Characterization of HeparinCharacterization of Heparin--derived Oligosaccharides derived Oligosaccharides using Ionusing Ion--Pair ReversedPair Reversed--Phase LC/ESIPhase LC/ESI--MS MS

Weibin ChenWeibin Chen, Catalin E. , Catalin E. DoneanuDoneanu, John C. , John C. GeblerGebler

Biopharmaceutical SciencesBiopharmaceutical Sciences

Waters Corporation, Milford, MAWaters Corporation, Milford, MA

ASMS 2009ASMS 2009Advances in Carbohydrate and Advances in Carbohydrate and ProteoglycanProteoglycan Analysis Analysis

June 3, 2009June 3, 2009

2

Adapted from J. Biol. Chem. 2004;279:2608-2615

-O3SO(2-O-Sulfate)

(6-O-Sulfate)

CH2OSO3-

NHSO3-

(N-Sulfonate)

1

23

45

6

Structures of Major Heparin Oligosaccharides Derived from Heparinase Digestion

A naturally-occurring, highly-sulfated linear polysaccharide consisting of a variably-sulfated repeating disaccharide unit

3

Molecular Weight Distributions of Heparin and Molecular Weight Distributions of Heparin and Low Molecular Weight Heparin (LMWH)Low Molecular Weight Heparin (LMWH)

From Chest 2001;119;64-94

Mean MW of Most Commercial Heparin: ~ 15 kDa

4

Partial Depolymerization by Chemical or Enzymatic Means

Mass Spectrometry for Domain Structural Distributions: Mass Spectrometry for Domain Structural Distributions: Analysis of Partially Analysis of Partially DepolymerizedDepolymerized Heparin Heparin OligosaccharidesOligosaccharides

Heparinase

MS:Composition

MS/MS:Isomeric Differentiation

5

Heparin essentially consists of a variably-sulfated repeating disaccharide unit — Enormous structural diversity

o Possible structural isomers of dp6: 1728

o Require a high-resolution separation technique

— Highly hydrophilic

o Restrictions in chromatographic

techniques

— Presence of sulfate groups

o Generating salt adducts with cations

o Loss of the groups during MS analysis n

Challenges in LC/MS Analysis of Heparin Challenges in LC/MS Analysis of Heparin Oligosaccharide Oligosaccharide -- a Structural Point Viewa Structural Point View

6

Chromatographic Techniques Coupled with MS Chromatographic Techniques Coupled with MS for the Analysis of Heparin Oligosaccharidesfor the Analysis of Heparin Oligosaccharides

Hydrophilic Interaction Chromatography (Amide-HILIC)— Naimy, H., Leymarie, N., Bowman, M., and Zaia, J. (2008)

Biochemistry 47, 3155-3161

Size Exclusion Chromatography (SEC)— Henriksen, J.; Ringborg, L. H.; Roepstorff, P. J. Mass Spectrom. 2004;

39, 1305

Ion-pair Reversed-Phase Chromatography (IRRP)— Thanawiroon, C.; Rice, K. G.; Toida, T.; Linhardt, R. J. J. Biol. Chem.

2004, 279 (4), 2608.— Henriksen, J., Roepstorff, P., Ringborg, L. H. Carbohydr. Res. 2006,

341, 382.— Korir, A. K., Limtiaco, J. F. K.; Gutierrez, S. M.; Larive, C. K. Anal.

Chem. 2008, 80, 1297.

7

IonIon--pair Reversedpair Reversed--Phase Chromatography Phase Chromatography

NH3+

NH3+

NH3+

+H3N

Heparin Oligomer

-3OS

-3OS SO3

_

CO2-

C18 ligand

BEHParticle

Ion-pairing reagent

+H3N

+H3N

8

IonIon--pairing Reagents Used for Heparin pairing Reagents Used for Heparin Oligosaccharide Analysis in LiteratureOligosaccharide Analysis in Literature

Tetrabutyl Ammonium Salt— Not volatile, not compatible with MS detection

Trialkyl Amines (Tributylamine or Tripropylamine)— Need to adjust pH to make the ion-pairing reagent charged

— Volatile acids used, mostly acetic acid

— Acetic Acid greatly suppresses the MS signals – detection limits

(J. Chrom. A, 2003, Vol.1014, P.215)

(JBC, 2004,vol279,P2608)

9

New RPIPNew RPIP--UPLC/MS Method to Improve the UPLC/MS Method to Improve the Performances of LC/MS MethodsPerformances of LC/MS Methods

Use UPLC instead of HPLC to improve separation efficiency— Smaller particles (1.7µm vs. 3.0µm) mean high speed, high

efficiency, and better resolutions

Novel additives in the mobile phases to improve the LC resolution and MS responses— Linear alkyl amines (hexylamine or pentylamine) as ion-pairing

reagent

— 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to adjust the pH of mobile phase

Time-of-flight mass spectrometer to offer high-resolution, high accurate mass measurements

10

Impact of MobileImpact of Mobile--Phase Acid Modifier on the Phase Acid Modifier on the MS Responses of dp6 in Negative Ion ModeMS Responses of dp6 in Negative Ion Mode

11

Alternative IonAlternative Ion--Pairing Reagents for IPRP Pairing Reagents for IPRP ChromatographyChromatography

Hexylamineb.p. 131°C

TripropylamineTPA

b.p. 156°C

TributylamineTBA

b.p. 216°C

TriethylamineTEA

b.p. 90°C

Propylamineb.p. 48°C

Butylamineb.p. 78°C

Pentylamineb.p. 104°C

Octylamineb.p. 175°C

N

N

N

NH2

NH2

NH2

NH2

NH2

12

Ion-pairing Reagent Buffering Acid Abbreviation

Proylammonium HFIP PPA

Butylammonium HFIP BTA

Pentylammonium HFIP PTA

Hexylammonium acetate/HFIP HXA

Octylammonium HFIP OTA

Tripropylammonium acetate/HFIP TPA

Tributylammonium acetate/HFIP TBA

The IonThe Ion--pairing and Buffer Systems pairing and Buffer Systems Investigated in Current StudyInvestigated in Current Study

13

Peak Capacity (P):

)/2w(wtt1P

12

12

+−

+=

MobileMobile--Phase Comparison: Peak Phase Comparison: Peak Capacity and MS Ion CountsCapacity and MS Ion Counts

dp6

dp8

Time (min)1.00 5.00 10.00

14

Impact of MobileImpact of Mobile--phase Modifiers on the Peak phase Modifiers on the Peak Capacity and MS Response: IonCapacity and MS Response: Ion--pairing Reagentspairing Reagents

2.0

4.0

6.0

8.0

6 8 10

Peak C

apacity

oligosaccharides (dp)

APPA BTA

PTA HXA

OTA TPA PPA BTA

PTA HXA

OTA TPA

20

60

100

140

6 8 10

MS

Io

n C

ou

nts

oligosaccharides (dp)

A

15

3.0

5.0

7.0

6 8 10 12 14 16 18

oligosaccharides (dp)

Pe

ak C

ap

acity

B5 mM PTA

15 mM PTA

25 mM PTA

40 mM PTA

5 mM PTA

15 mM PTA

25 mM PTA

40 mM PTA

0

200

400

600

6 8 10 12 14 16 18oligosaccharide (dp)

MS

Ion

Counts

B

Impact of MobileImpact of Mobile--phase Modifiers on the Peak phase Modifiers on the Peak Capacity and MS Response: PTA ConcentrationCapacity and MS Response: PTA Concentration

16

2.0

3.0

4.0

5.0

6.0

6 8 10 12 14 16 18oligosaccharide (dp)

Peak C

apacity

C

no HFIP10 mM HFIP25 mM HFIP50 mM HFIP

100 mM HFIP200 mM HFIP

C

100

300

500

700

6 8 10 12 14 16 18oligosaccharide (dp)

MS

Io

n

Counts

no HFIP10 mM HFIP25 mM HFIP50 mM HFIP

100 mM HFIP200 mM HFIP

Impact of MobileImpact of Mobile--phase Modifiers on the Peak phase Modifiers on the Peak Capacity and MS Response: HFIP ConcentrationCapacity and MS Response: HFIP Concentration

17

Experimental Conditions for the Analysis of Experimental Conditions for the Analysis of HeparinHeparin--derived Oligosaccharidesderived Oligosaccharides

LC Conditions— System: Acquity UPLC® System

— Column: 2.1mm X 150mm, BEH 1.7 µm

— Mobile Phases

o A: 15 mM PTA, 50 mM HFIP in H2O, pH 8.5

o B: 15 mM PTA, 50 mM HFIP in 75% Acetonitrile

— Gradient: 3% B /min

MS Conditions— Synapt HDMS™ Mass Spectrometer

— Cone Voltage: 25 V

— Collision Energy: 4 eV

18

IPRPIPRP--UPLC/ESI MS Analysis of a heparin UPLC/ESI MS Analysis of a heparin Oligosaccharide Mixture (dp6 Oligosaccharide Mixture (dp6 –– dp18)dp18)

*

A

B

dp6 dp8

dp10

dp12dp14

dp18

*

*

**

*

TIC

UV

19

Mass Spectra of Fully Sulfated dp6 and dp8 Mass Spectra of Fully Sulfated dp6 and dp8 OligosaccharidesOligosaccharides

3-

2-

3- 2- dp8

dp6

20

ESIESI--MS Spectrum of a Fully Sulfated MS Spectrum of a Fully Sulfated Hexasaccharides (dp6)Hexasaccharides (dp6)

m/z900 980 1060 1140

%

0

100

OO

O

O

[M+2PTA-2H]2-

[M+3PTA-2H]2-

[M+4PTA-2H]2-

1038.65

[M+5PTA-2H]2-

[M+6PTA-2H]2-

O

21

ESIESI--MS Spectrum of a Fully Sulfated dp18 MS Spectrum of a Fully Sulfated dp18 (27 Sulfate Groups) (27 Sulfate Groups)

m/z1500 1600 1700 1800

[M+11PTA-4H]4-

[M+13PTA-4H]4-

[M+15PTA-4H]4-

[M+17PTA-4H]4-

[M+19PTA-4H]4-

m/z1602 1603 1604 1605

monoisotopic,1601.981

22

Separation of Hexasaccharide Isomers with Separation of Hexasaccharide Isomers with Different Degree of SulfationDifferent Degree of Sulfation

dp6 w/9 Sulfates

dp6 w/ 8 Sulfates

dp6 w/ 7 Sulfates

dp6 w/ 6 Sulfates

dp6 w/ 5 Sulfates

23

Separation of Isomeric Hexasaccharides Separation of Isomeric Hexasaccharides Containing Eight Sulfate Groups (dp6, 8S)Containing Eight Sulfate Groups (dp6, 8S)

6.00 8.00 10.00

AU

0.002

0.006

0.010

0.014

6.00 8.00 10.00

%

0

100

XIC

UV

1 2

3

4

5

6

7

Time (min)

Time (min)

1 23

4

5

6

7

Rel

ativ

e In

tens

ity

24

Analyzing HeparinAnalyzing Heparin--derived Oligosaccharides in derived Oligosaccharides in both Negative and Positive Ion Modeboth Negative and Positive Ion Mode

ESI, Positive

ESI, Negative

25

Spectra of a Fully Sulfated Hexasaccharide Spectra of a Fully Sulfated Hexasaccharide from Negative and Positive Ion Modefrom Negative and Positive Ion Mode

ESI -

ESI +

26

ESIESI--MS Spectrum of a Fully Sulfated dp22 MS Spectrum of a Fully Sulfated dp22 (33 Sulfate Groups)(33 Sulfate Groups)

2100 2200 2300 2400 2500m/z

[M+29PTA+4H]4+

[M+32PTA+4H]4+

[M+35PTA+4H]4+

monoisotopic,2241.249

m/z2241 2243 2245

27

MS/MS Spectrum of Hexasaccharides without MS/MS Spectrum of Hexasaccharides without NN--acetyl Groupacetyl Group

m/z200 400 600 800 1000 1200

%

0

100

162.068

496.125B3

693.215

356.114Y2

657.194B4

320.093B2

338.103C2 / Z2

C4 / Z4675.205

Y3517.183

1092.262

1172.219

1012.306

994.295Y4

320.093B2

496.125B3

657.194B4

Y2356.114

Y3517.183

Y4693.215

A

28

MS/MS for Identification of NMS/MS for Identification of N--acetyl Group in acetyl Group in Hexasaccharides (dp6, 7S)Hexasaccharides (dp6, 7S)

m/z200 400 600 800 1000 1200

%

0

100

168.057

496.125B3

717.215*Z4

699.204*B4

320.093B2

338.103C2 / Z2

*C4/*Y4735.226

*Y3559.194

1054.316

1134.2791036.306

541.183*Z3204.082

*Z1

380.114*Z2

138.051 Z2338.103

*B4699.205

*C4735.226

*Z1204.082

*Z2380.114

29

UV Absorbance Calibration Plots of Fully Sulfated dp6, UV Absorbance Calibration Plots of Fully Sulfated dp6, dp8 and dp10 from 0.5 to 500 dp8 and dp10 from 0.5 to 500 µµg/mLg/mL

R2=0.9994

0

4000

8000

12000

100.0 300.0 500.0

dp6

dp8

dp10

Concentration (µg/mL)

Abs

prba

nce

R2=0.9997

R2=0.9983

A

30

MS Response Calibration Plots of Fully Sulfated MS Response Calibration Plots of Fully Sulfated dp6, dp8 and dp10 from 0.5 to 50 dp6, dp8 and dp10 from 0.5 to 50 µµg/mLg/mL

0

500

1500

2500

3500

10.0 20.0 30.0 40.0 50.0 60.0

Peak Area

MS Ion Counts

Concentration (µg/mL)

Res

pons

es

B

31

Analyzing Heparin Oligosaccharides in Positive Ion Analyzing Heparin Oligosaccharides in Positive Ion Mode With HFIP and Acetic Acid As Acid Modifiers in Mode With HFIP and Acetic Acid As Acid Modifiers in Mobile Phases Mobile Phases

HFIP

Acetic Acid

32

Comparison of Mass Spectra of dp12 from Comparison of Mass Spectra of dp12 from Mobile Phases with HFIP or Acetic AcidMobile Phases with HFIP or Acetic Acid

HFIP

Acetic acid

33

TinzaparinTinzaparin Sodium, API of Sodium, API of InnohepInnohep®®

http://www.innohepusa.com/FullPrescribingInformationforInnohep.pdf

34

IPRPIPRP--UPLC/MS Analysis of a LMWH, UPLC/MS Analysis of a LMWH, TinzaparinTinzaparin Sodium, API of Sodium, API of InnohepInnohep®®

dp6

dp8

dp10

dp12

dp14

dp18

dp16

dp20

dp4

dp22

dp6

dp8

dp10

dp12

dp14

dp18

dp16

dp20

dp4

dp22

35

Mass spectra showing the detection of dp14 Mass spectra showing the detection of dp14 from a standard and from a standard and TinzaparinTinzaparin SodiumSodium

dp14 std

dp14 from Tinzaparin

36

ConclusionsConclusions

A RPIP-UPLC/ESI MS method is developed for the analysis of heparin derived oligosaccharides— Improving chromatographic separation for the differentiation of

the oligomers between class sizes as well as isomeric oligosaccharides

— Enhancing MS responses of oligomers to allows accurate mass measurement of the intact heparin oligomers up to dp22

— Enabling the MS analysis in both negative and positive ion mode and obtaining MS/MS data for the location of N-Acetyl groups among isomers

The method provides a fast and practical approach to obtain in-depth molecular profile information for various heparin samples.