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
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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
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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.