HIGH FIELDPUSHER ION

MIRROR

ION DETECTIONSYSTEMQUADRUPOLE

AIR-COOLED TURBOMOLECULAR PUMPSOIL-FREESCROLL PUMP

6

LOCKMASS SPRAYANALYTE SPRAY

5

TRAP

ION MOBILITYSEPARATION

TRANSFER

HELIUM CELL

HIGH FIELDPUSHER ION

MIRROR

ION DETECTIONSYSTEMQUADRUPOLE

AIR-COOLED TURBOMOLECULAR PUMPSOIL-FREESCROLL PUMP

6

5

TRAP

ION MOBILITYSEPARATION

TRANSFER

HELIUM CELL

T-WAVEION GUIDE

DUAL STAGE REFLECTRON

S YNA P T G2: B R E A K T H ROUG H QUA N T ITAT IV E A N D QUA L ITAT IV E P E R FO RMA N C E FO R U P L C / M S A N D M S / M S (MS E) A P P L IC AT IO NSAlistair Wallace1, Jose Castro-Perez2, Hilary Major1, Yasuhiro Yamada3, Jason Wildgoose1, Martin Green1, Kevin Giles1, and John Hoyes1 1Waters Corporation, Floats Road, Manchester, UK, 2Waters Corporation, Milford, MA, US, 3Showa University, Tokyo, Japan

INT RODUCT IONWe demonstrate the ability of SYNAPT™ G2, with its

innovative QuanTof™ Technology, to provide high-

resolution, exact mass measurement, accurate isotope

ratios, enhanced dynamic range, and comprehensive

MS and MS/MS information, all at acquisition rates

compatible with ACQUITY UPLC® separations.

InstrumentationThe SYNAPT G2 System is an innovative hybrid

quadrupole IMS orthogonal acceleration time-of-flight

(oa-Tof) mass spectrometer providing a new level of

high-resolution, exact mass, tandem MS performance,

and the option to combine this with high-efficiency ion

mobility separations, as shown in Figure 1A.

SYNAPT G2 employs QuanTof Technology – a next-

generation oa-Tof architecture that integrates a series

of technological advancements, as shown in Figure

1B. QuanTof combines innovative high field pusher and

dual-stage reflectron designs with a novel ion detection

system in an optimized, folded, Tof geometry. This pro-

vides a new dimension of high-resolution, exact mass,

quantitative performance, which, crucially, is available

at acquisition rates compatible with UPLC® separations.

This new level of Tof performance means SYNAPT G2 is

the ideal platform for the most analytically-challenging

samples, for example in the analysis of complex

mixtures for proteomics and biomarker discovery; or

metabolite, impurity, and lipid profiling studies. Figure 1. (A) Schematic of the SYNAPT G2 System. (B) QuanTof Technology, the enabling next-generation Tof technology of SYNAPT G2. QuanTof’s high field pusher and dual-stage reflectron, which incorporates high-transmission parallel wire grids, reduces ion turnaround times due to pre-push kinetic energy spread, and improves focusing of high energy ions, respectively. These innovative technologies combine to provide the highest levels of Tof performance. The novel ion detection system combines an ultra-fast electron multiplier and hybrid ADC detector electronics to provide outstanding sensitivity and quantitative performance for both MS and the elevated data acquisition rates of HDMS™ analysis.

A

B

EXPERIMENTALSamples analyzed: Bile samples from rat dosed with

Ritonavir at 10 mg/kg

C37H48N6O5S2

UPLC conditions

LC system: ACQUITY UPLC

Column: ACQUITY UPLC HSS T3, 2.1 x 100 mm, I.D. 1.7 µm

Mobile phase A: 5 mM Ammonium Acetate, pH 5

Mobile phase B: MeCN

Gradient:

Time (min) Flow Rate %A %B Curve (mL/min)

1. Initial 0.5 98.0 2.0 n/a

2. 5.0 0.5 50.0 50.0 6

3. 9.0 0.5 40.0 60.0 6

4. 9.1 0.5 1.0 99.0 1

5. 12.9 0.5 1.0 99.0 1

6. 13.0 0.5 98.0 2.0 1

MS conditions

MS system: SYNAPT G2

Ionization mode: ESI positive

Acquisition mode: MSE

Capillary voltage: 1.5 kV

Cone voltage: 40.0 V

Trap collision energy: 6.0 V

Transfer collision energy: 4.0 V

Collision energy ramp: 15.0 to 25.0 eV

Trap/Transfer gas: Argon

Acquisition range: m/z 100 to 1200

DISCUSSIONThe combination of high chromatographic and mass resolution is essen-

tial for the comprehensive, confident analysis of very complex matrices,

for example in profiling of metabolites in biological (in vivo) samples.

High resolution and mass accuracy at the highest acquisition ratesFigure 2 demonstrates the ability of SYNAPT G2 to provide high

resolution (> 40,000 FWHM) at the fast spectral acquisition rates

required to keep pace with ACQUITY UPLC separations, which

typically deliver peak widths of less than 2 sec at half height. By

delivering up to 20 spectra/second, SYNAPT G2 ensures sufficient

points can be obtained to generate accurate LC peak profiles with

mass resolutions over 40,000 FWHM to maximize the ability to

better resolve compounds and provide exact mass measurement.

The ability of SYNAPT G2 to deliver high mass accuracy and accu-

rate isotope ratios significantly aids the confident identification of

small molecules through elimination of false positives, as shown

in Figure 3.

Figure 2. High resolution at 20 spectra/sec.

Theoretical Isotopic Distribution

Resolution > 40,000 FWHM

Acquired Isotope Distribution

Resolution > 40,000 FWHM

S

S

OH O

O

O

ONH

NH

NH N N

N

PrecisionQuanTof delivers exact mass accuracy with high precision across LC

peaks, which in turn provides high selectivity and confidence for the

detection and identification of components in complex mixtures.

In the case of complex (in vivo) matrices such as bile, urine, and

plasma, QuanTof’s selectivity provides more confident detection

of components in the presence of endogenous metabolites and the

dosing vehicle. This is demonstrated in Figure 4 where a window of

< 5 mDa is typically used to generate an extracted ion chromato-

gram for a drug and its metabolites.

Dynamic rangeSince complex (in vivo) samples can contain thousands of com-

ponents (drug-related metabolites and endogenous peaks) over

a wide dynamic range, it is important that high mass accuracy is

maintained across the concentration range. Figure 5 demonstrates

that SYNAPT G2 provides an in-spectrum dynamic range of more

than 4 orders of magnitude where mass accuracies for caffeine

(low concentration) and verapamil (high concentration) are

< 0.1 ppm and 1.5 ppm, respectively.

Comprehensive fragment ion analysisMSE is a patented data independent acquisition method, which pro-

vides a simple route to delivering comprehensive molecular (MS)

and fragment ion (MSE) information from every detectable compo-

nent in a complex mixture. The use of this rapid, information-rich

approach on SYNAPT G2 ensures high selectivity and accuracy (in

MS mode) for quantitative profiling and high mass resolution and

accuracy (in MSE mode) for identification and characterization, all

at acquisition rates of up to 20 spectra/sec. This is demonstrated

in Figure 6 for the analysis of Ritonavir in bile (from an in vivo

metabolite profiling study). All mass accuracies are < 0.5 mDa

(1.3 ppm RMS). A 1.0 mDa extracted ion chromatogram window

demonstrates the high precision across the entire chromatographic

peak. The accuracy of fragment ion data enabled unambiguous

structure assignment with MassFragment™ Software.

1 mDa XICHydroxylated Metabolites

1 mDa XICParent Drug

Figure 4. Extracted ion chromatogram windows at 1 mDa of parent drug and hydroxylated metabolites using profile data.

Figure 5. In-spectral dynamic range. Exact mass measurement of verapamil and caffeine at concentrations that differ by more than 4 orders of magnitude.

Results and Discussion

Introduce the analytical problem i.e. detection,

quantification, identification and characterization

of small molecule form complex rat bile sample.

Run through the importance of high resolution and

wide spectral dynamic range for the analysis of

complex mixtures

Introduce the importance of mass measurement

accuracy and stability.

Introduce the need for comprehensive MS/MS -

MSE

<0.1ppm1.5 ppm

OO

O

ON

N

Verapamil

O

O N

NNN

Caffeine

>4 orders in-spectrum dynamic range

Results and Discussion

Introduce the analytical problem i.e. detection,

quantification, identification and characterization

of small molecule form complex rat bile sample.

Run through the importance of high resolution and

wide spectral dynamic range for the analysis of

complex mixtures

Introduce the importance of mass measurement

accuracy and stability.

Introduce the need for comprehensive MS/MS -

MSE

<0.1ppm1.5 ppm

OO

O

ON

N

verapamil

O

O N

NNN

caffeine

>4 orders in-spectrum dynamic range

Figure 3. Elemental composition calculation for the parent drug Ritonavir showing accurate isotopic ratios reflected by the i-FIT™ (norm) value of 0, and high mass accuracy.

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

m/z100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725

%

0

100

2: TOF MS ES+ 9.81e3

268.1487

197.0750

140.0537 171.0959 266.1329

721.3204

426.1854

296.1429

427.1882

494.3260

0.30 mDa2.40 ppm

0.30 mDa1.70 ppm

0.10 mDa0.51 ppm

0.30 mDa1.12 ppm

-0.40 mDa-1.35 ppm

0.30 mDa0.70 ppm

-0.20 mDa-0.28 ppm

Time2.20 2.30 2.40 2.50

%0

100

m/z 721.3204

m/z 426.1854

m/z 296.1429

m/z 268.1487

m/z 197.0750

m/z 171.0959

m/z 140.0537

Figure 6. UPLC/MSE fragment ion spectrum of Ritonavir (C37H49N6O5S2) from a complex rat bile sample. Data was acquired from a UPLC peak width of 1.5 sec at half height. A 1 mDa window was used to generate extracted ion chromatograms (inset) and structures were automatically determined (MassLynx™ Application Manager, MassFragment Software) for each individual fragment ion.

UPLC/MSE

10 spectra/sec

> 40,000 FWHM

0.30 mDa RMS

1.30 ppm RMS

1 mDa window

CONCLUSIONSn The SYNAPT G2 System, with QuanTof Technology provides

high-resolution (above 40,000 FWHM) at the high spectral

acquisition rates required for UPLC/MS analysis, unlike

electrostatic ion trap or FT-MS-based mass analyzers.

n The exact mass, accurate isotope ratios and wide dynamic range

significantly aid the detection, quantitation, confirmation, and

identification of compounds from complex biological samples

using UPLC/MS.

n SYNAPT G2, with its patented MSE data-independent acquisition

strategy, provides a simple route to comprehensive exact mass

fragment ion information for every detectable precursor in

UPLC separations.

n Unlike other quadrupole time-of-flight or ion trap-based instruments,

the application of MSE and MassLynx Informatics (MassFragment

Software and MSE-dedicated application managers) with SYNAPT

G2 provides a unique route to simple, rapid, and comprehensive

exact mass MS/MS analysis for the quantitation, identification, and

characterization of peptides, lipids, and small molecule samples.

Waters, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. The Science of What’s Possible, SYNAPT, MassLynx, QuanTof, i-FIT, HDMS, and MassFragment are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2009 Waters Corporation. Printed in the U.S.A.May 2009 720003057EN LB-UM