i: Development of 9,-a=Mass SpectrometryCompatible LiquidChromatographyMobile Phases

0 • • • • • • • • • • • • • • • • • • •

Michael P. Balogh, Joseph P. Romano, Matthew J. Dilts,Jennifer M. Duker

Waters Chromatography, 34 Maple Street, Milford, MA 01757

,_ .' 2

'; introduction

Liquid chromatography coupled with mass spectrometry(LC/MS) provides.a powerful tool for compound andidentification studies. Unfortunately many commonly usedHPLC mobile phases are not compatible with massspectrometry. Modifiers such as phosphate buffers andhexane sulfonic acid were originally chosen bychromatographers strictly for their chromatographic benefits.Experience indicates more volatile buffers are required tomaintain performance of the mass spectrometer(l_3).Modifiers can be chosen, as presented in this work, thatpreserve the chromatographic benefits and develop amethod amenable to LC/MS use.

-,' 3

" ' Background• • • • • • • • • • • • ,0 • • • • • • • • • •

[] Chromatographers use vanous criteria whenchoosing materials compatible with theirseparation needs:- Extinction coefficient

- Solvent wavelength cutoff- Buffer capacity- Solubility- Selectivity

[] Mass spectrometrists have concerns forintroduction of a sample into the source of theinstrument:-Maximizing sensitivity performance-Background ions present in the spectra-Heat impact on El traceable spectra

[] Convert an established method from a non-volatilebuffer to one compatible with LC/MS.

[]Maintain chromatographic resolution and widerange of selectivity with near 100% aqueous tohigh organic gradient conditions.

[]Deliver good quality electron-impact (El)librarysearchable mass spectra.

5

Methods and Materials• • • • • • • • • • • • • • • • 0 • • • • • •

[]A method for determining chlorinated phenoxyacids in water by high performance liquidchromatography using a photodiode arraydetector, proposed by the US EPAenvironmental monitoring systems laboratory,is used to illustrate a method of adaptingexisting separations to LC/MS use(4)

"' 5

I'nstrumentation

• Waters Integrity LC/MS System controlled via Millennium 2010 ChromatographyManager Software- Model 717 plus Autosampler-Model 616 Low Dispersion HPLC Pump-Model 996 Photodiode Array UV Detector-Thermabeam Mass Detector

• HPLC Conditions

- 100 mM Ammonium Acetate: ACN (pH 6.9)-0.5% TEA:ACN (pH 7.3)

-95% aqueous held until 2 minutes followed by a linear gradient to 60% organicat 10 minutes.

-Flow rate - 0.250 ml/min

- 2mm x 15cm NovaPAK C18 Reverse-phase column• Acquisition Parameters

-Photodiode array- 210-310 nm at 1.2 nm resolution and 1 spectra/sec.-Mass spectrometer- El source 250 C with acquisition from 42 - 260 amu at 1

spectra/sec.

4

• 7

Experimental

[] Separations such as those presented here may simplyrequire substituting the salt of a weak acid and weak base

as a direct replacement of the original less volatile modifier.Using the Henderson-Hasselbalch equation buffercapacities can be determined based on pH.

- Additionally, the effect of the mobile phase choice on theresulting spectra and the effectiveness of library matchingusing the Palisades Probability-based Matching algorithmmust be considered.

[] Some attempts to develop methods for LC/MSdetermination (5)ofphenoxy acid herbicides have beenfound to yield poor chromatographic resolution and poorspectral quality among other problems.

• °

Buffer Capacity as a Function of pH(A.L.L Duchateau_ et. al., JChrom., 552, 1991)

30butter cal3ac=ty

13(in mM) C

18 B

12 A

1

oH..... i I I

2 3 4 5 6 7

(A) 0.05 M ammonium formate solution; (B) 0.05 M ammoniumacetate solution; (C) 0.05 M ammonium bicarbonate solution, pHadjustments done with acetic acid.

' 8

Original Method• • • • • • • • • • • • • • • • • • • • • • •

• Eight chlorinated phenoxy acid herbicides (including 5-hydroxydicamba)• Dual column (primary column and confirmation column)

• 0.025 M phosphate and acetonitrile gradient - 40 minutes plus 10-20minutes to re-establish initial conditions.

• Photodiode array only - no mass spectrometry.

= N

' I lli...._- ;_le _ oo _.''"o.,. _;°.,o 2,., o.,, _ o- _..'oa ................"

FI4;IIlilLIF e. _. 'rYI'II.-AI IIl'l (: t:llX(pplj_l¢'l(;B/_l ()lr • ¢.:IF_I)IIP A _NAI.YlI:f;

9

Conversion Method A• • • • O. • • • • • • • • • • • • • • • • • •

[] lOOmMammonium acetate:acetonitrilegradient[] Single 2mm NovaPAK C18 column

[]Photodiode array and particle beam mass spectrometer in-line.- Method A and Method B provided good chromatographic resolution and reduce

run time from 40 minutes to 15 minutes.

." 10

Conversion Method B- 0.5% triethylamine:acetonitrilegradient(same profileas MethodA)[]Single2ram NovaPAKC18 column- Photodiodearrayand particlebeammassspectrometerin-line.• MethodA and MethodB providedgoodchromatographicresolution

and reducedruntime from40 minutesto 15 minutes.

•' , 11

Results• • • • ,0 • • • 0 • • • • • • • • • • • • • 0

[] Examination of the mass spectra from each run shows apreference for Method A due to the reduced backgroundinterference and the enhanced library match capability.

_File Edit.. _Plot..Derived.. Library Help...................

i ii

12

Results

_File:_Edit _Plot.._Derived.Library Help..............:

: z .... 13

.....'Results

[] Photodiode array was used to examine thestandards for homogeneity which can be used tocorroborate ions found in the mass spectra thatdo not appear to be coherent with the compoundfound.

Results• • • 0 • 0 • • • 0 • • • • • • • • • • • • •

[] Mass spectrometry was used for positivecompound identification since the change inchemistry resulted in the two hydrophilic peaks(Picloram and Chloramben)eluting in reverseorder.

b._,.-_

, 15

Conclusion

[] The addition of mass spectrometry to proposedEPA Method 555 for the determination ofchlorinated phenoxy acid herbicides in waterachieves:-Simplification by reducing the system from two columns

to a single analytical column.

-The same level of specificity as a GC/MS method byusing LC/MS to produce El library-searchable spectra.

- Shortened run times and higher throughput.at lowernarrow-bore flow rates (40 minutes acqulsltnonto 15minutes),.

• _1_._'_"

':-"References,• • • • • • • • • • • • • • • • • • • • • • •

• Selection of Buffers and of an Ion-pairing Agent for Thermospray Liquid Chromatography-MassSpectrometricAnalysis of IonicCompounds,Duchateau, A. L., et. al., J Chmm., 1991,552, 605-612.

• Detection of Alkaloidsby Positive IonThermospray LC/MS Usinga NonconventionalIon-PairingAgent,Mesmer, M. Z., et. al., 41st ASMS Conference on Mass Spectrometry andAIlied Topics,San Francisco,CA, May, 1993.

• Determinationof AminoglycosideAntibioticsby Reversed-phase Ion-pair High-performanceChromatographyCoupled with PulsedAmperomelryand Ion Spray Mass spectrometry,McLaughlin,L. G.and Henion,J. D., J Chrom., 1992, 591,195-206.

• Method 555. Determination of ChlorinatedAcidsin Water by High Performance LiquidChromatographywith a PhotodiodeArray UltravioletDetector,Revision 1.0, August 1992, Eichelberger,J. W. and Bashe, W.J., EnvironmentalMonitoringSystemsLaboratory,Office of Research and Development,US EPA,Cincinnati, OH.

• Determination of Chlorinated Phenoxy Acidand Ester Herbicides in Soil and Water by LiquidChromatographyParticleBeam Mass Spectrometryand UltravioletAbsorptionSpectrophotometry,Kim, I.S., et. al., Anal. Chem. 1991,63,819-823.