Objective

The aim of the study presented is to illustrate the enhanced performance of the Waters® Micromass®

LCT Premier™ oa-TOF Mass Spectrometer. The data presented shows mass accuracy achieved over a twelve-hour period. Using negative ion mode electrospray, very good mass accuracy is achieved routinely with real time centroid data acquisition and lockmass correction. Acquisition of UV data in parallel is performed. The system used is comprised of a Waters Alliance® HT 2795 Separations Module, 2996 PDA Detector, Symmetry®

C18 Column and Micromass® LCT Premier™ oa-TOF Mass Spectrometer.

Introduction

The LCT Premier is a newly developed benchtop oa-TOF (orthogonal acceleration time of flight) mass spectrometer. New hardware and software control technology has been incorporated to meet the increased analytical demands in the pharmaceutical, environmental and clinical applications arenas. This study utilizes a plant extract at concentrations of 100 ng/µL. The extract contains a plethora of major and minor components of which exact mass measurement can be performed in one analysis. The high duty cycle of TOF is utilized for qualitative studies, generating full spectra at high mass accuracy (<3ppm RMS). The highly specific data generated provides an extra degree of information that aids interpretation of the data. Using real time exact mass centroid data acquisition, the evaluation of electrospray negative ion mode performance of the LCT Premier has been performed. The LCT Premier oa-TOF is presented in Figure 1 and the schematic of the LCT Premier with the analyzer in V geometry is illustrated in Figure 2.

M. McCullaghWaters Corporation, MS Technology Centre, Manchester, M23 9LZ

Figure 1. LCT Premier incorporating integral LockSprayFigure 1. LCT Premier incorporating integral LockSpray™™.

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EVALUATION OF LCT PREMIER™

NEGATIVE ION MODE ESI EXACT MASS MEASUREMENT PERFORMANCE

Experimental

Mass Spectrometer: Micromass LCT Premier oa-TOF

HPLC: Alliance HT 2795 Separations Module

HPLC

Column: Waters Symmetry C18

(250 mm x 4.6 mm, 5 µm particles) with guard column

(2 mm x 3.9 mm, 5 µm particles)

Column temperature: 35 ˚C

Flow: 1 mL/min - split 1:4

Mobile phase: A: H2O (0.2% HCOOH), B: MeCN

Gradient: 0-10 min: 15% B; 10-40 min: 15-30% B; 40-50 min: 30-15% B

MS

Ionization mode: ESI Voltage -ve = 2.7kV

Sample cone voltage: 100V

Reference mass: Leucine enkephalin, [M-H]=554.2615

Acquisition parameters: 100 - 1000 Da

1 spectrum/second

5500 FWHM

0.1 second inter scan delay

Figure 2. Oa-TOF schematic (V-mode > 5000 FWHM).

Figure 3. Schematic of the LCT Premier LockSpray electrospray source.

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Results and Discussion

Two plant extracts (Passiflora caerulea and edulis)were selected for analysis to evaluate the LCT Premier negative mode exact mass measurement performance. The BPI chromatograms obtained for the negative mode analysis of the two species are shown in Figure 4, where Passiflora edulis (A) and Passiflora caerulea (B) are presented. Visually comparing the two chromatograms, the difference in the profile of the two species is evident. This highlights the advantage of oa-TOF technology, where good sensitivity can be obtained with full exact mass spectral acquisition. A plethora of major and minor components have been detected in both extracts, and acquiring full spectra has ensured that a maximum amount of specific information can be obtained.

The data was acquired in centroid mode and mass corrected in real time. The reference mass Leucine enkephalin was sampled independently using the integral LockSpray source.

The reference compound Leucine enkephalin ionizes in negative mode to produce ([M-H]-=554.2615), and is sampled independently from the analyte spray to provide a lockmass. As shown in Figures 5 and 6, it is not seen in the analyte mass spectrum. Lockmass correction takes place automatically in real time and the independent sampling enhances the mass accuracy obtained. Both plant extracts were consecutively injected six times, each analysis time taking sixty minutes. This allowed the negative ion mode performance of the LCT Premier to be evaluated over a period of twelve hours. In Figure 5, examples of the exact mass spectra acquired over the twelve-hour period are illustrated for isoorientin. The manually selected spectra illustrate data where exact mass measurement has been achieved within –0.1 mDa to 0.2 mDa. In Figure 6, data acquired for orientin in the two plant extracts is shown. Example exact mass spectra acquired over the twelve-hour period are illustrated where exact mass measurement has been achieved within –0.2 mDa to 0.2 mDa range.

PASSIFLORA EDULIS 100NG/ML

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

%

0

100

%

0

100

PREMPASS300903_070 1: TOF MS ES-

PREMPASS300903_053 1: TOF MS ES-

A

B

Figure 4. Negative mode BPI chromatograms, acquired for analysis of a 100 ng/µL plant extract of Passiflora edulis (A) and caerulea (B).

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PASSIFLORA CAERULEA 100NG/ML

m/z100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

PREMPASS300903_074 1: TOF MS ES- 447.0927

PREMPASS300903_072 1: TOF MS ES- 447.0927

PREMPASS300903_070 1: TOF MS ES- 447.0927

PREMPASS300903_058 1: TOF MS ES- 447.0927

PREMPASS300903_057 1: TOF MS ES- 447.0929

PREMPASS300903_056 1: TOF MS ES- 447.0929

PREMPASS300903_055 1: TOF MS ES- 447.0926

174.9562

PASSIFLORA EDULIS 100NG/ML

m/z100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800

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100

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PREMPASS300903_073 1: TOF MS ES- 493.0982

PREMPASS300903_071 521 1: TOF MS ES- 493.0984

PREMPASS300903_070 1: TOF MS ES- 493.0980

PREMPASS300903_058 1: TOF MS ES- 493.0984

PREMPASS300903_057 1: TOF MS ES- 493.0978

PREMPASS300903_055 1: TOF MS ES- 493.0983

PREMPASS300903_054 1: TOF MS ES- 493.0982

Figure 5. Examples of negative mode exact mass spectrum of isoorientin acquired for the repeat analysis of Passiflora caerulea and edulis isoorientin over a period of twelve hours.

Figure 6. Examples of negative mode exact mass spectrum of orientin acquired for the repeat analysis of Passiflora caerulea and edulis over a period of twelve hours.

Table1: Exact mass measurement obtained over a twelve-hour period for isoorientin determined to be present in the plant extracts of Passiflora caerulea and edulis.

Table 2: Exact mass measurement obtained over a twelve-hour period for orientin determined to be present in the plant extracts of Passiflora caerulea and edulis.

1.82RMS PPMERROR

-3.1-1.4447.0914447.092772012

-0.2-0.1447.0926447.092766011

-3.1-1.4447.0914447.092760010

0.80.4447.0931447.09275409

1.10.5447.0932447.09274808

1.20.6447.0933447.09274207

-2.1-0.9447.0918447.09273606

10.4447.0931447.09273005

2.71.2447.0939447.09272404

-0.9-0.4447.0923447.09271803

-0.7-0.3447.0924447.09271202

2.11447.0937447.0927601

Error ppmError mDaIsoorientin[M-H]

MeasuredMass

Isoorientin[M-H]

CalculatedMass

Total AnalysisTime Mins

AnalysisNumber

1.95RMS PPMERROR

-2.2-1.1493.0900493.098272012

-1.7-0.8493.0974493.098266011

0a.80.4493.0986493.098260010

-3.0-1.5493.0967493.09825409

2.51.2493.0994493.09824808

-1.5-0.7493.0975493.09824207

-1.3-0.6493.0976493.09823606

-0.6-0.3493.0979493.09823005

-2.7-1.4493.0969493.09822404

0.00.0493.0982493.09821803

-2.7-1.4493.0969493.09821202

-1.8-0.9493.0973493.0982601

Error ppmError mDaOrientin[M-H+aCH2O2]

MeasuredMass

Orientin[M-H+CH2O2]

CalculatedMass

Total AnalysisTime Mins

AnalysisNumber

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Table 3: Exact mass measurement obtained over a twelve-hour period for isovitexin determined to be present in the plant extracts of Passiflora caerulea and edulis.

2.7RMS PPMERROR

-4.1-1.8431.0961431.097872012

-1.8-0.8431.0970431.097866011

-4.7-2.0431.0958431.097860010

-0.2-0.1431.0977431.09785409

-1.8-0.8431.0970431.09784808

00431.0978431.09784207

-2.81.2431.0966431.09783606

2.10.9431.0987431.09783005

-2.3-1.0431.0968431.09782404

-3.6-1.6431.0963431.09781803

3.41.5431.0993431.09781202

0.20.1431.0979431.0978601

Error ppmError mDaIsovitexin[M-H]

MeasuredMass

Isovitexin[M-H]

CalculatedMass

Total AnalysisTime Mins

AnalysisNumber

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Tables 1, 2 and 3 show the masses measured, ppm errors and RMS ppm errors for isoorientin, orientin, and isovitexin respectively. The data processing was performed automatically using OpenLynxTM. RMS ppm errors of less than 3 ppm were obtained in negative ion mode during the analysis period.

Conclusion

• Using plant extract samples, the flavonoids isoorientin, orientin, and isovitexin have been successfully detected over a period of twelve hours.

• Analysis has been performed in centroid acquisition mode and exact mass measurement performed in real time.

• 1.83 RMS ppm error obtained for negative mode acquisition of isoorientin.

• 1.95 RMS ppm error obtained for negative mode acquisition of orientin.

• 2.70 RMS ppm error obtained for negative mode acquisition of isovitexin.

• The LCT Premier oa-TOF with integral LockSpray and independent reference mass acquisition enables the routine acquisition of highly specific data.

• Increased confidence in analyte confirmation is achieved using exact mass measurement.

Waters, LCT Premier, Alliance, Symmetry, LockSpray and OpenLynx are trademarks of Waters Corporation.Trademarks are the property of their respective owners.©2004 Waters Corporation Produced in the U.S.A. November 2004 720000979EN MC-PDF

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