13C- and 15N-Isotope Analysis of Desphenylchloridazon by Liquid Chromatography-Isotope-Ratio Mass Spectrometry and Derivatization Gas Chromatography-Isotope-Ratio Mass Spectrometry

Article abstract of DOI:10.1021/acs.analchem.8b04906

The widespread application of herbicides impacts surface water and groundwater. Metabolites (e.g., desphenylchloridazon from chloridazon) may be persistent and even more polar than the parent herbicide, which increases the risk of groundwater contamination. When parent herbicides are still applied, metabolites are constantly formed and may also be degraded. Evaluating their degradation on the basis of concentration measurements is, therefore, difficult. This study presents compound-specific stable-isotope analysis (CSIA) of nitrogen- and carbon-isotope ratios at natural abundances as an alternative analytical approach to track the origin, formation, and degradation of desphenylchloridazon (DPC), the major degradation product of the herbicide chloridazon. Methods were developed and validated for carbon- and nitrogen-isotope analysis (δ¹³C and δ¹⁵N) of DPC by liquid chromatography-isotope-ratio mass spectrometry (LC-IRMS) and derivatization gas chromatography-IRMS (GC-IRMS), respectively. Injecting standards directly onto an Atlantis LC-column resulted in reproducible δ¹³C-isotope analysis (standard deviation <0.5‰) by LC-IRMS with a limit of precise analysis of 996 ng of DPC on-column. Accurate and reproducible δ¹⁵N analysis with a standard deviation of <0.4‰ was achieved by GC-IRMS after derivatization of >100 ng of DPC with 160-fold excess of (trimethylsilyl)diazomethane. Application of the method to environmental-seepage water indicated that newly formed DPC could be distinguished from "old" DPC by the different isotopic signatures of the two DPC sources.

Full text of DOI:10.1021/acs.analchem.8b04906

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Article
13C and 15N isotope analysis of desphenylchloridazon by
liquid chromatography isotope ratio mass spectrometry
(LC-IRMS) and derivatization-gas chromatography
isotope ratio mass spectrometry (GC-IRMS)
Aileen Melsbach, Violaine Ponsin, Clara Torrentó, Christina
Lihl, Thomas B. Hofstetter, Daniel Hunkeler, and Martin Elsner
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04906 • Publication Date (Web): 23 Jan 2019
Downloaded from http://pubs.acs.org on January 27, 2019
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13C and 15N isotope analysis of desphenylchloridazon by liquid
chromatography isotope ratio mass spectrometry (LC-IRMS)
and derivatization-gas chromatography isotope ratio mass
spectrometry (GC-IRMS)
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Aileen Melsbacha,‡, Violaine Ponsinc,‡, Clara Torrentóc,†, Christina Lihla, Thomas B. Hofstetterd,
Daniel Hunkelerc, Martin Elsnera,b,*
aHelmholtz Zentrum München, Institute of Groundwater Ecology, 85764 Neuherberg, Germany
bTechnical University of Munich, Chair of Analytical Chemistry and Water Chemistry, 81377 Munich, Germany
cCentre for Hydrogeology and Geothermics (CHYN), University of Neuchâtel, 2000 Neuchâtel, Switzerland
dEawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland
Abstract
Widespread application of herbicides impacts surface water and groundwater. Their metabolites (e.g., desphenylchloridzon
from chloridazon) may be persistent and even more polar than the parent herbicide, which increases the risk of groundwater
contamination. When parent herbicides are still applied, metabolites are constantly formed and may in addition be degraded.
Evaluating their degradation based on concentration measurements is, therefore, difficult. This study presents compound-
specific stable isotope analysis (CSIA) of nitrogen and carbon isotope ratios at natural abundances as alternative analytical
approach to track origin, formation and degradation of desphenylchloridazon (DPC), the major degradation product of the
herbicide chloridazon. Methods were developed and validated for carbon and nitrogen isotope analysis (δ13C and δ15N) of
DPC by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) and derivatization-gas chromatography-IRMS
(GC-IRMS), respectively. Injecting standards directly onto an Atlantis LC-column resulted in reproducible δ13C isotope
analysis (standard deviation < 0.5 ‰) by LC-IRMS with a limit of precise analysis of 996 ng DPC on-column. Accurate and
reproducible δ15N analysis with a standard deviation < 0.4 ‰ was achieved by GC-IRMS after derivatization of > 100 ng
DPC with 160-fold excess of (trimethylsilyl)diazomethane. Application of the method to environmental seepage water
indicated that newly formed DPC could be distinguished from “old” DPC by different isotopic signatures of the two DPC
sources.
Keywords: Compound-specific Isotope Analysis, multielement isotope analysis, herbicides, metabolites, plant protection
products, micropollutants, environmental fate
In many regions of the European Union, groundwater
is our most important drinking water resource and is
therefore constantly screened for contaminants1,2. In
recent years, there is growing concern about pollution by
persistent and mobile organic contaminants such as polar
compounds and their metabolites3-6. Metabolites are often
more persistent and polar than the parent compounds
resulting in a high leaching potential with an increased
risk to contaminate groundwater7. For some of them,
however, methods are lacking to demonstrate their origin,
formation and degradation. To evaluate their
environmental fate, conventional models rely on parent-
compound-to-metabolite-ratios. However, as pesticides
are still applied on the field, there is a constant formation
of persistent metabolites. Thus, the evaluation of
metabolite degradation with conventional models based
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on concentration measurements may lead to bias. Further
organic compounds, derivatization-GC-IRMS has been
brought forward as alternative strategy24,25,31,32. This
approach is chosen as the methylation of DPC enhances
its GC suitability. Methylation of a compound using
“mild” derivatization reagents (e.g., trimethyl sulfonium
hydroxide (TMSH), methanol/BF3) allows control over
the isotope ratio of the methyl group that is introduced.
Hence, the change in the 13C/12C composition of the
target analyte caused by the introduction of an additional
carbon atom can be corrected by equations stated in the
literature31,33,34. However, these mild reagents fail to
derivatize groups of low reactivity such as amino-,
amide-, or hydroxyl-groups.
bias is introduced, when one contaminant is formed from
at least two different sources (parent compound)8.
A representative compound for polar contaminants is
desphenylchloridazon (DPC). It is among the most
frequently detected micropollutants related to crop
production, exceeding concentrations of 10 µg/L in
natural water2,9-16
. DPC is formed by microbial
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degradation of the selective systemic herbicide
chloridazon (CLZ)16-19. CLZ is being applied in the
agricultural production of mangold, beetroot and sugar
beet20. Consequently, there is a constant formation of
DPC deriving from newly applied CLZ. DPC can be
transformed
to
methyl-desphenylchloridazon
transformation pathway and
Consequently, for compounds containing less reactive
groups an alternative strategy must be followed. For
13C/12C isotope analysis, liquid chromatography is the
method of choice 35-38. LC-IRMS has the advantage that
compounds can be analyzed directly without
derivatization, but the liquid chromatography presents the
challenge that carbon isotope measurements must be
conducted without organic eluents, which otherwise
would be converted to CO2 and would interfere with
13C/12C analysis of the analyte39,40. For nitrogen isotope
analysis such sensitive LC-IRMS is not possible, but here
GC-IRMS after derivatization by more reactive reagents
is an option, because for 15N/14N analysis control over
carbon isotope ratios is not required. To this end, the idea
of Kuhlmann41 is followed, where the methylation of
DPC with diazomethane is described. Further adaptions
(MDPC)9,21
.
Its
environmental fate, however, are still mostly unknown.
This study presents compound-specific stable isotope
analysis (CSIA) as an alternative approach to identify a
compound's origin and transformation by analyzing
stable isotope ratios at natural abundance22. As herbicides
deriving from different manufacturers may differ in their
13C/12C and/or 15N/14N isotopic signatures, isotope
analysis enables a distinction between different sources.
In particular, DPC contains the same nitrogen atoms as
its parent compound CLZ so that it is expected to show
also the same nitrogen isotope signature - provided that
the isotope ratio is not changed by isotope effects during
degradation. In contrast, only part of the carbon atoms of
CLZ are transferred to DPC, because it is formed by
cleavage of the phenyl-ring from the heterocyclic
pyridazine-ring (see structures in Table S1) so that DPC
may show a different carbon isotope signature compared
to CLZ. Carbon isotope analysis, however, may still be
particularly insightful, because changes in isotope ratios
of DPC may be detected by CSIA to deliver evidence
about formation and (bio)degradation of this persistent
metabolite. Since molecules with light isotopes are
usually degraded more rapidly than those with heavy
isotopes, transformation leads to an enrichment of heavy
isotopes in the fraction of remaining pesticide8. This
increase in the isotope ratio (e.g., 13C/12C) can therefore
give evidence of the degradation of the compound8. By
combining both elements in the form of a dual-element
isotope plot, further information about the reaction
mechanism of a compound’s degradation or its origin can
be gained23.
described
by
Mogusu
et
al.24
use
(trimethylsilyl)diazomethane (TMSD), a less explosive
substitute compared to diazomethane, to methylate polar
organic compounds42,43. For diazomethane and TMSD
the control over the isotope value of the additional carbon
atom is lost since no reproducible isotope effects are
expected31. As the methylation leaves the 15N/14N ratio
unaffected, however, this approach is well suitable for
nitrogen isotope analysis.
Following these two approaches, this study
demonstrates the feasibility of dual-element isotope
analysis of
a very polar and fairly ubiquitous
environmental contaminant using complementary
methods for LC-IRMS and GC-IRMS. The development
of a precise and true method44 for LC-IRMS and GC-
IRMS to measure 13C/12C and 15N/14N isotope ratios
of DPC is presented. The developed methods were
optimized and a feasibility study tested the applicability
to environmental seepage water to probe for formation of
DPC from different sources simulating a typical field
situation.
Even though methods for carbon- and nitrogen-isotope
analysis exist for several pesticides and their
metabolites8,24-30,
most
CSIA
methods
of
environmental compounds have focused so far on GC-
amenable compounds. CSIA is typically accomplished by
coupling gas chromatography (GC) to isotope ratio mass
spectrometry (IRMS). Like most polar organic
compounds, however, DPC is not amenable to GC as it
decomposes before reaching a boiling point (see Table
S1). To analyze the isotopic composition of such polar
EXPERIMENTAL / METHODS
Chemicals.
Desphenylchloridazon
(5-Amino-4-
chloro-3-pyridazinone, CAS no.: 6339-19-1) was
obtained from BASF (99.8%, Limburgerhof, Germany).
Methyl-desphenylchloridazon
(5-amino-4-chloro-2-
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methyl-3(2H)-pyradizone, CAS no.: 17254-80-7) was
3.9 mm  20 mm, 100 Å, Waters) and an Atlantis T3
column (3 µm, 3 mm  100 mm, 100 Å, Waters)
operated at 500 μL/min isocratically with a pH 2
phosphoric acid solution at room temperature. Isotopic
ratio measurements were carried out on a Delta V
Advantage IRMS coupled to the LC system by an Isolink
interface (Thermo Fisher Scientific). The eluting
compounds were quantitatively oxidized using oxidant
(90 g/L Na2S2O8) and phosphoric acid (1.5 M H3PO4),
each introduced at a flow rate of 30 μL/min in the
oxidation reactor held at 99.9 °C. Before use, the reagent
solutions were degassed in an ultrasonic bath under
vacuum for 30 min. To avoid re-uptake of CO2, all
solutions were continuously sparged with helium during
use. In order to avoid clogging in the system, an in-line
filter with a pore size of 5 μm (Vici, Schenkon,
Switzerland) was placed in front of the oxidation reactor
of the LC-IsoLink interface. The ion source was held at 2
× 10-6 mbar, the accelerating voltage was 3 kV, and ions
were generated by electron ionization at 124 eV. The
injection volume ranged between 10 and 100 µL. Peak
identification was based on retention times in comparison
with external standards. The LC-IRMS system and data
collection were controlled using Isodat 3.0 software
(Thermo Fisher Scientific).
purchased from LGC Standards GmbH (Wesel,
Germany). Chloridazon (≥98%, CAS no.: 1698-60-8) and
Acetochlor (96.3%, CAS no.: 34256-82-1) were sourced
from Chemos GmbH & Co. KG (Regenstauf, Germany).
Desethylatrazine (purity not available, CAS no.: 6190-
65-4) was produced by Synchem (Felsberg, Germany).
(Trimetylsilyl)diazomethane, 2.0 M dissolved in diethyl
ether (CAS no.: 18107-18-1, acute toxicity and health
hazardous), sodium persulfate (≥99.9%, CAS no.: 7775-
27-1) and phosphoric acid (≥85%, CAS no.: 7664-38-2)
were supplied by Sigma Aldrich (Merck KGaA,
Darmstadt, Germany), while methanol (≥99.9%, CAS
no.: 67-56-1) and acetone (≥99.9%, CAS no.: 67-64-1)
were received from Roth (Karlsruhe, Germany).
Ultrapure water was derived from a Millipore DirectQ
apparatus (Millipore, Bedford, MA, USA).
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EA-IRMS Measurement for Determination of
Reference Values. Carbon and nitrogen composition of
our in-house standards of CLZ, DPC and MDPC were
characterized by an elemental analyzer-isotope ratio mass
spectrometer (EA-IRMS) as described in Meyer et al.45.
A system consisting of an EuroEA (Euro Vector, Milano,
Italy) was hyphenated to a Finnigan MAT 253 IRMS via
a FinniganTM ConFlow III interface (Thermo Fisher
Scientific, Bremen, Germany). The standards were
calibrated against the organic referencing materials USG
40 (L-glutamic acid), USG 41 (L-glutamic acid) and
IAEA 600 (caffeine) provided by the International
Atomic Agency (Vienna, Austria).
Derivatization
Procedure
with
(Trimethylsilyl)diazomethane (TMSD). Derivatization
of DPC was accomplished based on the method of
Kuhlmann41 using diazomethane, as previous attempts
with TMSH and methanol / BF3 had been unsuccessful
(data not shown). However, due to the classification of
diazomethane as toxic and explosive, here the more stable
(trimethylsilyl)diazomethane (TMSD) was tested as a
less explosive substitute. Reaction of the target analyte
with TMSD forms diazomethane in situ, which
subsequently methylates the analyte (see Scheme 1) to
form MDPC. The derivatization of DPC with TMSD was
carried out offline in 20 mL headspace vials. A 250 mg/L
standard of DPC, dissolved in methanol, was used for
method development. Derivatization of the target analyte
was evaluated at different temperatures (50°C and 70°C,
Figure S5), by varying reaction times (data not shown),
and with different TMSD-to-analyte ratios. TMSD-to-
analyte ratios varied between 90 and 230, which
corresponds to 80 µL to 200 µL of a 2 M TMSD solution
in diethyl ether added to 1 mL of a 250 mg/L DPC
solution. After adding the TMSD, the vial was tightly
crimped and placed for 2 h into a heated water bath.
Afterwards, the methanol was evaporated until complete
dryness using a gentle stream of nitrogen. As tested with
standards, no nitrogen isotope fractionation was
introduced during evaporation. The residue was
reconstituted 3 times with acetone and transferred into a
GC vial with a 200 µL insert. The final reconstitution
volume for isotope measurements was 200 µL. The limit
of precise isotope analysis and the method’s trueness was
The carbon (δ13C) and nitrogen (δ15N) isotope values
are reported in per mil relative to PeeDee Belemnite (V-
PDB) and air, respectively, according to the equations 1
and 2:
13C/12CSample
-
13C/12CReference
δ13C =
(1)
13C/12CReference
15N/14NSample
-
15N/14NReference
δ15N =
(2)
15N/14NReference
For carbon analysis by LC-IRMS, δ13C values were
determined relative to our laboratory CO2 monitoring gas,
which was introduced at the beginning and the end of
each analysis run. δ15N values were determined
analogously relative to our laboratory N2 monitoring gas.
Both gases were previously calibrated against RM8563
(CO2) and NSVEC (N2), supplied by the International
Atomic Energy Agency (IAEA).
Isotope Analysis by LC-IRMS. High-performance
liquid chromatography (HPLC) was carried out on a
Dionex system consisting of an Ultimate 3000 HPLC
pump and an Ultimate 3000 autosampler (Thermo Fisher
Scientific). Chromatography was performed with an
Atlantis
T3
Sentry
guard
column
(3 µm,
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determined using varying concentrations of the DPC
standard (5 mg/L to 1000 mg/L).
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Scheme 1: Derivatization reaction of DPC with TMSD with methanol as a catalytic converter, the formation of the
by-product during derivatization is shown in blue; the difference between the methylation of the amino-group is
highlighted in red
GC-IRMS Conditions for Nitrogen Isotope
Analysis. For the analysis of δ15N isotope ratios, a GC-
IRMS system consisting of a TRACE GC Ultra gas
chromatograph (Thermo Fisher Scientific, Milan, Italy)
coupled with a Finnigan MAT 253 isotope ratio mass
spectrometer (IRMS) (Thermo Fisher Scentific, Bremen,
Germany) was used. Both instruments were linked via a
Finnigan Combustion III interface (Thermo Fisher
Scientific). The IRMS was operated at a vacuum of
2.1  10-6 mbar, an accelerating potential of 9 kV and an
emission energy of 2 mA. For combustion of the target
analyte, a NiO tube/CuO-NiO reactor (Thermo Fisher
Scientific) was used at a temperature of 1030 °C. The gas
chromatograph was equipped with a DB-1701 column
(J&W Scientific, Santa Clara, CA) with a length of 30 m,
an inner diameter of 0.25 mm and a film thickness of
1 µm. The instrument was operated with helium carrier
gas (grade 5.0) at a flow rate of 1.4 mL/min. Splitless
injection was performed into a splitless liner at 250 °C
(Thermo Fischer Scientific, Australia). The GC
temperature program started at 100 °C and was held for
1 min, followed by a temperature ramp of 25 °C/min to
240 °C, followed by another temperature ramp of
10 °C/min until the final temperature of 280 °C was held
for 5 min. In contrast, for on-column injection, the flow
and injector temperature were controlled by an Optic 3
device (ATAS, GL Science, Eindhoven, Netherlands)
equipped with a custom-made glass on-column liner.
Samples were injected using a PAL autosampler (CTC
Analytics AG, Zwingen, Switzerland). The ATAS
injector had an initial temperature of 50 °C, held for 300 s
and was then ramped with 4 °C/s to 250 °C. The split
flow started at 14 mL/min. After injection, the split flow
was set to 0 mL/min for 120 s and finally set to its initial
value of 14 mL/min. Simultaneously, the flow rate started
at 0.3 mL/min (held for 120 s) and was increased to
1.4 mL/min within 120 s. Meanwhile, the initial
temperature of the GC oven was set to 40 °C, held for
1 min, ramped by 25 °C/min to 240 °C, held for 0 min,
ramped with 10 °C and held for 5 min. The injection
volume ranged between 1 and 3 L for splitless injection
and 1 and 4 L for on-column injection. To control the
system and to verify the method, retention times and
isotope values were constantly monitored by bracketing
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samples with in-house standards of desethylatrazine
(DEA), acetochlor (ACETO) and MDPC.
(collected from a lysimeter site, described in detail by
Torrentó et al.47). Thereto, 30 μg/L CLZ (δ15N = -31.5
± 1.0 ‰) were spiked into 10 L seepage water that
contained 10 µg/L DPC (δ15N = -15.1 ± 1.0 ‰)
originating from another CLZ source from previous
experiments. The samples were then stored at 13 °C in
the dark over various periods of time (0 to 11 months).
Subsequently, the concentration of CLZ, DPC and
MDPC was measured with ultrahigh performance liquid
chromatography (UHPLC) (see the Supporting
Information II.2.). The nitrogen isotope values of DPC
were determined with derivatization-GC-IRMS. To this
end, samples were concentrated using the solid-phase
extraction procedure by Torrentó et al.48 (see the
Supporting Information II.3. and Figure S1). Prior to GC-
IRMS analysis, preparative HPLC was required as an
additional clean-up step. Method details are described in
the Supporting Information II.4. and Figure S2.
Correction Procedure of Isotope Values. All
reported isotope ratios are expressed as arithmetic means
of three replicate measurements with their respective
standard deviations (± σ). For LC-IRMS, calibration was
performed using in-house standards and monitoring gas
peaks allocated throughout the chromatograms. Trueness
of the LC-IRMS system was achieved by correction with
a bracketing method using a DPC standard (Table S2),
whose signature had previously been determined by EA-
IRMS.
For correction of δ15N isotope values, two approaches
were applied. In the first measurement campaign, as there
was no MDPC standard within the required concentration
range commercially available, a correction based on the
comparison with DEA and ACETO was used to test for
the trueness of isotope values after conversion to N2 in
the combustion furnace. The EA-IRMS values (Table S2)
of these standards were plotted against the measured GC-
IRMS values. The differences were used to correct values
of the derivatized DPC analyte. DPC was measured by
three laboratories (Table S3) to increase the accuracy and
thus reduce measurement errors deriving from other
analytical methods. In the second measurement
campaign, authentic MDPC synthesized by LGC
Standards GmbH was used so that the principle of
identical treatment by Werner and Brand46 could be
applied, and drifts during measurements as well as
differences within the combustion efficiency were
corrected directly.
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Results and Discussion
DPC-Carbon Isotope Analysis. To determine the
limit of precise isotope analysis of the LC-IRMS method,
a DPC standard was injected at concentrations between
2.8 and 133 nmol
C on column (Figure 1). A
chromatogram is shown in Figure S4. The limit of precise
isotope analysis was determined with the moving mean
procedure described by Jochmann et al.49 using an
uncertainty interval of ± 0.5 ‰. This limit obtained for
carbon isotope analysis of DPC measured by LC-IRMS
was 27.5 nmol C on column (996 ng DPC on column),
which corresponds to an injection of 50 µL of a 0.14 mM
(20 mg/L) solution of DPC. This value is within the range
of detection limits previously determined for other
Peak Identification and Quantification with GC-
compounds analyzed by LC-IRMS25,50
.
qMS. Gas chromatography
–
quadrupole mass
spectrometry (GC-qMS) measurements were carried out
to identify MDPC and any co-products generated during
derivatization. The instrumental set-up is described
within the Supporting Information II.1. One microliter of
a derivatized 250 mg/L solution was injected and
measured in scan mode. MDPC was identified using the
presence of mass-to-charge ratios 159 and 145 as
qualifier ions. Additionally, the retention time and spectra
were confirmed by measuring the non-derivatized
authentic standard of MDPC.
Isotope Ratios of Commercially Available
Chloridazon Products
- Source Fingerprinting:
Carbon and nitrogen isotope ratios of CLZ standards
from different suppliers (see Table S4) were analyzed to
check whether CLZ standards deriving from different
suppliers show different isotopic signatures as a result of
industrial production. All samples were measured with
the EA-IRMS method already described.
Evolution of Isotope Ratios Deriving from Different
Chloridazon Sources: The developed method was
applied to investigate whether it is possible to track DPC
deriving from different CLZ sources in seepage water
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The method showed good reproducibility of δ13C
values, with a mean value of -14.6 ± 0.5 ‰ for 80
individual injections of 27.5 nmol C of DPC on column
comprising different measurement sequences over a time
of 3.5 months (Figure 1b). A mean absolute offset of
+3.3 ‰ between the average value determined by LC-
IRMS and the EA value was measured. Such a difference
between LC-IRMS values and EA-IRMS values has been
previously observed for amino acids50,51, caffeine and
ethanol52, pharmaceuticals53, and bentazone31. Several
analyses in Flow Injection Analysis (FIA) mode (i.e.
bypassing the LC column) resulted in the same offset
between EA values and FIA-IRMS values (data not
shown). This observation suggests incomplete wet
oxidation of DPC rather than a chromatography-related
issue as a reason for this offset. Attempts to optimize
oxidation conditions neither led to a reduced offset, nor
to a higher intensity of the DPC peak. As the δ13C values
obtained by LC-IRMS were reproducible, the resulting
offset was constant and could be corrected accordingly.
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Derivatization of DPC – Nitrogen Isotope Analysis.
As shown in Figure 2, DPC derivatization resulted in
MDPC and its isomer 4-chloro-5-(methylamino)-3(2H)-
pyridazone as a major by-product, as well as a minor by-
product deriving from the reaction of TMSD with itself.
Both products were identified by GC-qMS. Additionally,
MDPC was verified using and authentic standard. For
method development and optimization purposes, the
yield of derivatized DPC was tested by GC-qMS for two
temperatures, 50 °C and 70 °C, maintaining the same
TMSD-to-analyte-ratio (expressed as molar ratio
(n(TMSD):n(analyte) ratio). Temperature dependence
was minor, indicating robustness of the method. A
slightly higher yield of the target analyte (derivatized
DPC) was achieved at a temperature of 70 °C (Supporting
Information III.2, Figure S5), thus, method validation at
the GC-IRMS was continued using this temperature for
derivatization. The ratio of the isomer to the target analyte
remained at approximately 1/10, unaffected by the
temperature. The recovery of derivatized DPC at 70°C
was approximately 65 %, which was quantified using an
authentic standard at different concentrations (R2 > 0.99,
data not shown).
Figure 1: a) Correlation of amount-dependency tests for
carbon isotope values as well as the amplitude by LC-IRMS.
Grey circles represent the average intensity for each amount
on column, while black diamonds represent the average
corresponding delta value of replicate measurements; The
limit of precise isotope analysis was determined following
the procedure described by Jochmann et al.49 and is shown
by the grey rectangle. The grey horizontal line stands for the
mean of all values with intensities above the gray rectangle,
b) Reproducibility of carbon isotope values (blue diamonds)
of DPC with LC-IRMS, the results are stated as the deviation
of the measured value from the value determined by EA-
IRMS (Δδ13C); the blue line shows the average carbon
isotope values ± 0.5 ‰ (dashed lines), the black line
represents the EA δ13C value of DPC ± 0.5 ‰ (dashed lines).
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Figure 2: Chromatogram of DPC derivatized with TMSD showing the derivatization products MDPC (red box) and the reaction
by-product 4-chloro-5-(methylamino)-3(2H)-pyridazon (grey box). 2,6-dichlorobenzamide (BAM) was used as an internal
standard. An authentic standard of MDPC was applied for peak identification.
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Figure 3a shows the measured δ15N isotope values of
conservative approach. The δ15N isotope values show a
deviation from the EA-IRMS value (Δδ15N) of -
1.6 ± 0.4 ‰ (black markers in Figure 3b) that can be
corrected for. Since the pure non-derivatized standard of
MDPC shows a similar off-set (red markers in Figure 3b),
we conclude that this deviation results from incomplete
combustion of the target analyte rather than from
isotopically sensitive branching due to formation of the
major by-product during derivatization.
250 mg/L DPC derivatized with increasing excess of the
derivatization reagent TMSD. A plateau of the δ15N
isotope value is reached at an excess of TMSD of greater
than 150 n(TMSD):n(analyte) indicating optimum
transformation of DPC to MDPC at this proportion.
Following the approaches of Reinnicke et al.31 and
Mogusu et al.24, further method validation was carried out
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with an excess of 160 n(TMSD):n(analyte) as
a
Figure 3: δ15N values of DPC in a) dependence on the excess of TMSD used for the derivatization procedure, b) the reproducibility
of δ15N values of derivatized DPC (black diamonds) and MDPC (red diamonds) measured with GC-IRMS; and c) δ15N values of
DPC and the amplitude (blue circles) in dependence on the amount of nitrogen of derivatized DPC injected onto the column to
determine the limit of precision – the amount of derivatized DPC equals the initial amount of DPC used for derivatization; black
diamonds show the δ15N isotope values using splitless injection, while the white diamonds show the precision gained with on-
column injections; data was corrected for the off-set caused by combustion efficiency; the grey rectangle marks the limit of precise
nitrogen isotope analysis. The result is stated as the deviation of the measured value from the value determined by EA-IRMS
(Δδ15N); the red line shows the average δ15N isotope value and its tolerated standard deviation of ±1 ‰ (red dashed line); (the black
line shows the target isotope value determined with the EA, while the dashed lines indicate the tolerated standard deviation of ±1 ‰
Figure 3c shows the nitrogen isotope values of DPC
derivatized with an excess of TMSD greater than
n(TMSD):n(analyte) = 150 (140 µL of a 2 M TMSD
solution on 1 mL of a 5 mg/L to 1000 mg/L analyte
solution) injected with two different injection techniques.
All values were corrected for the offset due to incomplete
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combustion. For comparison, the EA-IRMS reference
Supporting Information (Table S5). The spiked seepage
water was used to test whether a mixture of the nitrogen
isotope value of DPC deriving from the spiked CLZ and
the DPC already present in the water could be observed
over a defined time period, simulating a typical field
situation.
value is shown as black line. The limit of precise nitrogen
isotope analysis of DPC is, as expected, amplitude-
dependent. For splitless injection, this limit is equal to
31 nmol N derivatized DPC injected, corresponding to an
injection of 1.2 µg non-derivatized DPC. Additionally,
on-column injection was tested as a more sensitive
method. In accordance with the findings of Schreglmann
et al. for sensitive isotope analysis of atrazine 54, on-
column injections of the derivatized DPC resulted in a
decrease of the limit of precise isotope analysis by a
factor of 10 as shown in Figure 3b. Thus, 2.06 nmol N of
derivatized DPC on-column (100 ng DPC on column)
were sufficient for accurate results, which corresponds to
an injection of 1 µL of a 0.69 mM DPC-solution.
Concentration measurements of CLZ, DPC and MDPC
in the seepage water (Figure 5, upper panel and Table S6
in the Supporting Information) showed a significant
decrease in CLZ concentration (white) after 7 months (t1)
and concentrations below the limit of detection after
11 months (t2). Simultaneously, the DPC concentration
increased over time consisting of the initial DPC (shaded
grey) and newly formed DPC from degraded CLZ
(white). After 8 months, the concentration of DPC
remained constant (data not shown). The formation of
DPC from CLZ agrees with the findings of Buttiglieri et
al.16 and Schuhmann et al.21 in environmental samples,
where CLZ was degraded within the first 8 to 12 weeks
after application on an agricultural field.
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Isotope Ratios of Commercially Available
Chloridazon Products - Source Fingerprinting: δ13C
and δ15N EA-IRMS measurements of commercially
available CLZ products were used to investigate the
possibility to distinguish between different sources. The
results are shown as a dual-element isotope plot in Figure
4. There is a significant variability for both elements.
δ15N isotope values ranging from -5.7 ‰ to -32.0 ‰ were
measured (Table S4). As both, CLZ and DPC, contain the
identical N-atoms, the metabolite can be related to the
parent based on their nitrogen isotope compositions. This
highlights the potential of δ15N values of DPC to serve as
a fingerprint to retrace the parent compound CLZ.
The corresponding nitrogen isotope values are shown
in the panel below (Figure 5). Concomitant with the
disappearance of CLZ by reaction a shift in δ15N of DPC
towards the isotopic composition of the added CLZ (-31.5
±1.0 ‰) was observed. Formation of MDPC was small
(the ratio of MDPC to DPC was always smaller than
10 %) so that its influence on the DPC nitrogen isotope
and its contribution to the mass balance in the samples
can be neglected. Also, the interference of MDPC with
derivatized DPC on the nitrogen isotope value remains
within the uncertainty of the presented isotope analysis.
In the case that this ratio is greater in environmental
samples, fractionative HPLC can be used to separate the
two analytes prior to derivatization-GC-IRMS
(Supporting Information II.5).
In contrast to nitrogen isotope values of CLZ, the
detected variability of its δ13C values cannot directly be
used to conclude on the carbon isotope signature of DPC
because cleavage of the phenyl-ring causes differences in
the isotopic signature between parent compound and
metabolite.
As the initial nitrogen isotopic composition as well as
the concentrations of both DPC and CLZ are known, a
two sources-mixing model, based on the weighted
arithmetic mean of the isotope ratio, was applied to
investigate whether DPC nitrogen isotope values
accurately reflect the relative contribution of either
source. To this end, it is assumed that all additional DPC
is formed from CLZ and calculations were based on the
EA-IRMS values of the CLZ that was applied. The
differences between the measured points and the
calculated isotope values (dashed lines) of Figure 5
(lower panel) were less than 1 ‰ and thus within the
measurement uncertainty of the instrument. This
indicates that nitrogen isotope values of DPC did indeed
reflect the relative contribution of the DPC from different
origin and, therefore, the approach holds promise for
future source elucidation of the CLZ metabolite in field
samples.
Figure 4: Dual-Isotope plot of Chloridazon standards derived
from different suppliers.
Evolution of Isotope Ratios of DPC from Different
Chloridazon Sources: The developed method was applied
to DPC-containing environmental seepage water spiked
with CLZ. Its original composition is listed in the
We note that the mass balance does not close for DPC
formation from CLZ (Figure 5). Possible explanations are
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either (a) that part of the CLZ was degraded without
reproducible, precise and true carbon and nitrogen
compound-specific stable isotope analysis of DPC in the
µg/L –concentration range (996 ng and 100 ng DPC on
column for carbon and nitrogen isotope analysis,
respectively). Taking reported DPC concentration of
0.72 µg/L to 7.4 µg/L in surface and ground water into
account 16, the combination of the presented methods with
large-volume extraction as presented by Torrentó et al.48
enables the isotopic analysis of DPC in environmental
water samples. Thus, the application of the developed
forming DPC (potentially producing biomass) or (b) that
DPC was degraded via a so far unknown transformation
pathway that did not entail nitrogen isotope fractionation.
Evidence against the second hypothesis, however, is
given by our observation that after complete CLZ
degradation the concentration of DPC remained constant
(data not shown). While further investigations into this
matter are beyond the scope of this feasibility test, the
possibility to add also carbon isotope analysis to the
picture – as newly established in this contribution, but not
yet pursued in this feasibility test – provides an added
value to probe not only for formation of metabolites from
different sources, but also for their further degradation.
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methods brings forward
a basis for analysis of
environmental water samples from field surveys, and the
combination of the developed methods gives access to
dual-element isotope plots. Our study highlights the
potential of such plots to distinguish different sources.
Future DPC degradation studies may use such dual
element isotope information to obtain additional
information about transformation pathways of DPC and
underlying
mechanisms55.
Until
now,
only
transformation to MDPC is known, which was, however,
observed to occur on longer time scales than in our
experiment21. Additionally, as shown in the degradation
experiment of chloridazon, these methods can be used to
distinguish the source of DPC by measuring the nitrogen
isotope signature and to identify the mixing of DPC
deriving from different CLZ sources.
ASSOCIATED CONTENT
SUPPORTING INFORMATION
Further details on analyte properties, isotope standards,
analytical methods and method validation. The Supporting
Information is available free of charge on the ACS
Publications Website.
AUTHOR INFORMATION
Corresponding Author
*Phone: + 49 89 2180 78232; fax: + 49 89 2180 78255; e-
mail: m.elsner@tum.de
Present Addresses
†Grup MAiMA, Departament de Mineralogia, Petrologia
i Geologia Aplicada, Facultat de Ciències de la Terra,
Universitat de Barcelona (UB), C/ Martí i Franquès s/n,
08028, Barcelona, Spain.
Figure 5: Degradation of CLZ to DPC over time and the
resulting change of the δ15N value of DPC due to two
different sources of CLZ. Measured δ15N values are shown
as circles, while the dashed lines are the corresponding
calculated δ15N value based on the mixing of the two CLZ
sources originating from the initial δ15N of DPC t0 and the
spiked CLZ (initial δ15N shown as black dashed line). It is
assumed that the CLZ is degraded completely to DPC.
Samples were taken directly after spiking with CLZ (t0) and
after storage for 7 months (t1) and 11 months (t2).
Author Contributions
‡These authors contributed equally as first authors to this
work.
Notes
The authors declare no competing financial interest.
CONCLUSION AND OUTLOOK.
ACKNOWLEDGMENT
This study was supported by the project CRSII2_141805/1
from the Swiss National Science Foundation (SNSF). The
With LC-IRMS and GC-IRMS, this study brings
forward two complementary approaches to accomplish
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authors would like to thank the NPAC (UNiNE) and Jakov
Bolotin (Eawag) for their help in the laboratory as well as
Doris Ebert from BASF for providing the chemical DPC.
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6
ACS Paragon Plus Environment