Determination of the [15N]-nitrate/[14N]-nitrate ratio in plant feeding studies by GC–MS

molecules

Article

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Determination of the [ N]-Nitrate/[ N]-Nitrate Ratio

in Plant Feeding Studies by GC–MS

Sebastian Schramm, Maria Fe Angela Comia Boco, Sarah Manzer, Oliver König, Tong Zhang,

Fatima Tuz Zohora Mony, Adebimpe Naﬁsat Adedeji-Badmus, Brigitte Poppenberger and

Wilfried Rozhon *

Biotechnology of Horticultural Crops, TUM School of Life Sciences Weihenstephan, Technical University of

Munich, Liesel-Beckmann-Straße 1, 85354 Freising, Germany; seb.schramm@tum.de (S.S.);

mariafefeangela.boco@studio.unibo.it (M.F.A.C.B.); sarah.manzer@tum.de (S.M.); oliver.koenig@tum.de (O.K.);

ge52qug@mytum.de (T.Z.); fatima.mony@tum.de (F.T.Z.M.); adebimpe.adedeji@tum.de (A.N.A.-B.);

brigitte.poppenberger@wzw.tum.de (B.P.)

*

Correspondence: wilfried.rozhon@wzw.tum.de; Tel.: +49-8161-71-2023

ꢀ

ꢁꢂꢀꢃꢄꢅꢆꢇ

Academic Editor: Young Jin Lee

Received: 28 March 2019; Accepted: 17 April 2019; Published: 18 April 2019

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ꢁꢂꢃꢄꢅꢆ

Abstract: Feeding experiments with stable isotopes are helpful tools for investigation of metabolic

ﬂuxes and biochemical pathways. For assessing nitrogen metabolism, the heavier nitrogen isotope,

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[

N], has been frequently used. In plants, it is usually applied in form of [ N]-nitrate, which is

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assimilated mainly in leaves. Thus, methods for quantiﬁcation of the [ N]-nitrate/[ N]-nitrate ratio

in leaves are useful for the planning and evaluation of feeding and pulse–chase experiments. Here we

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describe a simple and sensitive method for determining the [ N]-nitrate to [ N]-nitrate ratio in leaves.

Leaf discs (8 mm diameter, approximately 10 mg fresh weight) were suﬃcient for analysis, allowing

a single leaf to be sampled multiple times. Nitrate was extracted with hot water and derivatized

with mesitylene in the presence of sulfuric acid to nitromesitylene. The derivatization product was

analyzed by gas chromatography–mass spectrometry with electron ionization. Separation of the

derivatized samples required only 6 min. The method shows excellent repeatability with intraday

15

and interday standard deviations of less than 0.9 mol%. Using the method, we show that [ N]-nitrate

declines in leaves of hydroponically grown Crassocephalum crepidioides, an African orphan crop,

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with a biological half-life of 4.5 days after transfer to medium containing [ N]-nitrate as the sole

nitrogen source.

Keywords: GC–MS; mesitylene; nitrate; nitrogen; feeding experiments; stable isotope

1

. Introduction

Nitrogen represents a very important fraction of a plant’s chemical composition. It is an essential

plant macro-nutrient, necessary to ensure correct development and growth. Nitrogen is a fundamental

constituent of amino acids, the building blocks of peptides and proteins. It is present in chlorophyll,

a crucial molecule in photosynthesis, and in the phytohormones auxin and cytokinin. In addition,

nitrogen is a constituent of the nucleobases present in DNA, RNA, and in many co-substrates, for instance

ATP (adenosine triphosphate), NADH (nicotinamide adenine dinucleotide), and UDP-glucose (uridine

diphosphate glucose), involved in a tremendous number of metabolic processes. It is also present in

polyamines, quaternary ammonium compounds [

glucosinolates [ ], benzoxazinoids [ ], some phytoalexins [

grown in nitrogen-rich substrates may contain more than 5% nitrogen in dry weight while plants grown

under nitrogen-limiting conditions contain still approximately 0.5–1.0% nitrogen in dry weight [10 11].

Plants take up nitrogen via the root system mainly in form of nitrate and ammonium. In most substrates

1], plant defense compounds including alkaloids [

2–5],

6,7

8

9], and in many other metabolites. Plants

,

Molecules 2019, 24, 1531; doi:10.3390 /molecules24081531

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nitrate is the predominant form but ammonium may be present at high levels in ﬂooded soils [12

and in grasslands [13]. Ammonium can be incorporated directly in the root into amino acids or under

some conditions, particularly at a high supply, may be transported to the shoot [14 15]. In contrast,

]

,

nitrate is mainly transported to the shoot and reduced by nitrate reductase to nitrite, which is in

turn further reduced by nitrite reductase to ammonium. Ammonium is incorporated by the actions

of glutamine synthetase and glutamate synthetase into glutamine and glutamate [16], respectively,

which serve as precursors for biosynthesis of further amino acids [17] and other nitrogen-containing

compounds. Since ammonium [18

–20] and nitrite [21] are toxic for plant tissues, nitrate reduction

is well regulated [22 24] to avoid accumulation of these intermediates. Consequently, nitrate may

–

accumulate in leaves under rich fertilization.

Suﬃcient nitrogen supply is crucial for high plant biomass production and yield, which has

been studied in many plant species including the cereals wheat [25

,

26], maize [27

–

29], oat [30],

35] and

and barley [31 32], oilseeds like canola [25] and sunﬂower [33], root crops like potato [34

,

sugar beet [36], vegetables including spinach [37], lettuce [10], and kohlrabi [11] and energy crops

like willow [38] and Miscanthus sinensis [39]. Even the legumes, which possess root nodules that

contain nitrogen-ﬁxing symbionts, show increased yields when grown in nitrate-rich substrates [40,41].

However, fertilizers must be applied at the demand of the plants since overfertilization causes severe

environmental problems including contamination of surface and groundwater with nitrate [42] and

is responsible for algal bloom in the sea [43]. In addition, overfertilization of vegetables like lettuce,

spinach, celery, and Chinese cabbage causes nitrate accumulation at levels of up to several g/kg

fresh weight [44–47]. Possible health impacts of a high dietary intake of nitrate are intensively and

controversially discussed [48–55].

Due to the importance of nitrate, a number of methods have been developed for quantiﬁcation of

nitrate in soils, growth substrates and samples from plants, animals, and humans. These methods

include spectrophotometry [56,57], ion sensitive electrode [58], ion chromatography [59], ion-pair

chromatography [60], gas chromatography [61], and capillary electrophoresis [62]. In addition, soluble

nitrogen-containing compounds, particularly the amino acids, can be quantiﬁed in plant tissues for

assessing the nutritional status of plants and ripening of fruits [63–66]. While these methods are useful

for evaluating plant nutritional status, they are of limited use for investigation of nitrogen ﬂuxes in

plants. Metabolic ﬂuxes are often investigated by so called pulse–chase analysis, where organisms

or cells are initially incubated with a labeled compound (the pulse), which will be incorporated into

the metabolites. Subsequently, the labeled compound is removed and replaced by the unlabeled

version. Depletion of the labeled molecule and its conversion to labeled metabolites is measured

over time during the so called ‘chase’ period. Due to their simple detection, classically radio isotopes

13

were used for such experiments. In case of nitrogen, the radionuclide N has been used for tracer

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studies [14

produced on site. Alternatives to radionuclides include stable isotopes, which also allow for performing

,67–

69]. However, the short ¹N half-life of 9.97 min [70] is a drawback since N must be

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ﬁeld studies. The two stable isotopes of nitrogen are N, which has an abundance in the atmosphere

of 99.634 mol%, and ¹⁵N with an atmospheric abundance of only 0.366 mol% [71]. The huge diﬀerence

in abundance makes ¹⁵N a good tracer for metabolic studies. This technique has, for instance, been

used to quantify nitrogen ﬁxation [72], estimate the nitrogen ﬂow in alfalfa plants [73], investigate

nitrogen assimilation and amino acid translocation in barley [68

,74,75], follow nitrogen remobilization

from vegetative organs in pea [76], study nitrogen metabolism in buds of Picea glauca [77], and observe

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nitrogen ﬂuxes in Arabidopsis thaliana seeds during the initiation of germination [78]. N labeling is

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also useful for obtaining metabolites and proteins [79] accessible for NMR spectroscopy since the

nucleus gives much sharper signals than the N nucleus [77].

N

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¹⁵N contents can be analyzed by elemental analysis–isotope ratio mass spectrometry

EA–IRMS) [80,81], gas chromatography coupled to a combustion and reduction unit and a

(

mass spectrometer [82], and by nuclear magnetic resonance spectroscopy (NMR) [77

,78,83–85].

Other methods, including emission spectroscopy [86] and IR spectroscopy [87,88], are rarely used.

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For feeding experiments with stable nitrogen isotopes [ N]-potassium nitrate is most commonly

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used. For designing an appropriate experimental setup, the kinetics of replacement of [ N]-nitrate

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by [ N]-nitrate (or vice versa) in the plant tissue are relevant. The methods mentioned above

measure either the total nitrogen content (EA-IRMS, emission spectroscopy), cannot analyze nitrate

(nitrate is non-volatile and cannot be analyzed directly by GC), or lack insensitivity for analysis of

small sample quantities (NMR) and are thus not suitable for rapid determination of the ratio of

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[

N]-nitrate/[ N]-nitrate in limited amounts of plant material.

Several methods for quantiﬁcation of nitrate in biological samples, mainly of human or animal

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origin, using [ N]-nitrate as an internal standard have been described. Green et al. presented a

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method for quantiﬁcation of nitrate by spiking the sample with [ N]-nitrate followed by reaction

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with benzene to nitrobenzene. The ratio of [ N] and [ N]-nitrobenzene was subsequently analyzed

by gas chromatography–mass spectrometry (GC–MS) to determine the nitrate concentration [89].

Similar procedures using other, more reactive aromatic compounds were also developed [90

Other methods made use of reaction of nitrate with pentaﬂuorobenzyl bromide and subsequent

GC–MS analysis of the obtained derivatives [93 94]. Here we tested whether these methods can

–92].

,

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be applied for determining the [ N]-nitrate/[ N]-nitrate ratio in plant material using GC–MS with

electron impact ionization. We found that mesitylene gives the best results as the derivatization

reagent. We optimized all the steps of the method including extraction, derivatization, and detection.

Possible interferences with plant metabolites were investigated and ﬁnally the method was validated.

The method presented can be a helpful tool for establishing appropriate conditions for ¹N feeding

and pulse–chase experiments for measuring the level of stable isotope labeled nitrate in plants.

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. Results

Several GC–MS-based methods for quantiﬁcation of nitrate in mammalian ﬂuids, mainly

urine, saliva, and plasma, have been reported. They depend on reaction of the nitrate ion

with 2,3,4,5,6-pentaﬂuorobenzyl bromide [93 96], benzene [89], 1,3,5-trimethoxybenzene [92],

or mesitylene [90 91] to form organic compounds, which can be sensitively analyzed by GC–MS.

,2,3,4,5-Pentaﬂuorobenzyl bromide reacts with nucleophiles under mild conditions. Although nitrate

–

,

1

is a poor nucleophile, it can substitute the bromine of 1,2,3,4,5-pentaﬂuorobenzyl bromide to form

a nitric acid ester. The other compounds mentioned above are nitrated via electrophilic aromatic

substitution: nitrate reacts ﬁrst with sulfuric acid to the nitronium cation, which subsequently combines

with the aromatic ring to yield the nitrated aromatic compounds (Figure 1A). In that case nitrogen

is directly linked to carbon and the obtained derivatives are very stable, which makes them highly

suitable for quantiﬁcation. The reaction is very speciﬁc for nitrate; the only side reaction that may

happen is sulfonation of the aromatic reagent. Since the nitro group has a strong inactivating eﬀect

on the aromatic system, double derivatization is not observed. Among the reagents mentioned

above, 1,3,5-trimethoxybenzene has, due to the activating eﬀect of the methoxy groups, the highest

reactivity. Mesitylene is, because of the methyl groups, slightly activated while benzene shows the

lowest reactivity among these reagents.

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To test the suitability of these reagents for determination of the [ N]/[ N]-nitrate ratio in plant

leaves by GC–EI–MS (gas chromatography–electron ionization–mass spectrometry), test reactions

were performed. Reaction products obtained with 1,3,5-trimethoxybenzene contained high levels

of acid, probably sulfonated reagent, and were thus unfavorable for analysis by GC–MS. Tests with

2

,3,4,5,6-pentaﬂuorobenzyl bromide revealed that only fragments with m/z 181 and 161 were visible

in the positive EI–MS spectrum. These ions had both lost the nitrogen and were therefore not

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suitable for distinguishing [ N] and [ N]-nitrate. This is in accordance with the literature, where

,3,4,5,6-pentaﬂuorobenzyl derivatives are usually analyzed by MS after electron capture negative ion

chemical ionization [93 96 98]. In addition, 2,3,4,5,6-pentaﬂuorobenzyl bromide reacts also with many

other compounds typically present in plants, for instance phenolics [99] and fatty acids [98]. In contrast,

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–

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mesitylene gave very promising results: a peak at m/z 148 was obtained for [ N]-nitrate (Figure 1B).

Molecules 2019, 24, 1531

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The small peak at m/z 149 with a height of approximately 10% of the m/z 148 peak originates from the

natural abundance of the ¹³C and N isotopes. The observed fragmentation pattern indicated that

the ionized molecule with an m/z of 165 reacted under loss of a hydroxyl radical to the base ion m/z

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148, which reacted further under loss of carbon monoxide to m/z 120. The latter ion decayed under

loss of hydrogen cyanide to m/z 93. This is in agreement with the fragmentation pattern observed for

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[

N]-nitrate, where peaks of m/z 149 and 121, one mass unit higher than the [ N]-containing ions,

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were observed. In addition, also for [ N]-nitrate m/z 93 was observed, conﬁrming that this ion does

not contain nitrogen.

Figure 1. Formation of nitromesitylene and MS spectra. (

nitronium cation, which reacts subsequently with mesitylene to form nitromesitylene. (

spectrum of [¹⁴N]-nitromesitylene. Neutral losses of a hydroxyl radical (

OH), carbon monoxide (CO),

A

) Nitrate reacts with sulfuric acid to the

B) EI–MS

·

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1

and hydrogen cyanide (HCN) are indicated. (

data are presented in Supplementary File S1.

C

) EI–MS spectrum of [ N]-nitromesitylene. Original

+

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For benzene, the molecular peak M of m/z 123 obtained by derivatization of [ N]-nitrate and of

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+

m/z 124 for [ N]-nitrate dominated the spectra (Figure 2). However, also [MH] showed a signiﬁcant

peak and thus the molecular peak was not suitable for determination of the [ N]/[ N]-nitrate ratio.

Apart from the molecular peak only the fragments m/z 107 (for N) and m/z 108 for ( N) were found

to be suitable for determining the [ N]/[ N]-nitrate ratio since all other fragments lacked nitrogen.

However, this signal is relatively weak. In addition, nitrobenzene also shows peak tailing and the

reagent benzene is toxic and carcinogenic [100]. Thus, benzene is clearly less suitable than mesitylene.

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Taking these results together, mesitylene might be a useful reagent for simple determination of the

N]/[ N]-nitrate ratio in plants. Thus, we decided to focus on optimizing the reaction and analysis

[¹⁵

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conditions for this reagent.

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Figure 2. Derivatization with benzene. (

) Mass spectrum of the derivatization product [ N]-nitrobenzene. (C) Chromatogram obtained by

A

) Chromatogram obtained by derivatization of [¹⁴N]-nitrate.

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(

B

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derivatization of [ N]-nitrate. (D) Mass spectrum of the derivatization product [ N]-nitrobenzene.

Original data are included in Supplementary File S2.

2

.1. Method Development

Formation of the nitronium cation, the ﬁrst step in nitration of mesitylene (Figure 1A), depends

on the sulfuric acid concentration. Thus, the reaction was performed in the presence of 65–85% (w/w)

sulfuric acid. In addition, the reaction time was varied between 5 and 20 min. A possible side reaction

to nitration of the aromatic ring is oxidation of the methyl residues, which happens particularly at

an elevated temperature [101,102]. Thus, the reactions were performed at room temperature. Since

the mixture was biphasic, the tubes were shaken vigorously to enhance the reaction. For reliable

quantiﬁcation of the reaction product nitromesitylene, 2,4-dinitrotoluene (DNT) was added to the

reaction. DNT is a suitable internal standard because the presence of two nitro groups deactivates the

aromatic system and thus DNT does not react with nitrate under the conditions tested here. In addition,

DNT was well separated from nitromesitylene by GC–MS and the fragment with m/z 165 can be used

for quantiﬁcation (Figure 3). However, DNT showed signiﬁcant peak tailing. Since DNT was only

needed during optimization of the method, the drawback of DNT peak tailing was considered as

acceptable. In the ﬁnal method DNT is not added to the samples and thus DNT peak tailing has no

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impact on the accuracy of [ N]/[ N]-nitrate ratio measurements.

Figure 3. 2,4-Dinitrotoluene as internal standard. (

A

) Chromatogram obtained by derivatization

of [ N]-nitrate with nitromesitylene containing 0.5 mM 2,4-dinitrotoluene. ( ) Mass spectrum of

,4-dinitrotoluene. Original data are included in Supplementary File S3.

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B

2

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A clear increase of the nitromesitylene signal was observed with the concentration of sulfuric acid

Figure 4A). At 65% and 70% no reaction was observed while little product was formed at 75%. For the

(

1

0 min and 20 min reactions the maximum was reached at 80% sulfuric acid and no further increase

of the signal was observed when 85% sulfuric acid was used. For the 5 min reactions, the maximum

was observed only for 85% sulfuric acid although a high yield was also obtained at 80%. These data

indicate that a sulfuric acid concentration of at least 80% and a reaction time of 10 to 20 min should

be used. The obtained reaction product was quite acidic since a lot of carbon dioxide was evolved

when the supernatant was treated with sodium carbonate. A possible explanation might be that some

sulfuric acid had dissolved in mesitylene. Experiments showed that this problem can be eliminated

by addition of water to the tubes after ﬁnishing the reactions. To enhance visibility of the phases a

small amount of indigo carmine (approximately 0.01%) was added to the water used for dilution of

the reaction mix, which stained the lower water/sulfuric acid phase intensively blue while the upper

organic phase remained colorless.

Figure 4. Optimization of the method. (

bars are the average and SD of three replicates, respectively. (

concentration. The dots and bars are the average and SD of three replicates, respectively. (

A

) Reaction time and sulfuric acid concentration. The dots and

) Amount of mesitylene and sulfuric acid

) Linearity

) Calibration curve for determination of

the [ N]-nitrate level using standards containing 0.2 mM nitrate. ( ) Stability of the reaction product.

The derivatized sample was injected after incubation for the indicated time. The blue dots represent

B

C

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in the range of 0 to 1 mM [ N]-nitrate and [ N]-nitrate. (

D

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E

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the measured content of [ N]-nitrate in mol% while the red dots represent the ratio of the area of

nitromesitylene to the area of the internal standard 2,4-dinitrotoluene (DNT). The dots and bars are the

average and SD of ﬁve measurements, respectively. (

sample was injected splitless or with a split ratio of 1, 2, 5, or 10. The injection volume was 1

for splitless injection, where 0.5 L was injected. Original data are presented in Supplementary File S4.

F) Testing diﬀerent injection modes. The same

µL except

µ

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For the former experiments, pure mesitylene was used, which served on one hand as reagent and

on the other hand as solvent. Mesitylene has a similar boiling point to the reaction product and thus

only small volumes could be injected to prevent signiﬁcant peak broadening of the nitromesitylene

peak. In addition, pipette tips were almost invisible since their refractive index (approximately 1.49) is

very similar to that of mesitylene (1.499). These disadvantages raised the question whether the amount

of mesitylene may be downsized. Reactions with diﬀerent volumes indicated that the highest signal

was obtained with 10 µL mesitylene (the total volume of the organic phase was kept constant at 200 µL

by adding heptane after incubation at room temperature for 20 min). At higher volumes, the signal

decreased slightly (Figure 4B). Using 80% or 85% sulfuric acid had no signiﬁcant eﬀect on the reaction.

Thus, 80% sulfuric acid and 10

µL mesitylene with a reaction time of 20 min and subsequent addition

of water (containing indigo carmine) and 190

µL heptane for extraction of the reaction product were

the most suitable conditions. Addition of heptane had also the advantage that pipette tips were well

visible in the mesitylene/heptane mixture.

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Reactions with diﬀerent concentrations of nitrate showed perfect linearity for [ N]-nitrate and

[¹⁵

N]-nitrate and, importantly, an identical detector response (Figure 4C). Also, calibration curves for

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analysis of the level of [ N]-nitrate were linear when mol% [ N]-nitrate was plotted on the x-axis and

the sum of the intensities of m/z 149 + 150 divided by the sum of the intensities of m/z 148 + 149 + 150

plotted on the y-axis (Figure 4D). The ion with m/z 150 had to be included to correct for nitrate that

had reacted with mesitylene containing a ¹³C atom. Without considering ion m/z 150, a polynomial

function of second order rather than a linear function was obtained as a calibration curve.

To investigate the stability of the reaction product a sample was derivatized and analyzed

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immediately as well as on the following four days. The calculated ratio of [ N]-nitrate to [ N]-nitrate

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given in mol% [ N]-nitrate (Figure 4E) as well as the ratio of the peak areas of the reaction product

nitromesitylene to the internal standard DNT were stable over the entire time tested. This conﬁrms

that the reaction product is stable for at least 80 h.

Initial analyses performed in the splitless injection mode showed relatively broad nitromesitylene

peaks with a long tail (Figure 4F). Overloading of the column could be excluded as a reason for peak

asymmetry since injection of small volumes gave the same result. However, analyses performed in the

split mode showed symmetric peaks even at low split ratios (Figure 4F). Thus, we decided to use an

injection volume of 1 µL and a split ratio of 1. Under these conditions sharp symmetric peaks with an

approximately equal peak area compared to splitless injection were obtained.

Separation could be achieved within 6 min using a VF-ms column, making the analysis quick.

Chromatograms of standards and leaf samples looked almost identical and showed no peaks interfering

with the nitromesitylene peak, conﬁrming that the method is highly speciﬁc and suitable for analysis

of leaf extracts (Figure 5).

Spiking experiments with plant extracts showed a matrix eﬀect, particularly when samples with

high levels of phenolic compounds were measured (Figure 6A). In the presence of 0.57 mM phenolic

compounds (calculated as ferulic acid equivalents in the Folin–Ciocalteu assay) a reduction of the

signal by approximately one third was observed. In the presence of high levels of phenolic compounds

(

3.51 mM ferulic acid equivalents) only approximately 20% of the signal intensity of the control was

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observed. Analysis of [ N]-nitrate and [ N]-nitrate showed that presence even of high phenolic

levels had no eﬀect on the mole fraction (Figure 6B). The matrix eﬀect may be caused by reaction

of nitrate with phenolic compounds in the presence of sulfuric acid. Phenolic compounds can be

eﬃciently removed by treatment with activated charcoal [103,104]. The sample containing a high level

of phenolic compounds was treated with two types of activated charcoal and with synthetic graphite.

Quantiﬁcation of the phenolic compounds after treatment with the adsorbents showed that both types

of activated charcoal removed phenolics eﬃciently (>0.02 mM residue) while graphite removed only

approximately half of the phenolics (1.91 mM after treatment). However, analysis showed that activated

charcoal purchased from Merck reduced the matrix eﬀect signiﬁcantly while activated charcoal from

Alfa Aeser and synthetic graphite had only a small eﬀect (Figure 6A). This indicates that also other

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compounds apart from the phenolics assayed with the Folin–Ciocalteu assay might be responsible

for quenching of the signal. Treatment with the adsorbents resulted in signiﬁcantly lower levels for

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[

N]-nitrate (Figure 6B). Thus, such a treatment is unsuitable for analysis. Since the matrix lowered

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the signal but did not change the measured [ N]-nitrate/[ N]-nitrate we decided to use the plant

extracts directly.

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Figure 5. Chromatograms. (A) Chromatogram of a standard containing 0.2 mM [ N]-nitrate.

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(B) Chromatogram of a standard containing 0.2 mM [ N]-nitrate. (C) Chromatogram of a plant sample

grown on medium containing [¹⁵N]-nitrate and shifted to medium containing [¹⁴N]-nitrate 3 days prior

analysis. (D) Chromatogram of a blank. Original data are presented in Supplementary File S5.

Figure 6. Matrix eﬀect of plant leaf extracts. (A) Samples containing diﬀerent levels of phenolic

compounds (measured in mM ferulic acid equivalents) were spiked with a mixture containing

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[

N]-nitrate and [ N]-nitrate to a ﬁnal concentration of 0.4 mM total nitrate and treated with the

indicated absorbents. Subsequently, the residual phenolics and the recovery rate of total nitrate were

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assayed using DNT as the internal standard. (

B) The [ N]-nitrate levels of the same samples shown

in (A). Original data are presented in Supplementary File S6.

15

The results for [ N]-nitrate presented above are the mole fraction in mol%. However, the mole

fraction (x in mol%) and the ration (r in mol/mol) are connected via the formula [105]:

r

+ r

x = 100 ∗ ₁

(1)

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Thus, if desired, the mole fraction can be converted to the ratio of [ N]-nitrate to [ N]-nitrate by

using the formula:

x

r =

(2)

1

00 − x

2

.2. Method Validation

15

The limit of detection (LOD) and limit of quantiﬁcation (LOQ) for [ N]-nitrate in a mixture with

N]-nitrate were 0.65 mol% and 2.2 mol%, respectively. The sample used for determining the LOD

[¹⁴

and LOQ contained 0.2 mM nitrate since concentrations of 0.1 to 0.6 mM are typically seen for leaf

extracts of Crassocephalum crepidioides (Supplementary File S7).

For assessing repeatability of the method two samples, one containing a medium and one a high

level of phenolic compounds, were analyzed on ﬁve consecutive days with ﬁve replicates on each day.

The calibration functions showed excellent linearity with Pearson correlation coeﬃcients exceeding

0

.999 on each day and excellent repeatability with relative standard deviations of 1.8% and 2.2% for

the slope and interception, respectively (Table 1). Also, the results obtained for the sample containing a

medium level of phenolics were highly repeatable with an intraday SD in the range of 0.25–0.81 mol%

and an interday repeatability of 0.87 mol% (Table 2). The results for the sample with a high content of

phenolics showed a slightly increased standard deviation with 0.51–2.27% for the intraday SD and

2

.18 mol% for the interday SD. This can be explained by the lower signal and thus decreased signal to

noise ratio.

Table 1. Interday repeatability of the calibration curve.

Calibration ²

Experiments ¹

a

b

r

Day 1

Day 2

Day 3

Day 4

Day 5

0.00866

0.00908

0.00895

0.00879

0.00890

0.10698

0.09956

0.10065

0.10223

0.10155

0.99915

0.99991

0.99988

0.99980

0.99982

Average

SD

RSD in%

0.00888

0.00016

1.78

0.10220

0.00286

2.80

1

Original data are presented in Supplementary File S8; ²Linear regression function of the type y = a

×

x + b; a, slope;

b, interception; r, Pearson correlation coeﬃcient.

Table 2. Intraday and interday repeatability.

Reproducibility

Experiment ¹

Average

mol%

SD

mol%

RSD

%

Repeats

0

.57 mM FA

Day 1

5

53.10

50.94

51.99

51.84

51.33

0.57

0.46

0.25

0.81

1.07

0.91

0.48

1.58

Day 2

Day 3

Day 4

Day 5

Interday

25

51.84

0.88

1.70

3

.51 mM FA

Day 1

5

52.37

48.75

48.49

49.86

50.49

0.51

2.27

2.12

0.99

0.98

4.65

4.37

1.99

1.97

Day 2

Day 3

Day 4

Day 5

Interday

25

49.99

2.00

4.00

1

Original data are presented in Supplementary File S8.

Molecules 2019, 24, 1531

10 of 22

14

15

2

.3. Application: Kinetics of [ N]-Nitrate/[ N]-Nitrate Replacement in Leaves of Crassocephalum crepidioides

To assess nitrate uptake of Crassocephalum crepidioides, an African orphan crop [ 106], plants were

4

,

15

grown hydroponically in modiﬁed Hoagland medium containing [ N]-nitrate as the sole nitrogen

source. Since a closed system was used, water was regularly added to maintain a constant volume.

In addition, samples were regularly analyzed for minerals and the medium was supplemented if

required (Figure 7A). Nitrate and phosphate were the only minerals that had to be supplemented

15

during the course of the experiment. After 29 days, the [ N]-nitrate containing medium was replaced

14

by [ N]-nitrate medium and from this moment on leaf samples were taken for analysis of foliar

1

5

14

15

[

N]-nitrate and [ N]-nitrate levels. Initially, a rapid decrease of [ N]-nitrate was observed while

15

at later time points a lower decline was observed. Regression analysis showed that [ N]-nitrate

decreased approximately exponentially with a biological half-life of 4.5 day (Figure 7B).

Figure 7. Kinetics of replacement of [¹⁵N]-nitrate by [¹⁴N]-nitrate in leaves of Crassocephalum crepidioides.

1

5

(A) Plants were grown hydroponically in liquid medium containing [ N]-nitrate as the sole nitrogen

1

4

source for 29 days. Subsequently, the medium was replaced by medium containing [ N]-nitrate as the

sole nitrogen source (time 0, indicated by a dotted line) and grown for a further 19 days. Samples of the

medium were taken at the indicated time points and analyzed for ions. If required, the medium was

15

14

supplemented for nitrate by adding [ N] or [ N]-potassium nitrate (blue arrows) or for phosphate

by adding potassium dihydrogen phosphate (red arrows). ( ) Leaf disc samples were taken at the

B

15

1

4

indicated time points for analysis of leaf [ N]-nitrate and [ N]-nitrate levels. The points and bars

represent the biological average and standard error (SE) of samples taken from four diﬀerent leaves.

Total nitrate concentrations in leaf material and original data are presented in Supplementary Files S9

and S10.

3

. Discussion

For assessing nitrogen metabolism and re-localization of nitrogen-containing metabolites,

the heavier nitrogen isotope, ¹⁵N, has been frequently used [68

72 78]. In plants it is usually

applied in form of nitrate. Nitrate is assimilated mainly in leaves, where it is reduced via nitrite as

the intermediate to ammonium, which is rapidly incorporated into amino acids [23 107]. Plants can

,

–

,

accumulate high levels of nitrate, which can be critical for re-localization studies since it may take some

time until the newly fed labeled nitrate has replaced the previously present unlabeled nitrate. Similarly,

in pulse–chase experiments it is important to know to which extent the tracer nitrate has replaced the

previously present species since that limits the extent of labeling in downstream metabolites. Thus, for

appropriate planning of feeding and pulse–chase experiments knowledge of the kinetics of nitrate

1

5

14

uptake and replacement of [ N]-nitrate by [ N]-nitrate (or vice versa) in leaves is very helpful.

15

14

The method for assessing the [ N]-nitrate/[ N]-nitrate in plant material described here is simple,

rapid and does not require special equipment apart from a conventional GC–MS system with EI

ionization. Nitrate is extracted with hot water and derivatized at room temperature with mesitylene

in the presence of sulfuric acid to nitromesitylene. The reaction product is highly stable and the

derivatized samples can be stored at room temperature for at least 80 h without any impact on the

Molecules 2019, 24, 1531

11 of 22

performance. The calibration shows perfect linearity and the method is highly reproducible with

intraday and interday SDs of less than 0.9 mol% for samples with moderate phenolic contents and less

than 2.3 mol% for samples with high phenolic contents. Ideally, the nitrate concentration in the plant

extract should be higher than 0.1 mM. At lower concentrations contamination with ubiquitously present

nitrate might become critical. The limit of detection and quantiﬁcation is 0.65 mol% and 2.2 mol% for

1

5

[

N]-nitrate, respectively. The reason for this relatively high detection limit is the natural abundance

13

15

of C of approximately 1.1%, which causes even in the absence of [ N]-nitrate a signal at m/z 149 of

approximately 10% of that at m/z 148, the ion obtained for derivatized [ N]-nitrate. Opportunities

14

to reduce the detection limit include using a high resolution mass spectrometer that can distinguish

15

13

the mass defect of [ N] and [ C] or analyzing derivatized samples by gas chromatography coupled

to combustion and reduction units and an isotope ratio mass spectrometer. However, for typical

15

applications using isotope-enriched [ N]-nitrate a normal GC–EI–MS system as used here is suﬃcient.

15

While the plant matrix had no eﬀect on the results for the [ N]-nitrate mole fraction, the signal

intensity was signiﬁcantly reduced, particularly in plant extracts containing high contents of phenolics.

Experiments with activated charcoal showed that the matrix eﬀect could be reduced. However,

15

treatment with charcoal had a slight but signiﬁcant impact on the results for the [ N]-nitrate mole

fraction. Thus, such a treatment was not useful. Interestingly, while charcoal treatment removed the

phenolics as assessed by the Folin–Ciocalteu assay completely, the matrix eﬀect was not completely

eliminated. That indicates that the nitronium ion may also react with other compounds present in the

plant extract apart from phenolics. Possible candidates are reducing compounds like ascorbic acid,

15

glutathione, or cysteine [108,109]. However, since the measured [ N]-nitrate mole fraction was not

aﬀected by the plant matrix this was not studied in more detail.

In summary, the presented method allows simple and reproducible determination of the foliar

N]-nitrate/[ N]-nitrate ratio even from small samples. The method can be helpful for establishing

[¹⁵

14

and evaluating feeding and pulse–chase experiments.

4

. Materials and Methods

4

.1. Reagents

Potassium nitrate (>99%) was purchased from Duchefa (Haarlem, the Netherlands).

N]-Potassium nitrate (>98 isotope purity), mesitylene, heptane, indigo carmine, 2,4-dinitrotoluene,

,3,5-trimethoxybenzene, toluene, 2,3,4,5,6-pentaﬂuorobenzyl bromide, ethyl acetate, acetone, sodium

[¹⁵

1

tetraborate, and benzyldimethyltetradecylammonium chloride were purchased from Sigma (St. Louis,

MO, USA). Sulfuric acid 96% and benzene were purchased from Carl Roth (Karlsruhe, Germany).

4

.2. Plant Material and Growth Conditions

Crassocephalum crepidioides accession Ilé-Ifè [106] was used in this study. Seedlings were

transplanted into hydroponic medium after 4 weeks of pre-cultivation on soil. Each hydroponic tank

15

harbored 6 plants and contained 4 L of modiﬁed Hoagland medium with [ N]-nitrate as the sole

nitrogen source. Since a relatively low nitrate concentration was required the calcium nitrate present

in Hoagland medium was replaced by calcium chloride (for medium composition and preparation

see Supplementary File S11). Thus, the medium contained a relatively high but constant chloride

concentration. Constant aeration of the medium was provided by air stones connected to an air pump.

◦

−1

−2

Plants were cultivated at 21 C with a 16 h light/8 h dark cycle and a light intensity of 100

µmol

·

s

·

m .

14

After 29 days in the initial medium plants were switched to medium containing [ N]-nitrate. Roots

were rinsed with deionized water before transfer to the other medium. Sampling of leaf discs was

performed over a 19-day period.

Plants used for preparation of nitrate-free leaf extracts used for investigating the matrix effect (see point

4.4.6.) were cultivated on low nutrient soil SP ED63 P (Patzer GmbH and Co. KG, Sinntal-Altengronau,

Germany; initial nitrate content: 555 mg/kg dry weight) for 12 weeks. The plants showed nitrogen

Molecules 2019, 24, 1531

12 of 22

deﬁciency symptoms three weeks prior to harvest and absence of nitrate in the leaf extracts was

conﬁrmed by ion-pair chromatography and by GC–MS.

4

.3. Sample Preparation and GC–MS

Leaf discs with 8 mm diameter (approximately 10 mg) were cut out using a hollow punch, placed

◦

into 2 mL safe lock tubes and stored at –20 C until analysis. Distilled water (150

µL) was added

and the tube incubated in a Thermomix shaker (Eppendorf, Hamburg, Germany) set to 1400 rpm

◦

and 95 C for 20 min. After centrifugation at 10,000 rpm for 2 min the nitrate concentration was

quantiﬁed by ion-pair chromatography (see Section 4.7). and the remaining extract diluted to a ﬁnal

nitrate concentration of 0.2–0.4 mM if required. A 2 mL safe lock tube was charged with 100

00 L 80% sulfuric acid, and 10 L mesitylene. The tubes were placed in a Thermomix shaker set

to 1400 rpm at room temperature for 20 min. Water (500 L) containing 0.01% indigo carmine and

90 L heptane were added and the tubes shaken vigorously prior centrifugation at 10,000 rpm for

0 s. Approximately 150 L of the supernatant was transferred into a 1.5 mL reaction tube containing

approximately 5 mg sodium carbonate and mixed vigorously. After centrifugation at 10,000 rpm for

µL extract,

6

µ

1

µ

3

µ

3

0 s, 80 µL of the clear supernatant was transferred into an autosampler tube.

A Varian 431-GC gas chromatograph equipped with a CP-8400 autosampler and connected to a

2

0

10-MS mass spectrometer (Palo Alto, CA, USA) was used. For analysis, a VF-5ms 30 m

×

0.25 mm

×

.25 m capillary column was used. Helium was used as carrier gas at a ﬂow rate of 1 mL/min and

µ

◦

a split ration of 10. The injector was set to 230 C. Injection (1

ratio of 1. The temperature program started with an isothermal step at 120 C for 1 min. Then the

temperature was raised linearly within 4 min to 200 C at a rate of 20 C/min. Finally, the temperature

was raised within 1 min to 260 C at a rate of 60 C prior returning to the initial conditions. The transfer

line was operated at 200 C, the ion trap at 160 C, and the manifold at 40 C. MS spectra were recorded

from 3.6 to 4.8 min and from m/z 50 to 200. For establishing the calibration curve the sum of the

intensities of the ions with m/z 149 and 150 was divided by the sum of the ions with m/z 148 + 149 + 150

and plotted on the y-axis while mol% of [ N]-nitrate was plotted on the x-axis. A detailed step-by-step

protocol is included in Appendix A.

µ

L sample) was performed at a split

◦

1

5

4

.4. Method Development

4

.4.1. Initial Testing of Diﬀerent Derivatization Methods

Derivatization with mesitylene was performed according to Dunphy et al. [90] except that no

internal standard was added. Derivatization with 1,3,5-trimetrhoxybenzen was performed as described

by Gutzki et al. [92] except that the reaction product was extracted with toluene rather than benzene.

For derivatization with 2,3,4,5,6-pentaﬂuorobenzyl bromide the protocols of Tsikas et al. [94] and of

Kage et al. [93] were applied. Derivatization with benzene was adapted from [89]. In brief, 100

µL of

sample was mixed with 600 L of 85% sulfuric acid and 200 L of benzene in 2 mL safe lock tubes.

µ

The tubes were shaken at room temperature in an Eppendorf Thermomixer (Hamburg, Germany) set

to 1400 rpm for 15 min. The upper layer was transferred to a tube containing approximately 5 mg

sodium carbonate, shaken vigorously and centrifuged at 10,000

directly analyzed. The reaction product with benzene was analyzed by GC–MS as stated above except

×

g for 30 s. The supernatant was

◦

that the temperature program was modiﬁed: isothermal at 100 C for 1 min, then a liner increase

◦

to 180 C within 4 min, and ﬁnally a linear increase to 260 C within 2 min prior returning to the

initial conditions. The other derivatization products were analyzed using the settings mentioned in

Section 4.4.2.

4

.4.2. Optimization of Reaction Time and Sulfuric Acid Concentration

14

Sample (100

µ

L; 1 mM [ N]-potassium nitrate) was mixed with 600

µL sulfuric acid of the

indicated concentration and 300

µL mesitylene containing 0.5 mM 2,4-dinitrotoluene as the internal

Molecules 2019, 24, 1531

13 of 22

standard. The mixture was derivatized and processed as stated above except that a reaction time of 5,

1

0, or 20 min was used. The clear supernatant was analyzed by GC–MS as described above except that

◦

the following GC settings were used: injector temperature 250 C; thermal program: isothermal at

1

◦

20 C for 1 min, then a linear increase to 230 C within 5.5 min, and ﬁnally a linear increase to 290 C

within 1.5 min prior returning to the initial conditions. MS spectra were recorded from 3.6 to 4.8 min

and from m/z 50 to 200.

4

.4.3. Optimization of Mesitylene Amount and Sulfuric Acid Concentration

14

Sample (100

acid and 5, 10, 20, 50, 100, or 150

room temperature for 20 min. The reaction was stopped by addition of 500

.01% indigo carmine. Subsequently, 50 L of 0.5 mM 2,4-dinitrotoluene dissolved in mesitylene

µ

L; 0.5 mM [ N]-potassium nitrate) was mixed with 600

L mesitylene was added. The mixture was shaken at 1400 rpm at

L of water containing

µL of 80% or 85% sulfuric

µ

0

µ

was added as the internal standard and heptane was added to complete the organic phase to 200

The supernatants were processed and analyzed as described in Section 4.4.2.

µL.

4

.4.4. Test for Linearity

14

15

Potassium nitrate solutions (100 µL of [ N]-potassium nitrate or 100 µL of [ N]-potassium

nitrate) of diﬀerent concentrations (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1 mM) were analyzed as

described in Section 4.4.3.

4

.4.5. Stability of the Reaction Products

A sample was derivatized with 20

µ

L of mesitylene as described in Section 4.4.3. The derivatized

solution was measured on ﬁve consecutive days with ﬁve replicates on each day. The derivatized

sample was stored at room temperature.

4

.4.6. Matrix Eﬀect

Nitrate-free plant extracts obtained from C. crepidioides leaves were spiked with a mixture of [ N]

14

15

and [ N]-potassium nitrate to a total concentration of 0.4 mM nitrate. The phenolic contents were

assayed as described in Section 4.8. [ N] and [ N]-nitrate were measured as described in Section 4.4.3.

15

14

4

.5. Method Validation

A sample containing 0.4 mM [ N]-nitrate was measured 9 times and the average and SD were

14

calculated. The LOD and LOQ were calculated by dividing the 3-fold and 10-fold SD by the slope of

the calibration curve, respectively.

For testing the repeatability, samples with a medium and high content of phenolics were spiked

14

15

with a mixture of [ N] and [ N]-potassium nitrate to a total concentration of 0.4 mM nitrate.

The samples were analyzed on ﬁve consecutive days with ﬁve replicates on each day. Every day a

calibration curve (see Appendix A for details) was established.

4

.6. Quantiﬁcation of Nitrate in Leaf Extracts by Ion-Pair Chromatography

Quantiﬁcation of nitrate by ion-pair chromatography was adapted from Chou et al. [60].

A Shimadzu 10A high performance liquid chromatography system (Shimadzu, Kyoto, Japan) was used.

The HPLC system consisted of a SCL-10A system controller, a FCV-10AL valve for eluent selection,

a LC-10AT pump equipped with DGU-14A inline degasser, a SIL-10A autosampler, a CTO-10ASvp

◦

column oven set to 25 C, a Nucleodur 100-5 C18ec 125

×

4.6 mm HPLC column (Machery-Nagel,

Düren, Germany), and a SPD-10A UV detector, which was operated at 213 nm. The eluent consisted

of 10 mM 1-octylamine set with phosphoric acid to pH 7.0 in 20% ACN (acetonitrile). For elution,

a ﬂow rate of 1 mL/ min was set and the injection volume was 25

µL. Samples were diluted with

eluent 1:10 prior to injection. Standards were dissolved in eluent and contained nitrate in the range

Molecules 2019, 24, 1531

14 of 22

of 0–10 mg/L. Chromatograms were evaluated with the Clarity software package (version 5.0.4.158,

DataApex, Prague, Czech Republic).

4

.7. Analysis of Cations and Anions in Hydroponic Media by Ion Chromatography

For analysis of cations the samples were diluted 1:50 by transferring 1 mL of sample into a

0 mL volumetric ﬂask and adding deionized water to the mark. An aliquot was transferred into

5

a polypropylene autosampler vial and 100

chromatograph consisted of a LC-10ADvp pump, a FCV-10ALvp valve for eluent selection, a SIL-10A

autosampler, a CTO-10ASvp column (Shimadzu), a Nucleodur 100-5 C18ec 4 3 mm pre-column,

an IC-Pak Cation M/D 3.9 150 mm column (Waters, Milford, MA, USA), and a 430 conductivity

µL was injected into the ion chromatograph. The ion

×

detector (Waters). Chromatograms were evaluated with the Clarity software package. The column

oven was maintained at 25 C. The eluent consisted of 3 mM nitric acid and 0.1 mM EDTA

◦

(ethylenediaminetetraacetic acid) and was delivered at a ﬂow rate of 1 mL/min. Standards contained

sodium (0–0.05 mM), ammonium (0–0.05 mM), potassium (0–0.25 mM), magnesium (0–0.25 mM),

and calcium (0–0.5 mM).

Anions were analyzed by diluting the samples 1:5 by transferring 1 mL of sample into a

5

mL volumetric ﬂask and adding deionized water to the mark. An aliquot was transferred into a

polypropylene autosampler vial and 100 L was injected into the ion chromatograph described above

except that an IC-PAK Anion HC 150 4.6 mm column (Waters) and a sodium gluconate/borate buﬀer

sodium gluconate 320 mg/L, boric acid 360 mg/L, disodium tetraborate 264 mg/L, glycerol 4 g/L,

µ

×

(

acetonitrile 12%, and 1-butanol 2%) delivered at a ﬂow rate of 1.6 mL/min were used. The standards

contained chloride (0–6.42 mM), nitrate (0–1.7 mM), phosphate (0–0.4 mM), and sulfate (0–1.81 mM).

4

.8. Determination of Total Phenolics in Leaf Extracts

The method was adapted from Veliogli et al. [110]. In brief, suitable volumes of sample or standard

(

0–50

total volume of 700

incubating at room temperature for 5 min, 250

µ

L of 0.1 mM ferulic acid) were transferred into 1 mL plastic cuvettes and water was added to a

L. Subsequently, 50 L of Folin–Ciocalteu reagent was added. After mixing and

L of 2 M sodium carbonate was added and mixed well.

µ

The absorbance was measured at 725 nm after incubation for 1 h at room temperature. The results

were expressed in mM ferulic acid equivalents.

Supplementary Materials: The following are available online at http://www.mdpi.com /1420-3049 /24 /8 /1531 /s1 :

Supplementary File S1: Data for Figure 1, Supplementary File S2: Data for Figure 2, Supplementary File S3: Data

for Figure 3, Supplementary File S4: Data for Figure 4, Supplementary File S5: Data for Figure 5, Supplementary

File S6: Data for Figure 6, Supplementary File S7: Determination of the LOD and LOQ, Supplementary File S8:

Data for Tables 1 and 2, Supplementary File S9: Data for Figure 7A, Supplementary File S10: Data for Figure 7B,

Supplementary File S11: Composition and preparation of modiﬁed Hoagland hydroponic medium, Supplementary

15

File S12: Standards for quantiﬁcation of [ N]-nitrate content.

Author Contributions: Conceptualization, S.S., B.P., and W.R.; methodology and validation, S.S., M.F.A.C.B., S.M.,

O.K., T.Z., F.T.Z.M., A.N.A.-B., and W.R.; writing—original draft preparation, all authors; writing—review and

editing, S.S., B.P., and W.R.; preparation of ﬁgures, S.S. and W.R.

Funding: A.N.A.-B. received a PhD scholarship from the Deutscher Akademischer Austausch Dienst (DAAD),

funding number 57381412. M.F.A.C.B. received a scholarship from the Erasmus + program and a scholarship

from the Er.Go (l’Azienda Regionale per il Diritto agli Studi Superiori dell’Emilia Romagna). The publication

costs were covered by the German Research Foundation (DFG) and the Technical University of Munich (TUM) in

the framework of the Open Access Publishing Program.

Acknowledgments: Thanks to Sabine von Tucher for critical reading of the manuscript.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Molecules 2019, 24, 1531

15 of 22

Appendix A

15

14

Step-by-Step Protocol for Determination of the [ N]-Nitrate/[ N]-Nitrate Ration in Leaves

Appendix A.1.1. Reagents

1

4

14

•

[ N]-Nitrate stock, 1 mM (transfer 101.1 mg [ N]-potassium nitrate into a 1000 mL volumetric

ﬂask, dissolve in distilled water and add distilled water to the mark. The solution is stable for one

◦

week at 4 C or for at least 2 years at −20 C).

15

[ N]-Nitrate stock, 1 mM (transfer 102.1 mg [ N]-potassium nitrate into a 1000 mL volumetric

ﬂask, dissolve in distilled water and add distilled water to the mark. The solution is stable for one

◦

week at 4 C or for at least 2 years at −20 C).

Sulfuric acid, 80% (w/w) (place 54 mL distilled water in a 250 mL ﬂask and cool with ice; add

in total 146 mL sulfuric acid, 96% (w/w) under cooling with ice slowly and in small aliquots.

The solution can be stored at room temperature inﬁnitely).

•

Mesitylene

Water containing 0.01% indigo carmine (dissolve approximately 10 mg indigo carmine in 100 mL

water and add 100

one month).

Heptane

µL 80% sulfuric acid; the solution can be kept at room temperature for at least

•

Eluent for ion-pair chromatography: 10 mM 1-octylamine phosphate pH 7.0 in 20% (v/v) ACN

transfer 1.29 g 1-octylamine and 156.4 g ACN (note: this corresponds to 200 mL ACN) into a

000 mL beaker and add approximately 700 mL distilled water. Set the pH with 4 M phosphoric

acid to 7.0. Transfer into a 1000 mL volumetric ﬂask and add distilled water to the mark. Filter

(

1

through a 0.22 or 0.45

at least 1 year).

µm nylon membrane ﬁlter. The eluent can be kept at room temperature for

Appendix A.1.2. Preparation of Standards

1

.

Mix the solutions indicated in Table A1 in 5 mL volumetric ﬂasks:

Table A1. Preparation of standards.

15

[¹⁴N]-Nitrate 1 mM µL

[¹⁵N]-Nitrate ¹^,²mol%

No.

[ N]-Nitrate 1 mM µL

St1

St2

St3

St4

St5

St6

St7

St8

St9

St10

0

10

20

1000

990

980

950

900

800

600

400

200

0

0.366

1.352

2.339

50

5.298

100

200

400

600

800

1000

2

10.299

20.093

39.820

59.546

79.273

99.000

1

The total nitrate concentration is 0.2 mM; Calculated for 99.634 mol% isotope purity, the natural frequency of ¹⁴N,

for [ N]-nitrate and 99 mol% isotope purity of [ N]-nitrate. Standards prepared from stocks with other purities

can be calculated using Supplementary File S12.

1

4

15

◦

2

.

Add water to the mark. The standards can be kept at –20 C for at least two years.

Appendix A.1.3. Extraction

Punch out leaf discs of 8 mm diameter using a hollow punch. These discs weigh approximately

0 mg.

1

.

1

Molecules 2019, 24, 1531

16 of 22

◦

2

3

4

5

.

Transfer the leaf discs into 2 mL safe lock tubes and store at –20 C until analysis.

◦

Add 150 µL distilled water, incubate in a shaker set to 1400 rpm and 95 C for 20 min.

Centrifuge at 10,000 rpm for 2 min.

Transfer the clear supernatant into a new tube and measure the nitrate content by ion-pair

chromatography or proceed immediately to derivatization. The extract can be stored at

for several days.

◦

−

20 C

Appendix A.1.4. Derivatization and Analysis

Transfer 100 L of leaf extract or standard (see Table A1) into a 2 mL safe-lock tube. Note: if

1

.

µ

the nitrate concentration was determined, dilute the extract to a ﬁnal nitrate concentration of

approximately 0.2 mM prior transferring 100 µL into the 2 mL safe lock tube.

Add 10 µL mesitylene.

Add 600 µL 80% sulfuric acid.

Incubate the tubes at room temperature in a shaker set to 1400 rpm for 20 min.

2.

3.

4.

5.

6.

7.

Add 500

Centrifuge at 10,000 g for 30 s.

Transfer 150 L of the upper, colorless organic phase into a 1.5 mL tube containing approximately

mg sodium carbonate. Avoid transferring any of the aqueous phase. Note: indigo carmine

µL water containing 0.01% indigo carmine and 190 µL heptane and mix again for 1 min.

µ

5

stains the lower aqueous phase intensively blue, which helps with recognizing the phases.

Shake the tubes vigorously for a few seconds.

Centrifuge at 10,000 g for 30 s.

8

9

.

1

0. Transfer 80 µL of the clear supernatant into an autosampler vial.

1. Analyze the samples by GC–MS using a VF-5ms 30 m 0.25 mm

×

0.25

µ

m capillary column and

◦

helium as carrier gas at a ﬂow rate of 1 mL/min and a split ratio of 1:10. The injector is set to 230 C.

Injection (1 µL sample) is performed with a split of 1. The temperature program is set according

◦

to Table A2. The transfer line is operated at 200 C, the ion trap at 160 C, and the manifold at

4

◦

0 C. MS spectra are recorded from 3.6 to 4.8 min and from m/z 70 to 170. Alternatively, SIM

(

selected ion monitoring) for m/z 148, 149, and 150 can be applied. Typical chromatograms and

MS spectra are shown in Figures 1 and 5, respectively. For evaluation the intensities of the ions of

m/z 148, 149, and 150 are used; the sum of the intensities of m/z 149 + 150 is divided by the sum of

15

the intensities of m/z 148 + 149 + 150. These values are plotted against the mol% of [ N]-nitrate

see Table A1). A linear curve is obtained.

(

Table A2. Colum oven program for GC–MS.

◦

Time min

Temperature C

Heating Rate C/min

0

1

5

6

120

200

260

0

20

60

—

Appendix A.1.5. Optional: Determination of the Nitrate Concentration by Ion-Pair Chromatography

1

.

Mix exactly 20 µL extract with 180 µL eluent and centrifuge at 10,000 rpm for 5 min.

2

.

Prepare standards according to Table A3.

Molecules 2019, 24, 1531

17 of 22

Table A3. Preparation of standards for nitrate quantiﬁcation.

No.

Final Nitrate Concentration in mM

Nitrate Stock 1 mM ¹µL

St1

St2

St3

St4

St5

St6

St7

St8

0

10

20

0.001

0.002

0.005

0.01

0.02

0.05

0.1

50

100

200

500

1000

1

The stock solution must be transferred into a 10 mL volumetric ﬂask and eluent for ion-pair chromatography

1

4

15

added to the mark. Usually 1 mM [ N]-nitrate is used but 1 mM [ N]-nitrate is also suitable.

3

.

Transfer 150

µL of the clear supernatant into an autosampler vial and analyze by ion-pair

chromatography using an HPLC system with the following settings:

•

Column: Nucleodur 100-5 C18ec 125 × 4.6 mm

Precolumn: Nucleodur 100-5 C18ec 125 × 4.6 mm

Injection volume: 25 µL

Column oven: 25 C

Detector: UV, 213 nm

◦

Eluent: eluent for ion-pair chromatography: 10 mM 1-octylamine phosphate pH 7.0 in 20%

(

v/v) ACN

•

Flow rate: 1 mL/min

Isocratic elution

Analysis time: 5 min

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1

Sample Availability: In this study only standard chemicals were used, which are available from the sources

indicated in the Materials and Methods section. Seeds of C. crepidioides Ilé-Ifè are available from the

corresponding author.

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