Research Article

Received: 14 November 2011 Revised: 12 February 2012 Accepted: 13 February 2012 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2012, 26, 1053–1060

Gas chromatography/isotope ratio mass spectrometry ofrecalcitrant target compounds: performance of differentcombustion reactors and strategies for standardization

SandraReinnicke1, Dieter Juchelka2, Sibylle Steinbeiss1, ArminMeyer1, AndreasHilkert2 andMartin Elsner1*1Institute of Groundwater Ecology, Helmholtz Zentrum Muenchen, Ingolstaedter Landstr. 1, 85764 Neuherberg, Germany2Thermo Fisher Scientific, Hanna-Kunath Str. 11, 28199 Bremen, Germany

RATIONALE: Compound-specific isotope analysis (CSIA) relies on continuous flow combustion of organic substances toCO2 and N2 in a miniature reactor to measure 13 C/12C and 15N/14N stable isotope ratios. Accurate analysis is wellestablished for many volatile hydrocarbons. In contrast, compounds which contain hetero and halogen atoms are lessvolatile and may be more recalcitrant to combustion.METHODS: This study tested carbon and nitrogen isotope analysis of atrazine, desethylatrazine (DEA), dichlobenil and2,6-dichlorobenzamide (BAM) by gas chromatography/isotope ratio mass spectrometry (GC/IRMS) with multiplereactor tubes of two different kinds (conventional CuO/NiO/Pt and a NiO tube/CuO-NiO reactor prototype).RESULTS: The advantages of the NiO tube/CuO-NiO reactor were the absence of an additional reduction reactor, thepossibility of routine reoxidation in nitrogen isotope analysis, and reliable atrazine and DEA measurements over severalhundred injections. In contrast, BAM analysis showed good accuracy for carbon, but notable variations in the trueness ofnitrogen isotope ratios. Accurate carbon and nitrogen analysis was nevertheless possible by bracketing samples withexternal compound-specific standards and subsequent offset correction.CONCLUSIONS: We conclude that instrument data should never be taken at its ’face value’, but must consistently bevalidated with compound-specific standards of the respective analytes. Copyright © 2012 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.6199

Compound-specific isotope analysis (CSIA) is routinely usedto monitor the carbon and nitrogen isotope composition of amultiplicity of organic compounds. Applications reachfrom biogeochemistry[1] to food[2] and fragrance analysis,[3]

detection of doping in sports,[4] to assessing the origin andfate of organic pollutants in the environment.[5,6] Automatedonline measurement using gas chromatography/combus-tion/isotope ratio mass spectrometry (GC/C/IRMS) is avaluable technique for studying volatile organic compounds.Once separated from a compound mixture, organic substancesare converted online into CO2, N2, and NOX in a combustionreactor consisting of a ceramic tube conventionally filled withCuO/NiO/Pt wires operating at 900 to 980 �C.[7,8] For nitrogenmeasurements, a reduction reactor is also required whichoperates at 650 �C and contains Cu wires to reduce NOX toN2.

[9] Isotope ratios are typically evaluated in comparison withpeaks of amonitoring gas, which is introduced at the beginningand the end of each chromatographic run and serves as amediator between runs.[10]

Compound-specific isotope applications often concernvolatile compounds such as low molecular weightchlorinated[11–14] or petroleum hydrocarbons.[12,14–18] Volatile

* Correspondence to: M. Elsner, Institute of GroundwaterEcology, Helmholtz Zentrum Muenchen, IngolstaedterLandstr. 1, 85764 Neuherberg, Germany.E-mail: martin.elsner@helmholtz-muenchen.de

Rapid Commun. Mass Spectrom. 2012, 26, 1053–1060

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compounds have relatively simple structures and theircarbon (13 C/12 C) isotope analysis is well established.[19]

Studies on these substances frequently report that (i)uncorrected GC/IRMS instrument values of underivatizedtarget compounds coincide with those from offlineanalysis or elemental analyzer (EA)/IRMS (an indicationof trueness); and (ii) reported standard deviations aretypically�0.3% for carbon and� 0.5% for nitrogen, accord-ing to the manufacturer ’s specification (an indication ofprecision).[11,19,20] Such results may seem to suggest thatcomplete conversion and accuracy (i.e., trueness and preci-sion) can be taken for granted when measuring theisotope ratios of new target compounds by GC/IRMS.This is convenient, because uncertainties are incorporatedinto the standard error, and additional corrections can beavoided.

In particular, if repeated analysis of compound-specificstandards can clearly establish the trueness and precisionof GC/IRMS isotope analysis for these compounds on agiven instrument,[11,12] this has consequences for thestrategy of standard bracketing. A standard mixture ofall compounds may then have to be measured only atthe beginning and at the end of daily measurement seriesas a quality control for consistent measurement conditions.Other protocols may employ more frequent bracketingwith such external standard mixtures. At present, noguidelines exist about which strategy to adopt underwhat circumstances.

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Over the years, GC/IRMS analysis has been extended tomore complex substances such as food ingredients andfragrance compounds,[21,22] steroids,[4,23] amino acids,[24,25]

and pesticides.[26–28] Compared with less polar compoundsof equal mass, these polar molecules have a strongertendency to sorption, influencing the peak shape during GCseparation. In addition, they have more complex chemicalstructures including the presence of heteroatoms. Potentially,this can make the combustion process more difficult, leadingto incomplete conversion and entailing systematic isotopefractionation. Compared with carbon isotope analysis,nitrogen (15N/14N) isotope analysis is particularly challeng-ing. The natural abundance of 15N is lower than that of 13 C,fewer nitrogen atoms are present in organic substances, twoN atoms are needed to form N2 and the ionization efficiencyof N2 is lower. To obtain a similar precision to that of13 C/12C analysis, theoretically a 50 times higher sampleamount is required,[29] leading to high substance loads thatcan affect peak separation and conversion efficiency.Such problems do not, however, only pertain to nitrogen. A

recent study in our lab showed that the carbon isotope ratiosof atrazine – a heteroaromatic compound containing C, Nand Cl – were not reproducible and showed poor accuracy(= poor precision +poor trueness[30]) when determined witha commercial NiO/CuO/Pt reactor operated at therecommended temperature of 940 �C. When the operatingtemperature was reduced to 800 �C, the precision improvedbut the carbon isotope values still showed an offset of 1.2 %compared with the results from EA/IRMS.[26,31] Bettertrueness, but higher limits for precise isotope analysis wereobtained with a self-made NiO reactor operated at1150 �C.[26] This can be problematic because (i) manyapplications require low limits for precise isotope analysiswhile (ii) the reproducibility and trueness of isotope ratiosare crucial for any isotope investigation, particularly inforensic studies.[32,33]

These recent results emphasize the need for furtherinvestigations addressing cases of such recalcitrant targetcompounds. Promising in this context are prototypes of anew commercial NiO tube/CuO-NiO combustion reactor

a)

b)

Figure 1. Different oxidation reactors testreactor operated at 940 �C and (b) prototypoperated at 1030 �C.

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developed by Thermo Fisher Scientific (Bremen, Germany).Compared with the conventional model, the new reactorcontains a Ni-tube with an oxidized surface that displays agreater surface area, and it is operated at 1030 �C instead of940 �C (Fig. 1). The greater active surface prolongs the lifetimeof the reactor, particularly for nitrogen isotope analysis. Mostimportantly, the new reactor design relies to a much greaterdegree on high-temperature conversion on a NiO surfacethan on oxidation by CuO. This eliminates the need for aseparate reduction reactor, because (i) NiO adds oxygenequivalents specifically to organic compounds so thatcontinuous oxygen bleed as with CuO is avoided;[34] and (ii)the Ni/NiO surface withdraws oxygen from nitrogen oxides(NOx) thereby acting like a reduction catalyst. Potentially,these surface properties may also lead to better conversionof target compounds that are difficult to convert with theconventional design.

The objective of this study was, therefore, to test the twodifferent reactors (conventional reactors with CuO/NiO/Pt-wires versus prototypes of a new NiO tube/CuO-NiO com-bustion reactor) for 13C/12C and 15N/14N isotope analysis ofselected target compounds that we identified as particularlydifficult to analyze. Specifically, we chose atrazine, itsmetabolite desethylatrazine (DEA), 2,6-dichlorobenzonitrile(dichlobenil) and its main metabolite 2,6-dichlorobenzamide(BAM). These compounds are environmental pollutants whichare frequently detected in groundwater. However, they are alsochallenging to analyze by GC/IRMS due to their complexstructure including C, N and Cl (Table 1). The performance ofthe reactors was carefully tested for the accuracy (= truenessand precision) of carbon and nitrogen isotope ratio values.To this end, compounds were characterized beforehand byEA/IRMS. Although the method does not guarantee absolutetrueness, bias was minimized by (i) careful calibration withinternational referencematerials and (ii) high compound purityeliminating extraneous carbon and nitrogen sources (seeExperimental section). The investigations were conducted atthe Helmholtz Zentrum Muenchen, Germany (laboratory 1),and selected measurements were repeated in the IRMSapplication laboratory of Thermo Fisher Scientific, Bremen,

ed: (a) the conventional NiO/CuO/Ptes of a new NiO tube/CuO-NiO reactor

y & Sons, Ltd. Rapid Commun. Mass Spectrom. 2012, 26, 1053–1060

Table 1. Structures of investigated compounds, carbon and nitrogen isotope ratio values determined by EA/IRMS and GC/IRMS in two different laboratories

Common name Dichlobenil2,6-Dichlorobenzamide

(BAM) AtrazineDesethylatrazine

(DEA)

Herbicide Metabolite of dichlobenil Herbicide Metabolite of atrazineStructure CN

Cl ClCl

NH2O

ClN N

N NN

H

CH3

CH3

H

CH3

Cl

N N

N NH2N

CH3

H

CH3

Cl

EA/IRMSd13C [%]a �25.8� 0.1 (n = 4) �26.4� 0.1 (n = 4) �28.6� 0.1 (n = 4) �29.2� 0.1 (n = 4)d15N [%]a 1.1� 0.2 (n = 4) �11.4� 0.2 (n = 4) �1.3� 0.2 (n = 4) �4.2� 0.2 (n = 4)

Stable Isotope Laboratory 1 (IGÖ, Munich, Germany), CuO-NiO reactord13C [%]b �26.4� 0.3 (n = 80)c �26.2� 0.1 (n = 80)c �30.3� 1.6 (n = 35)d n.a.f

d15N [%]b �0.5� 1.6 (n = 150)e �13.2� 1.0 (n = 150)e n.a.f

Stable Isotope Laboratory 1 (IGÖ, Munich, Germany), NiO tube/CuO-NiO reactord13C [%]b �25.4� 0.3 (n = 3) �26.4� 0.1 (n = 4) �28.5� 0.2 (n = 95)g �30.0� 0.6 (n = 94)g

d15N [%]b n.a.f �12.7� 1.2 (n = 160)h �2.1� 0.4 (n = 122)g �4.8� 0.5 (n = 122)g

Stable Isotope Laboratory 2 (Thermo Fisher Scientific, Bremen, Germany), NiO tube/CuO-NiO reactord13C [%]b �26.0� 0.1 (n = 6) �26.3� 0.2 (n = 6) �28.7� 0.2 (n = 5) n.a.f

d15N [%]b 0.7� 0.3 (n = 20) �14.3� 0.7 (n = 20) �1.6� 0.2 (n = 20) �4.6� 0.6 (n = 20)aUncertainties represent analytical errors for EA/IRMS; buncertainties represent standard deviation of all measurements;cdata of Fig. 2; ddata from Meyer et al.;[26] edata of Fig. 3; fnot analyzed; gvalues correspond to data of initial measurementsobtained with the first reactor of Fig. 4; hdata of Fig. 5.

GC/IRMS of recalcitrant target compounds

Germany (laboratory 2). The implications of our results arediscussed for the study of new target compounds, with a parti-cular focus on standard bracketing strategies.

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EXPERIMENTAL

Instrumentation

GC/IRMS systems each consisting of a TRACE GC Ultra gaschromatograph (Thermo Fisher Scientific, Milan, Italy)coupled to a Finnigan MAT 253 isotope ratio mass spectro-meter (IRMS) (Thermo Fisher Scientific, Bremen, Germany)via a Finnigan GC Combustion III interface (Thermo FisherScientific) were used for measurements. The emission currentwas set to 1.5 mA for carbon isotope analyses and 2.0 mA fornitrogen isotope analyses. The amount dependency of isotopemeasurements (’linearity’) was checked daily and was alwaysbetter than 0.06 %/V with the conductance limit (’sulfurwindow’) adjusted to maximum amplitude (1.5 turns open).No check for absolute sensitivity (in molecules per ion) waspossible in laboratory 1, since the instruments employed werenot equipped with a dual inlet system. Helium (grade 5.0)was used as carrier gas and liquid samples were injectedvia a GC Pal autosampler (CTC, Zwingen, Switzerland). Allinjections were conducted splitless (2 min) into a hot injector,held at 250 �C. The helium flow rate was set to 1.4 mL/minand the analytical column was a DB 5 (30 m� 0.25 mm;1 mm film; J&W Scientific, Folsom, CA, USA). Standardsolutions containing either atrazine and DEA or dichlobeniland BAM were analyzed using the following temperature

Copyright © 2012Rapid Commun. Mass Spectrom. 2012, 26, 1053–1060

programmes. Atrazine and DEA measurements started at150 �C (hold 1 min), ramped at 10 �C/min to 190 �C (hold1 min), followed by a ramp of 3 �C/min to 230 �C (hold 2 min)and finally ramped at 30 �C/min to 280 �C (hold 3 min). Fordichlobenil and BAM the GC program was set to 120 �C (hold1 min), ramp at 8 �C/min to 200 �C (hold 1 min) and ramp at15 �C/min to a final temperature of 280 �C (hold for 2 min).

Target values for carbon and nitrogen isotope ratioswere determined in a complementary approach by elementalanalyzer/isotope ratio mass spectrometry (EA/IRMS) usinga system consisting of an EuroEA (EuroVector, Milan, Italy)coupled to a MAT253 isotope ratio mass spectrometer (ThermoFisher Scientific) by a ConFlow III interface (Thermo FisherScientific). Calibration was performed with organic referencematerials as external standards (USGS 40 and USGS 41 (bothglutamic acid), as well as IAEA 600 (caffeine) for 15N/14N mea-surements, IAEA600, IAEACH3 (cellulose), IAEACH6 (sucrose)and IAEA CH7 (polyethylene) for 13C/12C analysis) providedby the International Atomic Energy Agency (IAEA, Vienna).

The d13C and d15N values are reported in per mill (%)relative to Vienna PeeDee Belemnite (V-PDB) and AIR-N2,respectively,[35,36] where d13C is expressed as:

d13C ¼�13C=12C

Sample�13 C=12C

Standard

�

13 C=12CStandard

(1)

and an analogous equation applies for d15N. The d13C andd15N values were evaluated relative to a laboratory standardgas (CO2 and N2, respectively) that was introduced at thebeginning and the end of each run. These standard gases were

(1)

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calibrated in a Finnigan MAT Delta S isotope ratio mass spec-trometer with dual inlet system (Thermo Fisher Scientific)against V-PDB and air, respectively, by use of internationalreference materials (RM 8562, RM 8563, RM 8564 (all of themCO2 gases) for CO2, and NSVEC (N2 gas) for N2). All the refer-ence standards were provided by the IAEA.

Oxidation reactors

In the first instance, an instrumental setup comprising a commer-cial oxidation reactor (Fig. 1(a)), consisting of a ceramic tubefilledwith CuO/NiO/Pt-wires (P/N 1040920, operated at 800 �C,940 �C and 980 �C) was used for carbon and nitrogen measure-ments. The reactorwas connected to a standard reduction reactorfilled with copper wires (P/N 1040820, 650 �C) for subsequentNOx reduction to N2 (all reactors from Thermo Fisher Scientific).For nitrogen isotope measurements the CuO/NiO/Pt reactorwas used as received, in preconditioned form from the manufac-turer, but without additional oxidation, because a high oxygenbleed during measurements would exhaust the reduction oven.This would lead to the formation of nitrogen oxide (14N16O atm/z 30) meaning that conversion into N2 would no longer bequantitative. For carbon isotope measurements, in contrast, theCuO/NiO/Pt reactor was initially oxidized for 4 h and theoxidation was routinely repeated after 40 to 60 measurementsto ensure sufficient oxidation capacity.Alternatively, in a second setup prototypes of a new NiO

tube/CuO-NiO reactor (Thermo Fisher Scientific, patentWO2009/121506 A1 pending) were used, consisting of a cera-mic tube filled with a Ni-tube and NiO/CuOwires (Fig. 1(b)).Since this reactor contains both an oxidation and a reductionunit, no extra reduction reactor was necessary for nitrogenmeasurements. In contrast to the conventional oxidation reac-tor, the NiO tube/CuO-NiO reactor was coupled to theseparation column via a silcosteel capillary (Restek, Belle-fonte, PA, USA). To this end, a solder joint was implementedbetween the silcosteel capillary and the (metallized) ceramicsurface. The operating temperature of the new reactor wasset to 1030 �C. The reactor was initially oxidized for 2 h at600 �C, and 12 h at 1030 �C. Repeated oxidation (30 min to1 h) was routinely performed after 20 to 50 measurements.

Chemicals

Atrazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine,97.7%,CAS: 1912-24-9)was suppliedbyTropitzsch (Marktredwitz,Germany), desethylatrazine (>99.9%, CAS No.: 6190-65-4),dichlobenil (2,6-dichlorobenzonitrile, >98%, CAS No.: 1194-65-6)and BAM (2,6-dichlorobenzamide, >98 %, CAS No.: 2008-58-4)were supplied by Riedel-de Haën distributed by Sigma Aldrich(Taufkirchen, Germany). Methanol (pestanal, 99.9%, Merck,Darmstadt, Germany) was used as solvent.

Figure 2. d13C values of dichlobenil and BAM standards withthe conventional oxidation setup using the NiO/CuO/Ptreactor operated at 940 �C (open diamonds) and 800 �C (filleddiamonds), respectively. Values for (a) dichlobenil and (b) BAM.Black lines represent the d13C values determined by EA/IRMS.

RESULTS AND DISCUSSION

13C/12C analysis of dichlobenil (2,6-dichlorobenzonitrile)and BAM (2,6-dichlorobenzamide) with the conventionalreactor design

In a recent study[26] we observed that the conventional reactordesign – i.e., CuO/NiO/Pt-wires threaded through a ceramictube – could not accomplish accurate 13 C/12C isotope

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analysis of atrazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine) when operated at 940 �C. The precision im-proved, but values remained systematically 1.2 % off whenthe reactor was operated at 800 �C, indicating incomplete con-version.[26,31] We therefore tested the conventional reactordesign for the carbon isotope analysis of two other relevantorganic substances that also have an aromatic structure andcontain both nitrogen and chlorine inside their molecular struc-ture, that is, the herbicide dichlobenil and its metabolite BAM.

In contrast to the results for atrazine, the conventionalNiO/CuO/Pt reactor gave true and precise d13C values fordichlobenil (�26.4� 0.3 %, n = 80) and BAM (�26.2� 0.1 %,n = 34) over a broad concentration range (Fig. 2), as repre-sented by the corresponding range of amplitudes. Theminimum amplitudes (m/z 44) required for precise 13C/12Canalysis of dichlobenilwere 250mVcorresponding to 1.6 nmolCon-column (Fig. 2(a)) and 500 mV for BAM corresponding to2.6 nmol C on-column (Fig. 2(b)), illustrating differences be-tween compounds. Different temperatures (800 �C and 940 �C)of the oxidation reactor caused no discernible differences inthe isotope ratios. These result correspond to typical textbookexamples of compound-specific carbon isotope analysis.[11]

15N/14N analysis of dichlobenil and BAM with theconventional reactor

Determining the 15N/14N values of dichlobenil and BAMusing aNiO/CuO/Pt reactor (used as received) at 980 �C gaveprecise ratios for dichlobenil (Fig. 3(a)) and BAM (Fig. 3(b))with d15N=�0.9� 0.5 % (n = 43) and d15N=�12.6� 0.4 %(n = 34), respectively. However, both compounds showed anoffset compared with the EA values of �2 % for dichlobeniland �1.2 % for BAM (Fig. 3). In contrast to carbon isotopeanalyses, we analyzed constant compound concentrations(26.3 nmol N in the presence of 184 nmol C) so that a decreasein the corresponding amplitudes directly indicates a decreasein conversion efficiency. Figure 3(b) illustrates that, after 34measurements, the nitrogen isotope ratios of BAM started to

y & Sons, Ltd. Rapid Commun. Mass Spectrom. 2012, 26, 1053–1060

Figure 3. d13C values (filled diamonds) and amplitudes(crosses) of (a) dichlobenil and (b) BAM using the conven-tional NiO/CuO/Pt reactor at 980 �C. Black lines give thed15N values determined by EA/IRMS. Dashed lines representoxidation events.

2

‰

GC/IRMS of recalcitrant target compounds

drift by a total of 5% towardsmore negative values, indicatinga decrease in the system performance. Surprisingly, the ampli-tudes of the m/z 44 peak remained constant (543� 3 mV,n=43), demonstrating that the loss of trueness of isotopeanalysis did not necessarily correlate with, and could not bedetected from, a decrease in conversion efficiency. Reoxidationof the reactor for 10 min extended the reactor lifetime in thelong run, as already observed for bentazone nitrogen isotopeanalyses.[28] Initially, however, a conditioning period wasrequired before precise 15N/14N analysis of dichlobenil andBAM was reestablished (Fig. 3, injections 44 to 60, 109 to 115).During the conditioning phases, amplitudes were at first smallbut then noticeably recovered, indicating that the conversionefficiency was influenced by the excess of oxygen in thesystem. After the conditioning phases the d15N values ofdichlobenil and BAM stabilized, but higher standard devia-tions and an offset in the isotope ratios compared with theEAvalues had to be accepted for both compounds.

0 100 200 300 400 500 600 700 800 900 1000 1100 1200

-36

-34

-32

-30

-28

-26

-6-4-20

δ13C

in ‰

δ15N

in

injection #

Figure 4. d13C and d15N measurements of atrazine (open tri-angles) and desethylatrazine (filled triangles) using five proto-types of a new NiO tube/CuO-NiO reactor design. Injections0 to 650 represent data determined with NiO tube/CuO-NiOprototype I. Solid vertical lines indicate the change to a newNiO tube/CuO-NiO prototype, and dashed vertical linesshow oxidation events. Horizontal black lines give the iso-tope ratios of atrazine and desethylatrazine determined byEA/IRMS. Gaps represent data points that were left out,because they corresponded to measurements of samplesrather than standards, or of substances other than atrazine.

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13C/12C and 15N/14N analysis of atrazine anddesethylatrazine (DEA) with prototypes of a new NiO tube/CuO-NiO reactor

For those cases where the conventional reactor showed unsa-tisfactory performance (i.e., d13C analysis of atrazine[26] andd15N analysis of BAM and dichlobenil) prototypes of a newNiO tube/CuO-NiO reactor were tested. No evident differ-ences in peak shape were observable between the reactorsunder the conditions employed in laboratory 1 (splitless injec-tion into hot injector). In addition, we extended the range ofanalyses including the d15N analysis of atrazine as well asthe d13C and d15N analysis of desethylatrazine (DEA), themain metabolite of atrazine. In addition to the analyses car-ried out in laboratory 1, independent nitrogen and carbon iso-tope measurements with the NiO tube/CuO-NiO reactorwere conducted for all target compounds in laboratory 2.All the measurements with the NiO tube/CuO-NiO reactorsetup conducted in this study are summarized in Table 1.The initial performance of the new NiO tube/CuO-NiO

reactor was excellent for carbon and nitrogen isotopemeasurements of atrazine standards over more than 300 mea-surements, showing an average of �28.5� 0.2 % (n = 95) for

Copyright © 2012Rapid Commun. Mass Spectrom. 2012, 26, 1053–1060

d13C and �2.1� 0.4 % (n = 122) for d15N (Fig. 4, injections 1to 350). Simultaneous measurement of DEA isotope ratiosgave average values of �30.0� 0.6 % (n = 94) for d13C and�4.8� 0.5 % (n = 122) for d15N (Fig. 4, injections 1 to 350).The concentrations varied between 20 and 500 mg L-1, withinjection volumes varying between 1 and 9mL The observedlimits for precise 15N/14N isotope analysis were amplitudesof ≥100 mV, corresponding to 5.6 nmol N of atrazine and6.4 nmol N of DEA on column, respectively. The highest load-ings tested were 23.1 nmol N (in the presence of 37 nmol C)and 26.6 nmol N (in the presence of 31.9 nmol C), correspond-ing to amplitudes at m/z 28 of 556� 83 mV and 544� 82 mV,respectively. The isotope ratios determined in laboratory 2were in perfect agreement with measurements conducted byGC/IRMS and EA/IRMS in laboratory 1. In laboratory 2, fornitrogen isotope measurements 1 mL of 750 mg L–1 atrazineand DEA solutions were injected (17.4 nmol N atrazine and19.9 nmol N of DEA on-column) giving amplitudes of2400� 72 mVand 2100� 63 mV. For carbon isotope measure-ments 1 mL samples of 50 mg L–1 (1.9 nmol C of atrazine on-column) were injected producing amplitudes of 3500� 42 mV.

After 350 injections (corresponding to a cumulative loadingof roughly 800 nmol atrazine as well as 800 nmol DEA on col-umn) the precision of the isotope ratios deteriorated in thelong-term series in laboratory 1, as represented by higherstandard deviations (Fig. 4). Lower helium gas flow throughthe reactor indicated clogging (presumably by formationof NiCl2 and CuCl, thus impairing the performance of thereactor (data not shown)).

Subsequently, four additional prototypes of the NiO tube/CuO-NiO reactors were tested (Fig. 4, injections 650 to 1160).They still showed good trueness, but significantly lower pre-cision than the first NiO tube/CuO-NiO reactor. Potentialreasons for this include not only formation of NiCl2 and CuCl,but also the possibility of (partial) thermolytic breakdown of

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compounds eluting from the GC column in the colder zones ofthe combustion reactor. Indeed, unpublished results of labora-tory 2 indicate that the positioning of the reaction zones playsa crucial role in the new reactor design, which was subject toongoing optimization during the time of our study. Althoughthe precision was therefore not optimized in all prototypes ofthe new design, the performance of the new design was greatlyimproved in comparison with the conventional NiO/CuO/Ptreactor. In addition to the obvious advantages – i.e., the abilityto operate without additional reduction reactor and to oxidizethe combustion reactor for extended series of nitrogen isotopemeasurements – the new NiO tube/CuO-NiO reactor designenabled us to obtain for the first time true and precise atrazineand DEA analysis over sequences of several hundred measure-ments in laboratory 1.

13C/12C and 15N/14N analysis of dichlobenil and BAMwith prototypes of a new NiO tube/CuO-NiO reactor

Analogous to the results with the conventional NiO/CuO/Ptreactor (Fig. 2), prototypes of the new NiO tube/CuO-NiOreactor also gave true and precise values for the d13C isotopeanalysis of dichlobenil and BAM (Table 1). In contrast, the per-formance of the NiO tube/CuO-NiO reactor for d15N isotopeanalysis for BAM showed poor accuracy, as demonstrated inFig. 5(a) (for comparison, data from the conventional reactoris shown again, Fig. 5(b)). A long-term series of BAM mea-surements in laboratory 1 showed an offset of d15N comparedwith the EA value (Fig. 5), which varied depending on thelifetime of the reactor. Although the NiO tube/CuO-NiO reac-tor seemed to have a longer lifetime than the conventionalNiO/CuO/Pt reactor, both reactors showed a similar perfor-mance, indicated by a conditioning phase after oxidationevents and a drift in offsets. Also, results obtained in labora-tory 2 showed d15N value of BAM with a systematic driftand an offset of �3 % (n = 10) compared with the EA/IRMSresults (Table 1). Different substance amounts on-columnwererequired for the two laboratories, depending on the instru-ment sensitivity. Minimum amplitudes around 300 mV in

0 20 40 60 80 100 120 140 160 180 200 220 240-20

-15

-10

-5

0

δ15N

in ‰

injection #

b)-20

-15

-10

-5

0 a)

δ15N

in ‰

Figure 5. d15N measurements of BAM using the two differentcombustion reactors. Panel (a) represents data obtained withthe NiO tube/CuO-NiO prototype at 1030 �C (gaps corre-spond to data points left out, because they correspond toexperimental samples rather than standards), panel (b) showsin comparison again the data obtained with the conventionalNiO/CuO/Pt reactor at 980 �C (same data as in Fig. 2b).

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laboratory 1 were obtained with 2 mL of a 500 mg L–1 BAMsolution (5.3 nmol N on-column) and, in contrast, in labora-tory 2 amplitudes of 270 mV were obtained with 0.5 mL of750 mg L–1 BAM solution (2 nmol N on-column). Contrastingwith BAM, the nitrogen isotope ratios of dichlobenil (d15N of0.7� 0.3 %, measurements from laboratory 2) showed goodtrueness, agreeing well with values determined by EA/IRMS.

Several critical points in the GC/IRMS system could beresponsible for the drift and the systematic deviation thatwas observed in the nitrogen, but not in carbon isotope ratiosobtained for BAM. (1) Active sites in the GC column, in con-nectors or even in colder parts of the combustion oven, canlead to peak distortion and isotope fractionation during thechromatographic process and during subsequent transfer.Such effects may be more pronounced for nitrogen, since sur-face complexation may involve specifically the NH2 group.(2) Incomplete combustion can lead to isobaric interferencefrom CO (m/z 28) in the nitrogen isotope measurement. Ifthe amount of CO is small, the d13C measurements wouldnot be noticeably affected. (3) Insufficient reduction of nitro-gen oxides can lead to incomplete conversion into N2. Expla-nations must clearly accommodate the facts (i) that deviationsfor nitrogen were observed in both laboratories, (ii) that nosuch effects were observed for carbon although the NiOtube/CuO-NiO reactor was operated in the same way (tem-perature, oxidation frequency) in C and Nmode, and (iii) thatresults of laboratory 2 gave better trueness for dichlobenilalthough dichlobenil and BAM contained the same numberof C, N and Cl atoms (meaning that they posed a similar oxi-dation and reduction demand on the reactor tube). Presently,none of the reasons can be excluded, and further investiga-tions are necessary to constrain possible reasons.

Strategies for referencing

Our data demonstrate the need for a referencing strategywhich corrects for substance-specific offsets of isotope ratios.Figure 6 clearly shows that it would not be possible, for exam-ple, to correct an offset in BAM nitrogen isotope ratios at the

0 50 100 150 200 250-20

-15

-10

-5

0

5

10

15

20

25

30

δ15N

in ‰

injection #

Figure 6. d15N values of BAM standards (filled diamonds)and different BAM samples from a degradation experimentcorresponding to different degrees of degradation (emptycircles) obtained with a NiO tube/CuO-NiO prototype.[37]

Black line represents the BAM standard value determinedby EA/IRMS and the dashed lines indicates oxidation events.

y & Sons, Ltd. Rapid Commun. Mass Spectrom. 2012, 26, 1053–1060

GC/IRMS of recalcitrant target compounds

end of a series with an offset determined at the beginning.Therefore, to integrate such a correction into daily analysis,the following referencing strategy was implemented follow-ing the ’identical treatment principle’ from Werner andBrand:[10] (i) to measure external standards before and afterthe sample to correct for a certain offset, and (ii) to use a refer-ence material which is identical to the target substances. Inthe following we present the application and the practicabil-ity of such a referencing strategy for BAM as an example.Figure 6 shows data from a biodegradation experiment ofBAM,[37] where samples (white circles) showed more positived15N values than the original reactant. The shift was differentbetween samples, but well reproducible for each sample, asindicated by the agreement of triplicates (clusters of threepoints in Fig. 6). The samples were bracketed by measure-ments of our compound-specific BAM isotope standard(black diamonds) at a similar concentration to the samples.Sample measurements were not conducted during the unsys-tematic drift of the nitrogen isotope ratios that was observedin the conditioning phase after each oxidation of the NiOtube/CuO-NiO reactor when surplus oxygen needed to bleedout (Fig. 6, injections 1 to 15, 49 to 64 and 155 to 175). Instead,they were started when the standard deviation of at least fivemeasurements of the BAM standard was within the analyticalerror (1 % for nitrogen analyses). Triplicate sample measure-ments were subsequently bracketed by triplicate standardmeasurements, and sample isotope ratios were corrected withthe offset of the mean value of standards measured beforeand after the sample. Figure 6 illustrates that with this proce-dure precise and true values can be obtained.

105

CONCLUSIONS

The combustion of organic target compounds in a continuoushelium flow inside a miniature reactor tube is the key step incompound-specific isotope analysis by GC/IRMS and has ledto a boost of new applications in many disciplines. Contrast-ing with the excess oxidation power that can be applied inoffline analysis or elemental analyzer reactors, however, thisprocess is fueled by just a few miniature CuO/NiO wires.Nonetheless, previous studies have reported excellent preci-sion and trueness, suggesting that complete conversion andtrue isotope ratios can be typically observed in GC/IRMSmeasurements. This study addressed organic target com-pounds that contain nitrogen and chlorine inside an aromaticstructure and may, therefore, be more difficult to convert thanthe typical range of substances. The study used a number ofcombustion reactors and tested two different reactor types(conventional NiO/CuO/Pt wires and NiO tube/CuO-NiOwires). The NiO tube/CuO-NiO reactor showed compellingadvantages over the conventional one: no additional reduc-tion reactor was necessary, reoxidation could be routinelyperformed for nitrogen isotope measurements, and reliableatrazine and DEA analysis was for the first time obtainedover several hundred measurements.Overall, however, notable variations in the trueness of

BAM nitrogen isotope measurements were observed withboth types of reactor tubes. Our data therefore demonstratethat (i) complete conversion and (ii) an absolute trueness ofisotope ratios in GC/IRMS may not be achievable for sometarget compounds. Consequently, instrument data should

Copyright © 2012Rapid Commun. Mass Spectrom. 2012, 26, 1053–1060

never be taken at ’face value’, but measurements mustcontinuously be validated with compound-specific isotopestandards of the respective analyte. Nonetheless, accurateisotope analysis may be achieved. In an exemplary case wedemonstrate that stringent bracketing with a compound-specific isotope standard allows for a continuous drift correc-tion so that accurate isotope ratios can be obtained. In futurestudies it is, therefore, recommended to routinely test theperformance of reactor tubes for ’tricky’ target substancesand to adapt the referencing strategy accordingly.

AcknowledgementsThis work was conducted in a Helmholtz Young InvestigatorGroup supported by theHelmholtz Initiative andNetworkingFund, as well as by funding from DFG (German NationalScience Foundation) within the priority program SPP 1315.We thank Giovanni Calderone for the isotope analysis ofdichlobenil, BAM, atrazine and desethylatrazine at ThermoFisher Scientific IRMS Application Laboratory. Three anon-ymous reviewers andGeorges Lacrampe-Couloume (Universityof Toronto) are acknowledged for critical constructive commentswhich improved the quality of the manuscript.

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