A NOVEL DUAL ELECTROSPRAY ION SOURCEINTERFACED TO AN ORTHOGONAL ACCELERATION-

TIME-OF-FLIGHT MASS SPECTROMETER COUPLED WITHLIQUID CHROMATOGRAPHY FOR EXACT MASS

MEASUREMENT OF SIX SYNTHETIC POLYMER ADDITIVES

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1Jonathan P.Williams, 1Kevin Giles, 1Ashley B. Sage, 2Terence Hunt, 2Anthony T. Jackson and 2James H. Scrivens1Micromass UK Ltd, Floats Road, Wythenshawe, Manchester, UK.

2ICI Research and Technology Centre, PO Box 90, Wilton, Middlesbrough, Cleveland, TS90 8JE

Abstract

A dual electrospray ion source interfaced to anorthogonal acceleration-time-of-flight mass spectrometer(oa-TOF-MS) is described. The novel design has beenused to analyse a six component synthetic polymeradditive mixture by liquid chromatography-massspectrometry for exact mass measurement confirmation.The interface provides a simpler, more robust method ofobtaining exact mass measurements through infusion of areference compound into a separate electrospray inletrather than post column addition presently used with asingle electrospray probe. The results show negligible orno inter-channel 'cross talk' and exact mass measurementsof < 5 ppm.

Introduction

Polymer additives have several key functions. They aidprocessing and finally characterize the formulatedcommercial product within the field of polymer chemistry.Typical additives act, for example, as light stabilizers,antioxidants, flame retardants or plasticizers.

Polymer additive analysis is important because ofpossible health and environmental risksthat can arise from the manufactureof certain plastic materials,which are intended tocome into contact, forexample, withfoodstuffs1.

The possibility of migration of the additives into foodstuffsneeds to be carefully monitored and therefore the levels ofadditives in the plastic need to be determined and wellregulated.

Often added at levels between 0.1-3% w/w, thedetermination of the fate of these additives during andafter use, their presence in competitor products andinherent purity presents a daunting analytical challenge.Therefore, precise identification is a crucial requirement intheir characterization. Synthetic polymer formulations arecomplex, therefore extraction procedures from thepolymer matrix usually precede chromatographicseparation.

Electrospray mass spectrometry and tandem massspectrometry have been previously employed on some ofthe additives studied2. Here high and low collisionenergies were used to produce nominal mass product ionspectra. From the results mechanistic and fragmentationpathways were proposed.

For this study, oa-TOF-MS has been convenientlyused with liquid chromatography

for the exact massmeasurement of the

same group ofadditivespreviouslyinvestigated2.

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Exact mass assignment by liquid chromatography oa-TOF-MS is presently achieved through post-columnaddition of a suitable reference material into thesolvent flow prior to entry into the ionization region ofthe mass spectrometer. The reference material isadded solely to be used as a single point 'lock mass'against which any subsequently acquired massspectra are mass measured.

However, post-column addition of a suitable referenceinto the solvent flow does have some practicallimitations that have to be manually addressed. Theseinclude reference signal intensity fluctuations withliquid chromatography gradients, the possibility ofmass interference with analyte material having thesame nominal mass and ion suppression of thereference compound with high concentrations ofanalyte response. These practical limitations havebeen overcome by the design of a novel dualelectrospray ionization source that samples both theanalyte and reference streams independently.

The ion source was interfaced to an orthogonal-acceleration time-of-flight mass spectrometer (oa-TOF-MS) and the design and functionality of the source isdescribed here. Evaluation of the dual spray sourcefor exact mass measurement was performed by LC-MS on a six component synthetic polymer additivemixture.

Together with the novelty of the design, exact massmeasurement of synthetic polymer additives does notappear to be cited in the literature and therefore havebeen investigated for a variety of such compounds ofmasses < 1500 Da.

Experimental

Samples. Pure synthetic polymer additives were used.The six polymer additives investigated consisted ofantioxidants and light stabilizers. Their structures andrelative molecular masses are shown in Figure 1. Theadditives were obtained from ICI (Wilton,UK) andused as received. 1 mg/mL stock solutions of eachadditive were prepared in (1:4,

dichloromethane:methanol). A solution containing theadditive mixture was prepared to a concentration of10 ng/µL of each component. All solvents used wereHPLC grade (Sigma-Aldrich, Poole, UK) and usedwithout further purification.

Cl

N

N

N

H O

H OCO C 18H 37

O

C

O

N N

C CN OO

R

R

R

R CH 2 O H

CH 3C CH 2 CO CH 2

O

O H

O H

2

TIN U V IN 327(M r 357)

IRG A N O X 1076(M r 530)

IRG A N O X 3114(M r 783)

=

H O STA N O X 03(M r 794)

CO

C

H O

O

4

IRG A N O X 1010(M r 1176)

O P

3

IRG A FO S 168(M r 647)

Figure 1. Structure of the six synthetic polymeradditives

Liquid chromatography

A Waters 2790 AllianceHT system (Waters Corp., Milford, MA,USA) provided the liquid flow at a rate of 300 µL/min to themass spectrometer. The liquid chromatographic separation wasperformed on a Waters Symmetry C8, 2.1 x 100 mm, 3.5 µmcolumn. A 10 µL injection of the solution containing the additivemixture was injected on-column. The following gradient profile,as shown in Table 1, was used for the separation:

Mobile phase A: H2O + 0.1% HCOOHMobile phase B: CH3OH + 0.1% HCOOH

After elution the liquid stream exiting the column was introducedinto the analyte channel of the dual electrospray source. SeeFigure 2.

Mass Spectrometry

Mass spectra were acquired on a LCTTM oa-TOF massspectrometer (Micromass UK Ltd, Manchester) which was fittedwith a novel two way electrospray ionization source(LockSprayTM), see Figure 2. The dual source housing fits directlyaround the Z SPRAYTM source of the LCT with the referencesprayer positioned perpendicular to the analyte sprayer. Nodesolvation gas is required for the reference sprayer since lowflow rates, typically < 10 µL/min, are used. The same ionizationvoltage is applied to both the reference and analyte spray tipswith an equal flow of nebulization gas assisting the liquid flowof each spray.

The spray from each capillary tip is sampled independentlythrough use of a rotating baffle, which is externally driven by astepper motor. The sprays are admitted to the sampling coneregion of the LCT through an aperture in the rotating baffle. Thetwo positions of the sampling rotor are indexed which allowsdata from each of the two sprays to be stored into separatefuctions within a single data file during an acquisition.

During the investigation the reference spray was sampled everyfive seconds. Mass spectra were collected in positiveelectrospray mode and acquired from 200-1250 Da at anacquisition rate of 1 spectrum/s with an inter-acquisition delayof 0.1s. The electrospray voltage was 3 kV and cone voltagesof 34V and 20V were applied to the analyte and referencematerials respectively during an acquisition.

Exact mass measurement was provided by infusing leucineenkephalin ([M+H]+= 556.2771) into the reference channel ofthe dual source. This 'lock mass' was infused at a flow rate of 5µL/min using a Harvard Apparatus (South Natick, MA,USA)Model 22 Syringe Pump at a concentration of 0.5 ng/µL (1:1,acetonitrile:H

2O).

Three replicate LC-MS acquisitions were made to obtain thedata. Data acquisition and processing was carried out usingMassLynxTM (V3.5).

Table 1. Gradient Profile

time(mins) A(%) B(%)

0 10 90

1 10 90

11 0 100

15 0 100

15.1 10 90

20 10 90

Figure 2. Schematic of the LockSpray setup

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Results

During an acquisition, ions from the two sprays are sampled independently and are stored in separate functionsin a single data file. Figure 3 shows the total ion chromatograms obtained from an acquisition. The bottom tracewas obtained from the analyte channel and the top trace from the reference channel.

Figure 4 shows spectra obtained from the analyte (bottom trace) and reference (top trace) data files during theelution of the peak at 1.82 minutes. These spectra clearly show that cross-talk between the two channels isnegligible. Fragment ions observed at m/z 645 and m/z 495 are consistent with previous investigations.

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 14.00Time0

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 14.00Time0

100

%

0

100

%

0

100

%

0.660.48

2.041.851.30 10.206.442.68 2.95

5.805.524.884.143.23 8.918.55

6.99 8.00 9.109.83

13.5913.1312.67

10.93 11.3011.94

14.33 14.78

0.660.48

2.041.851.30 10.206.442.68 2.95

5.805.524.884.143.23 8.918.55

6.99 8.00 9.109.83

13.5913.1312.67

10.93 11.3011.94

14.33 14.78

1.80

1.500.70

4.66

4.16

10.36

4.86

8.07

12.53

Figure 3. Total ion chromatograms obtained from reference channel (top) and analytechannel (bottom)

360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840m/z0

100

%

0

100

%

556.3

557.3

578.2

817.5

645.4

495.2812.5

646.4

818.5

819.5

Figure 4. Mass spectra obtained from each channel at the same point in time

The mass chromatograms obtained fromthe separation of the six componentmixture are shown in Figure 5. All of theadditives are well resolved with theexception of Irganox 3114 and Tinuvin327 where there is some degree of co-elution. This however posed no problem forexact mass measurement since they arewell resolved by their individual masses.

Typical background subtracted massspectra achieved are shown in Figure 6.Fragment ions observed with Irganox1076 from the protonated molecule areconsistent with previous investigations2.

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 16.00Time0

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 16.00Time0

100

%

0

100

%

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

12.51

1.83

12.51

1.83

10.3610.36

8.118.11

4.864.86

4.644.64

1.821.82

1.80

1.500.70

4.66

4.16

10.364.86

8.07

12.53

Hostanox 03

Irganox 3114

Tinuvin 327

TIC

Irganox 1010

Irganox 1076

Irgafos 168

Figure 5. Mass chromatograms showing polymer additive separation

300 400 500 600 700 800 900 1000 1100 1200m/z0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

12001201

1202

817

818

806

808

647

648

553

475554

358

360Tinuvin 327

Irganox 1076

Irgafos 168

Irganox 3114

Hostanox 03

Irganox 1010

picture caption - futura book oblique Size 9pt, leading 14pt

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Figure 7 shows an example of a 'lock mass' corrected exact mass spectrum achieved for Irgafos 168.

For exact mass measurement of each additive, five spectra from the reference data file were combined toproduce an averaged spectrum at the same point in time as the analyte data to be mass measured. The exactmass data obtained are shown in Table 2. The results gave mass errors < 5ppm from the theoretical mass,which would allow sufficient accuracy for assignment of an elemental formula.

200 300 400 500 600 700 800 900 1000 1100 1200m/z0

100

%

647.4618

648.4659

Figure 7. Exact mass spectrum obtained from Irgafos 168

Table 2. Exact mass measurement results for the polymer additives

Additive Mass measured Theoretical mass Deviation Deviation(mDa) (ppm)

Tinuvin 327 358.1692 0.6 1.7358.1679 -0.7 -2.0358.1698 1.2 3.4

Mean 358.1690 [M+H]+ 358.1686 0.4 1.1RMS 0.9 2.4

Irganox 1076 553.4589 -0.8 -1.4553.4577 -2.0 -3.6553.4581 -1.6 -2.9

Mean 553.4582 [M+Na]+ 553.4597 -1.5 -2.7RMS 1.5 2.7

Irgafos 168 647.4618 2.5 3.9647.4603 1.0 1.5647.4614 2.1 3.2

Mean 647.4612 [M+H]+ 647.4593 1.9 2.9RMS 2.0 3.0

Irganox 3114 806.5111 2.7 3.3806.5093 0.9 1.1806.5114 3.0 3.7

Mean 806.5106 [M+Na]+ 806.5084 2.2 2.7RMS 2.4 3.0

Hostanox 03 817.4637 -1.8 -2.2817.4633 -2.2 -2.7817.4685 3.0 3.7

Mean 817.4652 [M+Na]+ 817.4655 -0.3 -0.4RMS 2.4 2.9

Irganox 1010 1199.7760 2.2 1.81199.7753 1.5 1.31199.7799 6.1 5.1

Mean 1199.7771 [M+Na]+ 1199.7738 3.3 2.8RMS 3.8 3.2

Conclusions

The dual electrospray interface has been successfully interfacedto the LCT. The exact mass measurement results for six syntheticpolymer additives have been obtained with errors < 5 ppm,therefore allowing confirmation of empirical formulae. Thedesign allows exact mass measurements to be made moresimply and robustly than with post-column addition of thereference compound, principally through elimination ofsuppression effects, possible mass interference and LC gradienteffects which are sometimes inherent with post column addition.

References

1. JP Williams, Ph.D. Thesis, University of Wales Swansea, UK(2000).

2. AT Jackson, A Buzy, KR Jennings, JH Scrivens, Eur.MassSpectrom., 2, (1996) 115.

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