mA

U)

1

12

a

b

0

20

40

60

80

1001518.5880

z=3

Rel

ativ

e Ab

unda

nce 1517.9194

z=3

Peak 1

[M-3H]3-

1522.5926

1510 1515 1520 1525 1530 1535 1540 1545 1550

m/z

0

20

40

60

80

1001523.2603

z=3

1524.2618z=3

z=3

Rel

ativ

e Ab

unda

nce

Peak 2

1510 1515 1520m/z

[M-3H]3-

a

b

N H 2

ONH

N

N H 2

ONH

NMethylation


[M-4H]4-

Peak 1: RT 5.93 min


Peak 2: RT 6.13 min


[M-4H]4-

1688 1689 1690 1691 1692 1693 1694 1695 1696m/z

0

100

0

100

0

100

0

1001692.2498

z=41692.5007z=4

1691.7489z=4

1692.9972z=41691.5004

z=4 1693.5048z=4

1692.2515z=4

1692.4994z=41691.7504

z=41692.9998z=41691.4985

z=4 1693.7490z=4

1692.2498z=41691.7498

z=4 1692.7501z=4

1693.0035z=41691.5001

z=4 1693.7549z=4

1692.5012z=4

1692.2517z=4 1692.7518

z=41691.7488z=4 1693.0029

z=41691.5002z=4

Rel

ativ

e A

bund

ance



Peak 2: RT 5.76 min [M-4H]4-

Peak 1: RT 5.57 min


[M-4H]4-

1655 1656 1657 1658 1659 1660 1661 1662 1663m/z

0

100

0

100

0

100

0

1001656.7231

z=41662.2181

z=41661.7166z=4

1656.9744z=4

1662.7181z=41655.9773

z=4

1660.4620z=4

1660.2134z=4

1660.9648z=4

1659.9624z=4

1661.4653z=4

1660.9640z=41660.4652

z=4 1661.2151z=4

1660.2202z=4 1661.7079

z=41659.9653z=4

1660.7145z=41660.2139

z=4

1661.4708z=4

1659.9581z=4

Rel

ativ

e Ab

unda

nce

b

a

2 3 4 5 6 7 8

0

0

100

56

Ion current

UV




13.0 93 7




Rel

ativ

e Ab

unda

nce

Abs

orba

nce

(mA

U)

3

24

1

3

24

Time (min)




PN64732-EN 0616S

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