An Alternative LC-UV Procedure for the Determination of Prochloraz Residues in Fruits

Um método alternativo, utilizando cromatografia líquida com detecção espectrofotométrica, para análise de procloraz como o produto de degradação 2,4,6-triclorofenol em manga, mamão e laranja é descrito. Acetato de etila, acetona e diclorometano foram testados para a extração de procloraz das frutas. Após a extração, procloraz foi submetido a uma reação com cloridrato de piridina para gerar o derivado 2,4,6-triclorofenol. A análise foi realizada por cromatografia líquida com detector espectrofotométrico e por cromatografia gasosa com detecção por captura de elétrons. Recuperações médias de frutas fortificadas (0,1 e 0,2 mg kg) variaram de 80 a 94% com coeficiente de variação entre 5,6% e 12,6% (n=8). Os limites de detecção e quantificação foram 0,05 e 0,1 mg kg, respectivamente. O método alternativo foi aplicado a amostras de manga e mamão, as quais foram tratadas por imersão em solução da formulação de procloraz sob condições de laboratório. Além disso, amostras de frutas de mercados locais foram analisadas.


Introduction
The fungicide prochloraz, N-propyl-N- [2-(2,4,6trichlorophenoxy)-ethyl]imidazole-1-carboxamide, Figure 1, has been employed to control leaf scab and grey mold on fruits, vegetables and ornamentals. 1,2 n Brazil, prochloraz has been released by the legislation for field application on apple, orange, tomato, wheat, rice and ornamentals and also as a postharvest fungicide on mango and papaya. 3However, a potential problem affecting the quality of the fruit is the appearance of fungicide residues.So, regular monitoring of fruit for fungicide content is required.
The paper reports an alternative LC-UV method for the determination of prochloraz as its 2,4,6-trichlorophenol derivative in mango, papaya and orange samples.Additional objectives in this work were to compare this proposed method with the GC-ECD procedure and to apply it to analyse prochloraz in mango and papaya submitted to dip treatment with a prochloraz formulation under laboratory conditions, and in fruit samples taken from local markets.
LC-grade water was obtained by filtering deionized water through a 0.45 µm filter with a Waters Millipore (Milford, MA, USA) system.Methanol and water were degassed using a Branson 5200 (Branson Ultrasonic Corporation, Danbury, CT, USA) ultrasonic bath.
Certified standards of prochloraz (98.4% pure) and 2,4,6-trichlorophenol (99.5% pure) were purchased from Dr. Ehrenstorfer (Augsburg, Germany).The individual stock solutions of the analytes were prepared by diluting 1.0 mg of the standards in 10.0 mL of methanol to obtain a concentration of 100 µg mL -1 .The working standard solutions were prepared by diluting the stock solutions as required.
Gas chromatographic analyses were carried out using a Varian 3300 gas chromatograph equipped with an electron-capture detector (ECD), an on-column injector, and a connected Varian 4290 reporting integrator.The megabore column was a ZB-1701 fused-silica column (30 m x 0.53 mm i.d., 1.25 µm; Zebron-Phenomenex, Torrance, CA).The injector and detector were operated at 240 °C and 300 °C, respectively.The oven temperature was programmed as follows: 140 °C for 1 min, increasing to 265 °C at 10 °C min -1 and holding for 10 min.Nitrogen was the carrier (2 mL min -1 ) and makeup (28 mL min -1 ) gas.

Sample preparation
Fruit samples (mango, orange and papaya) were purchased from local markets.The fruits were triturated separately using a household blender, homogenized and stored in individual jars at -18 °C until analysis.

Extraction and derivatization procedures
For hydrolysis of prochloraz residues in 2,4,6trichlorophenol, a derivatization reaction proposed by De Paoli et al. has been used: 4 (i) extraction procedure: a 5 g portion of the fruit sample was weighed into a glassstoppered flask.10.0 mL of acetone were added and the flask was shaken for 20 min on a mechanical shaker (Thermolyne, Dubuque, Iowa, USA); (ii) derivatization procedure: a fraction of the extract (1 mL) was transferred to a test tube.A 1 g portion of dry pyridine hydrochloride was added and the test tube sealed with a stopper and heated to 220 °C for 90 min in a glycerine bath.The test tube was cooled and 10 mL of water was added.The aqueous solution was extracted two times with 5 mL of diethyl ether:n-hexane (1:4, v/v), and the organic phase was transferred into another test tube.5 mL of 0.1 mol L -1 of KOH was added.The test tube was shaken for 1 min and the upper phase discarded; 5 mL of 1 mol L -1 HCl were added to the aqueous phase and this was extracted two times with 5 mL of toluene.An aliquot (1 µL) was injected into the GC-ECD system.For LC-UV analysis, an aliquot of 2 mL of the toluene phase was taken to dryness under a gentle stream of nitrogen.Residues were redissolved in 2 mL of methanol and an aliquot (20 µL) was injected into the LC-UV system.

Recovery studies
Recovery studies were carried out with untreated mango, orange and papaya samples.Samples of 20 g of each fruit were spiked with appropriate volumes of prochloraz standard solution.The fortified fruits were left to stand for a few minutes before extraction to allow the spike solution to penetrate the fruits.Recovery assays were performed at 0.1 and 0.2 mg kg -1 .At each fortification level eight replicates were analysed.The extraction and derivatization procedures described above were followed.

Treatment conditions
The experiments were performed on mature mango (Mangifera indica L.) and papaya (Carica papaya L.) under laboratory conditions.To carry out this study, mango and papaya were sorted to eliminate those with defects and selected for uniform size.Mango and papaya samples were placed separately in plastic boxes (20 fruits per box).The treatments were carried out in duplicate during 3 min, by dipping the samples in aqueous suspensions of SPORTAK 450 CE ® (450 g L -1 ) at the dose: 110 mL per 100 L of water. 13The fruits were left to dry at room temperature and stored for 29 days at 10 °C and 85-90% relative humidity (RH).Samples were taken before the SPORTAK 450 CE ® application and also at 0, 7, 14, 29 days after application.

Chromatographic conditions
In preliminary experiments, taking into account the unsatisfactory peak shape when prochloraz was injected directly into the LC-UV system a derivatization procedure, already established for the determination of prochloraz in apple, sugar beet root and leaves, wheat, wheat straw and tomato using GC-ECD, was tested. 4Therefore, LC-UV analyses of prochloraz as its 2,4,6-trichlorophenol derivative were conducted on a conventional LiChrospher 100 RP-18 reversed-phase column.To evaluate the mobile phase, different ratios of methanol-water were tested with respect to optimal peak form, separation efficiency and short elution time.Methanol (70%) in water using the isocratic mode with a flow rate of 0.8 mL min -1 shows the best conditions with respect to the analysis of the prochloraz derivative.The UV-Vis detector was operated at 220 nm.The identification of prochloraz as 2,4,6trichlorophenol was carried out by comparison of the retention time obtained with the corresponding 2,4,6trichlorophenol certified standard.Figure 2A shows the chromatogram of prochloraz submitted to the derivatization reaction with pyridine hydrochloride and Figure 2B shows prochloraz injected directly into the LC-UV system.Figure 3 shows the chromatograms of the fruit control samples and fortified papaya sample.The total running time of the LC-UV analysis was 10 min.
Since, prochloraz degrades when it is injected directly without derivatization into the gas chromatograph, the   procedure based on the one proposed by De Paoli et al. 4 was used for its derivatization, Figure 4.With relation to the GC-ECD analysis, during the optimization of the chromatographic conditions, different initial temperatures (90, 110, 120 and 140 °C) were tested.From this, an initial temperature of 140 °C proved to be the most suitable with respect to peak form.Figure 5 shows the chromatograms of an orange control sample, fortified orange sample and standard solution of the pesticide studied.The total running time of GC-ECD analysis was 10 min.

Extraction procedure
Preliminary investigations were performed for choosing the extraction solvent.Dichloromethane, acetone and ethyl acetate were tested.Acetone was selected, since it presented the highest recoveries (80-100%) for extraction of the compound from mango, orange and papaya.Despite the suitable recoveries (79-89%), the use of dichloromethane in the method is not favorable, because of environmental concerns.The recovery tests using ethyl acetate were in the range of 70-79%.For the recovery experiments untreated fruit samples were used.Recoveries were calculated by comparison with the appropriate working standard solutions.A 20 g portion of untreated fruit was fortified at two different concentrations (0.1 and 0.2 mg kg -1 ) and quantified using the external standard method.The results of the average recoveries ranged from 80% to 94%, with relative standard deviation (RSD) values of 5.6% to 12.6%, as can be seen in Table 1.Each recovery analysis was repeated 8 times.The precision and accuracy were considered adequate for the validation of the method. 11Standard solutions were injected after every ten samples to monitor changes in the chromatographic conditions.The chromatograms of the fruit extracts were satisfactory, without any interference in the retention time of the fungicide for both techniques.The amounts of 2,4,6-trichlorophenol obtained were corrected with the following factor to convert to the amounts of prochloraz [mol.wt. of prochloraz (376.7)/ mol wt. of 2,4,6triclhorophenol (197.5)= 1.91].The Brazilian legislation 3 establishes maximum residue limits (MRLs) for prochloraz for papaya, mango and orange matrices.The MRL values are 1.0, 0.2 and 0.5 mg kg -1 , respectively.
Table 2 shows some differences between the method studied and the De Paoli et al. 4 and Lafuente and Tadeo 5 methods.The comparison emphasizes the recovery values,   coefficients of variation, extraction and clean-up procedures.Also these methods require large volumes of solvent and large amounts of sample.The present method has comparable results at the same level of concentration (De Paoli et al. 4 ) and emphasizes the reduced number of steps involved in the analytical procedure (Lafuente and Tadeo 5 ).On the other hand, the chromatographic peak for the unhydrolysed prochloraz obtained by Lafuente and Tadeo was symmetric and relatively broad.

Linearity
Under the chromatographic conditions described, good linearities and correlation coefficients were achieved for the compound studied.Replicates (n=3) of six standard pesticide solutions of different concentrations were found to be linear in the range from 0.4 to 5.0 µg mL -1 for both chromatographic techniques.The equations for the calibration curves were y=11942.35x+239.66for LC-UV and y=24974.94x-40.73 for GC-ECD.The determination coefficients obtained for the prochloraz were 0.9997 (LC-UV) and 0.9991 (GC-ECD).

Limits of Detection (LOD) and Quantification (LOQ)
The criteria established by Thier and Zeumer 11 to find LOD and LOQ were used in this study for both chromatographic techniques.The LOD for prochloraz was 0.05 mg kg -1 .The LOQ was determined as the lowest concentration of the compound that gives a response that could be quantified with RSD of the less than 20% and a recovery at least 70%.Thus, the LOQ value for this compound was 0.1 mg kg -1 .

Prochloraz degradation
In this study, prochloraz residues were detected in all mango and papaya samples submitted to the dip treatment with an aqueous suspension of SPORTAK 450 CE ® (prochloraz as active ingredient) using the LC-UV procedure.For mango, the initial concentration of 9.5 mg kg -1 (0 day) decays to 7.2 mg kg -1 (7 days), 3.4 mg kg -1 (14 days), and dropped to 0.1 mg kg -1 in 29 days.For papaya, the initial concentration of 12.5 mg kg -1 (0 day) decays to 7.5 mg kg -1 (7 days), 2.1 mg kg -1 (14 days), and dropped to 0.4 mg kg -1 in 29 days.On the basis of the MRLs established by the Brazilian legislation for these matrices (1.0 mg kg -1 for papaya and 0.2 mg kg -1 for mango), these fruits can be considerable acceptable for human consumption 29 days after treatment, considering this experiment was done under laboratory conditions.

Real samples
The LC-UV method was applied to the analysis of the 126 real fruit samples (mango, orange and papaya) obtained

Table 1 .
Recovery of prochloraz from fortified fruits employing LC-UV and GC-ECD a n=8 analyses.