Waters Oligonucleotide Analysis Solutions

©2017 Waters Corporation 1COMPANY CONFIDENTIAL

Oligonucleotide Analysis SolutionsFrom Characterization to QC


Optimized LC-MS Workflows for Oligonucleotide Characterization and QC Analysis

SEPARATIONS DETECTION & INFORMATICS

MassPREP StandardQuality reference material

Oligonucleotide Columns with BEH TechnologySuperior temperature and pH stability; exceptional sample resolution and column life

Bio-inert flow path and the benefits of UPLC resolution, sensitivity, and throughput for optimal performance

Characterization and High Res Monitoring: ACQUITY UPLC H-Class Bio with TUV and Xevo G2-XS QTof

+ProMass HR

+ProMass or

ProMass HR

UPLC-MS

UPLC-HRMS

QC Analysis with Mass Detection: ACQUITY UPLC H-Class Bio with TUV and ACQUITY QDa Mass Detector

UPLC-HDMS

+ProMass HR

Characterization with ion mobility: ACQUITY UPLC H-Class Bio with TUV and SYNAPT G2-Si


Tools for Achieving Optimal Oligonucleotide Separations


ACQUITY H-Class Bio UPLC Liquid Chromatography System

Unrivaled UPLC resolution, sensitivity and throughput for optimum peak clarity and selectivity

Quaternary Solvent Manager (QSM) with Auto ●Blend Plus™ for fully automated multi-solvent blending

Bio-inert flow path with excellent corrosion resistance - uniquely suited for the high ionic strength aqueous conditions commonly used for biomolecule separations

*Note: The ACQUITY H-Class Bio is our recommended LC system for oligonucleotide analysis, though other Waters LC systems may be used


Industry Leading Waters Oligonucleotide Columns with BEH* Technology

ACQUITY UPLC columns for optimum speed, performance and throughput – BEH, C18, 130Å, 1.7µm Particles:

o 50 x 2.1 mm, 1.7 µm columno 100 x 2.1 mm, 1.7 µm column

Xbridge™ HPLC/UHPLC columns for routine analysis and lab scale purification– BEH, C18, 130Å, 2.5µm Particles:

o 50 x 2.1 mm, 2.5 µm columno 50 x 4.6 mm, 2.5 µm columno 50 x 10 mm, 2.5 µm column

Unique Patented *Bridged Ethyl Hybrid (BEH) Technology delivers N-1 oligo resolution and superior column life at elevated pH values and temperatures – widely considered the industry gold standard


Longevity of BEH Particles vs. Silica for Oligo Separations at High pH & Temp.

Accelerated High pH Stability Test of Competitive Columns


Waters MassPREP™ Oligonucleotide Standard

Note: Every batch of ACQUITY and XBridge Oligonucleotide column packing material is QC Tested with this standard to ensure performance and consistency

High quality oligonucleotide reference material for calibration, troubleshooting and system suitability.

P/N 186004135


++++ -

--

-

- ---+C18 sorbent

Ion Pairing Reverse Phase(IP-RP) Chromatography for Optimal Separations

Reversed Phase interaction with ion-pairing reagent- Hydrophobicity of the nucleobases +charge-charge interaction (oligo backbone)

Reversed-phase interaction without Ion-Pairing reagent- hydrophobicity of the nucleobases

0 minutes 100 minutes 10

1520

2535

3015 20

25

35

30

TEA+

TEA+ layer on the column surface


Ion Pairing Reagents for Oligonucleotide LC-MS Analysis

Gong, L. and J. S. O. McCullagh (2014). "Comparing ion-pairing reagents and sample dissolution solvents for ion-pairing reversed-phase liquid chromatography/ electrospray ionization mass spectrometry analysis of oligonucleotides." Rapid Communications in Mass Spectrometry 28(4): 339-350.

TEA-HFIP was first ion pairing system for high efficiency LC/MS analysis of oligonucleotidesApffel, A., et al. (1997). "Analysis of Oligonucleotides by HPLC−Electrospray Ionization Mass Spectrometry." Analytical Chemistry 69(7): 1320-1325.

Waters scientists expanded on these capabilities over the following decade + of research

Apffel, A., et al. (1997). "New procedure for the use of high-performance liquid chromatography–electrospray ionization mass spectrometry for the analysis of nucleotides and oligonucleotides." Journal of Chromatography A 777(1): 3-21.

Gilar, M., et al. (2002). "Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:: Retention prediction." Journal of Chromatography A 958(1–2): 167-182.

Fountain, K. J., et al. (2003). "Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry." Rapid Communications in Mass Spectrometry 17(7): 646-653.McCarthy, S. M., et al. (2009). "Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA." Analytical Biochemistry 390(2): 181-188.


Range of Ion-Pairing Reagents Used Today for Oligonucleotide Analysis

Ion Pairing Agent Buffering Acid AbbreviationTriethylammonium Acetate TEAATriethylammonium Bicarbonate TEABDimetylbutylammonium Acetate DMBAATributylammonium Acetate TBAATripropylammonium Acetate TPAAHexylammonium Acetate HAATriethylammonium Hexafluoroisopropanol TEA-HFIP

Selectivity and resolution are impacted by choice We have found TEA-HFIP to be the most effective for single-stranded

oligo analysis in terms of achieving optimal LC-MS sensitivity and resolution, and it is used throughout this presentation


Triethylammonium HFIP Reagent

Triethylammonium Acetate Reagent

0 30Minutes

13 14 15 1618

20

21 22 26

29

25

28

30

27

24

23

19

171211

UV 2

60 n

m

10 32

24

11 12 13 14 15 16+17

18+19

20

21 2223

25

28

29

30

Minutes

UV 2

60 n

m

T

G

GG

C

G

A

TTT

C

C

TGTTAAGT

TEAA vs. TEA-HFIP for IP-RP Separation of an Oligo Mixture


Recent work: Alternative HFIP LC/MS Buffer systems

400 mM HFIP, pH 8.0

25 mM HFIP, pH 9.5

50 mM HFIP, pH 9.0

Triethylamine

Butylamine

Dibutylamine

Cost

$

$$$$$

$$$ 10x less

20x less

“standard”


Injection # 48 98 153 203 248 317 365 429Peak 4/5Peak 3/5Peak 2/5

Peak 1/5

BEH Columns Perform well under High pH

OST BEH, C18, 135Å, 1.7µm50 x 2.1 mm column

buffer exchange

Peak 4/5Peak 3/5Peak 2/5

Peak 1/5

Relative retention time to peak 5 (35 nt)15mM BA:50mM HFIP, pH 9.0

1 2 3 4 5

Stable chromatography over 400 injections using butylamine/ 50mM HFIP buffer


Peak Width (W50) Inter assay Intra assay

Mean < 0.08 min 0.08 minS.D. < 0.003 0.002R.S.D (%) < 3.5 2.49

Hour 29, inj # 53

Hour 124, inj # 243

Hour 193, inj # 421

Ruggedness of BEH Columns

Average Peak width (W50) vs. Hours at pH 9.0

stable column performance in harsh conditions

15nt 20nt 25nt 30nt 35nt

N-1

N-1

N-1

Highly Reproducible and robust column performance over 200 hours at pH 9.0, 60 C using butylamine/ 50mM HFIP buffer


Troubleshooting Oligonucleotide LC-MS- Quality of Mobile Phase Matters!

LC-MS grade solvents and additives should be used MS-grade TEA purchased fromSigma (Fluka 65897-50ML-F)

MS-grade HFIP purchased from Sigma (Fluka 42060-50ML)


Troubleshooting Oligonucleotide LC-MS- Mobile Phases Need to be Fresh!

We recommend mobile phases be prepared daily to prevent MS signal loss

8.84E7

0.2556

5.87E7

0.2563

1.13E7

0.2609

1st injection

Base-line

+ 9 hours

~30% drop in MS response

+ 2.5 days

~87% drop in MS response

Remake Buffer

Recover MS response

1.43E8

0.213

TUV

MS

TIME


Troubleshooting Oligonucleotide LC-MS- System Cleanliness Matters!

Contaminatedsystem

Clean system

Contaminatedsystem

Clean system

Systems previously used for aqueous based separations may require a multi-step cleaning protocol

LC-HRMS/HDMS systems will require extended flushing with 50:50 H2O:MeOH, 0.1%FA to reduce phosphate adduct from cleaning procedure


Recent Work: A simple, highly effective protocol for reducing the accumulation of metal adducts

Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/ mass spectrometry analysis

Robert E. Birdsall, Martin Gilar, et.al. Volume 30, Issue 14 | 30 July 2016 (pages 1667–1679)

This manuscript by Robert Birdsall, et.al., was featured on the cover of RCM in July, 2016. It presents a simple, highly effective solution to a historic problem with oligonucleotide analysis - the accumulation of metal adducts over time, which degrade the MS signal and lead to inconsistent performance.


Waters Oligo Applications Notebook

Download @ waters.com/oligos

Contains 24 application notes illustrating the oligonucleotide separations performance and productivity that can be achieved using Waters column chemistries, reagent standards, HPLC & UPLC systems, and LC-MS workflows:

• UPLC-MS• UPLC-HRMS• UPLC-HDMS


UPLC-MSScreening for Oligo ID and Purity

with the ACQUITY QDa Mass Detector

Our most deployable and scalable solution Easy-to-use with a low maintenance requirement

Quickly confirm product ID & assess purity


The ACQUITY® QDa™ Mass Detector

Revolutionary innovative design focused on ease of use for analysts

Empowering analytical chemists everywhere with orthogonal mass detection – added information with every sample

Compact, robust and affordable - built for constant use with a wide variety of conditions

Seamlessly integrates with Empower and MassLynx based HPLC and UPLC™ systems


The QDa is Easily Deployed – Like adding an optical detector

+ QDa

QDa Mass Detector

Empower® and MassLynx™ Control Minimal operator training requiredQualification available110/220V operationWorkhorse - minimal maintenance


Automated Start Up and CalibrationEnsures Reproducible Performance

Automated resolution and calibration with each start-up ensures mass information is accurate, precise and reproducible– No tuning required!

ESI source optimized for UPLC ensures chromatographic resolution, sensitivity and throughput is preserved

Graphic QDa™ monitor display enables easy viewing and adjustment of system parameters


Familiar Graphical User Interfaces for Ease-Of-Use and Fast Adoption

Interface identical to that of a PDA for setup, data viewing and reporting

Very little training needed Quick addendum to current lab

SOP

MassLynx delivers additional functionality

Ability to deconvolute data Export to ProMass HR for

assignments


Disposable Sample Aperture and Capillary for Easy Maintenance

Sample Aperture:As simple as replacing a detector lamp

Capillary:

No cutting or assembly required


ACQUITY QDa in Summary

A breakthrough mass detector that enables analytical chemists / chromatographers to collect mass data on a routine basis

Delivers greater insight into every peak; enhances monitoring and streamlines workflows for improved productivity

Easily deployed, simple to maintain, compact, robust and affordable!


TUV

15 nt20 nt 25 nt

30 nt35 nt

QDa Proof-of-Principle with Waters MassPREP™ OST Standard

nt Avg. MW [M-4H]-4 [M-5H]-5 [M-6H]-6 [M-7H]-7 [M-8H]-8 [M-9H]-9 [M-10H]-10 [M-11H]-11 [M-12H]-12 [M-13H]-13 [M-14H]-14 [M-15H]-15 [M-16H]-16 [M-17H]-17

15 4,500.9 1124.2 899.2 749.1 642.0 561.6 499.1 449.1 408.2 374.1 345.2 320.5 299.1 280.3 263.820 6,021.9 1504.5 1203.4 1002.6 859.3 751.7 668.1 601.2 546.4 500.8 462.2 429.1 400.5 375.4 353.225 7,542.9 1884.7 1507.6 1256.1 1076.5 941.9 837.1 753.3 684.7 627.6 579.2 537.8 501.9 470.4 442.730 9,063.8 2265.0 1811.8 1509.6 1293.8 1132.0 1006.1 905.4 823.0 754.3 696.2 646.4 603.2 565.5 532.235 10,584.8 2645.2 2116.0 1763.1 1511.1 1322.1 1175.1 1057.5 961.2 881.1 813.2 755.0 704.6 660.5 621.6

green = observable charge states

QDa50 pmol on-column

Multiple charge states observed per oligonucleotide

Detection readily observed with low pmol column loads

Quantification based on UV Channel with MS providing mass confirmation P/N 186004135


Robust Reproducible Performance

15 nt 20 nt25 nt

30 nt35 nt

646.5

696.2

754.4

823.1

905.41006.2 1131.8

n=3 [M-8H]-8 [M-9H]-9 [M-10H]-10 [M-11H]-11 [M-12H]-12 [M-13H]-13 [M-14H]-14 [M-15H]-15

Expected m/z 1132.0 1006.1 905.4 823.0 754.3 696.2 646.4 603.2

Average m/z 1132.1 1006.1 905.4 823.1 754.3 696.2 646.5 603.4

Delta +0.1 0.0 0.0 +0.1 0.0 0.0 +0.1 +0.2

%RSD 0.01 0.01 0.00 0.00 0.01 0.01 0.02 0.01

All data within QDa mass accuracy specification: +/- 0.2

TIC Trace:50 pmol on-column

Observed charge states for 30nt oligo: (-8 to -14)


MaxEnt1

Deconvolution

Manual Deconvolution with MaxEnt1

30 nt35 nt

Calculated Average Mass of 30nt oligo = 9,063.8 Da

9,064.0 Da

+Na

Deconvoluted Spectrum

9,086.0 Da

Raw ESI Spectrum

646.5

696.2

754.4

823.1

905.41006.2 1131.8

MaxEnt1 deconvolution is available within MassLynx, and provides for manual deconvolution of both nominal mass and high resolution data.


Enabling Automated Data Workflowswith ProMass and ProMass HR

“Automated” deconvolution and reporting package Supports both nominal mass and high resolution mass spec (HRMS) data Enables higher throughput workflows

+

Znova™ deconvolution for nominal mass data (Quadropole)

Respect™ deconvolution for high res data (QTof) – only available with Promass HR


ProMass Workflow

MassLynxAcquisition

• Acquisition of LC-MS(/MS) data

• Automatically calls ProMass Bridge

ProMass“Bridge”

• Informatics tool that enables the transfer of data from MassLynx to ProMass for automated batch processing

ProMassProcessingand Reporting

• Automated deconvolution

• Assesses / confirms target mass and purity

• Assignment of impurity peaks

• Report Generation


ProMass: Deconvolution Parameters

Peak width is set to encompass the width at the base of most peaks from the input ESI mass spectra. A typical Peak Width value is 2-3 m/z units.

Merge Width determines the m/z window around the detected peaks where intensities are summed (merged) to compute a centroided value. Typically, this is set to 10 - 25% of the Peak Width parameter (e.g., 0.2-0.5 for 2 m/z unit peaks)


ProMass: Batch Processing Parameters

Results Tab Reporting Tab


ProMass: Report Generation

Summary Report

ESI Mass Spectrum

Deconvoluted Spectrum

Magnified Deconvolution

Deconvolution Peak Reports

Note:• Requires dongle to process

(can not remote in)• PDF also available

• A top-level summary page that shows results from multiple runs and provides hyperlinks that allows users to quickly drill down into individual results

• Target mass features allows one to automatically search for one or more target masses from the LC-MS data


ProMass: ZNova Mass Accuracy based on Waters MassPREP™ Oligonucleotide Standard

n=3 nt 15 nt 20 nt 25 nt 30 nt 35Expected Mass (Da) 4,500.9 6,021.9 7,542.9 9,063.8 10,584.8

Observed Mass (Da) 4,500.8 6,022.0 7,543.3 9,063.8 10,585.3

mass -0.1 0.1 0.4 0.0 0.5

SD 0.06 0.17 0.20 0.10 0.06

15 nt20 nt

25 nt

30 nt35 nt50 pmol

on-column Triplicate Analysis

2 minute window centered around each peak used for combined spectra

QDa mass data deconvoluted with Promass Znova™ algorithm

Mass errors within 1.0 Da of expected


Demonstrating QDa Enabled Mass Confirmation & Impurity Profiling

Upper Strand (50 pmol)

TUV, 260 nm QDa, 410-1250 m/z

Upper Strand, MW 6693.1 Da5’-UCGUCAAGCGAUUACAAGGTT-3’

Lower Strand, MW 6607.0 Da5’-TTCCUUGUAAUCGCUUGACGA-3’

Sample: Two distinct 21nt ssRNA oligos representing the complementary upper and lower strands of a siRNA duplex (10 pmol/uL)

Time (min)

Flow (ml/min)

A B C D

Initial 0.200 82.0 18.0 0.0 0.0

4.00 0.200 80.0 20.0 0.0 0.0

4.01 0.200 50.0 50.0 0.0 0.0

6.00 0.200 50.0 50.0 0.0 0.0

6.01 0.200 82.0 18.0 0.0 0.0

4 Minute Gradient: 0.5%/min


Summary Report of Batch Processing Results with Interactive Display

Target Mass: 6693.1 Da (Upper Strand)

Data for 48 samples processed in < 10 min

100% match with expected results -displayed in easy-to-use color-coded format

Multiple target masses can be searched

96-well plate format

Click for interactive sample report (following slide)

©2016 Waters Corporation 38

Interactive ProMass Sample Report

Total Ion Chromatogram

Blue Text = Hyperlinks to data views

Raw ESI Spectrum Deconvoluted Spectrum


Data Analysis (upper strand): Retention Time and Mass Accuracy

2

2.1

2.2

2.3

2.4

2.5

2.6Retention Time (min)

Vial position

Rete

ntio

n tim

e (m

in)

N=42 Retention time (min)

Average 2.44S.D. 0.01

%RSD 0.57

A1 A3 A5 A8 B2 B4 B7 C1 C3 C6 C8 D2 D5 D7 E1 E4 E6 E8 F3 F5 F7-1

-0.8-0.6-0.4-0.2

00.20.40.60.8

1Mass Error (Da)

Vial position

Mas

s er

ror

(Da)

N=42 Mass (Da)Expecte

d 6693.1Average 6693.3

S.D. 0.3

Consistent retention times across all 42 samples

All data within 1 Dalton of expected mass


ProMass: Spectral Analysis for Purity

A1 A3 A5 A8 B2 B4 B7 C1 C3 C6 C8 D2 D5 D7 E1 E4 E6 E8 F3 F5 F780828486889092949698

100% Abundance in Spectrum

Vial position

Spec

tral

abu

ndan

ce (

%)

N=42 % Abundance


R.S.D. 1.87

A5

E6

N=40 % Abundance


R.S.D. 0.84

Non target masses other than predicted adducts are calculated as impurities


UPLC-HRMSCharacterization and HRMS Monitoring

with the Xevo G2-XS Qtof

Robust High Resolution MS Enhanced Sensitivity for Characterization Work

MS/MS for Sequence Confirmation


Demonstrating High Res Performance using the MassPREP™ Oligonucleotide Standard

Instrumentation LC: Waters ACQUITY® H-Class Bio MS: Xevo G2-XS Qtof

Column Chemistry ACQUITY UPLC OST BEH C18 Column 130Å, 1.7

µm, 2.1 mm X 50 mm (P/N: 186003949)

Waters MassPREP Oligonucleotide Standard Reconstituted to 5 pmol/µL in pure H2O 1 µL of sample injected /LC-MS analysis

Method 15 min Ion Pairing RP gradient (TEA/HFIP) Data collected by TUV and by ESI- MS


MassPREP™ Oligonucleotide Standard:TUV and TIC comparisonLoad: 5 pmol on-column

TUV

TIC


MassPREP™ Oligonucleotide Standard: Raw ESI Spectra for 25nt Oligo

4-

3-5-6-7-8-

9-

10-

11-

12-

M3- charge state isotopic distribution

Resolution: > 30K

5 pmol on-column

+Na+

2508 2530m/z


Promass HR: Respect™ Deconvolution Mass Accuracy based on MassPREP Standard

Deconvoluted spectrum for 30nt

OST Standard

Expected Mass

Observed Mass

Mass Accuracy

(ppm)

15nt 4498.7348 4498.7300 -1.07

20nt 6018.9650 6018.9730 1.33

25nt 7539.1952 7539.1990 0.50

30nt 9059.4254 9059.4210 -0.49

35nt 10579.656 10579.6260 -2.79

Avg. 1.24

Mass accuracy: < 5 ppm

Promass HR includes the Respect™ deconvolution algorithm for processing high resolution MS data


MassPREP Oligonucleotide Standard:-Determining Limit of Detection (LOD)

TUV

TIC

Load: 20 fmol on-column

Detection limited by UV signal, not MS sensitivity


Evaluating performance with complementary ssRNA strands

Upper Strand, MW 6693.1 Da5’-UCGUCAAGCGAUUACAAGGTT-3’

Lower Strand, MW 6607.0 Da5’-TTCCUUGUAAUCGCUUGACGA-3’

1 µL injected for LC-MS analysis.

Sample: Two distinct 21nt ssRNA oligos representing the complementary upper and lower strands of a siRNA duplex (10 pmol/uL). 10 minute gradient for high resolution chromatogram

Oligo sourced from IDT


Upper Strand:10 Min High-Resolution Chromatogram

N

N-1 N+1

N-1 = 5’-rUrCrGrUrCrArArGrCrGrArUrUrArCrArArGrGrTT-3’Average mass = 6386.9 Da; ProMass HR observed mass:6386.8 Da

N = 5’-rUrCrGrUrCrArArGrCrGrArUrUrArCrArArGrGrTT-3’Average mass = 6693.1 Da; ProMass HR observed mass:6693.0 Da

N+1 = 5’-rGrUrCrGrUrCrArArGrCrGrArUrUrArCrArArGrGrTT-3’Average mass = 7038.3 Da; ProMass HR observed mass:7038.7 Da

A ssRNA sequence 5’-UCGUCAAGCGAUUACAAGGTT-3’ with a double thymine overhang was separated from the base deletion (N-1) and base insertion (N+1) forms using a 10 min high resolution separation gradient from 13% B to 21% B.


Upper Strand: Extracted TIC (XIC) of N (Target), N+1 and N-1 species

N+1

N (Target)

N-1XIC’s

time


Upper Strand:QTof ESI Raw Spectrum

4-

3-

5-6-

7-

8-

9-

M3- charge state isotopic distribution.

2199 2205m/z


Summary Report of Batch Processing Results with Interactive Display

Promass HR Respect™ algorithmn used

Target Mass: 6689.9550 Da (Upper Strand)

Data processed for all 48 samples in < 10 minutes

100% match with expected results -displayed in easy-to-use color-coded format


Interactive ProMass Sample Report

Total Ion Chromatogram Raw ESI Spectrum Deconvoluted Spectrum

Blue Text = Hyperlinks to Data Views


Oligo MS/MS Sequence Confirmation

Collision of oligonucleotides with gas molecules at elevated energies produces characteristic fragment ions at each residue

McLuckey Fragmentation Schemec, y, & w ions predominate with QTof fragmentation


LC-MS/MS Sequence Confirmation - Upper Strand (M4- Peak Deconvoluted*)

y2

c2

w2

y3

c3

w3

y4

c4

w5

y5

c5w4

y6

c7

w7

y7

w6

y8c9

y9

c8

y11 y12y10

c10

c16

w13

c15

c12 y14c14

Y17y19c18

y20

-G

-G

M

y16

y18

c6

w8

w9 w10

Y13

-G

w14

U C G U C A A G C G A U U A C A A G G T T5’- -3’

5 pmol on-column

*Deconvolution using MaxEnt-3, which is available within Mass Lynx


UPLC-HDMSCharacterization with Ion Mobility using the

SYNAPT G2-Si Qtof

Ion Mobility brings an added dimension of separation that translates into greater spectral clarity and enhanced

selectivity and sensitivity, especially for low level impurities


Charge State / Drift Time Stripping – Reduces spectral complexity; makes data more suitable for deconvolution– Observe fragment ion series for each IMS resolved charge state

Wideband Enhancement– Improve the resulting signal intensity (ca. 5-10 fold) of MS/MS spectra for low

level impurities

Time Aligned Parallel (TAP) Fragmentation– Separation of primary fragment ions via ion mobility enables selection of

specific primary fragment ions for secondary fragmentation - Pseudo MS3

3 Ways MS/MS - Ion Mobility Spectrometry (IMS) Can Enhance Selectivity and Sensitivity


Charge State / Drift Time Stripping Improves Clarity of Resulting MS/MS Spectra

Analysis of PEG 4450 via HDMS

DriftScope™ data showing the gas-phase separation power of the SYNAPT HDMS System. Components with different charge states are separated via ion mobility, thus enabling the examinations of different (minor) components in the PEG material.

Waters Application Note:Characterizing Polyethylene Glycol (PEG) by SYNAPT High Definition Mass Spectromet ry (HDMS).Petra Olivova, Weibin Chen, and John C. Gebler

Mass spectrum showing the ions with +2 charge state. Improved mass spectral clarity makes data more suitable for charge state deconvolution algorithms.


Wideband Enhancement


Time Aligned Parallel (TAP) Fragmentation


In Summary - Our LC-MS Workflows for Oligonucleotide Analysis:

UPLC-MS ACQUITY QDa

HPLC-HRMSXevo G2-XS QTof

UPLC-HDMSSYNAPT G2 Si QTof

Mass Accuracy – Deconvoluted Data +/- 1.0 Dalton < 5 PPM

(~0.038 Da)< 5 PPM

(~0.038 Da)Typical Column Load 50 pmol 5-10 pmol 5-10 pmolLimit of Detection < 10 pmol 20 fmol 20 fmolMass Confirmation Impurity Profile Impurity ID MS/MS Sequencing

(in source)

Cost $ $$$ $$$$


To Learn More Visit: WWW.Waters.com/oligos

or Contact your local Waters Account Representative

Thank You

http://www.waters.com/oligos

Science

Waters Oligonucleotide Analysis Solutions