XEVO TQ MS PHARMACEUTICAL APPLICATION NOTEBOOK

2

The Changing Face of LC/MS: From Experts to Users .................................................................................3

Improving MS/MS Sensitivity using Xevo TQ MS with ScanWave ........................................................... 11

Simultaneous Confirmation and Quantification using Xevo TQ MS: Product Ion Confirmation (PIC) .............................................................................................................15

Novel Dual-Scan MRM Mode Mass Spectrometry for the Detection of Metabolites during Drug Quantification ...............................................................................................19

Data-Directed Detection and Confirmation of Drug Metabolites in Bioanalytical Studies ...................................................................................................................... 23

Improving Qualitative Confirmation using Xevo TQ MS with Survey Scanning .......................................27

A Novel Method for Monitoring Matrix Interferences in Biological Samples using Dual-Scan MRM Mode Mass Spectrometry ....................................................................................31

Rapid, Simple Impurity Characterization with the Xevo TQ Mass Spectrometer ..................................... 35

XEVO TQ MS: PHARMACEUTICAL AP PLICATIONS

THE CHANgINg FACE OF LC/MS: FROM EXPERTS TO USERSRobert S. Plumb Senior Applications Manager, Pharmaceutical Business Operations, Waters Corporation

Michael P. Balogh Principal Scientist, MS Technology Development, Waters Corporation

3

Researchers and practitioners from various

disciplines and sub-disciplines within chemistry,

biochemistry, and physics regularly depend on

mass spectrometric analysis. Pharmaceutical

industry workers involved in drug discovery and

development rely on the specificity, dynamic

range, and sensitivity of mass spectrometry

(MS). Particularly in drug discovery, where

compound identification and purity from

synthesis and early pharmacokinetics are

determined, MS has proved indispensable.

Biochemists expand the use of MS to protein, peptide,

and oligonucleotide analysis. Using mass spectrometers,

they monitor enzyme reactions, confirm amino acid

sequences, and identify large proteins from databases

that include samples derived from proteolytic fragments.

They also monitor protein folding, carried out by means

of hydrogen-deuterium exchange studies, and important

protein-ligand complex formation under physiological

conditions.

Clinical chemists, too, are adopting MS, replacing the

less-certain results of immunoassays for drug testing and

neonatal screening. So are food safety and environmental

researchers. They and their allied industrial counterparts

have turned to MS for some of the same reasons: PAH

and PCB analysis, water quality studies, and to measure

pesticide residues in foods. Determining oil composition,

a complex and costly prospect, fueled the development of

some of the earliest mass spectrometers and continues to

drive significant advances in the technology.

Today, the MS practitioner can choose among a range

of ionization techniques that have become robust and

trustworthy on a variety of instruments with demon-

strated capabilities.

Two decades ago, mass spectrometry was the preserve

of experts and skilled technicians: the instrumentation

required constant attention and adjustment. At this time

LC/MS was in it infancy and atmospheric pressure ioniza-

tion (API) source interfacing was just beginning. Samples

4

Advances in chromatography

Interfacing Liquid Chromatography with Mass Spectrometry

(LC/MS) allows analytical chemists access to about 80

percent of the chemical universe unreachable by Gas

Chromatography (GC); it is also responsible for the phe-

nomenal growth and interest in mass spectrometry

in recent decades.

A few individuals can be singled out for coupling LC with

MS. Beginning arguably in the 1970s, LC/MS as we know

it today reached maturation in the early 1990s. Many of

the devices and techniques we use today in practice are

drawn directly from that time.

In its simplest form, liquid chromatography relies on the

ability to predict and reproduce – with great precision –

competing interactions between analytes in solution (the

mobile or condensed phase) being passed over a bed of

packed particles (the stationary phase). The development

of columns, packed with a variety of functional moieties

in recent years, and of the solvent delivery systems, able

to precisely deliver the mobile phase, has enabled LC to

become the analytical backbone for many industries.

Continued advances in performance since then, including

development of smaller particles and greater selectivity,

also saw the meaning of the acronym change to high-

performance liquid chromatography (HPLC). In 2004,

further advances in instrumentation and column technol-

ogy achieved significant increases in resolution, speed,

and sensitivity in liquid chromatography. Columns packed

with smaller particles – 1.7 µm – and instrumentation with

specialized capabilities designed to deliver the mobile

phase at pressures up to 15,000 psi (1,000 bar) came to be

known as UltraPerformance® (UPLC®) technology. Much of

what is embodied in this current technology was predicted

by investigators such as Prof. John Knox in the 1970s.

requiring analysis were passed from the requesting scien-

tist to these “experts for analysis,” the samples would be

analyzed, processed, interpreted and the results returned

via a written report.

Two decades later, both the users and the capabilities of

LC/MS have changed significantly. Now mass spectrom-

eters and LC/MS systems are ubiquitous in the analytical

laboratory, especially in the pharmaceutical industry.

These instruments are used by a wide variety of scientists

for a diverse range of tasks, from purity screening in

medicinal chemistry, to the quantification of drugs in

blood, and the identification of proteins for biomarker

discovery.

The usability of current mass spectrometry platforms has

improved dramatically – scientists are now able to oper-

ate the systems remotely via the Internet; they can carry

out complex, data-dependent tasks such as purification

and peptide fragmentation; they are able to use to open

access systems where a non-analytical chemist can queue

samples for analysis and have the results emailed to them

without ever having to know or concern themselves about

the LC/MS process.

Recent reports put the number of LC/MS systems sold per

year in excess of 2500 units. This large number of units

sold each year is also reflected in the increased number of

users. In 1980, the number of scientists attending ASMS

was around 1250; by 2002 this had risen to greater than

4000 with a growth rate of 10 percent per year. This

growth in LC/MS users occurred because of the increase

in the number of samples analyzed each year per user,

creating larger and larger amounts of high-quality data.

More and more, this data is being turned directly into

information or knowledge so that decisions are made in

real-time. Many of these new users have little interest in

becoming expert mass spectroscopists and are instead

looking for the instrumentation itself to decide the appro-

priate experiments to be performed as well as to interpret

the data automatically and recommend a course of action

(pass/fail, pure/impure).

5

Advances in mass spectrometry

Mass spectrometers can be smaller than a coin, or they

can fill very large rooms. Although the various instrument

types serve in vastly different applications, they neverthe-

less share certain operating fundamentals. The unit of

measure has become the dalton (Da), displacing other

terms such as amu. 1 Da = 1/12 of the mass of a single atom

of the isotope of carbon-12 (12C).

Once employed strictly as qualitative devices – adjuncts

in determining compound identity – mass spectrometers

were once considered incapable of rigorous quantification.

But in more recent times, they have proven themselves

as both qualitative and quantitative instruments. A mass

spectrometer can measure the mass of a molecule only

after it converts the molecule to a gas-phase ion. To do so,

it imparts an electrical charge to molecules and converts

the resultant flux of electrically-charged ions into a pro-

portional electrical current that a data system then reads.

The data system converts the current to digital informa-

tion, displaying it as a mass spectrum.

The ions required in mass spectrometry can be created in

a number of ways suited to the target analyte in question:

by laser ablation of a compound dissolved in a matrix on a

planar surface such as by MALDI; by interaction with an

energized particle or electron such as in electron ioniza-

tion (EI); or as part of the transport process itself, as we

have come to know electrospray ionization (ESI), where

the eluent from a liquid chromatograph receives a high

voltage resulting in ions from an aerosol.

The ions are separated, detected, and measured according

to their mass-to-charge ratios (m/z). Relative ion current

(signal) is plotted versus m/z producing a mass spectrum.

Small molecules typically exhibit only a single charge: the

m/z is therefore some mass (m) over 1, with the “1” being

a proton added in the ionization process [represented

by M+H+ or M-H+ if formed by the loss of a proton], or, if

the ion is formed by loss of an electron, it is represented

as the radical cation [M+.]. Larger molecules can capture

charges in more than one location within their structure.

Small peptides typically may have two charges [M+2H+],

while very large molecules have numerous sites, allowing

simple algorithms to deduce the mass of the ion repre-

sented in the spectrum.

The general term atmospheric pressure ionization (API)

includes the most notable technique, electrospray ioniza-

tion (ESI), which itself provides the basis for various

related techniques capable of creating ions at atmospheric

pressure rather than in a vacuum. The sample is dissolved

in a polar solvent (typically less volatile than that used

with GC) and pumped through a stainless steel capillary

that carries between 500 and 4000 V. The liquid forms an

aerosol as it exits the capillary at atmospheric pressure,

and the desolvating droplets shed ions that flow into the

mass spectrometer, induced by the combined effects of

electrostatic attraction and vacuum.

The mechanism by which potential transfers from the

liquid to the analyte, creating ions, remains a topic of con-

troversy. In 1968, Malcolm Dole first proposed the charge

residue mechanism, in which he hypothesized that as a

droplet evaporates, its charge remains unchanged. The

droplet’s surface tension, ultimately unable to oppose the

repulsive forces from the imposed charge, explodes into

many smaller droplets. These Coulombic fissions occur

until droplets containing a single analyte ion remain. As

the solvent evaporates from the last droplet in the reduc-

tion series, a gas-phase ion forms. In 1976, Iribarne and

Thomson proposed a different model, the ion evaporation

mechanism, in which small droplets form by Coulombic

fission, similar to the way they form in Dole’s model. It

is possible that the two mechanisms may actually work

in concert: the charge residue mechanism dominant for

masses higher than 3000 Da while ion evaporation domi-

nant for lower masses.

The mass analyzer is the heart of the instrument and is

a means of separating or differentiating introduced ions.

Both positive and negative ions (as well as uncharged,

neutral species) form in the ion source. However, only one

polarity is recorded at a given moment.

6

The modern mass spectrometer

Modern instruments can switch polarities in milliseconds,

yielding high fidelity records. As well as separating the

ions, modern mass spectrometers can trap and fragment

ions (MS/MS or MSn) to produce a wealth of information

about the molecule’s structure. Other instruments such as

magnetic sector instruments, hybrid quadrupole time-of-

flight (Q-ToF), and ion cyclotron (ICR) mass spectrometers

can record the mass of a compound to 1 ppm, allowing for

the elemental composition of a molecular ion or fragment

ion to be deduced.

The increased sensitivity afforded by modern mass

spectrometry over other forms of detection, such as

UV and fluorescence, comes from the selectivity and

specificity of the MS and MS/MS process. During these

experiments, specific ions are allowed to pass through the

analyzer and reach the detector. During a multiple reac-

tion monitoring (MRM) MS/MS experiment, only ions that

undergo a specific fragmentation are allowed to reach the

detector; while this reduces the number of ions reaching

the detector, it all but eliminates the noise, resulting in

superior signal-to-noise ratio. This dramatically improves

assay sensitivity and specificity. The vast majority of

quantitative experiments are performed on quadrupole-

based instruments; whereas ion traps and accurate mass

instruments are preferred for structural elucidation

experiments.

Single quadrupole mass spectrometers require a clean

matrix to avoid the interference of unwanted ions, and

they exhibit very good sensitivity. Triple quadrupole, or

tandem, mass spectrometers (MS/MS) add to a single

quadrupole instrument an additional quadrupole, which

can act in various ways. One way is simply to separate and

detect the ions of interest in a complex mixture by the

ions’ unique mass-to-charge (m/z) ratio. Another way that

an additional quadrupole proves useful is when used in

conjunction with controlled fragmentation experiments.

Such experiments involve colliding ions of interest with

another molecule (typically a gas like argon). In such an

application, a precursor ion fragments into product ions,

and the MS/MS instrument identifies the compound of

interest by its unique constituent parts.

An ion trap instrument operates on principles similar to

those of a quadrupole instrument. Unlike the quadrupole

instrument, which filters streaming ions, both the ion trap

and more-capable ion cyclotron instruments store ions

in a three-dimensional space. Before saturation occurs,

the trap or cyclotron allows selected ions to be ejected,

according to their masses, for detection. A series of

experiments can be performed within the confines of the

trap, fragmenting an ion of interest to better define the

precursor by its fragments. Dynamic range is sometimes

limited in ion trap instruments and the finite volume/

capacity for ions limits the instrument’s range, especially

for samples in complex matrices.

The tandem quadrupole mass spectrometer

Tandem quadrupole and ion trap instruments have

become the workhorses of modern analytical LC/MS(MS).

The two capabilities have been incorporated into one

instrument platform to produce a linear ion-trap instru-

Figure 1. The Xevo™ TQ Mass Spectrometer.

7

ment that has all the structural characterization benefits

of ion trap mass spectrometers, with the quantitative

capabilities of tandem MS instrumentation.

These instruments have become popular with scientists

who are required to perform more than one type of exper-

iment (quantitative and qualitative) during the course of

their work and require the flexibility to perform it on the

same analytical platform. These tasks include impurity

identification and quantification and discovery DMPK,

where both dosed parent concentration and metabolite

characterization are required.

These instruments, while sounding ideal, do have some

drawbacks especially when using modern high-resolution

chromatography such as UPLC. Here, the chromato-

graphic peak widths are so narrow (1 to 2 seconds)

that there is not sufficient time for these ion trap mass

spectrometers to select the ions for trapping, fragment

the ion, and measure them to produce enough data points

to accurately define the peak. Although the collection of

MS/MS spectra can be performed with a standard tandem

quadrupole MS instrument while still correctly defining

the LC peak, sensitivity is compromised due to the low

duty cycle of the instrument.

A new direction for tandem quadrupole MS: The Xevo TQ

Along with the need to improve the utility and flex-

ibility of tandem quadrupole MS instrumentation are

the requirements to improve its data processing and

monitoring capabilities. The recent introduction of a new

iteration of the tandem quadrupole mass spectrometer,

using traveling-wave technology1, holds the potential to

resolve many of these issues. The Waters® Xevo™ TQ

Mass Spectrometer employs traveling-wave technology

that improves MS capabilities by performing simultane-

ous, multifunctional data acquisition, such as MRM and

product ion acquisition, all within a timescale compatible

with sub-2 µm UPLC. The instrument is equipped with

a modern tool-free source that simplifies the process of

routine maintenance and cleaning; instrument workflow is

also simplified with automated tuning, method generation

wizards, as well as real-time data checking functionality

that prevents sample waste if the analytical run fails for

any reason. The Xevo TQ MS’s software also features an

interactive LC/MS method database, QuanPedia™, that

ensures that the analyst selects and uses the correct

method parameters.

The T-Wave™ collision cell, originally introduced by Waters

in the Quattro Premier™ and used in the SYNAPT™ family

mass spectrometers, is employed to allow functionality

such as rapid MRM switching, fast 20-msec positive ion/

negative ion switching, and minimized crosstalk. This

functionality makes the instrument ideal for rapid method

development or use in a drug discovery environment for

development of multi-component assays.

Figure 3. MS software advancements such as QuanPedia allow users to choose pre-defined tasks for ease of operation.

Figure 2. A tool-free source ensures easy access for any user to perform rapid maintenance.

8

Xevo TQ MS: Benefits for pharmaceutical laboratories

In drug discovery, mass spectrometers often serve a dual

purpose as both a quantitative and a qualitative instrument

in DMPK departments. The Xevo TQ MS provides the

flexibility to not only perform both of these tasks, but also

to achieve them in the same analytical run or even with

the same eluting peak. This ScanWave™ functionality is

achieved by maximizing the duty cycle of the instrument.

In conventional mass spectrometers, the selected ions

enter from the first quadrupole (Q1) into the collision cell

where they are fragmented. The resulting fragmented ions

exit the collision cell and are transferred through the third

quadrupole (Q3) to the detector. This quadrupole (Q3) can

act either as a selective filter, as in MRM mode, only allow-

ing ions with a specific m/z value to pass through to the

detector, or it can scan across the entire m/z value range

providing a full spectrum.

This full-scan mode is particularly useful when performing

structural analysis; unfortunately, conventional instrumen-

tation suffers from poor duty cycle. This is because the ions

exit the collision cell simultaneously regardless of their m/z

value; as the third quadrupole scans, it can only measure or

detect one m/z value at a time. Therefore, for a scan speed

of 1000 m/z per second over a mass range of 1000 Da and

a 2-second-wide peak, the instrument will only spend 2 x

1/1000 second measuring each m/z value.

The Xevo TQ MS uses a novel collision cell design to

improve full scan sensitivity. In the last third of the colli-

sion cell, the fragmented ions are accumulated behind a

DC barrier to effect ion enrichment. These ions are then

released and contained between the DC barrier and an RF

barrier at the end of the collision cell. The RF barrier is

gradually reduced, ejecting the ions from the collision cell

to the third resolving quadrupole. These ions are ejected

according to their m/z ratio, with the heavier ions being

ejected first. To improve the duty cycle of the instrument,

the final quadrupole (Q3) is scanned in synchronization

with the ejection of the ions from the collision cell, thus

increasing the number of ions reaching the detector and

hence increasing sensitivity.

This increased scan sensitivity can be used to address

several business and scientific needs in pharmaceutical

analysis. The acquisition of this high-duty-cycle acquisition

scan can be triggered from a standard MS experiment to

provide structural information on the identity of an LC peak.

n In the field of bioanalytical analysis, the functional-

ity can be employed to confirm identity of a peak by

Product Ion Confirmation (PIC), which is carried out

within an MRM analysis. PIC works by taking one high

quality, high sensitivity spectra after the apex of a peak

and before the “touch-down” of a chromatographic peak.

This does not affect the fidelity or accuracy of the peak

quantification but allows for the acquisition of a product

ion spectra to confirm the identity of the peak.

n In the disciplines of metabolite identification and impu-

rity analysis, MS experiments such as constant neutral

loss or common fragment ion analysis are often em-

ployed to detect ions that are related to the parent API

molecule, or to look for particular metabolites that may

be toxic. Once the peaks of interest have been detected,

a second analytical run is often required to obtain

MS/MS structural information. The Xevo TQ MS can

utilize its ability to perform either constant neutral

loss or common fragment ion analysis and then rapidly

switch to high-sensitivity MS/MS to obtain structural

information. This capability removes the need for a sec-

ond analytical run, and, hence, improves productivity.

Figure 4. Daughter ion scans obtained with ScanWave, top, and without, bottom.

ScanWave DS

DS

O

O O

OO H

OH

O HO

OCH 3

OH

NH 2

C H 3

C l

C l

NH

NH

O

NH

O

O H

NH

O

NHC H 3

CH 3

CH 3

O H

OH O H

NH

O

NH

OO

NH 2

OH

O

CO O H

Fragmentation

0

1306

Daughter Ion Scan

ScanWaveDaughter Ion Scan

O

O O

OO H

OH

O HO

OCH 3

OH

NH 2

C H 3

C l

C l

NH

NH

O

NH

O

O H

NH

O

NHC H 3

CH 3

CH 3

O H

OH O H

NH

O

NH

OO

NH 2

OH

O

CO O H

O

O O

OO H

OH

O HO

OCH 3

OH

NH 2

C H 3

C l

C l

NH

NH

O

NH

O

O H

NH

O

NHC H 3

CH 3

CH 3

O H

OH O H

NH

O

NH

OO

NH 2

OH

O

CO O H

11441332

100

m/z200 400 600 800 1000 1200

%

100

%

0

144

170

725371

144

1306725

99

Recent regulatory guidelines on bioanalysis have placed

greater emphasis on the measurement of ion suppression

and control of the matrix, and measurement of drug-

related metabolites. The functionality of the Xevo TQ

MS facilitates rapid switching between matrix molecules

and analytes of interest. Phospholipids are a class of

endogenous molecules that have been associated with

ion suppression. These molecules can be monitored by

measuring the parents of the common fragment ion m/z

184, which is associated with the choline polar head

group. The Xevo TQ MS allows the simultaneous MRM

monitoring of the compound(s) of interest and precursors

of m/z 184, allowing rapid and reliable method develop-

ment. This capability can also be employed to monitor the

background ions during the course of a clinical trial, to

evaluate any differences between patients due to pheno-

type, gender, age, or diet. This information provides extra

confidence in the results and allows anomalies to

be explained.

Ease-of-use and performance extends the utility of LC/MS

The usability and functionality of mass spectrometers

have improved greatly over the last 15 years. Not only

have these instruments become more sensitive and

capable of performing multiple experiments simultane-

ously, they have also become easier to use – thus

improving instrument up-time and laboratory productiv-

ity. The advent of fast electronics and novel collision cell

designs has allowed high-sensitivity, full-scan MS/MS

experiments to be performed at the same time as high-

sensitivity MRM quantitative experiments. Concurrent

with an increase in MS capabilities has been the move

from analysis by an expert MS scientist to analysis by a

user who is tasked with answering a specific question,

such as to determine whether a product can be shipped or

to monitor food safety. Improvements in ease-of-use and

intelligent software features that help the general user be

successful in their task will continue to push LC/MS adop-

tion even wider into the general analytical community.

Figure 5. Monitoring of a model drug, alprazolam, and matrix effects of phospholipids, in a single analysis.

Acknowledgements

The authors would like to thank Paul Rainville and Marian Twohig for their scientific contributions.

Reference

1. The traveling wave device described here is similar to that described by Kirchner in U.S. Patent 5,206,506 (1993).

10

11

IM P ROV INg M S / M S S ENSIT IV I T y US INg X E V O T Q M S w IT H S C A N wAV E

Marian Twohig, Peter Alden, Gordon Fujimoto, Daniel Kenny, and Robert S. Plumb Waters Corporation, Milford, MA, U.S.

INT RODUCT ION

Tandem quadrupole mass spectrometry (MS) combined with liquid

chromatography (LC) – and, in particular, UltraPerformance LC®

(UPLC®) – has become the technology of choice for high sensitivity

quantitative analyses such as bioanalysis in the pharmaceutical

industry. The high selectivity and specificity of multiple reaction

monitoring (MRM) analysis gives rise to excellent signal-to-noise

ratios for the analysis of compounds in complex matrices. Full-scan

acquisitions are also used to provide useful information for struc-

tural elucidation in MS and MS/MS modes.

Conventional tandem quadrupole MS instruments have limited

sensitivity in full-scan mode due to poor duty cycle. The Waters®

Xevo™ TQ Mass Spectrometer with ScanWave™ functionality

delivers significant duty cycle improvements that provide enhanced

sensitivity in scanning acquisition modes.

ScanWave experiments are performed at up to 10,000 amu/sec,

making it possible to characterize narrow chromatographic peaks

better. This has become a necessity since the advent of sub-2 µm

column particle technology where narrow chromatographic peaks

can be 2 seconds wide or less.

ScanWave defined

The Xevo TQ MS employs a unique concept in collision cell technology.

Based on a novel use of Waters’ proven T-Wave™1 collision cell, the new

ScanWave™ mode of operation enhances both MS scan and product ion

data. ScanWave operation is based upon two concepts (Figure 2).

The first is that the front and back of the collision cell are indepen-

dently controlled, which allows fragmentation and accumulation

of ions to occur in the front of the gas cell while previously-

accumulated ions are simultaneously ejected from the back of the

gas cell. This provides 100 percent sampling efficiency.

Ejection of ions from the gas cell is mass dependent, although low

resolution. This low-resolution behavior allows for high space-

charge capacity without degradation of performance.

The second concept behind ScanWave is that it links the low-

resolution ion ejection from the gas cell with scanning of the

final-resolving quadrupole (MS2). This enables an intelligent ion

delivery where ions are presented to the final quadrupole when

they are actually needed, rather than continuously as in traditional

tandem quadrupole instruments.

This novel ion delivery technique provides significant duty cycle

improvements that in turn result in enhanced signal in scanning

acquisition modes. Since the scanning quadrupole (MS2) is the

device performing the mass analysis, it is not necessary to perform

a separate calibration. Scan rates, mass accuracy, and mass resolu-

tion are all identical to that for operation in traditional scanning

acquisition modes.

Figure 1. Unique T-Wave and ScanWave-enabled collision cell technology for the very best MS/MS data.

12

Figure 2. Schematic depicting a ScanWave experiment, where ions are accumulated before being sequentially ejected.

Significant increases in sensitivity using ScanWave

The data shown in Figure 3A are chromatograms for the conven-

tional product ion scan, DS, and for the enhanced product ion scan,

using ScanWave DS, produced from the UPLC/MS/MS analysis

of vancomycin, a glycopeptide antibiotic, with m/z 725 for the

[M+2H]2+ in positive ion electrospray mode. The chromatograms

have been superimposed and the vertical axes are displayed on

the same scale.

A factor of 6X signal enhancement is observed for the largest

chromatographic peak, number 5, when ScanWave DS is used.

Figure 3. Chromatogram A shows ScanWave product ion scan (ScanWave DS, green trace) versus the regular product ion scan (DS, red trace) of vancomycin, [M+2H]2+ m/z 725. In B, the ScanWave DS chromatogram is shown with the x-axis plotted in scan number.

In the conventional product ion scan mode, peaks 1, 2, 4, and 5 are

detected. When the same sample is analyzed using ScanWave DS,

the resulting signal enhancement improved the level of sensitivity

and the total number of peaks detected. In addition to the peaks that

were found in this sample using the conventional product ion scan,

spectra can be obtained for peaks 3, 6, 7, and 8.

Modern high resolution chromatography using sub-2 µm column

particles produces peaks with widths of 1 to 3 seconds at the base.

To accurately define these peaks, a high duty cycle/scan speed mass

spectrometer is required.

Figure 3B shows the same chromatogram plotted with scan number as

the x-axis. The scan speed of both the ScanWave DS and the conven-

tional product ion scan experiments was 5000 amu/sec. This allowed

more than 10 data points for the mass range 90 to 1455 amu to be

collected across chromatographic peaks which were 3 seconds wide.

Ejection Region

DC Barrier RF Barrier

Storage Region

PotentialTo Scanning Quadrupole

(MS2)

Traveling Wave

Traveling Wave

Traveling Wave

Traveling Wave

Low m/z Ion

High m/z Ion

Intermediate m/z Ion

Time2.80 3.00 3.20 3.40 3.60 3.80

%

0

100

2, 3

4

5

8

71

6

Scan550 600 650 700 750 800 850

%

0

100

ScanWave DS of 726ES+ TIC

1.25e8

2.88

3.73

A B

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

13

Figure 4 shows a mass spectrum of the largest chromatographic peak

(number 5) shown in Figure 3A. ScanWave DS of the doubly-charged

ion m/z 725 resulted in the major singly-charged fragments m/z 100,

m/z 144, and m/z 1306.

The data illustrates that the Xevo TQ MS is capable of acquiring high-

quality spectral data while operating at the high scan speeds required

to characterize narrow UPLC peaks.

Figure 4. ScanWave DS spectrum for vancomycin, [M+2H] 2+ m/z 725.

CONCLUSION

The enhanced sensitivity of the Xevo TQ MS in ScanWave mode allows

users to better characterize low-level components in their samples.

ScanWave technology allows ions to be accumulated, separated,

and ejected according to their m/z. The final quadrupole scanning is

synchronized with ion ejection from the collision cell such that the

ions of a given mass-to-charge ratio are delivered to the quadrupole

when it is ready to scan this m/z value. This results in a more efficient

instrument duty cycle and better sensitivity in scanning acquisitions.

In this application note, ScanWave technology has allowed the

peak detection of the vancomycin sample in MS/MS mode to be

significantly improved. When ScanWave DS mode was used, spectra

could be obtained for chromatographic peaks that were previously

not detected by the conventional product ion scan.

Reference

1. The traveling wave device described here is similar to that described by Kirchner in U.S. Patent 5,206,506 (1993).

100

%

0200 400 600 800 1000 1200 1400

1306

144

217

1143725m/z

ScanWave DS of 725ES+ 3.64e6

Waters, UPLC, and ACQUITY UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2008 Waters Corporation. Printed in the U.S.A.October 2008 720002828EN LB-CP

14

15

S IM U LTA N EOUS CO N F I RMAT IO N A N d QUA N T I F IC AT IO N US INg X E V O T Q M S: P RO dU C T IO N CO N F I RMAT IO N ( P IC )

Marian Twohig, Gordon Fujimoto, Joanne Mather, and Robert S. Plumb Waters Corporation, Milford, MA, U.S.

INT RODUCT ION

Tandem quadrupole mass spectrometers are used extensively in the

pharmaceutical industry for analyte quantification. This is primarily

performed by multiple reaction monitoring (MRM) as the matrices

are complex and the specificity of MRM gives the best signal-to-

noise ratios.

As well as performing quantification, these instruments are often

used for initial qualitative information, with the instrument operated

in scan mode. This information is used to confirm the identity of the

peak of interest that is being quantified.

In complex matrices, situations can arise where closely-related

compounds, e.g., metabolites or matrix interferences, can give rise

to signals even in MRM mode. This can lead to ambiguity and may

require a second qualitative experiment. Product ion confirmation

provides a means of verifying that the signal from the MRM peak is

from the compound of interest.

With conventional instrumentation, these experiments require

separate full-scan analyses. Many conventional tandem quadrupole

MS instruments are unable to perform MRM and scan experiments

simultaneously, in the timeframe of an LC peak, while maintaining

data quality. The Waters® Xevo™ TQ Mass Spectrometer is equipped

with a novel collision cell design. The collision gas is always on,

allowing both quantification (MRM) and characterization to be

performed simultaneously on the peak as it elutes from the LC or

UPLC® column while maintaining good data quality.

The new ScanWave™ mode of operation allows ions within the

collision cell to be accumulated and then separated according to

their mass-to-charge (m/z) ratio. Synchronizing the release of these

ions with the scanning of the second quadrupole mass analyzer

greatly improves duty cycle, which significantly enhances the signal

intensity of full-scan spectra for both MS and product ions.

Figure 1. Xevo TQ Mass Spectrometer with the ACQUITY UPLC® System.

EX PERIMENTAL

Product ion confirmation on Xevo TQ MS

The Xevo TQ MS can simultaneously acquire a product ion con-

firmation (PIC) scan along with an MRM chromatogram to obtain

additional information about an eluting peak. A PIC scan is enabled

in the MRM method, where a scan is used to collect either:

n MS scan

n Enhanced MS scan using ScanWave mode

n Product ion scan

n Enhanced product ion scan using ScanWave DS mode

In PIC mode, the Xevo TQ MS will switch from MRM to scan after

the apex of an LC peak as long as a minimum intensity threshold

is achieved. The trigger to start will occur after four consecutive

downward scans have been detected. If the minimum intensity

criteria is met, an MS or MS/MS spectrum is acquired using the

final resolving quadrupole (MS2) to perform the scan before

switching back to MRM mode (Figure 2). The threshold ensures

that the PIC scan is of sufficient quality to be beneficial to the user.

16

The high data collection rate of the Xevo TQ MS is such that the

area of the MRM peak can still be accurately determined, since PIC

is triggered after the peak top is detected and the definition of the

peak itself is not affected. Consequently, quantitative and qualita-

tive data are acquired simultaneously.

Figure 2. Schematic showing Product Ion Confirmation (PIC) switching after the peak top.

Figure 3 shows an example of an MRM chromatogram (3A) obtained

from the quantification of the corticosteroid fluticasone, m/z 501.

Qualitative confirmation of the peak of interest is provided by the

resulting PIC spectrum operated in ScanWave DS mode (3B).

The scan range for the PIC is selected by the software, in this

case m/z 40 to 511.

Figure 3. Chromatogram from the analysis of fluticasone, with MRM 501 > 293, and an example of the ScanWave DS PIC spectrum.

A PIC spectrum using ScanWave DS is displayed in Figure 4A. Here

it is been compared with a PIC spectrum using conventional product

ion scan (DS), 4B, and a combined spectrum (20 scans) from a

ScanWave DS of fluticasone, 4C. The spectral quality is maintained

when a PIC spectrum in ScanWave DS mode (4A) is compared to a

combined ScanWave DS spectrum (4C).

The data show that a four-fold signal enhancement was observed

when ScanWave DS mode (4A) is used to collect the PIC spectrum

compared to a conventional product ion spectrum (4B). This is due

to the more efficient duty cycle that is achieved in ScanWave mode.

This extra sensitivity available with ScanWave mode allows for high

quality spectra to be obtained even at low levels.

Switches here and acquires PIC Scan

Switches back to MRM data acquisition

MRM Trace

B

m/z40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

%

0

100 293

205

109185155

121147

275

265

251

217235

313

501361333 389 481

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

Flu_1_9_015d 1: MRM of1 Channel ES+ 501.3 > 293.2 (Fluticasone)

4.09e7

MRM forFluticasone

PIC spectrumfrom MRM peakat Rt = 1.80 min

O

O

OH

CH3

H

CH3

F

F

H

CH3

OS

F

O

CH3

A100

17

Figure 4. Spectrum shows a comparison of a PIC spectrum for ScanWave DS, a regular product ion PIC spectrum and a combined spectrum acquired by ScanWave DS for fluticasone m/z 501 (Vertical axis linked).

CONCLUSION

The Xevo TQ MS can be used to perform quantification of fluti-

casone with simultaneous characterization of the MRM peak as it

elutes from the chromatographic system. This eliminates the need

for separate injections when qualitative confirmation of MRM peaks

is required and reduces the total analysis time in these situations.

When used routinely, product ion confirmation increases user

confidence in qualitative results from complex matrixes, and thus

reduces the need for re-analysis.

m/z50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500

%

0

100

%

0

100

%

0

100Flu_1_9_015d 2 (1.813)

1: Product Ions of 501 ES+501.3 > 293.2 (Fluticasone)

5.26e7

293

205

109185155135

275

251217

313

501361333 389 481

Flu_1_9_014d 2 (1.814)

1: Product Ions of 501 ES+501.3 > 293.2 (Fluticasone)

5.26e7

293

205109 155

275251

313

359333 389

Flu_1_9_017d 961 (1.796)ScanWave DS of 501ES+

5.26e7293

205

109

95155121 185

275

251217

313

501361333 389 481

PIC spectrum ScanWave DS

PIC spectrum DS

Spectrum ScanWave DS

A

B

C

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

Waters, UPLC, and ACQUITY UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2008 Waters Corporation. Printed in the U.S.A.October 2008 720002829EN LB-CP

18

19

NOV E L dUA L-S C A N M RM MO d E MA S S S P EC T ROM E T Ry FO R T H E d E T EC T IO N O F M E TA BO L IT E S dU R INg d RUg QUA N T I F IC AT IO N

Paul D. Rainville, Jose Castro-Perez, Joanne Mather, and Robert S. Plumb Waters Corporation, Milford, MA, U.S.

INT RODUCT ION

The measurement of the levels of circulating drugs and their metab-

olites is important information in the development of new therapies.

Drug levels in biofluids are used to determine the bioavailability of

a drug. Additionally, elucidation of drug metabolite information is

vital due to the fact that they can often be toxic at certain levels,

have a greater pharmacodynamic effect than the parent drug, inter-

fere with concomitant medication, and impact liver function.

These two different pieces of information are normally acquired in

separate analytical experiments, resulting in increased laboratory

workload and reduced efficiency. Therefore the ability to determine

drug concentration and obtain metabolite structural information

during a single analysis is not only faster but more cost effective.

In the case of low sample volumes, e.g., pediatric studies, this

capability is critical for laboratories to obtain required quantitative

and qualitative data.

The Waters® Xevo™ TQ Mass Spectrometer is a tandem quadrupole

system equipped with a novel collision cell design that allows full-

scan MS and quantitative multiple reaction monitoring (MRM) data

to be acquired in a single analytical run.

Here, we present a method whereby full-scan MS and MRM data

can be acquired in a single run to determine the levels of a model

pharmaceutical in urine and utilize the associated full-scan data to

determine its related metabolites.

EX PERIMENTAL

Human urine was collected from volunteer individuals eight hours

after dosing with 400 mg of ibuprofen. The samples were stored

frozen prior to analysis. Samples were prepared by centrifugation

at 13,000 RCF for 5 minutes and diluted with water. Samples were

then injected onto the UPLC®/MS/MS system.

LC conditions

LC system: Waters ACQUITY UPLC® System

Column: ACQUITY UPLC BEH C18 Column

2.1 x 50 mm, 1.7 µm

Column temp.: 40 °C

Flow rate: 600 µL/min

Mobile phase A: 0.1% NH4OH

Mobile phase B: ACN

Gradient: 5% to 95% B/2 min

20

MS conditions

MS system: Waters Xevo TQ MS

Ionization mode: ESI negative

Capillary voltage: 2000 V

Cone voltage: 15 V

Desolvation temp.: 550 °C

Desolvation gas: 1000 L/Hr

Source temp.: 150 °C

Scan range: m/z 100 to 500

Collision energies: MRM data 7 V, full-scan data 3 V

MRM transition: m/z 205 > 161

RESULTS

Determining drug concentration and drug metabolites are both

important aspects in developing a new medicine. This experiment was

designed such that the levels of ibuprofen in urine were measured by

MRM mass spectrometry and full-scan MS data was collected to detect

the associated metabolites during a single injection.

The unique collision cell design of the Xevo TQ MS, which is continu-

ously filled with collision gas, enables it to operate with rapid

switching between MS and MS/MS data acquisition modes. This

occurs in timeframe that is compatible with the fast chromatography

and narrow peaks generated by the ACQUITY UPLC System: the

Xevo TQ MS is capable of operating at up to 10,000 Da/sec and can

correctly define the very sharp peaks produced by UPLC.

In this dataset, greater than 12 scans were acquired for the MRM channel

of ibuprofen while also obtaining full scan MS data. Peaks widths were

on the order of 2.4 seconds measured at peak base (data not shown).

Figure 2 shows the chemical structure of ibuprofen and some of its

major in vivo metabolites. Figure 3 displays the MRM transition

data for ibuprofen in the urine samples and also the simultaneously

acquired full-scan data. The full-scan data were then mined for

potential metabolites resulting from ibuprofen. Figure 4 shows

extracted ion chromatograms (XIC) that were generated relating

to the ketone glucuronide (m/z 411), glucuronide (m/z 381), and

hydroxy glucuronide (m/z 397) metabolites.

Figure 2. Ibuprofen and some of its associated metabolites.

Figure 3. MRM of ibuprofen and full-MS scan data acquired from subject urine.

O

CH2 OH

CH3

CH3

CH3

O

O-Gluc

CH3

CH3

HO

O

O-Gluc

CH3

CH3

CH3

OH

O

O-Gluc

CH3

CH3

CH3

OH

O

O-G luc

CH3

CH3

CH3

1-Hydroxy IbuprofenGlucuronide

3-Hydroxy IbuprofenGlucuronide

2-Hydroxy IbuprofenGlucuronideIbuprofen Glucuronide

Ibuprofen MRM

Diluted urine patient 1Full scan

Diluted urine patient 2Full scan

21

Figure 4. XIC of ibuprofen metabolites and full-scan data.

Further confirmatory product ion MS experiments revealed

several diagnostic fragment ions, such as m/z 193 and 175 for

the glucuronide acid moieties, m/z 221 for the aglycone, and

m/z 113 for ibuprofen itself (Figure 5).1

Figure 5. Product ion spectra of ibuprofen metabolites.

A further advantage to this acquisition approach is that it provides the

scientist with the ability to visualize the differences between subject

matrix using the full-scan MS. These differences may be related to

several factors: diet, sex, age, or the state of an individual’s health.

Thus the full scan data could be additionally utilized for the detection

of biomarkers. Further, the full-scan MS data could be interrogated in

the future if new information is required about the metabolism of the

compound – without the need to re-run the samples.

KetoneXIC m/z 411

GlucuronidesXIC m/z 381

Hydroxy glucuronidesXIC m/z 397

Full scan

Ketone

Glucuronides

Hydroxy glucuronides

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

22

Waters, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2008 Waters Corporation. Printed in the U.S.A.October 2008 720002832EN LB-CP

CONCLUSION

In this application note, we have demonstrated that the Xevo TQ MS

can acquire full-scan and MRM channel data to determine the level

of a model pharmaceutical compound in urine and its metabolite

information in a single analysis. The speed of the Xevo TQ MS

proves to be highly compatible with the high resolving power of the

ACQUITY UPLC System.

The benefits of this technique are realized in several ways. First,

the ability to gather full-scan data along with MRM channel data

enables scientists to collect multiple dimensions of information

about a sample in a single run – maximizing the resource utilization

of a laboratory that otherwise would have been performing multiple

experiments to gain the same information. Second, coupling this MS

technique with UPLC ensures a faster analysis. Finally, the richness of

the data acquired by full-scan MS allows that information to be mined

in multiple ways, giving researchers more confidence in their deci-

sions as they direct their drug discovery and development studies.

Reference

1. Plumb R, Rainville P., et al. Rapid Communications in Mass Spectrometry. 2007; 21: 4079-85.

23

dATA- d I R EC T E d d E T EC T IO N A N d CO N F I RMAT IO N O F d RUg M E TA BO L IT E S IN B IOA NA Ly T IC A L S T U dI E S

Robert S. Plumb and Paul D. Rainville Waters Corporation, Milford, MA, U.S.

INT RODUCT ION

LC/MS/MS analysis has become the analytical method of choice for

the accurate quantification of pharmaceutical compounds or active

metabolites in biological fluids. The specificity and selectivity

provided by tandem quadrupole MS in multiple reaction monitoring

(MRM) mode allows for rapid high-sensitivity analysis, often in the

pg/mL range. The data produced by LC/MS/MS analysis provides

drug concentration data that is critical to successful drug discovery

and development.

Recent U.S. FDA Guidance, “Industry Safety Testing of Drug

Metabolites,” provides recommendations to industry on when and

how to identify and characterize drug metabolites whose non-

clinical toxicity needs to be evaluated. The aim of these guidelines

is to ensure that variations in metabolic profiles across species are

both quantitatively and qualitatively measured.1

The Waters® Xevo™ TQ Mass Spectrometer is capable of operating

at acquisition speeds up to 10,000 Da/sec, which aids in the

adequate characterization of very sharp chromatographic peaks

produced by the ACQUITY UltraPerformance LC® (UPLC®) System.

The Xevo TQ MS is equipped with a novel collision cell design that

is continuously filled with collision gas, allowing rapid switching

between MS and MS/MS modes in a single analytical run.

This new collision cell is capable of enhanced high-sensitivity

operation in MS/MS mode. In this Scanwave™ mode of operation,

ions are constrained in the final third of the collision cell using both

a DC and RF barrier. These ions are then ejected from the collision

cell, in a controlled manner, from high to low m/z in synchronization

with the scanning of the final resolving quadrupole. This increases

the duty cycle of the instrument.

In this application note, we illustrate the ability of the Xevo TQ MS,

in Survey Scan mode, to detect drug metabolites “on the fly” using

a common diagnostic fragment ion.

EX PERIMENTAL

Rat plasma was spiked with ibuprofen and related major

metabolites. Samples were then precipitated using 2:1 acetonitrile

to sample (v/v). The sample was evaporated to dryness and

reconstituted in 9:1 water/methanol (v/v). The sample was then

injected onto the UPLC/MS/MS system.

LC /MS conditions

LC system: Waters ACQUITY UPLC® System

Column: ACQUITY UPLC BEH C18 Column

2.1 x 50 mm, 1.7 µm

Column temp.: 40.0 °C

Flow rate: 600 µL/min

Mobile phase A: 0.1 % NH4OH

Mobile phase B: Acetonitrile

Gradient: 5% to 95% B/2 min

Figure 1. Xevo TQ Mass Spectrometer.

24

MS system: Waters Xevo TQ MS

Ionization mode: ESI negative

Capillary voltage: 2000 V

Cone voltage: 15 V

Collision energy: 7 eV

RESULTS

The superior efficiency of the ACQUITY UPLC System produces extremely

narrow peaks, 2 seconds or less at the base. These narrow peaks require

a fast data capture rate mass spectrometer to accurately define the peak.

Figure 2 shows the MRM peak for ibuprofen using the transition m/z

205 to 161. The peak is 1.2 seconds wide at the base, and the high data

capture rate of the Xevo TQ MS allows for more than 60 scans across the

peak. This facilitates the accurate definition of the chromatographic peak,

even if several MRM transitions are employed during analysis.

Figure 2. UPLC/MS/MS of ibuprofen using the MRM transition m/z 205 to 161.

Recent FDA guidelines have recommended that, during human

clinical trials, the concentration and identity of any metabolites

with an exposure of greater than 10% of the dosed compound must

be determined. Mass spectrometry can detect and identify drug

metabolites by various means. One method is to utilize Survey

Scan mode. In this mode of operation, the MS is set to monitor a

diagnostic fragment ion from the parent drug compound.

The use of a common fragment ion requires the mass spectrometer

to scan the first quadrupole (Q1) while monitoring for a fixed m/z

with the final resolving quadrupole (Q3). Ibuprofen gives rise to

several distinctive product ions, m/z 113, 133, and 161.2 Figure 3

illustrates Xevo TQ MS operation in Survey Scan mode.

In this example, the common fragment ion of m/z 113 was

monitored by the resolving quadrupole. When a peak, containing

a m/z of 113, was detected the MS switched to collect product ion

data on the precursor ion containing the m/z 113. Peaks that exceed

a user-defined detection threshold are used to trigger the acquisi-

tion of product ion data.

Figure 4 illustrates the MS/MS spectra obtained for the peak detect-

ed at 0.66 minutes. In this example, we can see that the precursor

peak m/z value is 397. The m/z 397 produces major fragment ions

at m/z 113, 175, 193, and 221.

Figure 3. Survey Scan: precursors of m/z 113 switching to product ion scan.

Figure 4. Survey Scan ScanWave DS spectrum of peak eluting at 0.66 minutes.

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

%

0

100 0.82

Scan1820 1840 1860 1880 1900 1920 1940 1960

%

0

100 0.81

25

The m/z values and MS fragment pattern confirm the identity of

this peak as the O-glucuronide metabolite of ibuprofen.2 The data

acquired for the peak eluting with a retention time of 0.88 minutes

are shown below in Figure 5.

Figure 5. ScanWave DS of peak eluting at 0.88 minutes with a m/z value of 381.

This peak was determined to have a m/z value of 381. Resulting

fragment ions produced from the product ion MS/MS were

m/z 113, 161, 175, 193, and 205. This data confirmed that this

peak was related to ibuprofen and, with the precursor ion m/z value

of 381, was confirmed as the glucuronide conjugate of ibuprofen.2

Thus with one simple analytical experiment, along with the knowl-

edge of the fragmentation pattern of the ibuprofen, the metabolites

could be detected and the structure confirmed.

CONCLUSION

The quantification of pharmaceutical compounds in biological fluids

is a regulatory requirement as part of any new drug submission,

e.g., IND, CTX. More recently, these regulations have required that

drug metabolites with an exposure greater than 10% of the active

pharmaceutical be quantified and characterized. The Xevo TQ MS

can perform data-directed MS/MS experiments, allowing metabolite

structural confirmation using common fragment ions within a UPLC

peak timeframe.

References

1. U.S.FDA. Guidance for Industry, “Safety Testing for Metabolites.” http://www.fda.gov/CDER/GUIDANCE/6897fnl.pdf

2. Plumb R., Rainville P, et al. Rapid Communications in Mass Spectrometry. 2007; 21: 4079-85.

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

Waters, ACQUITY UltraPerformance LC, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2008 Waters Corporation. Printed in the U.S.A.October 2008 720002833EN LB-CP

26

27

IM P ROV INg QUA L ITAT IV E CO N F I RMAT IO N US INg X E V O T Q M S w IT H SU RV E y S C A NNINg

Marian Twohig, Andrew Aubin, Michael Jones, and Robert S. Plumb Waters Corporation, Milford, MA, U.S.

INT RODUCT ION

On a conventional tandem quadrupole mass spectrometer, the

search for unknowns generally requires multiple injections: one

injection in full-scan LC/MS mode followed by a second injection

for targeted LC/MS/MS experiments. This results in increased time

required to obtain the necessary data, in addition to the time the

analyst needs to construct the MS/MS methods.

Real-time data-directed switching simplifies this experimental

approach. In data-directed mode, a full spectrum LC/MS run is

collected, with an LC/MS/MS experiment triggered if the signal in

the LC/MS survey meets preset criteria.

Modern Linear Ion Trap (LIT) mass spectrometers allow the col-

lection of MS, MRM, and MS/MS data in the same analytical run,

enabling quantitative and qualitative data to be obtained simultane-

ously. The duty cycle of these instruments when switching between

MS and MS/MS modes is typically 2 to 3 seconds. With modern

high-resolution, sub-2 µm particle chromatography, such as UPLC,®

peak widths of 2 to 3 seconds are now commonplace, thus with

these LIT MS systems this results in just 1 to 2 points across the

peak giving poorly defined peaks and possibly missed components.

The Waters® Xevo™ TQ MS is capable of scan speeds up to 10,000

amu/sec. Consequently, it is possible to employ a number of scan

functions in a single run while still maintaining good peak charac-

terization with no loss in data quality.

EX PERIMENTAL

Survey Scans on Xevo TQ MS

Survey Scans on the Xevo TQ MS allow intelligent switching of

LC/MS and LC/MS/MS data in one run, thus improving productivity.

Conventional MS or ScanWave™ MS scanning experiments can be

used to trigger MS/MS experiments in real time as the peaks are

eluting from the LC column. A more targeted screen can also be

performed using parent ion or neutral loss spectral acquisition,

to screen for compounds that have common structural features.

Conventional product ion or enhanced product ion spectra

(ScanWave) data can be generated for all the components present

in these complex samples. In ScanWave mode, duty cycle improve-

ments result in signal enhancement in scanning acquisition modes,

which facilitates the detection of low-level impurities.

An example of a survey scan for the active pharmaceutical

ingredient (API) quetiapine, an antipsychotic medication, is

shown in Figure 2, where the initial survey function is ScanWave

MS switching to ScanWave DS.

Figure 1. Xevo TQ Mass Spectrometer with ACQUITY UPLC.

28

Figure 2. Shown is an example of a Survey Scan of quetiapine (m/z 384) where the initial ScanWave MS Survey function switches to ScanWave DS mode.

In Figure 3, quetiapine (C21H25N3O2S) was analyzed in survey

scan mode. The structure-characteristic fragments of quetiapine1,2

are m/z 253 (C15H13N2S) and m/z 279 (C17H15N2S). In the above

example, a Precursor Ion Scan (m/z 253) was used to trigger the

acquisition of a ScanWave product ion scan (ScanWave DS),

generating a full product ion spectrum for the compounds

potentially related to quetiapine.

More than 20 compounds were observed to have the fragments

m/z 253 and m/z 279 as well as another signature fragment

m/z 221 (C15H13N2). Shown in Figure 4 are spectra from the

chromatographic peaks at retention times 7.86 min, 9.95 min,

10.13 min, 14.01 min, 15.94 min, and 17.52 min, respectively.

Figure 3. Survey precursor scan of m/z 253 (lower trace) switching to ScanWave DS.

Included are the API, quetiapine, and the previously-characterized1,2

quetiapine carboxylate and bis (dibenzo) piperazine, as well as three

unknown compounds that have similar fragmentation patterns.

This information was obtained from one Survey experiment without

the need for extra confirmatory MS/MS analyses. This allows the

analyst to acquire important structural information in a single run.

SN+

NH

S

N

N N

O

OH

m/z300 350 400 450 500 550 600 650

%

0

100 384

m/z100 200 300 400 500 600

%

0

100 253

158 384

ScanWave MS

ScanWave DS

m/z 384 Quetiapine

Diagnostic Fragment m/z 253

MS/MS of m/z 384

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100 8.41

7.86

6.11

2.68 7.18

9.26

17.15

15.9414.01

11.0412.42 18.27

9.79

9.277.87

8.38

17.51

14.00

Precursors of m/z 253

ScanWave DS

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

29

Figure 4. UPLC/MS/MS spectra in ScanWave DS mode from selected chromatographic peaks shown in Figure 3 (top).

CONCLUSION

The Waters Xevo TQ MS, with its unique collision cell design, where

the collision gas is always on, facilitates the simultaneous acquisition

of MS and MS/MS data in one LC/MS run. Its high scan speed allows

for these experiments to be performed with sufficient points across

the peak to accurately define the narrow peaks produced by UPLC.

This capability facilitates data-directed experiments, where real-time

switching between MS and MS/MS allows more information to be

acquired from a single injection. This reduces the need for separate

experiments and accelerates the process of structural identification

and unknown compound determination.

References

1. Xu H, Wang D, Sun C, Pan Y, Zhou M. Identification of an unknown trace level impurity in bulk drug of Seroquel by high-performance liquid chromatography combined with mass spectrometry. J Pharm Biomed Anal. 2007 Jun 28; 44(2): 414-20. Epub 2007 Mar 7.

2. Bharathi Ch, Prabahar KJ, Prasad ChS, Srinivasa Rao M, Trinadhachary GN, Handa VK, Dandala R, Naidu A. Identification, isolation, synthesis and characterisation of impurities of quetiapine fumarate. Pharmazie. 2008 Jan; 63(1): 14-9.

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

02_Sur_QT_3_10_001 2054 (7.856) 2: Auto ScanWave DS 482.46ES+ 8.93e7253

221143

279482

02_Sur_QT_3_10_001 2636 (9.958) 2: Auto ScanWave DS 383.84ES+ 8.93e7253

221158 210 279

02_Sur_QT_3_10_001 2683 (10.132) 2: Auto ScanWave DS 428.56ES+ 5.92e7253

221202

279

02_Sur_QT_3_10_001 3759 (14.007) 2: Auto ScanWave DS 456.48ES+ 7.02e7253

221 279456

02_Sur_QT_3_10_001 4297 (15.946) 2: Auto ScanWave DS 510.38ES+ 8.43e7253

221 279510

02_Sur_QT_3_10_001 4734 (17.524) 2: Auto ScanWave DS 505.53ES+ 3.88e7253

221279

505

Unknown

Quetiapine

Quetiapine Carboxylate

Unknown

Unknown

Bis (dibenzo) Piperazine

Waters and UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2008 Waters Corporation. Printed in the U.S.A.October 2008 720002838EN LB-CP

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

Waters, ACQUITY UPLC, UltraPerformance LC and UPLC are registered trademarks of Waters Corporation. XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2007 Waters Corporation. Printed/Produced in the U.S.A.Xxxxx 2007 72000XXXXEN XX-XX

30

31

A NOV E L M E T HO d FO R MO NIT O R INg MAT R I X IN T E R F E R EN C E S IN B IO LOgIC A L SAM P L E S US INg dUA L-S C A N M RM MO d E MA S S S P EC T ROM E T Ry

Paul D. Rainville, Joanne Mather, and Robert S. Plumb Waters Corporation, Milford, MA, U.S.

INT RODUCT ION

Development of a fast, sensitive, and robust bioanalytical

LC/MS/MS assay is essential for cost-effective and compliant

processing of samples in biological fluids.

However, any bioanalytical assay can be hampered by sample

matrix effects. Components such as drug metabolites, proteins,

and phospholipids within biological matrices frequently interfere

with the robustness and sensitivity of the assay. Furthermore,

regulatory authorities now require that matrix effects be deter-

mined in these assays.

Therefore, it would be very advantageous to actively monitor and

characterize the presence of matrix components coeluting with the

compound of interest during LC/MS/MS assay method development.

Significant time and effort could be saved by ensuring that compo-

nents in the matrix are well resolved from the analyte of interest.

However, conventional tandem quadrupole mass spectrometers

cannot acquire multiple reaction monitoring (MRM) data while

acquiring full-scan data at speeds fast enough for the narrow

chromatographic peaks generated by modern separations techniques

such as UPLC.®

In this application note, we describe the ability of a novel

UPLC/MS/MS platform, the Waters® Xevo™ TQ Mass Spectrometer,

to monitor potential matrix interferences in plasma while monitoring

a pharmaceutical compound of interest. Unique to the Xevo TQ MS

platform is its ability to switch between MS and MS/MS modes in a

UPLC run that typically generates peak widths of 2 to 3 seconds.

EX PERIMENTAL

Alprazolam was spiked into rat plasma at a concentration of

10 ng/mL and then precipitated with acetonitrile using a 2:1

acetonitrile/plasma ratio. The sample was then centrifuged at

13,000 RCF for 5 minutes. The supernatant was removed and

injected onto the UPLC/MS/MS system.

LC conditions

LC system: Waters® ACQUITY UPLC® System

Column: ACQUITY UPLC BEH C18 Column

2.1 x 50 mm, 1.7 µm

Column temp.: 40 °C

Flow rate: 600 µL/min

Mobile phase A: 0.1 % NH4OH

Mobile phase B: MeOH

Gradient: 5% to 95% B/2 min

32

MS conditions

MS System: Waters Xevo TQ MS

Ionization mode: ESI positive

Capillary voltage: 1000 V

Cone voltage: 25 V

Desolvation temp.: 500 °C

Desolvation gas: 1000 L/Hr

Source temp.: 150 °C

Scan range: m/z 100 to 1000

Collision energies: High 20 V, low 3 V

MRM transition: m/z 309 > 281

RESULTS

As previously stated, the large number of components found in

matrices commonly employed in bioanalysis, such as plasma and

urine, can pose a significant problem when developing and validat-

ing a quantitative bioanalytical method. Techniques such as solid

phase extraction (SPE) and high resolution chromatography are often

employed to reduce their effects.1,2

Plasma, for instance, has many endogenous compounds that can

interfere with the pharmaceutical compound undergoing quantifica-

tion. Some of the major interferences in plasma samples that cause

ion suppression or enhancement are phospholipids, specifically

variants containing the choline head group. Consequently, scientists

developing bioanalytical methods often will monitor phospholipids

as they can be a major source of matrix effects.

Parent or precursor ion scanning of the indicative choline fragment

ion (m/z 184, in positive ion mode) is commonly used to monitor

phospholipids (Figure 2).

Figure 2. Fragmentation pattern for a phospholipid containing the choline head group.

CH3

CH3

CH3

N+

O

O

O-

P

O

O

O

O

O

R R'

184

CH3

CH3

CH3

N+

O

O

O-

P

O

O

O

O

O

R R'

33

Figure 3 shows the MRM channel for alprazolam above a scan result

for precursor m/z 184. In this example, we can see that UPLC meth-

odology facilitates excellent resolution of the analyte of interest

from the choline-containing phospholipids.

Figure 3 further illustrates the differences between the two scans

of the two lots of plasma, indicating that each contains different

choline-containing phospholipids. This variance in plasma lot A and

lot B exemplifies the reason six different lots of matrix are required

to be analyzed during the bioanalytical method validation process.3

Figure 3. Monitoring of the model drug alprazolam and matrix effects contributed by choline-containing phospholipids in a single analysis.

Recognizing that there may be other compounds in the matrix that

could potentially interfere, and thus become a source of matrix

effects, a full MS scan was acquired from the same injection as

the MRM channel (Figure 4). Acquiring full-scan data gives the

bioanalytical scientist the tools to observe more potential matrix

interferences in the samples.

Figure 4. Simultaneous monitoring of the model drug alprazolam and matrix in the m/z 100 to 1000 range in a single analysis.

Figure 4 indicates the presence of other coeluting analytes from

the matrix. The coeluting analytes have the potential to introduce

matrix effects to the analysis of alprazolam, and may impact the

quantification of the analyte of interest. The scientist is now in a

position to adjust the conditions of the assay method, taking into

account the interfering components.

The unique capability of the Xevo TQ MS to rapidly switch between

MS and MS/MS modes facilitates simultaneous monitoring of

coeluting components as well measuring the compound of interest,

even when UPLC peaks are typically 2 to 3 seconds wide.

MRM alprazolamplasma lot A

MRM alprazolamplasma lot B

Parents of m/z 184plasma lot A

Parents of m/z 184plasma lot B

MRM alprazolamplasma lot A

MRM alprazolamplasma lot B

MS scan m/z 100 to 1000plasma lot A

MS scan m/z 100 to 1000plasma lot B

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

34

CONCLUSION

In this application note, we have shown the novel ability of the

Waters Xevo TQ MS coupled with an ACQUITY UPLC System to

acquire quality full-scan and MRM data in a single analysis. Using

this technique, we monitored potential matrix interferences present

in protein-precipitated plasma while monitoring the MRM transition

for a model pharmaceutical.

Interferences due to coelution of matrix components can thus be

detected “on the fly” in early method development, which reduces:

n Method development time

n Method variability

n Problematic occurrences during validation

Thus utilizing the Xevo TQ MS enables researchers to increase the

quality of the final MS/MS method while developing a fully-detailed

scan record should reviewing the data be of interest.

References

1. Zhang S, Chen G. Journal of Chromatographic Science. 2008; 46: 220-224.

2. Chambers E, Diehl D, Lu Z, Mazzeo J. Journal of Chromatography B. 2007; 852 (1-2): 22-34.

3. Guidance for Industry, Bioanalytical Method Validation. U.S. FDA. 2001.

Waters, UPLC, and ACQUITY UPLC are registered trademarks of Waters Corporation. Xevo and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2008 Waters Corporation. Printed in the U.S.A.October 2008 720002830EN LB-CP

35

R A P I d, S IM P L E IM P U RIT y C HA R AC T E R Iz AT IO N w IT H T H E X E V O T Q MA S S S P EC T ROM E T E R

Robert S. Plumb, Michael D. Jones, and Marian Twohig Waters Corporation, Milford, MA, U.S.

INT RODUCT ION

The detection and characterization of impurities and degradation

products of an active pharmaceutical ingredient (API) are regulatory

filing requirements. The detection and identification of impurities

not only ensures medicine safety but can also be used as a finger-

print for patent protection and counterfeit drug analysis.

Impurity characterization and identification are normally carried

out using information-rich analytical techniques such as NMR and

LC/MS. Analysis by LC/MS provides parent ion mass from full-scan

MS and structural information from the fragments generated in

MS/MS experiments. With traditional tandem quadrupole instrumen-

tation, the generation of this data requires multiple experiments to

obtain MS and MS/MS information.

Modern Linear Ion Trap (LIT) mass spectrometers allow the collection

of MS, multiple reaction monitoring (MRM), and MS/MS data in the

same analytical run, allowing quantitative and qualitative data to

be obtained simultaneously. However, the duty cycle of these instru-

ments when switching between MS and MS/MS modes is typically

2 to 3 seconds. With modern high-resolution, sub-2 µm column par-

ticle chromatography such as UPLC,® peak widths of 2 to 3 seconds

are now commonplace. With these LIT MS systems, this would result in

just 1 to 2 points across the peak, with the peaks either poorly defined

or missed completely; thus slower, lower-resolution LC systems must

be used, resulting in reduced throughput and lower data quality.

The Waters® Xevo™ TQ Mass Spectrometer is equipped with a novel

collision cell design that is continuously filled with collision gas,

allowing rapid switching between MS and MS/MS modes. The Xevo TQ

MS is capable of operating at up to 10,000 Da/sec and can correctly

define the very sharp peaks produced by UPLC, with more than 10

points across a 2-second-wide peak, even on a multi-scan experiment.

This new collision cell is capable of enhanced high-sensitivity

operation in MS/MS mode. In this mode of operation, ions are

constrained in the final third section of the collision cell using both

DC and RF barriers. These ions are then ejected from the collision

cell, in a controlled manner, from high to low m/z in synchronization

with the scanning of the final resolving quadrupole. This increases

the duty cycle of the instrument, resulting in enhanced sensitivity

that is ideal for the detection and characterization of low-concentra-

tion impurities that may result in toxic effects.

EX PERIMENTAL

To evaluate the performance of this system, the impurities of the

common pharmaceutical drug quetiapine, used to treat biopolar

disorder, was investigated using UPLC/MS/MS.

LC /MS conditions

LC system: Waters ACQUITY UPLC® System

Column: ACQUITY UPLC BEH C18 Column

2.1 x 50 mm, 1.7 µm

Column temp.: 65 °C

Flow rate: 800 µL/min

Mobile phase A: 20 mM Ammonium bicarbonate pH 10

Mobile phase B: Acetonitrile

Gradient: 15% to 95% B/18 min

36

MS system: Waters Xevo TQ MS

Ionization mode: ESI positive

Capillary voltage: 30 V

Collision energy: 15 eV

RESULTS

The unique collision cell design allows the Xevo TQ MS to be oper-

ated in several different modes of operation: full scan MS, MRM,

as well as MS/MS mode. As the collision cell is continuously filled

with collision gas, the instrument can rapidly switch between MS

and MS/MS in the same analytical run. This allows MRM and MS

scans to be performed in the same run. Combined with the high scan

rate, this allows for rapid survey scans to be performed, such as MS

neutral loss or parent ion, before switching to MS/MS.

This high data-capture rate allows for the accurate definition of the

peak, even with the very narrow peaks produced by UPLC. Figure 2

shows the UPLC/MS chromatogram produced in the analysis of an

API batch of quetiapine at a concentration of 1 µg/mL. Here we can

see that impurity peaks are 2 to 4 seconds wide at the base. The

data shown in Figure 3 illustrates the number of scans achieved in

MS and MS/MS modes.

Figure 2. UPLC/MS analysis of quetiapine at 1 µg/mL.

Maximizing LC peak definition

In this example, the Xevo TQ MS was operated in ScanWave MS

mode, switching to ScanWave DS (daughter ion scan) mode when a

peak was detected above a user-defined threshold. In this mode of

operation, the instrument selects the most intense peak in the MS

spectrum and acquires MS/MS data on this peak before returning to

MS mode. Since the collision cell is continuous filled with collision

gas, there is a no delay in switching between MS and MS/MS modes.

Figure 3. Rapid data collection is performed simultaneously in both MS and MS/MS modes.

We can see from this data that the instrument has acquired 9 points

across the peak in MS mode, and 15 points across the peak in

MS/MS mode – despite the fact that the peak is only 2 seconds wide

at the base. This high data-capture rate enables the Xevo TQ MS to

perform high quality, data-dependent MS-to-MS/MS experiments in

a UPLC timeframe with sufficient data points to accurately define

the peak. This dual mode of operation can also be used to acquire

full-scan MS data simultaneously with MRM data, or to detect a peak

with precursor ion scanning before switching to MS/MS mode.

Time5.00 10.00 15.00 20.00

%

0

1009.81

9.240.29

8.375.65

17.26

10.90

17.10

15.79

13.88

17.50

17.66

18.07

19.38

Scan2870 2880 2890 2900 2910

%0

100 17.58

17.49

340 360 380 400 420 440 460 4800

%

100

Scan15.61

16.6317.23

17.78

17.57

MS MS/MS

37

Precursor ion scanning

The detection of new impurities, degradation products, or, in a DMPK

study, drug metabolites, is often confounded by the signal from the

matrix. To detect and visualize these analytes, the analytical chem-

ist can use the specificity of the mass spectrometer.

Since compounds can undergo fragmentation as a result of the

degradation or metabolism process, the use of simple, predicted MRM

transitions for common degradation/metabolism pathways may result in

the non-detection of a potentially toxic impurity, degradation product,

or metabolite. A more comprehensive way to detect these compounds

is to monitor for the common fragment ions of the molecule of interest.

The Xevo TQ MS’s Survey Scan functionality utilizes the fast data-

capture rate of the instrument to facilitate the collection of precursor

ion data as well an MS/MS spectrum of the peaks detected.

This functionality was used to evaluate a commercially-purchased API

sample of quetiapine. The MS/MS spectra of quetiapine revealed that

it gave rise to three major product ions having m/z values 221, 253,

and 279. This data was used to detect drug-related impurities in the

API batch by performing a Survey Scan analysis on each of these ions.

Figure 4. Survey Scan UPLC/MS analysis of quetiapine for ion m/z 279 in positive ion mode.

The data collected for the parent ion chromatogram of m/z 279 is

displayed in Figure 4. Here, we can see the presence of seven major

peaks, six impurities and the quetiapine active peak, eluting with a

retention time of 9.86 minutes. A similar analysis using the com-

mon fragment ion m/z 221 to trigger data collection produced the

chromatogram shown in Figure 5. With this fragment ion, a total of

12 peaks were detected and MS/MS data acquired.

Figure 5. Survey Scan UPLC/MS analysis of quetiapine for ion m/z 221 in positive ion mode.

The MS/MS spectra obtained from the peak eluting with a retention time

of 5.6 minutes is displayed in Figure 6. This impurity has a m/z value of

400 amu and has been identified as the S-Oxide impurity of quetiapine.

Figure 6. AutoScanWave MS/MS spectrum of S-Oxide of quetiapine eluting at 5.6 minutes.

Time2.50 5.00 7.50 10.00 12.50 15.00 17.50

%

0

100 9.86

9.29

5.69

8.42

6.46

10.94

15.1017.63

17.91

Time2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00

%

0

100 8.398.38

5.64

8.40

9.25

17.15

9.84

9.89

9.90

10.12

13.9910.72

12.2817.25

18.28

m/z100 150 200 250 300 350 400

%

0

100 221

221

196

176

158

211247

222

239

279

253

38

ScanWave technology

The detection of low-level impurities is becoming increasingly

important, especially when monitoring potential genotoxins.

Collection of the MS/MS spectrum from Survey Scan experiments,

either precursor ion or common neutral loss, can be performed in

two modes of operation: standard MS/MS or ScanWave MS/MS.

As described previously, ScanWave technology allows for

increased sensitivity in the collection of MS/MS data. This increase

in sensitivity is illustrated by the MS spectra obtained for the

desthanol impurity of quetiapine (Figure 7). The top spectrum is

obtained in standard MS/MS mode, while the lower spectrum is

obtained in ScanWave MS/MS mode.

In this example, we can see that the ScanWave MS/MS data is

13 times more sensitive than that in standard MS/MS mode. This

increase is essential for the correct confirmation or identification

of low-level impurities.

Figure 7. Comparison of standard and ScanWave MS/MS sensitivity.

CONCLUSION

n The Xevo TQ MS provides unrivaled levels of sensitivity

and functionality.

n The high data-capture rates of the instrument, and its unique

collision cell design and ScanWave technology, allows the

maximum amount of data to be collected in one analytical run.

n This reduces the number of experiments needed to make a deci-

sion, allowing impurities to be detected and identified quicker,

and making maximum use of instrumentation.

n The rapid switching between MS and MS/MS possible with the

Xevo TQ MS allows the collection of qualitative data and quanti-

tative data in the same analytical run.

n The instrument’s high data-capture rate ensures that, even with

the narrow peaks of 2 to 3 seconds produced by today’s modern

sub-2 µm particle LC systems, sufficient points can be collected

across for accurate quantification.

n The use of ScanWave technology ensures that even the lowest-

level peaks are detected and MS/MS spectra acquired, ensuring

comprehensive impurity detection.

Waters, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2008 Waters Corporation. Printed in the U.S.A.October 2008 720002831EN LB-CP

m/z100 125 150 175 200 225 250 275 300 325 350

%

0

100

%

0

100

2: Auto Daughters 340.72ES+ 9.07e5279

221

161177

253

2: Auto ScanWave DS 340.39ES+ 1.21e7221

210206168

253

247228 279

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

PUBS:

AAPSAir, Water & Environment InternationalEuropean BioPharmaceutical ReviewEuropean Pharmaceutical ReviewDrug Discovery WorldRapid Communications in Mass SpectrometryTouch BriefingsWell Characterized BioPharmaceuticalFood Safety EuropeNext Generation PharmaceuticalSpectroscopy Asia

Job # & CC: WATR9752_B

Job Name: WATR9752_B_XevoNew_DBmec.indd Date: 06-13-08Live: 7”x10” Smallest Trim: 8.25”x10.875” Largest Trim: 8.5”x11.75” Bleed: 8.875”x12 Page: 1 Rev: 1Stage: mech Release: 06-13-08 DB AD: MM CW: GK PM: DG Scale: 100%Color: 4c Gutter: — Other: ST: DB

APP

RO

VALS C-LO

MECH PROOF

MK: ______________________ PM: ______________________ AB: ______________________

AD: ______________________ CW: ______________________ RM:______________________

QC: ______________________ CD: ______________________

Initials / Date Initials / Date Initials / Date

Client / Date: ________________________________________________

©2008 Waters Corporation. Waters, The Science of What's Possible, XEVO, IntelliStart, and ACQUITY UPLC are trademarks of Waters Corporation.

[ REACTION ]

Introducing XEVOTM TQ MS, the new tandem quadrupole mass spectrometer from Waters, featuring IntelliStartTM technology for simplified method development. This automated system solution delivers powerful quantitative capability with very low limits of detection —to the widest range of scientists. All this, along with ACQUITY UPLC® compatibility by design, means you get enhanced performance with dramatically improved speed, productivity, and efficiency. Run more samples per day and more compounds in a single analysis. And make critical, time-sensitive decisions with confidence. Find your answer at www.waters.com/xevo

WHEN ANALYZING COMPLEXSAMPLES WITH XEVO TQ MS,WILL YOU BE MORE IMPRESSEDWITH HOW POWERFUL IT IS?

OR HOW ACCESSIBLE IT IS?

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

Waters, ACQUITY UltraPerformance LC, ACQUITY UPLC, UltraPerformance LC, and UPLC are registered trademarks of Waters Corporation. Xevo, Scan-Wave, T-Wave, SYNAPT, Quattro Premier, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2008 Waters Corporation. Printed in the U.S.A.November 2008 720002826EN LB-CP

www.waters.com/xevo