Trimethylation Enhancement Using Diazomethane TR en Di Enables Enhanced Detection of Glufosinate and 3 Methylphosphinico Propionic Acid From Complex Canola Samples

Trimethylation enhancement using diazomethane
(TrEnDi) enables enhanced detection of glufosinate
and 3-(methylphosphinico)propionic acid from
complex canola samples
Christian A. Rosalesa,c, Krysten L. Sheedya,c, Karl V. Wasslena,c, Jeffrey M.
Manthorpea,b,c*, Jeffrey C. Smitha,b,c*
a) Department of Chemistry Carleton University, Ottawa, Ontario K1S 5B6, Canada
b) Institute of Biochemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada
c) Carleton Mass Spectrometry Centre, Carleton University, Ottawa, Ontario K1S 5B6,
Canada
*to whom correspondence should be addressed – jeff.smith@carleton.ca,
jeff.manthorpe@carleton.ca
Keywords: herbicide, glufosinate, glufosinate-ammonium, 3-

(methylphosphinico)propionic acid, diazomethane, TrEnDi, mass spectrometry, tandem
mass spectrometry, MRM
https://doi.org/10.26434/chemrxiv-2023-vl805 ORCID: https://orcid.org/0000-0003-1410-5797 Content not peer-reviewed by ChemRxiv. License: CC BY-NC 4.0
Abstract:
Over the past century, agriculture practices have transitioned from manual
cultivation to the use of chemical herbicides for improved crop yields. The most common
weedkiller glyphosate (GLY) has been used exponentially worldwide, leading to the
emergence of GLY-resistant weeds. This has prompted a need for effective alternatives,
leading to the development and increasing use of phosphinothricin, also known as
glufosinate (GLUF). As the agricultural application of GLUF rises, the potential for long-
term residual exposure in the food chain increases, highlighting the need for improved
analytical strategies for its detection, as well as its main breakdown product 3-
(methylphosphinico)propionic acid (MPPA). Chemical derivatization strategies have been
developed to improve the detection of GLUF and MPPA via liquid chromatography
tandem mass spectrometry (LCMS) analyses. In this article, we expand the use of
trimethylation enhancement using diazomethane (TrEnDi) to quantitatively derivatize
these analytes into permethylated GLUF ([GLUFTr]+) and MPPA ([MPPATr+H]+).
Comparing [GLUFTr]+ and [MPPATr+H]+ to underivatized counterparts, TrEnDi yields 2.8-
fold and 1.7-fold improvements in reversed-phase chromatographic retention,
respectively, while MS-based sensitivity is enhanced 4.1-fold and 11.0-fold, respectively.
Initial analytical improvements were performed on commercial standards; however,
successful analyte derivatization (with >99% yields) was also demonstrated on a
commercial herbicide solution imparting consistent analytical enhancements. To
investigate the benefits of TrEnDi in a bona fide agricultural scenario, the quantities of
GLUF and MPPA were determined in aqueous extracts from field-grown canola plants
before and after TrEnDi derivatization. In their underivatized forms, GLUF and MPPA
were undetectable in all field samples whereas [GLUFTr]+ and [MPPATr+H]+ were readily
quantifiable using the same analysis conditions. Our results demonstrate that TrEnDi
continues to be a useful tool to enhance the analytical characteristics of organic
molecules that are traditionally difficult to detect.
Introduction:
Advancements in biotechnology during the 20th century led to the development of
non-transgenic crops that showed selective resistance to herbicides as well as transgenic
crops that were genetically engineered to be insusceptible to specific non-selective
herbicides. The non-transgenic herbicide approach has proven to be effective,1–3 however
demonstrates inferior crop market share and weed regulation compared to more
economical transgenic-based herbicides.4 An example of this is glyphosate (GLY), the
non-selective, broad-spectrum herbicide with 8.6 billion kilograms being applied globally
since 1974. GLY is currently the most widely used herbicide, amassing a 15-fold increase
since the introduction of “Roundup Ready” genetically modified GLY-resistant crops in
1996.5 Regrettably, annual and exponential use of GLY-containing solutions have given
rise to 38 known species of GLY-resistant weeds,6 prompting a need to invent similarly
effective transgenic herbicides.
An alternative to GLY is the non-selective, broad spectrum, contact herbicide
phosphinothricin (2-amino-4-methyl-phosphino-butyric acid), or glufosinate (GLUF),
originally discovered through the study of Streptomyces viridochromogenes and S.
hygroscopicus.7,8 Introduced in 1984, GLUF has been primarily sold and distributed as an
ammonium salt under brands such as Basta®, Challenge®, Finale®, Ignite®, and Liberty®,
where it is applied following the emergence of crops to restrict the growth of a wide range
of weeds and to desiccate crops prior to harvest. In 2022, GLUF had an annual global
market size of $422 million USD, with projections forecasting an increase to $816 million
USD by 2029.9 Phytotoxic L-phosphinothricin is released within the plant through the
metabolism of the tripeptide bialaphos (comprised of L-phosphinothricin and two L-alanine
residues) via hydrolytic cleavage of the two L-alanine residues.10,11 Being a phosphinic
acid analogue of glutamic acid, GLUF has been found to be an active inhibitor of the
enzyme glutamine synthetase (GS), which plays a critical role in the metabolism of amino
acids and detoxification of ammonia.7,12 Inhibition of GS results in the accumulation of
reactive oxygen species and ammonia, where lipid peroxidation and plant death occurs. 13
Toxicological studies of GLUF in mammals have shown slight toxicity through oral
exposure, exhibiting inhibition of GS in brain, liver, and kidney tissues, partially halting
ammonia metabolism;14 however, its exposure indicated no teratogenicity, mutagenicity,
or carcinogenicity.15 Due to the mobility and persistence of GLUF and its metabolite 3-
(methylphosphinico)propionic acid (MPPA) in soils and waters,16,17 their environmental
fate and prevalence in the food chain are unpredictable, raising questions regarding their
impact on human health following long-term residual exposure.18,19
The zwitterionic and amphoteric properties of GLUF and MPPA coupled with their
low masses, high boiling points, lack of chromophores, and high polarities20 has led to the
use of hydrophilic interaction liquid chromatography (HILIC)21 and the Quick Polar
Pesticides (QuPPe) method22 for their analysis. Alternatively, chemical derivatization
coupled with gas chromatography (GC) or liquid chromatography (LC) mass spectrometry
(MS) has proven to be an advantageous analytical strategy for the improved analysis of
GLUF. Derivatization of GLUF using N-isopropoxycarbonyl methyl ester provides
improved sample volatility for GC,23 9-fluorenylmethylchloroformate (FMOC-Cl) enables
improved analyte selectivity on C18 columns,24 p-nitrobenzoyl chloride (PNBC), dansyl
chloride, and 4-chloro-3,5-dinitrobenzofluoride (CNBF) allows for ultraviolet (UV)
detection,25–27 while acetate/acetic anhydride and trimethyl orthoacetate provide a simple
and rapid derivatization method for GLUF and MPPA.28,29 Despite progress in this area,
the potential for trace amounts of GLUF and MPPA in environmental and food matrices
highlights the need for new derivatization strategies that are simple and instantaneous,
at the same time improving both the chromatographic and MS properties of these
analytes.
In recent years, trimethylation enhancement using diazomethane (TrEnDi) has
emerged as a rapid derivatization technique to increase the MS- or tandem MS (MS2)-
based sensitivity of amine containing biomolecules through the formation of fixed,
permanent positive charge(s). TrEnDi has been used to derivatize peptides and lipids
using ethereal diazomethane (DZM),30,31 13C-labeled DZM,32 and gaseous DZM using an
in situ variant of TrEnDi.33 To improve MS and MS2-based analyses of amino acid-like
molecules, TrEnDi has been used to permethylate GLY and aminomethylphosphonic acid
(AMPA), leading to significant improvements in their analysis using LCMS.34 This work
builds upon these findings, employing TrEnDi to derivatize GLUF and MPPA to >99%
yields, imparting enhanced LC retention, greater MS sensitivity, and lower limits of
detection (LOD) and quantitation (LOQ). The benefits of TrEnDi are demonstrated on
standard solutions, a commercial herbicide solution, as well as on aqueous extracts of
field-grown canola plants to accurately determine the residual quantities of GLUF and
MPPA within relevant environmental samples.
Experimental:
Chemicals and Materials. GLUF, MPPA, caffeine, and tetrafluoroboric acid diethyl ether
complex (HBF4•Et2O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC
grade water with 0.1% formic acid, HPLC grade acetonitrile with 0.1% formic acid, and
methanol were purchased from Fisher Scientific (Hampton, NH, USA). Potassium
hydroxide and diethyl ether were purchased from Caledon Laboratories Ltd.
(Georgetown, ON, Canada). Liberty® 200 SN (BASF, Ludwigshafen, Germany)
glufosinate-ammonium herbicide solution (200 g/L) was acquired as a gift from a local
agriculturalist. Field-grown canola plant samples were obtained from cropland in Douglas,
Ontario, Canada.
Production of Ethereal DZM and Derivatization of Analytes via TrEnDi. Due to the
explosive and toxic properties of DZM, its preparation and use for analyte derivatization
should be carried out in a chemical fume hood with proper ventilation behind a
polycarbonate blast shield. N-methyl-N-nitroso-p-toluenesulfonamide was prepared as
described previously.35 A solution of N-methyl-N-nitroso-p-toluenesulfonamide (5.0 g,
0.02 mol) in 50 mL of diethyl ether was added dropwise (1 drop/sec) from a 125 mL
separatory funnel into a MilliporeSigma DZM generation glassware (Product No.
Z202509) containing a stirred solution of potassium hydroxide (5 g, 0.09 mol), 8 mL
ethanol, and 10 mL water, forming gaseous DZM. The KOH solution was suspended in a
68 °C oil bath on a heated stir plate, with the other half of the DZM generation glassware
containing isopropyl alcohol and dry ice to condense ethereal DZM into a 100 mL round-
bottomed flask. The reaction proceeded for ~1 h before completion, with the ethereal
DZM being carefully sealed and stored at –20 °C until used for analyte derivatization.
For TrEnDi derivatization of GLUF and MPPA standards, Liberty® herbicide
samples, and canola samples, separate solutions were concentrated to dryness using a
stream of N2 (g) in 2 mL HPLC vials. 40 μL methanol was added to dry samples containing
GLUF (5 nmol standards and Liberty® samples, 100 μL aqueous canola sample aliquot)
or MPPA (5 nmol standard samples, 100 μL aqueous canola sample aliquot). Prior to
derivatization, 12 μL (1.2 μmol) of 1.0 M HBF4•Et2O solution in methanol was added.
Using a flame-polished glass pipette, 1 mL of ethereal DZM was transferred into standard
and Liberty® sample vials, with 0.5 mL being used for canola samples. TrEnDi-derivatized
samples were dried down to completeness using a stream of N2 (g) and reconstituted in
water + caffeine internal standard (0.5 μM) prior to analysis via LCMS, or methanol for
analysis using nano-electrospray (ESI)-MS and MS2 and immediately stored at 4 °C.
Reverse Phase High-Performance Liquid Chromatography. 20 µL of each sample
was injected and separated using a Dionex Ultimate 3000 HPLC (Thermo Fisher
Scientific, Waltham, MA) equipped with a 4.6 x 100 mm Agilent Eclipse Plus C18 3.5 µm
column operating at room temperature. Analytes were eluted over a 12.5 min gradient
running at 0.6 mL/min using eluent A (water with 0.1% formic acid) and eluent B
(acetonitrile with 0.1% formic acid): 0 min, 0.5% (B), 1.5 min, 0.5% (B), 7.0 min, 100%
(B), 9.8 min, 100% (B), 10.0 min, 0.5% (B), 12.5 min, 0.5% (B).
ESI-Multiple Reaction Monitoring (MRM) Analysis. The HPLC eluent was introduced
into a QTrap 4000 hybrid triple quadrupole linear ion trap mass spectrometer (Sciex,
Framingham, MA) fitted with a Turbo V ionspray source held at 375 °C in positive ion
mode with a 5000 V ionization voltage, curtain gas of 20, nebulizing gas of 30, and drying
gas of 10. Analysis of GLUF and MPPA was also performed in negative ion mode using
the same parameters with an ionization voltage of -4500 V (Figure S1). Samples were
analyzed using optimized MRM transitions at 20 Hz (50 ms each). Analyte and internal
standard MRM transition pair parameters can be found in Table 1.
Table 1: MRM parameters used for LCMS analyses for data in Figures 3–5.
Precursor Ion Product Ion

Transitions DP (V) EP (V) CE (V) CXP (V)
(m/z) (m/z)
[GLUF+H]+ 182.0 136.0 42.0 4.4 18.6 22.0
[GLUF+H] + 182.0 56.0 42.0 5.4 43.0 9.0
[MPPA+H]+ 153.0 135.2 49.0 5.0 14.0 10.0
[MPPA+H] + 153.0 78.9 43.0 5.0 29.0 13.0
Tr
[GLUF ] * + 252.0 193.0 41.0 4.1 21.0 9.0
[GLUFTr]+ * 252.0 93.0 41.0 5.0 46.0 14.0
Tr
[MPPA +H] + 55.0
181.0 149.0 6.0 16.0 9.0
**
[MPPATr+H]+ 55.0
181.0 93.0 6.0 29.0 9.0
**
Caffeine *** 195.1 138.1 75.0 10.0 28.0 10.0
Caffeine *** 195.1 110.1 75.0 10.0 28.0 10.0
* TrEnDi-derivatized GLUF
** TrEnDi-derivatized MPPA
*** Internal standard
NanoESI-MS and MS2 Analysis. To verify that TrEnDi resulted in >99% yields of
permethylated GLUF ([GLUFTr]+) and MPPA ([MPPATr+H]+) and to determine optimal
MRM transition pairs, direct infusion nanoESI-MS and MS2 was used. Six µL of 100 µM
samples were inserted into separate Proxeon nanoESI emitters (Thermo Fisher Scientific,
Odense, Denmark). Prepared samples were analyzed in positive ion mode by direct
infusion using an AB Sciex QStar XL mass spectrometer equipped with a nanoESI source
(Sciex, Framingham, MA) using a mass range of m/z 10–300, source potential of 1400 V,
declustering potential of 30 V, and focusing potential of 120 V.
Canola Samples. Samples from two separate canola fields (referred to as Field 1 and
Field 2) were collected and analyzed. Canola variety L345PC + Lumiderm was planted in
Field 1 on May 14, 2021, at a rate of 4 lbs/acre and Field 2 on April 24, 2021, at a rate of
5 lbs/acre. Field 1 was sprayed on June 24, 2021 with Liberty® and liquid ammonium
sulfate at rates of 1 L/acre with a watering rate of 15 gallons/acre. Field 2 was sprayed
on June 1, 2021, with Liberty® and liquid ammonium sulfate at rates of 1 L/acre and 2.5
L/acre, respectively, with a watering rate of 12 gallons/acre. Quarterly seasonal rainfall
data estimated 268 mm of precipitation between time of herbicide treatment and plant
harvesting.36 Samples were collected by pulling 5 canola plants randomly from both fields
on September 10, 2021. Canola plants were compartmentalized and separated into
stems, seeds, and shells, being stored in freezer bags at –20 C until analyte extraction.
Three stem samples, three seed samples, and three shell samples were prepared from
plants obtained from Field 1, followed by Field 2, for a total of 18 samples. 2.0045 g ±
0.0018 g of canola stems, 2.0046 g ± 0.0028 g of canola seeds, and 2.0052 g ± 0.0029
g of canola shells were weighed out and placed into separate 20 mL glass vials. 10 mL
of HPLC grade water was added to each vial and agitated using a MultiTherm shaker
(Benchmark, Sayreville, NJ) for 30 min at room temperature and 1000 rpm, followed by
1 min of handheld agitation. The aqueous canola extract samples were passed through
non-sterile 0.22 μm, 25 mm nylon syringe filters, and three 1 mL aliquots of the filtrate
were divided equally between 3 separate Eppendorf® tubes. The aqueous canola filtrates
were concentrated using a Savant SVC-100H SpeedVacTM Concentrator for 2 h. Sample
volumes for concentrated canola extracts were measured and collected into a single
Eppendorf® (3-fold concentrated samples) and stored at 4 °C prior to TrEnDi
derivatization and LCMS analysis. See Figure S2 for a diagram illustrating the
experimental workflow including analyte extraction and TrEnDi derivatization of the
canola samples. Canola sample datasets were extrapolated using Dixon’s Q test to
identify the presence of measurement outliers with a 95% confidence interval.37
Results & Discussion:
ESI-MS and MS2 Analysis of GLUF and MPPA.
To verify analyte derivatization and determine optimal LC-MRM transition pairs, 100
µM (2.0 nmol) analytical standards of GLUF and MPPA were independently analyzed via
ESI-MS and MS2 (Figure 1). High-resolution MS analysis of unmodified GLUF and MPPA
revealed protonated and sodiated ions (Figures 1a, 1c). Protonated forms of GLUF (m/z
182.0505) and MPPA (m/z 153.0332) were subjected to MS2 analysis to determine
quantification and identification transition pairs and enable MRM analysis. [GLUF+H]+
fragmented to form dominant ions at m/z 136.0547 and 56.0505 used for MRM transition
pairs for quantification and identification, respectively, whereas m/z 135.0256 and
78.9989 were used for quantification and identification of [MPPA+H]+, respectively
(Figures 1b, 1d). The [MPPA+H]+ fragment ion at m/z 107.0303 appears to be slightly
more intense than m/z 78.9989 in the high-resolution MS spectrum (Figure 1d); however,
further comparison of the two ion intensities via LC-MRM analysis indicated a greater
signal response for m/z 78.9989.
Historically, LC-MRM analyses of GLUF and MPPA have been performed in both
positive and negative polarities using mobile phase additives for improved analyte
ionization and detection.38,39 Although methods to analyze GLUF and MPPA exist that do
not require derivatization, these analytes can be challenging to determine using reversed-
phase HPLC due to their zwitterionic character, high polarity, and lack of UV-vis
absorption or fluorescence. However, chemical derivatization approaches do exist to
improve the analytical characteristics of GLUF and MPPA as described above.39 In this
work, positive ion mode LC-MRM was used to conduct a head to head comparison of
[GLUF+H]+ and [MPPA+H]+ to [GLUFTr]+ and [MPPATr+H]+, respectively, to directly
establish the analytical enhancements and MS-based sensitivity gains that are afforded
by TrEnDi.
Figure 1: High-resolution MS spectra of 100 µM GLUF and MPPA. a) [GLUF+H]+ MS,
b) [GLUF+H]+ MS2, c) [MPPA+H]+ MS, d) and [MPPA+H]+ MS2. Mass spectra indicate
parent ions (labelled with ionization mechanism indicated) as well as quantification
(labelled with “Q”) and identification (labelled with “q”) fragment ions used for LC-MRM
analysis.
Formation of [GLUFTr]+ and [MPPATr+H]+.
We have previously reported in situ TrEnDi (iTrEnDi) derivatization to enhance the
LCMS-based analysis of GLY and AMPA yielding improved HPLC retention and improved
ionization, boasting 12 and 340-fold gains in MS-based sensitivity, respectively, following
permethylation.34 Due to the structural similarities between GLY/GLUF and AMPA/MPPA,
both TrEnDi and iTrEnDi chemistry were demonstrated to form GLUF and MPPA
permethylated products in >99% yields (Scheme 1). It was determined that both GLUF
and MPPA were sufficiently soluble in methanol and that this solvent enabled the direct
addition of ethereal DZM; for simplicity TrEnDi was used for the experiments in this work.
Molar excess quantities of HBF4•OEt2 and N-methyl-N-nitroso-p-toluenesulfonamide
were used during TrEnDi derivatization to ensure quantitative and reproducible formation
of [GLUFTr]+ and [MPPATr+H]+.
Scheme 1: TrEnDi derivatization of GLUF and MPPA into [GLUFTr]+ and [MPPATr+H]+,
respectively.
TrEnDi derivatization of GLUF resulted in the complete modification of the
monomethyl phosphonate, carboxylic acid, and amino moieties, rendering a fixed,
permanent positive charge on the derivatized analyte. Methylation of carboxylic acid and
amino functional groups to produce a permanently charged ion ameliorate the potential
of alkali metal adduct formation, yielding a consolidated [GLUFTr]+ peak of greater signal
intensity and removing the need for acidic mobile phase modifiers typically used to
promote analyte protonation. Although TrEnDi completely methylates MPPA in a similar
manner, no fixed charge is present on [MPPATr] requiring protonation to form
[MPPATr+H]+.
To enable a comparative LC-MRM study of unmodified versus TrEnDi-modified forms
of GLUF and MPPA, high-resolution ESI-MS spectra of [GLUFTr]+ and [MPPATr+H]+ were
obtained to verify their parent masses and confirm high reaction yields followed by MS 2
analyses to determine their fragmentation behavior (Figure 2). MS of [GLUFTr]+ and
[MPPATr+H]+ yielded parent ions of m/z 252.1422 and m/z 181.0590, respectively
(Figures 2a, 2c). MS2 yielded fragment ions used for MRM transitions: [GLUFTr]+
(quantification, 252.0 → 193.0; identification 252.0 → 93.0) and [MPPATr+H]+
(quantification, 181.0 → 149.0; identification, 181.0 → 93.0) (Figures 2b, 2d).
Figure 2: High-resolution mass spectra of 100 µM [GLUFTr]+ and [MPPATr+H]+. a)
[GLUFTr]+ MS, b) [GLUFTr]+ MS2, c) [MPPATr+H]+ MS, d) and [MPPATr+H]+ MS2. Mass
spectra indicate parent ions (labelled with ionization mechanism indicated) as well as
quantification (labelled with “Q”) and identification (labelled with “q”) fragment ions used
for LC-MRM analysis.
[GLUFTr]+ and [MPPATr+H]+ Demonstrate Enhanced Analytical Characteristics.
LC-MRM analysis of [GLUFTr]+ and [MPPATr+H]+ demonstrated improved
reversed-phase chromatographic retention and MS-based sensitivity, inherently providing
lower limits of detection and quantification for permethylated species when compared to
their unmodified counterparts (Figure 3). Using reversed-phase chromatography, the
relatively hydrophilic unmodified analytes elute at the beginning of the mobile phase
gradient. [GLUF+H]+ eluted at 1.69 min, roughly the same as the dead volume of the
HPLC system, and [MPPA+H]+ eluted at 3.04 min. Following TrEnDi derivatization,
modified analytes demonstrated a notable increase in hydrophobicity through a significant
delay in retention, with retention times of 4.66 min ([GLUFTr]+) and 5.21 min
([MPPATr+H]+). These later elution times enable greater separation from polar
interferences and sample matrix. Additionally, both analytes demonstrated improved
chromatographic peak shapes with higher peak heights enabling enhanced identification
and lower detection limits in complex samples.
Figure 3: LC-MRM analyses of equimolar quantities of a) 0.02 nmol [GLUF+H]+ vs
[GLUFTr]+ and b) 0.1 nmol [MPPA+H]+ vs [MPPATr+H]+ demonstrating the sensitivity
enhancements of TrEnDi-modified analytes compared to their underivatized
counterparts.
To ascertain the extent to which sensitivity is reproducibly improved following
TrEnDi derivatization, analytical standards of GLUF and MPPA were derivatized in
triplicate and compared to unmodified analytes via LC-MRM. Figure 3 illustrates a
comparison of equimolar quantities of [GLUF+H]+ to [GLUFTr]+ (1 µM, 0.02 nmol) and
[MPPA+H]+ to [MPPATr+H]+ (5 µM, 0.1 nmol) to assess MS-based sensitivity
enhancement for the TrEnDi-modified analytes. Peak areas were normalized to a solution
of caffeine (0.5 uM, 0.01 nmol) used as an internal standard. Following modification,
[GLUFTr]+ and [MPPATr+H]+ yielded 4.1-fold and 11.0-fold enhancements in MS sensitivity
over their unmodified counterparts with 5.9% and 2.9% relative standard deviations
(RSDs), respectively, demonstrating the feasibility of improved quantitation for both
TrEnDi-derivatized compounds.
To enable quantitation and determine the analytical figures of merit for both
unmodified and TrEnDi-derivatized GLUF and MPPA, calibration curves for all analytes
were constructed (Figure S3). All calibration curves contained 5-6 data points, were
constructed within the linear dynamic range of the LC-MRM instrument, and had linearity
R2>0.992. The lower and upper ranges of the calibration curves varied based on the
expected concentrations of the analytes from biological samples. Comparing the
permethylated to unmodified calibration curves, the sensitivity was greater for modified
analytes. To calculate LODs and LOQs, the signal standard deviation of a blank injection
±0.5 min from a respective analyte elution time was multiplied by three (LOD) or ten (LOQ)
and divided by the slope of the calibration curve for each analyte. The LOD and LOQ for
[GLUF+H]+ were 4.8 fmol (43.5 ng/L) and 16.0 fmol (145.0 ng/L), respectively and for
[MPPA+H]+, 11.6 fmol (88.0 ng/L) and 38.6 fmol (293.2 ng/L), respectively. The LOD and
LOQ for [GLUFTr]+ were 1.1 fmol (13.5 ng/L) and 3.6 fmol (45.1 ng/L), respectively, and
for [MPPATr+H]+, 1.8 fmol (16.8 ng/L) and 6.1 fmol (55.9 ng/L), respectively. These results
clearly demonstrate that TrEnDi results in an improved LOD and LOQ for these analytes
and highlights the role that TrEnDi may play in their analysis in biological, environmental,
and forensic-based samples.
Derivatization of GLUF and MPPA in a Complex Herbicide Solution.
With optimized TrEnDi conditions established for permethylation of GLUF and
MPPA in standards, the derivatization chemistry was further tested using a herbicide
solution, representing a more complex sample matrix. A stock solution of Liberty® 200 SN
herbicide was created and diluted prior to TrEnDi, as described above. TrEnDi
successfully formed [GLUFTr]+ in the herbicide mixture with no unmodified or
undermethylated products identified. LC-MRM chromatograms of unmodified and
modified GLUF in Liberty® were acquired in chemical triplicate and overlayed (Figure 4a).
Normalized peak areas were used to compare the sensitivity of [GLUFTr]+ to [GLUF+H]+
and to ascertain reproducibility of TrEnDi in a complex sample. A 4.5-fold increase in
sensitivity of [GLUFTr]+ over [GLUF+H]+ was determined in Liberty® with an RSD of 12.6%,
demonstrating similar signal response improvements to experiments on analytical
standards (Figure 3). This demonstrates that TrEnDi is a powerful and reproducible tool
to boosting the sensitivity of GLUF analysis in simple and complex mixtures.
Interestingly, [MPPA+H]+ was not identified in the herbicide solution prior to
derivatization, whereas [MPPATr+H]+ was detected and quantifiable following TrEnDi at
5.9 nM (0.1 pmol, 1.1 µg/L) (Figure 4b). To ensure MPPA was present in Liberty® at
residual and unquantifiable amounts in the herbicide mixture prior to derivatization and
not a byproduct of TrEnDi chemistry, a control experiment was performed where a
standard solution of GLUF was derivatized at 1 µM and 10 µM to analyze for the potential
formation of [MPPATr+H]+ (Figure S4). Upon derivatization of both standard solutions, no
[MPPA+H]+ or [MPPATr+H]+ was identified, demonstrating that TrEnDi does not form
MPPA as a byproduct. Ensuring successful derivatization of both analytes in varying
sample complexities is critical for the application of TrEnDi to real-world sample analyses
as it holds the potential to enable quantitation of trace levels of GLUF and MPPA not
previously detectable.
Figure 4: Overlayed chromatograms of Liberty® herbicide solution (200 g/L) before and
after TrEnDi derivatization for a) [GLUF+H]+ and [GLUFTr]+, demonstrating improved
sensitivity for the permethylated product in a complex matrix. b) [MPPA+H]+ is
undetectable prior to TrEnDi [identical to the background trace (black)], whereas
[MPPATr+H]+ is quantifiable following TrEnDi.
Quantitation of [GLUFTr]+ and [MPPATr+H]+ in Canola Samples.
To demonstrate the feasibility of TrEnDi with real-world samples, genetically
modified canola samples from two fields (Field 1 and Field 2) were acquired following
treatment with Liberty® and liquid ammonium sulfate. For the purpose of this study, 5
canola plants were harvested, separated into stems, seeds, and shells and combined. A
simple aqueous extraction technique was performed on all canola samples with minimal
sample handling enabling the analysis of GLUF/MPPA before TrEnDi and
[GLUFTr]+/[MPPATr+H]+ following TrEnDi derivatization (Figure 5). [GLUFTr]+ and
[MPPATr+H]+ were the only analytes detectable and quantifiable in Field 1, with all 4
analytes being below the LOD in Field 2 (Figure S5). As a result, only Field 1 data is
detailed herein. Figures 5a and 5b show normalized peak areas of [GLUFTr]+ and
[MPPATr+H]+ extracted from Field 1 canola stems, seeds, and shells in chemical triplicate
(TrEnDi performed three times on three different aliquots of the same biological material)
from one of three biological sample sets. Figure 5c shows the normalized peak areas of
[GLUFTr]+ and [MPPATr+H]+ from all Field 1 canola stem, seed, and shell sample
measurements. To determine the absolute amounts of GLUF and MPPA on canola
samples, measurements were compared to calibration curves, with derivatized samples
being analyzed in chemical triplicate to ensure reproducibility. Additionally, the recovery
of unmodified GLUF and MPPA were determined using standards analyzed in triplicate
to confirm no analyte loss through use of syringe filters during extraction, yielding 107.5
± 3.8% and 94.7 ± 2.6% recoveries, respectively.
Figure 5: a,b) Average normalized peak areas of [GLUFTr]+ and [MPPATr+H]+ from Field
1 canola stems, seeds, and shells extracts analyzed in chemical triplicate from one of
three biological sample sets and c) from all respective biological sample measurements.
Unmodified GLUF and MPPA were not detected in any field samples, denoted on the
histograms by asterisks (*).
The time between herbicide treatment and sample extraction/analysis was substantial
[approximately 2 months (Field 1) and 3 months (Field 2)] with over 25 cm of precipitation
falling during that time. We hypothesize that the extended exposure of Field 2 samples to
the environment (i.e., precipitation, wind) explains why unmodified or modified GLUF and
MPPA were undetectable. For canola stem extract samples only, internal standard values
required a correction due to an isobaric interference (details in Supporting Information as
well as Figure S6). Furthermore, MS-based sensitivity gains were unable to be obtained
for [GLUFTr]+ and [MPPATr+H]+ due to the absence of unmodified GLUF and MPPA in all
samples. Despite this, quantities of permethylated products calculable from all canola
extracts.
Chemical triplicate data post-TrEnDi of canola stems, seeds, and shells from one of
three biological sample sets found [GLUFTr]+ at 3.4 µg/L, 2.9 µg/L, and 3.8 µg/L,
respectively, with [MPPATr+H]+ at 2.5 µg/L, 57.8 ng/L, and 1.8 µg/L, respectively.
Normalized peak area RSDs for the above findings ranged between 0.7-17.6%,
highlighting reproducibility when using TrEnDi. The average concentrations of [GLUFTr]+
on canola stems, seeds, and shells from all biological sample measurements were
determined to be 4.3 µg/L, 2.9 µg/L, and 4.3 µg/L, respectively, with [MPPATr+H]+ at 2.0
µg/L, 0.1 µg/L, and 1.8 µg/L, respectively. Normalized peak area RSDs ranged between
2.7-50.4%. Although higher RSD values are reported, biological data sets introduce a
larger range of reproducibility due to the potential for differences in rainfall exposure for
each individual canola plant, parts thereof, as well as variation in herbicide application. A
comprehensive table containing the concentrations of [GLUFTr]+ and [MPPATr+H]+ in all
sample types with associated errors can be found in Table 2.
Table 2: [GLUFTr]+ and [MPPATr+H]+ quantities measured on canola stem, seed, and
shell sample extracts.
Analyte Canola Sample Type

Concentration of Mass of analyte Moles of analyte on
extract (µg/L) on plant (µg/kg) plant material
(pmol/g)
Canola Stems
Biological Sample Set (1 of 3)
[GLUFTr]+ 3.4 ± 0.1 5.7 ± 0.18 22.6 ± 0.7
[MPPATr+H]+ 2.5 ± 0.2 4.2 ± 0.37 23.2 ± 2.1
Canola Seeds
[GLUFTr]+ 2.9 ± 0.02 4.8 ± 0.036 19.1 ± 0.1
[MPPATr+H]+ 0.06 ± 0.01 0.096 ± 0.017 0.5 ± 0.09
Canola Shells
[GLUFTr]+ 3.8 ± 0.08 6.3 ± 0.15 24.9 ± 0.6
[MPPATr+H]+ 1.8 ± 0.3 3.0 ± 0.52 16.5 ± 2.9
Canola Stems
All 3 Biological Sample Sets
[GLUFTr]+ 4.3 ± 1.0 7.1 ± 1.7 28.1 ± 6.6
[MPPATr+H]+ 2.0 ± 0.3 3.3 ± 0.42 18.2 ± 2.3
Canola Seeds
[GLUFTr]+ 2.9 ± 0.08 4.9 ± 0.13 19.3 ± 0.5
[MPPATr+H]+ 0.1 ± 0.07 0.22 ± 0.11 1.2 ± 0.6
Canola Shells
[GLUFTr]+ 4.3 ± 0.8 7.2 ± 1.4 28.4 ± 5.5
[MPPATr+H]+ 1.8 ± 0.2 3.0 ± 0.39 16.7 ± 2.2
To increase the reproducibility in canola sample sets, Dixon’s Q Test was
performed to identify measurement outliers. When performed, no data points were
omitted when using a 95% confidence interval. Therefore, the determined quantities of
GLUF and MPPA on the canola plants that were analyzed were well below the regulatory
maximum residue limit of 400 μg/kg from Health Canada,40 the European Union (EU),41
and the United States Environmental Protection Agency (US EPA).42
TrEnDi results in improved MS-based sensitivity of GLUF and MPPA following
derivatization leading to enhanced detection at low concentrations in various complex
samples. In the examples highlighted here, unmodified analytes were below the LOD and
would have been concluded to have not been present. We have demonstrated that
TrEnDi may be integrated into the sample preparation and analysis pipeline in a facile
manner.
Conclusion:
TrEnDi provides a rapid and instantaneous method to derivatize GLUF and MPPA,
quantitively forming permethylated products for both analytes. Following modification,
significant LCMS-based enhancements are observable through the formation of
[GLUFTr]+ and [MPPATr+H]+. Reversed-phase retention is low for [GLUF+H]+ (1.69 min,
dead volume) and [MPPA+H]+ (3.04 min), whereas retention significantly improves
following TrEnDi for [GLUFTr]+ (4.66 min) and [MPPATr+H]+ (5.21 min). Furthermore, MS-
based sensitivity is notably boosted for both permethylated analytes, with a 4.1-fold and
11.0-fold sensitivity enhancement for [GLUFTr]+ and [MPPATr+H]+, respectively. The LOD
and LOQ for each analyte were also improved following TrEnDi with an LOD of 1.1 fmol
(13.5 ng/L) and and LOQ of 3.6 fmol (45.1 ng/L) for [GLUFTr]+ and an LOD of 1.8 fmol
(16.8 ng/L) and an LOQ of 6.1 fmol (55.9 ng/L) for [MPPATr+H]+. To apply TrEnDi to a
more complex system, a Liberty® herbicide solution was derivatized where [GLUFTr]+ was
formed in >99% yield. [MPPA+H]+ was not detectable in Liberty®; however, following
TrEnDi, [MPPATr+H]+ was detected and quantified. For a final evaluation, canola stem,
seed, and shell extracts were prepared from field-grown and field-treated canola plants
and were derivatized using TrEnDi. After sample preparation and 3-fold concentration of
all canola samples, [GLUF+H]+ and [MPPA+H]+ were not identified in any samples,
whereas [GLUFTr]+ and [MPPATr+H]+ were quantifiable in all Field 1 samples. These
findings demonstrate the utility of TrEnDi for improved small molecule analysis via LCMS.
Associated Content:
The supporting information (SI) is available free of charge on the ACS Publications
website at DOI: 10.1021/jasms.XXXX and contains additional chromatograms, diagrams,
and calibration curves, as referenced in the main article.
Author Information:
Corresponding Authors
* Email: jeff.smith@carleton.ca. Phone: +1 613-520-2600 ext. 2408
* Email: jeff.manthorpe@carleton.ca. Phone: +1 613-520-2600 ext. 1711
Author Contributions
C.A.R. and K.V.W. were responsible for the experimental design and method
development. C.A.R. and K.L.S. conducted the experimental work. C.A.R. and K.L.S.
were responsible for managing the laboratory environment. J.M.M. and J.C.S. were
responsible for funding the project and oversight of all activities. C.A.R. was responsible
for writing the initial draft of the manuscript and all co-authors contributed to editing and
formatting the manuscript.
Notes
The authors declare no competing financial interests. The authors have no current or past
relationship with any producers of glufosinate-ammonium-based herbicide mixtures sold
residentially or commercially.
Acknowledgements:
J.C.S. and J.M.M. would like to gratefully acknowledge the Natural Sciences and
Engineering Research Council of Canada, the Canada Foundation for Innovation, the
Ontario Research Fund and Carleton University for research funding. C.A.R.
acknowledges the Ontario Graduate Scholarship program and both C.A.R. and K.L.S.
thank Carleton University for internal scholarships.
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Trimethylation Enhancement Using Diazomethane TR en Di Enables Enhanced Detection of Glufosinate and 3 Methylphosphinico Propionic Acid From Complex Canola Samples

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Trimethylation Enhancement Using Diazomethane TR en Di Enables Enhanced Detection of Glufosinate and 3 Methylphosphinico Propionic Acid From Complex Canola Samples

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Trimethylation enhancement using diazomethane

(TrEnDi) enables enhanced detection of glufosinate

and 3-(methylphosphinico)propionic acid from

complex canola samples

Christian A. Rosalesa,c, Krysten L. Sheedya,c, Karl V. Wasslena,c, Jeffrey M.

Manthorpea,b,c*, Jeffrey C. Smitha,b,c*

a) Department of Chemistry Carleton University, Ottawa, Ontario K1S 5B6, Canada

b) Institute of Biochemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada

*to whom correspondence should be addressed – jeff.smith@carleton.ca,

Keywords: herbicide, glufosinate, glufosinate-ammonium, 3-

leading to the development and increasing use of phosphinothricin, also known as

(methylphosphinico)propionic acid (MPPA). Chemical derivatization strategies have been

trimethylation enhancement using diazomethane (TrEnDi) to quantitatively derivatize

these analytes into permethylated GLUF ([GLUFTr]+) and MPPA ([MPPATr+H]+).

Comparing [GLUFTr]+ and [MPPATr+H]+ to underivatized counterparts, TrEnDi yields 2.8-

fold and 1.7-fold improvements in reversed-phase chromatographic retention,

respectively, while MS-based sensitivity is enhanced 4.1-fold and 11.0-fold, respectively.

Initial analytical improvements were performed on commercial standards; however,

successful analyte derivatization (with >99% yields) was also demonstrated on a

commercial herbicide solution imparting consistent analytical enhancements. To

continues to be a useful tool to enhance the analytical characteristics of organic

molecules that are traditionally difficult to detect.

Advancements in biotechnology during the 20th century led to the development of

non-transgenic crops that showed selective resistance to herbicides as well as transgenic

crops that were genetically engineered to be insusceptible to specific non-selective

herbicides. The non-transgenic herbicide approach has proven to be effective,1–3 however

economical transgenic-based herbicides.4 An example of this is glyphosate (GLY), the

since the introduction of “Roundup Ready” genetically modified GLY-resistant crops in

rise to 38 known species of GLY-resistant weeds,6 prompting a need to invent similarly

effective transgenic herbicides.

An alternative to GLY is the non-selective, broad spectrum, contact herbicide

phosphinothricin (2-amino-4-methyl-phosphino-butyric acid), or glufosinate (GLUF),

originally discovered through the study of Streptomyces viridochromogenes and S.

metabolism of the tripeptide bialaphos (comprised of L-phosphinothricin and two L-alanine

acids and detoxification of ammonia.7,12 Inhibition of GS results in the accumulation of

ammonia metabolism;14 however, its exposure indicated no teratogenicity, mutagenicity,

(methylphosphinico)propionic acid (MPPA) in soils and waters,16,17 their environmental

impact on human health following long-term residual exposure.18,19

Pesticides (QuPPe) method22 for their analysis. Alternatively, chemical derivatization

GLUF. Derivatization of GLUF using N-isopropoxycarbonyl methyl ester provides

improved sample volatility for GC,23 9-fluorenylmethylchloroformate (FMOC-Cl) enables

improved analyte selectivity on C18 columns,24 p-nitrobenzoyl chloride (PNBC), dansyl

chloride, and 4-chloro-3,5-dinitrobenzofluoride (CNBF) allows for ultraviolet (UV)

detection,25–27 while acetate/acetic anhydride and trimethyl orthoacetate provide a simple

In recent years, trimethylation enhancement using diazomethane (TrEnDi) has

emerged as a rapid derivatization technique to increase the MS- or tandem MS (MS2)-

based sensitivity of amine containing biomolecules through the formation of fixed,

in situ variant of TrEnDi.33 To improve MS and MS2-based analyses of amino acid-like

yields, imparting enhanced LC retention, greater MS sensitivity, and lower limits of

standard solutions, a commercial herbicide solution, as well as on aqueous extracts of

MPPA within relevant environmental samples.

(Georgetown, ON, Canada). Liberty® 200 SN (BASF, Ludwigshafen, Germany)

polycarbonate blast shield. N-methyl-N-nitroso-p-toluenesulfonamide was prepared as

described previously.35 A solution of N-methyl-N-nitroso-p-toluenesulfonamide (5.0 g,

separatory funnel into a MilliporeSigma DZM generation glassware (Product No.

Z202509) containing a stirred solution of potassium hydroxide (5 g, 0.09 mol), 8 mL

For TrEnDi derivatization of GLUF and MPPA standards, Liberty® herbicide

derivatization, 12 μL (1.2 μmol) of 1.0 M HBF4•Et2O solution in methanol was added.

Reverse Phase High-Performance Liquid Chromatography. 20 µL of each sample

standard MRM transition pair parameters can be found in Table 1.

Precursor Ion Product Ion

permethylated GLUF ([GLUFTr]+) and MPPA ([MPPATr+H]+) and to determine optimal

declustering potential of 30 V, and focusing potential of 120 V.

Manthorpea,b,c, Jeffrey C. Smitha,b,c