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.

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

CH 3

H

C H 3

F

F

H

C H 3

OS

F

O

C H 3

A100

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