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

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2004; 18: 2629–2636

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1667

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 ionization

mass spectrometry. II. Bromopropylate, trichlorphon,

parathion-methyl and tebuconazole

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

Received 23 June 2004; Revised 4 September 2004; Accepted 4 September 2004

The presence of degradation products of bromopropylate, trichlorphon, parathion-methyl and

tebuconazole in white and red wines elaborated from musts spiked with commercial formulations

of the pesticides was studied. Must and wine were subjected to solid-phase extraction followed by

gas chromatography with electron ionization mass spectrometric detection. a-Bromophenylphenyl-

methanol, aminoparathion, acetylaminoparathion-oxon and dichlorvos have been identified as

degradation products of bromopropylate, parathion-methyl and trichlorphon in wines, respec-

tively. Moreover, the presence of additives and impurities of the formulations in elaborated wines

has also been found. Copyright # 2004 John Wiley & Sons, Ltd.

Wine is a very important product for the economy and trade

of many countries. The pesticides used to control parasitosis

in vineyards could be persistent enough to reach the must

and be subjected to the physicochemical and biological pro-

cesses that occur during the wine-making process. Many

degradation products originate in this process, and their rela-

tive amounts have not yet been established. Their determina-

tion is necessary to adopt reliably maximum residues levels

in future and to have tracers at disposal to be aware of the use

of labile pesticides.

To this aim, red and white grape musts were spiked with

commercial formulations of bromopropylate, parathion-

methyl, trichlorphon and tebuconazole; wines were made

according to a traditional procedure and wine extracts were

analyzed by gas chromatography/electron ionization mass

spectrometry (GC/EI-MS). This work is a continuation of a

previous study devoted to different compounds.1

4,40-Dibromobenzophenone and 4,40-dibromobenzilic acid

have been identified as degradation products of bromopro-

pylate in matrices such as water and honey, and analytical

methods for them have been proposed.2–5 The degradation

rate, its mechanism, and the degradation products for

parathion-methyl have been mainly studied in aqueous

samples.6–12 The residues of parathion-methyl and two

related compounds, parathion-oxon and 4-nitrophenol, have

also been studied during the processing of milk and yogurt.13

Dichlorvos, chloral and phosphonic acid dimethyl ester have

been mentioned as degradation products of trichlorphon in

drinks and human urine;14 artifacts arising from the GC

determination have also been observed.15 A study dealing

with the thermal degradation of trichlorphon has been

published.16 For tebuconazole, it has been reported only

that degradation preferably yields trizole-containing

compounds.17

In the present work, the must and wine samples were

extracted by using polymeric phase cartridges and the

presence of degradation products in the extracts was

investigated by gas chromatography with mass spectro-

metric detection. The obtained EI spectra are shown and

interpreted. Moreover, attention is paid to the impurities and

additives of the commercial formulations, which have been

detected in low concentrations in the wines produced.

EXPERIMENTAL

Material and chemicalsPesticide standards (99% minimum purity) were obtained

from Promochem (Wesel, Germany) and Riedel de Haen

(Hannover, Germany). The commercial formulations Folicur

25 EW (tebuconazole 25%) from Bayer (Leverkusen,

Germany), Neoron 50 LE (bromopropylate 50%) from Novar-

tis (Basel, Switzerland), Parakey metil-35 (parathion-methyl

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.

35%) from I.Q. Key (Monterrey, Mexico) and Dipterex 50 LE

(trichlorphon 50%) from Bayer were also used. The commer-

cial formulations were dissolved in acetone at a concentration

of 200 mg/L for their injection for analysis by GC. Residue

analysis grade solvents were provided by Labscan (Dublin,

Ireland). Ultrapure water was obtained from a Milli-Q plus

apparatus from Millipore Corp. (Bedford, MA, USA). For

solid-phase extraction (SPE), Lichrolut EN 200 mg cartridges

were supplied by Merck (Darmstad, Germany).

Addition of pesticides and thewine-making processThe microvinification and spiking have been described in

detail previously.1 The formulations were added to the musts

to obtain an initial concentration of 10 mg/L for each pesti-

cide referred to the total volume of must; at this stage the

musts also contained matter in suspension. After liquid-solid

partitioning, it was expected that the concentration levels of

the pesticides in the liquid phase (must) would reflect the

levels present in real musts. Then, the musts were inoculated

with yeasts to carry out the alcoholic fermentation; for red

wine elaboration, a malolactic fermentation was also per-

formed. Next, both wine types were racked several times, sta-

bilized by cold and bottled. For each pesticide, the

microvinifications were done in duplicate. The must samples

were collected one day after the addition of the pesticides.

Wine samples were collected two days after bottling.

SPE and GC/MS determinationThe analytes were isolated from must and wine samples by

SPE on Lichrolut EN cartridges following the procedure pro-

posed in the first manuscript in this series;1 the GC/MS con-

ditions are also detailed there. Sample volumes of 10 mL were

used, diluting with 40 mL of water in the must analysis.

Cartridges were eluted with 3 mL of ethyl acetate.

Chromatograms and mass spectra were recorded by

coupling a Hewlett–Packard (Avondale, PA, USA) 6890 gas

chromatograph equipped with a 30 m� 0.25 mm� 0.25mm

HP5MS column to an HP5973 mass-selective detector (single

quadrupole mass spectrometer). Electron ionization (EI) mass

spectra were collected by scanning betweenm/z50 and 515. To

avoid quantitation errors derived from the sample matrix, the

calibration standards consisted of extracts from wine (or

must) spiked with known amounts of pesticides.

RESULTS AND DISCUSSION

Impurities and degradation products foundThe compounds whose structures are related to the active

ingredients, bromopropylate, parathion-methyl and trichlor-

phon, detected in the formulations as well as in the extracts,

are mentioned below. For tebuconazole, related compounds

were not found.

BromopropylateTable 1 summarizes the characteristic fragmentations for five

related compounds observed in the formulation and extracts.

Two compounds were identified by the interpretation of their

mass spectra. So, compounds BroI and BroIII are considered

to be the 2-propyl ester of the 2-bromobenzoic acid and the

alcoholic aromatic moiety of bromopropylate, respectively.

Compound BroIV was a common degradation product

of bromopropylate: 4,40-dibromobenzophenone. Figure 1

shows the structures of bromopropylate and the identified

compounds.

Compound BroVI, eluted in the tail of the bromopropylate

chromatographic peak, was not identified but its structure

could be very similar to that of bromopropylate as a

consequence of the similarity of retention times and fragment

ions (m/zvalues); perhaps, the difference between them lies in

the alcohol that esterifies the acid group. With respect to

compound BroII, the ion at m/z 183 and the corresponding

isotopic signal at m/z 185 were the only ions observed.

Parathion-methylTable 2 shows the compounds related to parathion-methyl

found in the formulation and in must and wine extracts.

Compounds ParI and ParIIwere simple derivatives of dithio-

phosphoric acid, compound ParIV was aminoparathion. All

of them were identified from their mass spectra by compari-

son with spectral libraries.

The structure of compound ParVIwas analogous to that of

a degradation product of fenitrothion found in wines.1 It

corresponds to an acetylated derivative from aminopar-

athion-oxon. As happened for fenitrothion, the intermediate

degradation products were not detected in the extracts either.

Figure 2 shows the compounds identified. Compound ParIII

was not identified; the interpretation of the EI mass spectrum

suggested that it was a compound similar to parathion-

methyl in which the substituents of the aromatic ring are

different.

TriclorphonThree related compounds were observed in the formulation

(see Table 3). Compound TriII was the known degradation

product dichlorvos, whose spectrum was very similar to

that of trichlorphon except for the lack of the ion at m/z 139,

characteristic of trichlorphon. It is often accepted that a low

amount of dichlorvos arises from the thermal degradation

of triclorphon in the injection port of the chromatograph; in

this work most of the dichlorvos is a degradation product

from trichlorphon and a byproduct of the formulation man-

ufacturing, as was ascertained by the injection of trichlorphon

standards.

The spectra of triclorphon and compound TriIII contained

many common fragment ions, although their relative

abundances were different, and the molecular ion of both

compounds was the same,m/z 256. At first it was thought that

the structure of compound TriIII should be that of triclor-

phon, modifying the position of the Cl atom and OH group.

Thus, compound TriIII was assumed to be phosphonic acid

(2,2-dichloro-1,1-hydroxychloroethyl) dimethyl ester or

phosphonic acid (2,2,2-hydroxydichloro-1-chloroethyl)

dimethyl ester. However, the review of the possible

fragmentations for the proposed compounds indicated

that compound TriIII could not contain the OH group, since

the a-cleavages adjacent to the C–P bond—(CH3O)2P

( O)C(OH)Cl ion at m/z 173 or (CH3O)2P( O)CHCl ion at

m/z 156—were not discerned in the spectrum. Then it was

assumed to be a compound analogous to dichlorvos (both

Copyright # 2004 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 2629–2636

2630 J. J. Jimenez et al.

derived from the phosphoric acid) to which H and Cl atoms

were added to the double bond (see Fig. 3); from the two

possible assignments, compound TriIII is likely to be

phosphoric acid (2,2,2-trichloroethyl) dimethyl ester because

in this case a H-rearrangement is not necessary to form the

(CH3O)2P( O)CH(OH) ion (m/z 139).

Finally, the structure of compound TriI could not be

elucidated. It was ascertained only that it was a phosphoric

acid derivative.

Occurrence of the degradationproducts and impuritiesTable 4 shows the GC peak areas of each of the compounds

found in wine and must extracts. Data are the mean of two

microvinifications. For bromopropylate, a byproduct of the

formulation whose amount decreased during the wine-mak-

ing process, compound BroI, was found in the musts and

wines. Two degradation products originated in the white

wine making process were detected. One of them, an alcohol

derivative of bromopropylate (BroIII), appeared in very low

concentration; in fact, its chromatographic peak could not be

integrated in the total ion current chromatogram, while the

unknown compound BroII was observed only in the spiked

must.

Table 1. Retention times and fragment ions for the compounds related to bromopropylate found in wines and in the commercial

formulation

Time (min) Formulation occurrence Ion (m/z) Relative abundance (%) Fragmentation

11.44 (BroI) Yes 242 12 Mþ; 2-bromobenzoic acid, 2-propyl ester227 4 Mþ –CH3

200 62 Br–C6H5–COOH183 100 BrC6H4CO155 28 BrC6H4

129 2 BrC4H2

77 27 C6H5

18.21 (BroII) No 183 100 Higher m/z, not identified30.15 (BroIII) Yes 262 98 Mþ; a-bromophenylphenylmethanol

261 100 Mþ –H245 8 Mþ –OH183 37 BrC6H4CO105 48 C6H5CO77 19 C6H5

31.45 (BroIV) Yes 338 22 Mþ; dibromobenzophenone259 24 Mþ –Br183 100 BrC6H4CO155 51 BrC6H4

105 42 C6H5CO77 30 C6H5

39.55 (BroV) Yes 426 1 Mþ; bromopropylate383 1 Mþ –CH(CH3)2

339 64 Mþ –CO2CH(CH3)2

323 1 Mþ –CO2CH(CH3)2 –O260 8 (BrC6H4)C(OH)(C6H4)243 1 (BrC6H4)C(C6H4)183 63 BrC6H4CO155 27 BrC6H4

39.64 (BroVI) Yes 381 14 Higher m/z, not identified, two Br atoms339 46 two Br atoms261 6 One Br atom185 61 One Br atom155 20 BrC6H4

104 8 C6H4CO*76 10 C6H4

Mþ: molecular ion.*: supposed.

Figure 1. Structures of bromopropylate and related

compounds.

Pesticide impurities in the wine-making process 2631


Figure 4(A) shows the chromatogram of a wine extract. The

concentration of bromopropylate decreased more than 98%

during the wine elaboration. The concentration of bromo-

propylate in must had already decreased sharply, too: 85 and

76% for red and white grape musts, respectively. Presum-

ably, the fungi and bacterial flora from the vineyards and the

hydrolysis reactions are more important than the fermenta-

tion process for the bromopropylate degradation.

The concentration of parathion-methyl in wine was also

low in comparison with the added concentration, with a

global loss during the process used to make wine close to

91%. In contrast to bromopropylate, the fermentation step

contributed notably to the concentration decrease because in

the spiked musts the concentration loss was slight, 15 and

32% for red and white wines, respectively. The impurities

ParI and ParII were detected in must but they did not persist

in wine.

Two degradation products of parathion-methyl were

discovered in wines: aminoparathion (ParIV) and acetyla-

minoparathion-oxon (ParVI). These two compounds were

also detected in the commercial formulation, but, as they

were not observed in the must extracts, they must have been

produced during the vinification. The relatively high

amounts in wine corroborate this supposition. Compound

ParIV was only present in white wine whereas compound

ParVI was detected in both types of wine. The degradation

products were more abundant in white wine than in red

wine. Figure 4(B) shows the chromatogram of a white wine

extract.

In relation to triclorphon, the occurrence of dichlorvos

(TriII) and TriIII was noted in must and in wine. The amount

of compound TriIII decreased during the process of red wine

production whereas it remained virtually constant during the

white wine making. The dichlorvos peak area increased

during the wine elaboration, as can be observed in Table 4,

also being more abundant in red wine. The dissipation of

triclorphon was higher than 90%. Figure 4(C) shows the m/z

109 extracted ion chromatogram, a common ion for the parent

Table 2. Retention times and fragment ions for the compounds related to parathion-methyl found in wines and in the commercial

formulation

Time (min) Formulation occurrence Ion (m/z) Abundance relative (%) Fragmentation

6.36 (ParI) Yes 156 53 Mþ; phosphorothioic acid, O,O,S-trimethyl ester141 13 Mþ –CH3

126 18 Mþ –CH2O110 100 (CH3O)2POH

7.09 (ParII) Yes 172 100 Mþ; phosphorodithioic acid, O,S,S-trimethyl ester157 18 Mþ –CH3

141 21 Mþ –OCH3

125 99 Mþ –SCH3

109 23 Mþ –OCH3 –S93 76 (CH3O)2P

12.28 (ParIII) Yes 266 79 Higher m/z, not identified251 5 Mþ –CH3

235 2 Mþ –CH3O203 19 Unknown158 2 Unknown142 8 Unknown125 38 (CH3O)2PS*109 7 (CH3O)2PO*93 100 (CH3O)2P*

20.03 (ParIV) Yes 233 100 Mþ; aminoparathion-methyl217 1 Mþ –NH2

201 9 Mþ –CH3OH124 84 H2N(C6H4)S108 37 H2N(C6H4)O93 16 (CH3O)2P

21.74 (ParV) Yes 263 98 Mþ; parathion-methyl246 10 Mþ –OH233 8 Mþ –NO200 11 Mþ –NO2 –OH137 8 OC6H4NO2 –H125 89 (CH3O)2PS109 100 (CH3O)2PO

22.61 (ParVI) Yes 273 69 Mþ; acetylaminoparathion-oxon derivative258 100 Mþ –CH3

241 8 Mþ –CH3OH217 6 Mþ –CH3 –CH3CN164 9 Mþ –(CH3O)2PO148 21 Mþ –(CH3O)2PO2

Mþ: molecular ion.*: supposed.



Figure 2. Structures of parathion-methyl and related compounds.

Table 3. Retention times and fragment ions for the compounds related to trichlorphon found in wines and in the commercial

formulation

Time (min) Formulation occurrence Ion (m/z) Abundance relative (%) Fragmentation

6.02 (TriI) No 151 1 Higher m/z, not identified128 2 Unknown126 3 (CH3O)2P( O)OH*110 100 (CH3O)2POH*95 23 Unknown80 64 Unknown59 20 Unknown

8.01 (TriII) Yes 220 7 Mþ; dichlorvos185 37 Mþ –Cl145 12 (CH3O)2P(OH)Cl109 100 (CH3O)2P( O)79 19 CH3OPOH or CH3OP( O)H

9.69 (TriIII) Yes 256 3 Mþ; phosphoric acid, (2,2,2-trichloroethyl) dimethyl ester (**)221 14 Mþ –Cl185 2 Mþ –Cl –HCl145 6 (CH3O)2P(OH)Cl139 79 (CH3O)2P( O)–C(OH)H109 100 (CH3O)2P( O)79 11 CH3OPOH or CH3OP( O)H

11.52 (TriIV) Yes 256 1 Mþ; trichlorphon221 5 Mþ –Cl185 12 Mþ –Cl –HCl145 31 (CH3O)2P(OH)Cl139 20 (CH3O)2P( O)–C(OH)H109 75 (CH3O)2P( O)79 100 CH3OPOH or CH3OP( O)H

Mþ: molecular ion.*: supposed.**: proposed.



compound and the degradation products. This chromato-

gram was used to give their relative amounts because of the

low level of residues present which did not allow the

integration of the peaks in the total ion chromatogram.

The behaviour of triclorphon differed from that of the

degradation products. The parent compound was more

abundant in white wine whereas the degradation products

were more abundant in red wine. However, the existence of

matrix effects that alter the mass transference of the analytes

from the injection port towards the chromatographic column

and affect the efficiency of the SPE could influence these

aspects.

Finally, degradation products of tebuconazole were not

detected in wines and musts. The concentration of tebuco-

nazole in the must decreased up to 15 and 36% for the red and

white wine production processes, respectively, but the loss

was higher in the final wine, 73 and 83%, respectively. On the

other hand, the GC peak area for tebuconazole increased in

the wine extracts in comparison with must extracts, while the

concentration was lower in wines. This demonstrates the

matrix effect to the quantification of this pesticide, either in

the gas chromatograph (as mentioned above) or in the SPE

step.

In contrast to the previous work,1 the amounts of the

degradation products and original active ingredients were

not always higher in the white wine. The higher occurrence of

residues in white wine is often assumed as a consequence of

the absence of the malolactic fermentation and the minor

presence of matter in suspension that can adsorb the

compounds.

In addition, the experimentation has shown that additives

and/or excipients of the formulations can also be found in

wines, in relatively high amounts. For example, this is the

case of an unknown compound from the bromopropylate

formulation, whose mainm/zvalues and relative abundances

in electron ionization were 70 (18), 86 (9), 98 (100), 112 (19), 126

(20), 140 (4), 154 (3), 168 (3), 180 (1), and 197 (19). In the

formulation, the GC peak area of this compound was 10.7%

relative to bromopropylate; in final wine, the area of this peak

was three times higher than that of bromopropylate. This

compound is marked with the letter ‘a’ in Fig. 4(A).

The same was observed in the tebuconazole extracts. Now,

a relatively intense chromatographic peak with characteristic

m/z values in EI: 55(16), 72 (57), 87 (100), 100 (52), 114 (13), 128

Figure 3. Structures of trichlorphon and related com-

pounds.

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)

Compound

Red wine White wine

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

Bromopropylate(BroI) 211 24 329 164(BroII) — — 61 —(BroIII) — — — xx(BroIV) — — — —(BroV) 1801 329 3502 1235Bromopropylate (1.5) (0.4) (2.4) (1.3)(BroVI) — — — —

Parathion-methyl(ParI) xx — — —(ParII) xx — xx —(ParIII) — — — —(ParIV) — — 2003 2032(ParV) 19 964 10.232 12 447 34 476Parathion-methyl (8.5) (0.9) (6.8) (0.9)(ParVI) — xx — 1056

Trichlorphon (m/z 109 ion chromatogram)(TriI) — — — —(TriII) 98 270 — 98(TriIII) 96 51 20 23(TriIV) 845 152 1004 321Trichlorphon (0.8) (0.2) (0.9) (0.8)

TebuconazoleTebuconazole 17 237 (8.5) 26 974 (2.7) 10 279 (6.4) 43 045 (1.7)

—¼not detected; xx¼detected, not quantified.



Figure 4. Chromatograms from wine extracts. See tables for peak identification. (A) Extract from a

white wine whose must was treated with bromopropylate (total ion current chromatogram). Peak (a):

unknown excipient from the commercial formulation. (B) Extract from a white wine whose must was

treated with parathion-methyl (total ion current chromatogram). (C) Extract from a red wine whose must

was treated with trichlorphon (m/z 109 ion chromatogram).



(4), 142 (9), 156 (7), 170 (4), and 199 (10), was attributed toN,N-

dimethyldecanacetamide. As can be seen in the chromato-

gram (Fig. 5), the amount of this compound is apparently

above the amount of tebuconazole in wine.

CONCLUSIONS

Five degradation products together with some impurities

have been found and characterized by GC/EI-MS in must

and wine samples after producing white and red wines from

spiked musts. Most of the degradation products are present in

wines in lower amounts than the residues of the pesticides.

Some impurities of the commercial formulations can be

found in wines that are produced. Their low amounts in the

formulations entail that many of them can be only detected in

must; in addition, their concentrations decrease during the

vinification.

Some additives of the formulations can persist throughout

the production process and are easily detected in the wine

extracts owing to their relatively high amounts.

AcknowledgementThe authors thank Junta de Castilla y Leon for providing

funds (Project VA126-01).

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Figure 5. Total ion chromatogram from an extract of white wine whose must was spiked

with tebuconazole.



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