ELSEVIER Analytica Chimica Acta 338 (1997) 63-68

ANALYTICA CHIMICA ACTA

Analysis of pyraclofos in crops by the Swedish National Food Administration gas chromatographic multiresidue methodology

Antonio Valverde”‘*, Tuija Pihlstrijmb, Ame Anderssonb

” Grupo Re.siduo.7 de Plaguicidas, Departamento de Quimica Fkica, Bioquimica y Quimica Inorgcinica, Facultad de Ciencius Experimentales, Universidad de Almeria. 04071 Almenb, Spain

h Nationul Food Administration, Box 622, S-751 26 Uussala, Sweden

Received 6 March 1996; revised 23 July 1996; accepted 9 September 1996

Abstract

The National Food Administration (NFA) gas chromatographic (GC) multiresidue method was assessed to determine residues of pyraclofos in fruits and vegetables. The relative retention times of pyraclofos in the FPDIOV- 1701 and TSDLSE-30

detector/column systems used for GC analysis (using parathion as reference) were 2.11 and 2.04, respectively. The corresponding relative response factors were 0.34 and 0.40. The mass spectrum obtained for pyraclofos using a MSD/PAS- 1701 GC system suggests the use of the peaks at m/z 360 (molecular and base peak), 194 and 139 to confirm trace levels of pyraclofos by gas chromatography with mass selective detection (GUMSD) in SIM mode. Recovery values obtained with the complete NFA/GC multiresidue method for pyraclofos from tomato and strawberry samples spiked at levels of 0.05 or

0.40mg/kg ranged from 72% to 111%. The limit of quantification of pyraclofos was stated as 0.05 mg/kg, but levels of 0.02 mg/kg could be determined from the TSD/SE-30 chromatographic response. The elution profile of pyraclofos on a GPCl SX-3 Biobeads column was also determined in this work.

Keywords: Gas chromatography; National Food Administration; Pyraclofos; Crops; Pesticides

1. Introduction

Pesticide residue analysis has its main application in the monitoring of foodstuffs destined for human

consumption. This type of analysis is usually carried out by official laboratories from national regulatory agencies, and its principal function in most countries

is the enforcement of legislated national maximum residue limits (MRLs). Laboratory manuals published

* Corresponding author. Tel: +34 50 21.5309: fax: +34 50

215070.

by different agencies [1,2] and recent reviews on this general area of analysis [3,4] show that most pesticide residue analyses are performed using multiresidue methods involving solvent extraction, clean-up, determination by gas chromatography (GC), and confirmation by MS.

The main analytical method used in the Swedish monitoring of pesticide residues in fruits and

vegetables is a capillary gas chromatographic multi- residue method, developed by the National Food Administration (NFA), in which samples are prepared for analysis by ethyl acetate extraction and GPC clean-up [5]. With this method, the NFA can detect,

0003.2670/97/$17.00 Copyright (:, 1997 Elsevier Science B.V. All rights reserved

PI/ SOOOX-2670(96)00442-4

64 A. Valverde et al./Analytica Chimica Acta 338 (1997) 6368

quantify and confirm residues of more than 160 pesticides [6], the routine confirmation process being

carried out by gas chromatography with mass selective detection (GC/MSD) and using powerful MS ChemStations and MS libraries.

2. Experimental

2.1. Apparatus and reagents

Such as it has been already described [7], besides confirming residues of those pesticides included in

the GC-Multiresidue method, the use of GC/MS in the pesticide monitoring programme allows the NFA to identify “unknown analytical responses” (UARs).

Sometimes, these UARs are identified as a pesticide or a metabolite not introduced in the monitoring programme, a substance occurring naturally in the

sample, or a contaminant introduced during the analytical process. However, in several cases, the investigation of an UAR can lead to the identifica-

tion of a new pesticide, for which references on residue analysis cannot be found. In such cases, the

results obtained in the tests carried out to introduce the new pesticide in the NFA/GC-multiresidue

method can be of interest to be published in the open literature.

Gas chromatographs: Varian Vista 6000 equipped

with TSD and FPD (P mode) detectors. Hewlett- Packard HP-5890 equipped with quadruple mass selec-

tive detector (MSD) HP-5970 and MS ChemStation HP G1030A including the HP G1038A (HP pesti- cide) and HP G1035A (Wiley 130 K) MS libraries.

GC Columns: TSD - SE-30 fused silica capillary column, Restec 1176B (25 mx0.25 mmx0.25 pm). FPD - OV-1701 fused silica capillary column, Restec

15953 (25 mx0.32mmx0.25 urn). MSD - PAS-1701 silica capillary column, Hewlett-Packard (25 mx0.32 mmx0.25 urn).

Pyraclofos, (RS)-[0-l-(4-chlorophenyl)pyrazol-4- yl-O-ethyl S-propyl phosphorothioate], is the com- mon name of the insecticide recently introduced by

Takeda (Tokyo, Japan) as code No “TIA-230” and trade mark “Voltage” or “Boltage” [8,9]. The

structure of pyraclofos is shown in Fig. 2 (molecular ion). It is an asymmetric organophosphorus insecti-

cide, cholinesterase inhibitor [ IO,1 I], used to control Lepidoptera, Coleoptera, Acarina and nematodes in fruits, vegetables and other crops [8,9,12]. Details of its use, mode of action and toxicity have been

reported by Kono [8] and Tomlin [9]. Pyraclofos is already registered in different countries, and some

examples of MRLs established by Japan and Spain for it in a variety of commodities are as follows:

0.05 mg/kg (potato/Japan), 1 .OO mg/kg (citrus/Spain),

2.00 mg/kg (tomato/Spain), 5.00 mg/kg (tea/Japan). However, no reports have been still published on the analysis of its residues in crops.

GPC apparatus: Precision metering pump, Eldex A-30-S (0.05-l .5 ml/min flow rate range); Three-way valve single key, Omnifit, with sample loop of 1 ml in

Teflon; Bio-Beads SX-3 (200-400 mesh) GPC column (400 x 10 mm). A scheme of the GPC system is shown in Fig. 1 of the reference [13].

Rotary vacuum evaporator: Rotavapor Btichi.

Universal food cutter: CUT-O-MAT (Kneubiihler, Switzerland).

Homogenizer: Waring blender with explosion-

proof motor. Filter: Glass fibre filter, 9 cm diameter (Sartorius).

Microfilter acrodisk CR PTFE, 0.45 mm (Gelman).

Pyraclofos standard: 99% purity was obtained from Takeda (Tokyo, Japan). Pyraclofos standard solution

of 24.20mgll was prepared in acetone. Standard solutions for chromatographic analysis and recovery tests were prepared by suitable dilution with ethyl

acetate-cyclohexane ( 1 + 1). Solvents: Acetone, cyclohexane and ethyl acetate,

pesticide residue grade (Merck). Sodium sulphate: Pesticide residue grade (Merck).

2.2. Chromatographic analysis

The objective of this paper is to show the first Analysis of pyraclofos was carried out in the FPD/

results obtained on the analytical behaviour of OV-1701 and TSD/SE-30 detector/column systems

pyraclofos in each step of the NFA/GC-Multiresidue (both columns were fitted to the same injector) and

method, since residues of this pesticide have been using the following operating conditions: splitless

recently identified and confirmed in some samples injection time, 60 s; injection volume, 3 ~1; helium as

from the Swedish Surveillance Sampling Programme carrier gas and nitrogen as make-up gas with inlet for imported fruits and vegetables. pressure that elutes parathion at 10.3 min on the OV-

A. Vulverde et al./Analytica Chimica Acta 338 (1997) 6348 65

6000 :i!&;;;;*, - Iy*t,?t8 ,458, 52847

M/Z -> 50 100 150 200 250 300 350 400 150 500 5io

Fig. 1. Mass spectra obtained at 1.5.36min from the GC/MSD

total ion current scan chromatogram corresponding to (a) a

strawberry sample from the NFA surveillance sampling program

1992-93; (b) a pyraclofos standard solution of 24.20mg/l.

1701 column and at 10.7 min on the SE-30 column;

injector temperature, 250°C; detector temperature, 300°C; oven temperature programme, 1 min at 90°C 30”C/min to 180°C 4”C/min to 260°C and held for

6min. Quantitative evaluations were done by com- paring the peak areas for the analytical solutions with those for standard solutions. The final result of each

analysis was the mean value of the FPD and TSD results.

The GC MSD/PAS- 1701 operating conditions were

as follows: splitless injection time, 1 min; injection

volume, 4 ~1; injector temperature, 250°C; GC-MSD interface temperature, 280°C; the oven temperature programme was the above described, but now the 180°C and 260°C temperatures were held for 0.5 and 7.5 min, respectively. The mass spectrometer para- meters were: scan mass range, 50-550D; scan rate,

1.3 scan/s; solvent delay, 2.5 s. The parameters for the

quadrupole mass filter were set automatically by the

Autotune programme.

2.3. GPC elution projile

The chromatographic conditions used to determine

the GPC elution profile of pyraclofos on the SX-3 Biobeads column were the same than those used in

the GPC clean-up step of the NFA/GC-Multiresidue

method (5): injection volume, 1 ml; eluent mixture, ethyl acetate-cyclohexane (1 +l); eluent flow rate, 1 ml/min. The GPC column was calibrated with a

standard solution of isofenfos of 2.58 mg/l, obtaining a “break point” of 17.5 ml (this break point is the starting point of the pesticide fraction collected in the

NFA/GC-Multiresidue method, and it is set at the point where about 2% of isofenfos in the calibrating

solution is eluted). The elution profile of pyraclofos

was determined performing two runs with a pyraclo- fos standard solution of 1.45 mg/l, and collecting fractions in 1 .O ml increments from 16.5 to 37.5 ml in

the first run and from 18.5 to 26.5ml in the second one. In all instances, individual fractions were analyzed by GC/TSD and GUFPD, making calibra-

tion standard with aliquots of the standard solution injected in the GPC column.

2.4. Evaporation test

The evaluation of the losses of pyraclofos during the evaporation of standard solutions on vacuum

evaporator was carried out as follows: 5 ml of pyraclofos standard solution were transferred to a 250ml round-bottomed flask, evaporated just to

dryness on a rotary vacuum evaporator at 37°C and

redissolved with 5 ml ethyl acetate-cyclohexane (l+l). This solution was analyzed by GUTSD and GC/FPD, and the chromatographic responses were compared with those obtained for the original standard solution. This evaporation test was per- formed with three different pyraclofos standard

solutions of 0.151, 0.302 and 0.605 mg/l.

2.5. Extraction, GPC clean-up and recovery study

The extraction and clean-up procedures assessed to analyze residues of pyraclofos were those described

66 A. Valverde et al./Analytica Chimica Acta 338 (1997) 63-68

in the NFA/GC-Multiresidue method for fruits and

vegetables [5]. A brief description of these proce- dures is as follows: Weigh 75 g of thoroughly

homogenized sample and blend with 200ml ethyl acetate and 40g sodium sulphate for 3 min. Filter the

solvent phase by suction through a glass fibre filter with a 20 g sodium sulphate layer, and dry the filtrate

by shaking with 30g sodium sulphate. Transfer 100ml of the ethyl acetate layer to a 250ml round- bottomed flask and concentrate to approximately 1 ml on rotary vacuum evaporator at 37°C. Transfer the

concentrate quantitatively to a graduated test tube, adjust the volume to 2.5ml with ethyl acetate and

then to 5.0ml with cyclohexane. Filter the extract through a 0.45 mm microfilter by suction with a 10 ml

syringe. Inject 1 ml extract onto the GPC column and elute with ethyl acetate-cyclohexane (l+ 1) at a flow rate of 1 .O ml/min. Collect a 20 ml fraction, starting at

the “break point” of the GPC column, and

concentrate to about 1 ml on rotary vacuum evapora- tor. Transfer the concentrate quantitatively to a graduated test tube and adjust the volume to 5 ml

with ethyl acetate-cyclohexane (l+l). The extract so obtained contains 1.5 g sample per ml and is ready to be analyzed by GC/TSD and GUFPD.

Pyraclofos recovery study was conducted on spiked samples of tomato and strawberry. Tomato

samples were spiked at two different levels, 0.05 and 0.40mg/kg, whereas strawberry samples were only

spiked at 0.05 mg/kg. Three replicates of each spiked sample and one check sample of each crop were

analyzed.

The retention time value obtained for pyraclofos in

the GUMSD chromatographic conditions was 15.36 min, and the mass spectrum determined at this

time from the total ion current scan chromatogram of a pyraclofos standard solution of 24.20 mg/l is shown in Fig. l(b) (Fig. l(a) shows the mass spectrum

determined at the same time for a strawberry sample from the NFA surveillance sampling programme 1992-93, in which the presence of pyraclofos was

suspected). This mass spectrum, which was not found in the HP-Pesticide and Wiley libraries, can be easily

explained after looking at the molecular structure of pyraclofos and the typical fragmentation pattern of this class of compounds [14,15]. The major frag-

mentation pathways proposed for pyraclofos are indicated in Fig. 2. Likewise, the mass spectrum in

Fig. l(b) suggests the use of the peaks at m/z 360 (molecular and base peak), 194 and 139 to confirm trace levels of pyraclofos by GUMSD in Selected Ion

Monitoring (SIM) mode [ 161. Results obtained in the tests carried out to

determine the elution profile of pyraclofos on the SX-3 Biobeads column are given in Table 1. These results indicate that pyraclofos starts to elute at 3ml after the “break point” of the column and that it is

eluted within a volume fraction of 4ml. Results of the evaporation test are shown in Fig. 3.

In Fig. 3, the chromatographic response (peak area units) of the pyraclofos solutions obtained after the

3. Results and discussion

Retention time values of pyraclofos in the SE-30

and OV-1701 columns under the used chromato-

graphic conditions were about 21.40 and 21.45 min, respectively. The relative retention times for pyra- clofos, using parathion as reference, were 2.04 (SE- 30) and 2.11 (OV-1701), whereas the corresponding relative response factors were 0.40 (SE-30) and 0.34 (OV-1701). FPD and TSD responses for pyraclofos standard solutions were linear (r’ >0.99) over the assessed concentration range of 0.15 to 1.98 mg/l, with sensitivities of about 2.3 x lo4 and 3.0x lo4 peak area units per mg/l, respectively. Fig. 2. Major fragmentation pathways of pyraclofos

A. Valverde et al./Analytica Chimica Acta 338 (I 997) 634X 67

Table I Table 2

Pyraclofos recoveries obtained by using the NFAIGC-multiresidue

methodology

Elution profile of pyraclofos on the GPC/SX-3 Biobeads column

(10x400 mm). Eluent mixture: ethyl acetate-cyclohexane (l+l) at

a flow of 1 ml/min (Column break point: 17.5 ml)

Elution volume Pyraclofos recovered (%) Mean

fraction (ml) recovery (%)

1st run 2nd run

18.5-19.5 -

19.5-20.5 -

20.5-21.5 11.3 13.0 12.15

21.5-22.5 43.1 42.9 43.00

22.5-23.5 33.3 34.4 33.85

23.5-24.5 7.1 7.4 7.25

24.5525.5

25.5-26.5 -

Total 94.8 97.7 96.23

Sample Spiking Recoveries

level (mg/kg) (%)

Mean C.V.

rec. (70) (70)

Tomato 0.05 82.6182.7184.3 x3.2 I.1

Tomato 0.40 92.2191.2l90.6 91.3 3.3

Strawberry 0.05 I 10.7/90.7/72.1 9 1.2 21.2

0 2 4 6 6 IO 12 14 16

peak wea unlls lx 10001/brtors avopor.

x FPO/OV-I701 0 TSO/SE-30

Fig. 3. Chromatographic response (peak area units) of pyraclofos

standard solutions: after evaporation test (Y axis) versus before

evaporation test (X axis).

Finally, we must mention that pyraclofos recov-

eries in Table 2 were obtained using a just-prepared GPC column and a just-cleaned GC system. This is important because some preliminary recovery tests

(not reported here), carried out with a GPC column

and a GC system very used, showed that pyraclofos can present, in such conditions, a strong “matrix effect”, which was not presented by other many

pesticides in the same conditions. In these prelimin- ary tests, recoveries from 108% to 224% were

obtained, but these became close to 100% when GC calibrations were done with pyraclofos standard solutions prepared in blank sample extracts. Appar- ently, the magnitude of this “matrix effect” did not

depend on the matrix since it was greater when the concentration of pyraclofos in the sample extracts was smaller.

Acknowledgements

evaporation test, have been plotted versus the chromatographic response of the corresponding

original standard solution. Linear regression analysis led to obtain a coefficient of determination (r2) of 0.999 and a slope of 1.05; it is to say, the average recovery of pyraclofos in the evaporation test was

105%.

Antonio Valverde thanks CICYT: AL1950172 for

the financial support received to make his contribu- tion to this paper.

References

Recovery values obtained for pyraclofos from

spiked tomato and strawberry samples by using the complete NFA/GC-Multiresidue method are given in

Table 2. The limit of quantification of the analytical method for pyraclofos can be stated as 0.05 mg/kg, but levels about O.O2mg/kg could be clearly deter- mined from the TSD/SE-30 chromatographic re- sponse.

111

I21

[31

[41

L51

FDA, Pesticide Analytical Manual, Vol. I, Food and Drug

Administration, Washington, DC, 1994.

General Inspectorate for Health Protection, Analytical

Methods for Pesticide Residues in Foodstuffs, Ministry of

Public Health, Welfare and Span, Bilthoven, Netherlands

1996.

P.T. Holland and C.P. Malcolm, in T. Cairns and J. Sherma

(Eds), Multiresidue Analysis of Fruits and Vegetables.

Emerging Strategies for Pesticide Analysis. CRC Press,

Boca Raton, FL, 1992.

J. Sherma, Anal. Chem., 67 (1995) IR-20R. A. Andersson and H. Palsheden, Var Foda, (1996) in press.

68 A. Valverde et al./Analytica Chimica Acta 338 (1997) 6348

[6] A. Andersson and T. Bergh, Fresenius’ J. Anal. Chem., 339

(1991) 387-389.

[7] G. Blomkvist, Var FGda, 38 (1986) 125-131.

[8] Y. Kono, Jpn. Pestic. inf., 53 (1988) 27-31.

[9] C. Tomlin (Ed.), The Pesticide Manual, The British Crop

Protection Council, Surrey, The Royal Society of Chemistry,

Cambridge, UK, 1994.

[lo] Y. Kono, Y. Okada and Y. Sato, Appl. Entomol. Zool., 20

(1985) 443449.

[ 1 l] Y. Kono, Y. Manabe and Y. Sato, Appl. Entomol. Zool., 21

(1986) 363-369.

[12] ICI-ZELTIA, Voltage un nuevo insecticida de ICI-Zeltia

contra Trips en cultivos, Documentation Tecnica, Septiem-

bre 1992, ICI-Zeltia, Madrid, 1992.

[13] A. Andersson and B. Ohlin, Var Wda, 38 (1986) 79-109.

[14] J.P.G. Wilkins, Pestic. Sci., 29 (1990) 163-181.

[15] R.M. Silverstein, G.C. Bassler, and T.C. Morril,

Spectrometric Identification of Organic Compounds, Wiley,

New York, 1991.

[16] T. Cairns, J. Assoc. Off. Anal. Chem., 75 (1992) 591-593.