Determination of impurities in pesticides and their degradation products formed during the...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2004; 18: 657–663

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1383

Determination of impurities in pesticides and their

degradation products formed during the wine-making

process by solid-phase extraction and gas

chromatography with detection by electron impact

mass spectrometry. I. Vinclozoline, procymidone

and fenitrothion

Juan Jose Jimenez*, Jose Luis Bernal, M. Jesus del Nozal, Elena Arias and Jose BernalDepartment of Analytical Chemistry, Faculty of Sciences, University of Valladolid, Prado de la Magdalena s/n, 47005 Valladolid, Spain

Received 29 September 2003; Revised 16 January 2004; Accepted 19 January 2004

The presence of degradation products of vinclozoline, procymidone and fenitrothion, and of impu-

rities from their commercial formulations, was studied in white and red wines elaborated from

spiked must. After solid-phase extraction the nature of the residues was established by gas chro-

matography with mass spectrometric detection. The structures of several degradation products and

impurities are discussed and elucidated on the basis of their electron impact spectra. In elaborated

wines the concentrations of the degradation products and impurities are lower than those of the

original active ingredients. Aminofenitrothion and acetylamino-formyl-fenitrothion-oxon are the

main residues of fenitrothion in wine. For dicarboximides, an alcohol derivative of vinclozoline

was found in addition to 3,5-dichloroaniline. Copyright # 2004 John Wiley & Sons, Ltd.

The determination of pesticide residues in wine is an impor-

tant factor to assess the wine quality and to propose, in the

near future, reliable maximum residue limits after determin-

ing the concentrations usually present. To this end, the occur-

rence of persistent degradation products and related

compounds should also be taken into account.

The aim of the present work, which is part of a broader

project, was to determine the evolution of the acaricides

vinclozoline, procymidone and fenitrothion from grape must

to wine. In order to identify the degradation products, a

simple and common sample preparation method for pesti-

cide residue analysis by gas chromatography was used.

Commercial pesticide formulations were added to must to

simulate the levels of the residues that would have arisen

from vineyard treatment; the residues in the must and wine

made up from that must were extracted by solid-phase

extraction on styrene-divinylbenzene cartridges, which have

been demonstrated to be useful for isolation of pesticides of

different polarities and functionalities.1

Not much attention has been paid in the literature to the

incidence of pesticide degradation products in grape

products. Some degradation products of the pesticides

considered here in various matrices have been described.

For fenitrothion, many compounds have been identified and

studied in soil, urine and water;2–7 a summary of the

microbial metabolites and their mutagenicity has also been

published.8 With respect to the dicarboximides vinclozoline

and procymidone, 3,5-dichloroaniline is often cited as a

degradation product in water, plants, must and wine.9–13 In

this case, the fate of dicarboximides14 and their degradation

products15,16 in wine has been published.

In the present work, extracts from white and red grape

must, and from wine elaborated by traditional microvinifica-

tion procedures, were analyzed by gas chromatography with

detection by electron impact mass spectrometry. The mass

spectra of the degradation products and impurities from the

formulation found in the extracts are shown, and their

possible structures and relative amounts are also discussed.

EXPERIMENTAL

Materials and chemicalsPesticide standards (99% minimum purity) were obtain-

ed from Promochem (Wesel, Germany) and Riedel de

Haen (Hannover, Germany). The commercial formulations

Kenolex 50WP (Procymidone 50%) from Kenogard S.A.

Copyright # 2004 John Wiley & Sons, Ltd.

*Correspondence to: J. J. Jimenez, Department of AnalyticalChemistry, Faculty of Sciences, University of Valladolid, Pradode la Magdalena s/n, 47005 Valladolid, Spain.E-mail: [email protected]/grant sponsor: Junta de Castilla y Leon; contract/grant number: VA126-01.

(Barcelona, Spain), Ronilan FL (Vinclozoline, 50%) from

BASF AG (Limburgerhof, Germany), and Folitihion 50 LE

(fenitrothion, 50%) from Bayer (Leverkusen, Germany)

were also used. Residue analysis grade methanol and ethyl

acetate were provided by Labscan (Dublin, Ireland). Ultra-

pure water was obtained from a Milli-Q-plus apparatus

from Millipore Corp. (Bedford, MA). For solid-phase extrac-

tion, Lichrolut EN 200 mg cartridges were supplied by Merck

(Darmstadt, Germany). UVAFERM BC and UVAFERM CS2

dry yeasts were purchased from E. Begerow Gmbh & Co

(Langenlonsheim, Germany).

Addition of pesticides; wine-making processMicrovinifications in 16 L glass containers were conducted

with white grapes of two mixed varieties, Godella and

Dona Blanca, and with red grapes of the Garnacha variety.

Grapes were crushed and pressed by a pneumatic press

and then the SO2 content of the must was adjusted; in the ela-

boration of white wine, the must was cold settled for 24 h. The

amount of commercial formulation required to achieve a con-

centration of 10 mg/L of each pesticide was added. Next, the

musts were inoculated with dry yeasts to start the alcoholic

fermentation: UVAFERM CS2 (Saccharomyces cerevrisae) in a

ratio of 10 g yeast/100 L must to make red wine, and UVA-

FERM BC (S. bayanus) in a ratio of 5 g yeast/100 L must to

make white wine. For red wine elaboration the lees were

removed after the fermentation and a malolactic fermenta-

tion was performed; the wine was then obtained by pressing.

Both wine types were racked several times, stabilized by cold,

adjusted with sodium metabisulfite, and bottled.

The microvinifications were spiked with only one pesticide

each time. For each pesticide the microvinifications were

done in duplicate. The analyzed must samples were collected

one day after the addition of the pesticides. Wine samples

were taken two days after bottling.

SPE of pesticides in must and wineWine samples were analyzed according to a slight modifica-

tion of the procedure reported previously.1 Briefly, it con-

sisted of elution of wine (10 mL) through a Lichrolut EN

cartridge, previously conditioned with 10 mL of methanol

and 10 mL of water. Then 6 mL of water were poured onto

the cartridge to clean up the extract, and were eluted and dis-

carded. Finally, the cartridge was dried by sucking nitrogen

through it; the solid phase was soaked in 3 mL of ethyl acetate

for 3 min, and the extract was then eluted by gravity.

For the analysis of must samples, 10 mL of sample were

diluted with 40 mL of water, and the analytes in the mixture

were extracted as described above.

Determination by GC/MSChromatograms and spectra were recorded by coupling a

Hewlett-Packard (Avondale, PA, USA) 6890 gas chromato-

graph equipped with an HP7683 injector and a 30 m�0.25 mm HP5MS capillary column (coated with a 0.25 mm

film of 5% phenylmethylpolysiloxane) to an HP5973 mass-

selective detector (single quadrupole mass spectrometer).

The oven temperature was programmed as follows: initial

temperature 508C (1 min), a 158C/min ramp to 1308C, a

48C/min ramp to 1708C, then 2.48C/min ramp to 2408C,

and finally a 158C/min ramp to 2708C (15 min). The carrier

gas (He) flow rate was constant at 1 mL/min, measured at

508C. Pulsed splitless injection of a 1mL sample volume was

performed at 2308C with a pressure pulse of 25 psi for

1 min; the purge valve was also opened at 1 min. The transfer

line was heated at 2808C. Mass spectra were scanned in

electron impact mode from m/z 50–515 (3.15 scans/s) after

a solvent delay of 5.50 min. Source and quadrupole tempera-

tures were 220 and 1008C, respectively. The electron multi-

plier voltage was maintained at 235 V above the Autotune

value.

Quantitation of the pesticides in the extractsLinear regression analysis was used to generate the calibra-

tion equations in the 1–50 mg/L range. To avoid quantitation

errors derived from the sample matrix, the calibration stan-

dards consisted of extracts from wine (or must) spiked with

known amounts of pesticides.1,17 Good fits, r2 > 0.990, were

always achieved. Pesticide stock solutions (1000 mg/L) and

working solutions were prepared in acetone.

Figure 1. Electron impact spectra of the compounds related

to vinclozoline.

Copyright # 2004 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 657–663

658 J. J. Jimenez et al.

RESULTS AND DISCUSSION

Degradation products and impurities foundVarious compounds structurally related to the studied

pesticides were found in bottled wines; some of these were

also detected in the commercial formulations used to spike

the musts. Solutions in acetone of the formulations (200 mg

formulation/L) were also prepared and analyzed by

GC/MS. In any case, the amounts of these compounds

(degradation products or impurities) in the formulations

were minimal in comparison with those of the pesticide itself;

in terms of peak areas, the abundance of each of these im-

purities or degradation compounds was lower than 10�3

to 10�4%.

VinclozolineFor vinclozoline, a transformation product, 3,5-dichlorani-

line (VinI), was observed in the commercial formulation

and in the sample extracts. However, 3-(3,5-dichlorophe-

nyl)-5,5-dimethyl 2,4-oxazolidinedione (VinII) was detected

only in the formulation. Both compounds were identified

from their mass spectra (Fig. 1) by comparison with spectral

libraries.

In the extracts from wine elaborated from the must treated

with vinclozoline, a compound (VinIV), which eluted after

vinclozoline, was detected; the chromatographic peak of this

compound was badly tailed, and the highest mass ion

detected in the spectrum was at m/z 259, lower than that of

the molecular ion of vinclozoline. This suggested the

possibility of an alcoholic compound for which molecular

ions are not always observed. Based on the spectrum (Fig. 1),

compound VinIV might be an oxazolidinedione with a

structure similar to that of vinclozoline. The allylic group in

VinIV could have been replaced by a hydroxyl, and the ion at

m/z 259 could then have arisen from an a-cleavage at the

tertiary alcohol. Figure 2 shows the structure of the proposed

degradation products detected by GC, and Table 1 shows the

proposed interpretations of several fragment ions.

ProcymidoneTwo degradation products of procymidone were observed in

the wine extracts. One of these was the corresponding

dichloraniline (ProI), which was expected because it is a

known and common degradation product of procymidone

and vinclozoline.9,14 Unlike the vinclozoline case, dichloroa-

niline was not found to be an impurity of the commercial

formulation used to spike the must. The spectrum of com-

pound ProII (Fig. 3) had the isotopic pattern characteristic

of a single chlorine atom in the structure. The interpretation

of the mass spectrum (Table 2) suggested that ProII was a

product resulting from the dechlorination of procymidone.

Figure 4 shows the structures of procymidone and of the pro-

posed degradation products.

FenitrothionTable 3 shows the compounds related to fenitrothion found in

must and wine extracts, and also in the commercial formula-

tion. A compound (FenIV), which eluted at a retention time

very close to that of fenitrothion, was detected; their spectra

were also similar, which suggested that compound FenIV

was an isomer of fenitrothion (FenV), in which the positions

of the groups substituted on the aromatic ring were different.

The spectra of two compounds detected in the formulation

(FenI and FenII), and of one compound detected in wine

extracts (FenIII), contain practically the same fragment ions

(m/z values) but with different abundances. Moreover, in all

three spectra, the highest mass ions were at m/z 247 (Fig. 5).

Table 1. Retention times and fragment ions for the

compounds related to vinclozoline found in wines and in the

commercial formulation

Time (min)Formulationoccurrence

Ion(m/z) Fragmentation

10.55 (VinI) Yes 161 Mþ; 3,5-dichloroaniline125 Mþ –HCl90 Mþ –HCl –Cl

20.42 (VinII) Yes 273 Mþ; dimethylatedvinclozoline

255 Mþ –H2O201 Mþ –CO2 –CO

21.91 (VinIII) Yes 285 Vinclozoline, Mþ

241 Mþ –CO2

212 Mþ –CO2 –CH CH2 �2 H198 Mþ –CO2 –CH CH2 –CH3 –H187 Cl2–C6H3–NCO178 Mþ –CO2 –CH CH2 –HCl159 Cl2–C6H3–N145 Cl2–C6H3

124 Cl–C6H3–N

23.90 (VinIV) No 275 Mþ;methylhydroxyvinclozolinederivative*

259 Mþ –CH3 –H or –O231 Mþ –CO2

216 Mþ –CO2 –CH3

71 O C–C( O)CH3

*Molecular ion not observed; Mþ: molecular ion.

Figure 2. Structures of vinclozoline and related compounds.

GC/MS analysis of pesticide residues in wine 659


The commonality of molecular ions, fragmentations and

retention times suggests that compounds FenI, FenII and

FenIII are isomers. The interpretation of the spectrum of the

most abundant isomer in wine, compound FenIII, suggested

that this compound was aminofenitrothion (see Table 3), a

degradation product originating from reduction of feni-

trothion and also found previously in other matrices.5 The

different relative abundances of the molecular ions could be

indicative of the isomers; the ortho- and para-thiophosphate-

aniline molecular ions are expected to be more stable than the

meta-substituted compounds as a consequence of greater

delocalization of the positive charge. Thus, for compounds

FenI and FenII, it is presumed that the amine group is bonded

to the ring in the meta-position. The resonance phenomenon

also can account for the high abundances of the ions atm/z 122

and 138 for compound FenIII (para-substituted).

Compound FenVI was detected only in wine samples;

its structure was assigned from the interpretation of the

main fragment ions shown in Table 3 and is thought to be

an acetamide of the aminofenitrothion-aldehyde (Fig. 6).Figure 3. Electron impact spectra of the compounds related

to procymidone.


compounds related to procymidone found in wines and in

the commercial formulation

Time (min)FormulationOccurrence Ion Fragmentation

10.49 (ProI) No 161 Mþ; 3,5-dichloroaniline125 Mþ –HCl90 Mþ –HCl –Cl

23.41 (ProII) Yes 249 Mþ; monodechlorinatedprocymidone

96 C5H6NO67 C3HNO

27.99 (ProIII) Yes 283 Procymidine, Mþ

268 Mþ –CH3

255 Mþ –CO212 Mþ –CO –CH3 – CH2¼CH2

145 Cl2–C6H3

124 Cl–C6H3–N

Mþ: molecular ion.

Figure 4. Structures of procymidone and related compounds.



The origin of compound FenVI is assumed to be the

aminofenitrothion-aldehyde that subsequently is acetylated

to form the amide, while the methyl group is oxidized to

an aldehyde group. However, the proposed intermedia-

tes fenitrothion-aldehyde and aminofenitrothion-aldehyde

were not observed in the extracts.

Finally, compound FenVII, an impurity in the formulation,

contained the same fragment ions as fenitrothion, but with

very different relative abundances, i.e., m/z 125 (100), 260 (81)

and 277 (13). The great difference in retention times argues

against assignment of this compound as a simple isomer of

fenitrothion; its structure was not elucidated.

Occurrence of the degradation products andimpuritiesTable 4 shows the GC peak areas of each of the compounds

found in wine and must extracts. The non-detection of

some impurities in musts is understandable owing to their

minimum amounts present in the original formulation.

For vinclozoline, compound VinIVwas the most abundant

degradation product in wine, while the dichloroaniline (VinI)

appeared to be a minor compound with two origins, the

formulation and the degradation process. However, the latter

process predominated since the peak area increased notably

in the wine extract. Figure 7(A) shows the chromatogram of a

red wine extract. The amount of vinclozoline residues was

higher in white wine than in red, probably because in the

process used to make white wine the malolactic fermentation

was not used and there were fewer settling steps.

Dichloroaniline (ProI) was the main degradation product

of procymidone, in contrast to the vinclozoline case, and

mainly in white wine. The presence of compound ProI was

only a consequence of the transformation of the active

ingredient, not an impurity. Dechlorinated procymidone

(ProII) was present at relatively lower concentrations

(Fig. 7(B)). Higher amounts of these residues were also

observed in white wine.


compounds related to fenitrothion found in wines and in the

commercial formulation

Time (min)FormulationOccurrence Ion Fragmentation

20.25 (FenI)21.99 (FenII)

Yes 247 Mþ; two isomers* ofcompound FenIII

22.37 (FenIII) No 247 Mþ; aminofenitrothion231 Mþ –NH2

215 Mþ –CH3OH184 Mþ –CH3O –S or Mþ –CH3O

–CH3OH138 Mþ –(CH3O)2PO125 (CH3O)2PS122 Mþ –(CH3O)2PS

22.79 (FenIV) Yes 277 Mþ, fenitrohtion isomer23.53 (FenV) Mþ, fenitrothion

260 Mþ –OH247 Mþ –NO109 (CH3O)2PO93 (CH3O)2P or (CH3)PS(CH3)

23.78 (FenVI) No 287 Mþ; acetylaminofenitrothion-aldehyde derivative

272 Mþ –CH3

255 Mþ –CH3OH178 Mþ –(CH3O)2PO162 Mþ –(CH3O)2PO2

149 Mþ –(CH3O)2PO –CHO125 (CH3O)2PO2

27.16 (FenVII) Yes 277 not identified

*Proposed; Mþ: molecular ion.

Figure 5. Electron impact spectra of the compounds related

to fenitrothion.



For fenitrothion, the degradation products and impurities

were also more abundant in the elaboration of white wine;

even the occurrence of a minor fenitrothion isomer (FenIV)

was detected. The abundances (peak areas) of compounds

FenI, FenII, FenIV and FenVII, impurities present in the

formulation, were reduced during the vinification, suggest-

ing that they were not degradation products. Compounds

FenIII and FenVI, not detected in the formulation, could be

monitored in white and red wines to test for the use of

fenitrothion in those cases in which the latter had not been

detected in wine (Fig. 7(C)).

Regarding the amounts of the different compounds

present in the analyzed samples, the peak areas give an

indication of the trends. However, it must be noted that these

are semi-quantitative data as a consequence of the possibly

different extraction efficiencies. Furthermore, the chromato-

graphic signal can be affected by the influence of the matrix in

the injection port of the gas chromatograph.1,17,18 In any case,

the amounts of the impurities and degradation products

detected in bottled wines seemed to be always lower than

those of the original active ingredients.

Finally, the losses of acaricides during the elaboration of

red wine varied between 93–94% for fenotrothion and

vincolozoline compared with 89% for procymidone, where-

as, for white wine, the losses were 82–85% for fenitrothion

and vinclozoline, and 76% for procymidone.

Figure 6. Structures of fenitrothion and related compounds.

Table 4. Occurrence of pesticides and related compounds in must and wines: peak areas (thousands of counts) of the

compounds in wine extracts. Concentrations (mg/L) for the active ingredient are expressed in brackets (n¼ 2)

Red wine White wine

Compound Peak area in must Peak area in wine Peak area in must Peak area in wine

VINCLOZOLINE(VinI) 14 200 19 125(VinII) — — — —(VinIII) vinclozoline 260.437 (7.31) 11.051 (0.55) 322.773 (8.23) 29.281 (1.51)(VinIV) — 3.004 — 7.062

PROCYMIDONE(ProI) — 825 — 4.757(ProII) — 255 244 122(ProIII) procymidone 531.813 (9.30) 25.287 (1.07) 432.933 (8.83) 43.348 (2.40)

FENITROTHION(FenI) 517 — 4.026 952(FenII) 356 22 295 32(FenIII) — 95 — 1.397(FenIV) 135 — 170 14(FenV) fenitrothion 86.235 (6.01) 10.684 (0.67) 93.455 (6.98) 37.643 (1.82)(FenVI) — 286 — 602(FenVII) 153 — 489 —

—¼not detected.



CONCLUSIONS

The GC-amenable degradation products of the acaricides vin-

clozoline, procymidone and fenitrothion, formed during typi-

cal vinification processes for red and white wines, have been

characterized. A likely hydroxylated derivative of vinclozo-

line, dichloroaniline, aminofenitrothion and a compound

related to aminofenitrothion-aldehyde are the main degrada-

tion products found in young wines for the three pesticides.

The amounts of the degradation products are substantially

lower in comparison with the remaining amounts of the

original acaricides. White wines contain acaricide concentra-

tions two or three times higher than red wines. The impurity

and degradation product content is also higher in white

wines. The different residue levels for white and red wines

might be attributed to the different wine-making processes.

AcknowledgementThe authors thank Junta de Castilla y Leon for providing

funds (Project VA126-01).

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Figure 7. Total ion current chromatograms from wine

extracts. See tables for peak identification. Extracts from

(a) a red wine whose must was treated with vinclozoline; (b) a

white wine whose must was treated with procymidone; and

(c) a white wine whose must was treated with fenitrothion.



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