Pestic. Sci. 1997, 49,56È64

Liquid and Gas Chromatographic Multi-residue Pesticide Determination in Animal Tissues

Aurora Navas Dj az,* Angeles Garcj a Pareja & Francisco Garcj aSanchez Departamento de Qu•mica Anal•tica, Facultad de Ciencias, Universidad de Malaga, 29071-Malaga, Spain (Received 13 June 1995; revised version received 17 April 1996; accepted 16 August 1996)

Abstract: A liquid chromatography multi-residue method with photometric detection has been developed. The method is applicable to the quantitative deter- mination of organochlorine (tetradifon, dicofol, chlorfenson, chlorobenzilate), organophosphorus (fenitrothion, azinphos-ethyl) and carbamate (pirimicarb) pesticides in animal tissues. The extracted residues are cleaned up by gel- permeation chromatography. A further fractionation on silica Sep-Pack car- tridges is included in the procedure. A gas chromatographic method with electron-capture detection for the analysis of the same pesticides was carried out and the results in the two cases compared. Lower detection and quantitation limits and similar recoveries of pesticides from spiked pig liver and brain samples were obtained by the LC method.

Key words: liquid chromatography, gas chromatography, multi-residue pesti- cides, animal tissues

1 INTRODUCTION by various element-sensitive detectors, leading to reli- ance on mass spectrometry because of its ability to help Pest control in modern agriculture includes treatment to deÐne the structure. This has especially been true in of crops pre- and post-harvest with a variety of chemi- the case of the combination of gas chromatography cals, such as herbicides and insecticides in the pre- with mass spectrometry (GC/MS). One of the obvious harvest stage and with fungicides and rodenticides in Ðelds of application for this technique was the analysis the storage stage of the total harvest process. Conse- of pesticide residues in food of animal and of plant quently, the residue analysis of herbicides, insecticides, origin.12,13 rodenticides and fungicides for quality control purposes In the present study, an alternative LC method is is of great interest. developed for the multi-residue analysis of pesticides Determination of pesticide residues is usually carried that display UV-visible absorbance, in samples of out by gas chromatography (GC). Liquid chromatog- animal origin. Pesticide residues are usually not only raphy (LC) has also been used for pesticide residue monitored in crops, fruits and vegetables, but also in analysis, but most of the methods only deal with one animal tissues where residues may occur by transmis- class of pesticide, such as carbamates,1,2 sion through the food chain. As part of the extensive organochlorine3h5 or organophosphorus.6,7 Only in a safety evaluation of agrochemicals, studies to establish few cases have several classes of pesticide been deter- the levels of parent compounds and their metabolites mined using one system8,9 or LC been used in com- which may be passed via the food chain into the edible bination with GC for multi-residue analysis.10 LC o†ers portions of animal tissues11,14,15 are carried out by some unique advantages for residue analysis11 and its industry. potential for multi-residue analysis has not yet been During the development of the liquid chromatog- totally exploited. Trace levels of pesticides, using a raphy method described in this current study, the fol- multi-residue screening procedure, have been detected lowing pesticides were examined: organochlorines [tetradifon (4-chlorophenyl 2,4,5-trichlorophenyl * To whom correspondence should be addressed. sulfone), dicofol (2,2,2-trichloro-1,1-bis(4-chlorophenyl) 56 Pestic. Sci. 0031-613X/97/$09.00( 1997 SCI. Printed in Great Britain Multi-residue pesticide determination in animal tissues 57 ethanol), chlorfenson (4-chlorophenyl 4-chloro- 2.1.2 Reagents benzenesulfonate) and chlorobenzilate (ethyl 4,4@- Acetone, hexane, chloroform were analytical reagent dichlorobenzilate)], organophosphorus (fenitrothion grade (Merck). Methanol and acetonitrile were Lichro- (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) and solv gradient grade (Merck). Water was distilled and azinphos-ethyl (S-(3,4-dihydro-4-ozobenzo[d][1,2,3,] deionized or Lichrosolv grade. Anhydrous sodium triazin-3-ylmethyl) O,O-diethyl phosphorodithioate)) sulfate (Carlo Erba, Milano, Italy) was treated by and carbamates (pirimicarb (2-dimethylamino-5,6- heating overnight at 350¡C. Pesticides were purchased dimethylpyrimidin-4-yl dimethylcarbamate)). The from Dr Ehrenstorfer, Augsburg, Germany or Riedel-de results obtained by the LC method are compared with HaeŽn, Hannover, Germany. The solutions were pre- those produced using a multi-residue gas chromato- pared by dissolving in methanol. Working standard graphic method with electron-capture detection. solutions were prepared by dilution in methanol. Safety considerations: appropriate precautions should be used to avoid inhalation of chloroform vapour.

2 EXPERIMENTAL METHOD 2.2 Methods 2.1 Materials 2.2.1 Extraction from liver 2.1.1 Instruments The weighed tissue (20 g) was chopped, dried by admix- ture with anhydrous sodium sulfate (5 g) and homoge- L iquid chromatograph: Pump, L-6200 (Merck-Hitachi, nized with a kitchen mixer for 10 min with Darmstadt, Germany); autosampler AS-4000 (Merck- chloroform ] acetone (1 ] 1 by volume; 100 ml). The Hitachi); detectors: (a) L-4250 UV-visible (Merck- extract was Ðltered through a glass Ðbre Ðlter (No. 3) Hitachi); software D-6000 HPLC Manager (Merck- and washed with chloroform ] acetone (1 ] 1by Hitachi); (b) diode-array detector Model ABI-1000S volume; 2 ] 10 ml). This extract was evaporated to (Applied Biosystems, Ramsey, NJ); software Labcalc dryness in a rotary vacuum evaporator at 35¡C (Galactic, Salem, NH). and redissolved in hexane ] chloroform ] acetone L C column: Lichrospher 100 RP-18, 125 ] 4 mm ID, (75 ] 20 ] 5 by volume) up to 10 ml. 5 km spherical particle (Merck). 2.2.2 Extraction from brain Clean-up columns: (1) Glass column 500 ] 15 mm The weighed tissue (20 g) was chopped, dried by admix- ID, slurry packed with Bio-Beads SX-3 (10 g dry), ture with anhydrous sodium sulfate (5 g) and homoge- 200È400 mesh (Bio-Rad Labs., Watford, UK) nized with a kitchen mixer for 10 min with in hexane chloroform acetone (75 20 5by ] ] ] ] chloroform ] acetone (1 ] 1 by volume; 100 ml). Total volume) to a bed height of 360 mm. The mobile phase homogenization was achieved through the use of an was supplied by gravity. The column was calibrated ultrasonic probe (Branson SoniÐer 250) for 15 min at with each pesticide eluted with hexane ] maximum power, the blend centrifuged at chloroform acetone (75 20 5 by volume). Frac- ] ] ] 4000 rev min~1 for 1 h and the supernatant transferred tions of 1 ml and increments to 120È140 ml were col- to a Ñask. The contents were evaporated to dryness in a lected. The pesticides eluted at 50È100 ml. The Ðrst rotary vacuum evaporator at 35¡C and redissolved 30 ml of eluant were discarded and the next 80 ml were in hexane ] chloroform ] acetone (75 ] 20 ] 5by collected. (2) Sep-Pack silica (Waters Assoc., Hartford, volume) up to 10 ml. UK) 1 g cartridge. Gas chromatograph: Konik Model KNK-3000 (Konik 2.2.3 Clean-up Instruments, Barcelona, Spain) equipped with electron- The extracts (5 ml) were transferred to the gel per- capture detector. Operating conditions: splitless injector meation column and the pesticides eluted with 250¡C, detector 350¡C. Fused silica columns: BP5 hexane ] chloroform ] acetone (75 ] 20 ] 5by (SGE) stationary phase 5% diphenyldimethylsiloxane, volume). The Ðrst 30 ml of eluant were discarded, the 25 m ] 0É22 mm ID, 0É25 km Ðlm thickness; main- next 80 ml collected and concentrated to 1 ml using a tained at 210¡C for 1 min, increased to 250¡C at rotary vacuum evaporator at 35¡C and then transferred 2¡C min~1 and kept for 10 min; He carrier gas, to a Sep-Pack silicagel cartridge (the Sep-Pak cartridge 10É5mlmin~1;N makeup, 70 ml min~1. pre-washed with hexane ] chloroform ] acetone 2 (75 20 5 by volume; 10 ml)) and eluted with Kitchen mixer: Osterizer LiqueÐer-Blender (Wisconsin, ] ] hexane chloroform acetone (75 20 5by USA) with a metal container. ] ] ] ] volume; 25 ml) at a 10 ml min~1 Ñow rate. The eluant Rotary vacuum evaporator: Buchi with thermostatic was collected and evaporated to dryness with a rotary water-bath and vacuum pump. vacuum evaporator at 35¡C. The residue was dissolved 58 Aurora N. D•az, Angeles G. Paveja, Francisco G. Sa nchez in methanol (5 ml), Ðltered through a membrane Ðlter linear regression plots of standard curves to determine (0É2 km) and analyzed by liquid chromatography. concentration of pesticides in experimental samples were made. The calibration graphs (n \ 8) were 2.2.4 L iquid chromatography linear between 2 and 10 kgml~1 for pirimicarb, 0É5 and The cleaned-up extract was chromatographed using 10 kgml~1 for fenitrothion and dicofol, 0É1 and gradient elution: water ] acetonitrile ] methanol 10 kgml~1 for chlorfenson and tetradifon, 0É5 and (30 ] 30 ] 40 by volume, wavelength 245 nm, 0È3 min) 15 kgml~1 for chlorobenzilate and 5 and 15 kgml~1 to water ] acetonitrile ] methanol (30 ] 30 ] 40 by for azinphos-ethyl. volume, wavelength 230 nm, 3È10 min) to water ] acetonitrile ] methanol (20 ] 20 ] 60 by volume, wavelength 230 nm, 10È20 min) at a 1 ml min~1 Ñow rate. Standard solution and analytical sample (10 kl) 3 RESULTS AND DISCUSSION were injected into the liquid chromatograph. For each sample, peak area responses of fenitrothion (1É55), 3.1 Liquid chromatography azinphos-ethyl (1É75), chlorfenson (2É81), chlorobenzilate (3É43), tetradifon (4É79) and dicofol (7É67) were mea- UV-visible detection is the most commonly used detec- sured at retention times relative to pirimicarb (2É40 min). tor employed for residue analysis by LC. To optimize Using measured responses, linear regression plots of detection, the absorption spectra of the pesticides were standard curves to determine concentration of obtained. The maximum absorption wavelength of each pesticides in experimental samples were made. The pesticide was optimized representing the absorption calibration graphs (n \ 8) were linear between 0É1 spectra as a contour plot. Figure 1 shows the contour and 10 kgml~1 for pirimicarb and azinphos-ethyl, plot of several spectra collected during chromato- 0É5 and 20 kgml~1 for fenitrothion and tetradifon, 0É3 graphic elution using a time interval in the events and 20 kgml~1 for chlorfenson, 0É5 and 15 kgml~1 for program of the diode-array detector of 20 spectra per chlorobenzilate and 0É6 and 20 kgml~1 for dicofol. minute. From this, a wavelength program providing optimum resolution of each pesticide component can be 2.2.5 Gas chromatography deduced. Table 1 lists the wavelengths selected. Standard solutions and analytical samples (1 kl) were Optimization of chromatographic parameters was injected into the gas chromatograph Ðtted with BP5 performed by searching for the separation of the peaks capillary column. For each sample, peak area responses corresponding to each pesticide, allowing a separate of fenitrothion (1É30), azinphos-ethyl (4É86), chlorfenson peak integration. In Table 2, the retention time tR , (2É15), chlorobenzilate (2É65), tetradifon (4É42) and capacity factor k and separation factor a, achieved dicofol (1É49) were measured at retention times relative under two di†erent conditions, A and B, are indicated. to pirimicarb (4É39 min). Using measured responses, In conditions A, a gradient elution at a constant Ñow

Fig. 1. Contour plot of the chromatographic elution of (1) pirimicarb, (2) fenitrothion, (3) azinphos-ethyl, (4) chlorfenson, (5) chlorobenzilate, (6) tetradifon and (7) dicofol at 5 kgml~1 each. Conditions: 10 kl injected, Ñow rate 1 ml min~1. Multi-residue pesticide determination in animal tissues 59

TABLE 1 Liquid Chromatography Conditions

Mobile phase composition (% by volume)

Conditions T ime (min.) W avelength (nm) Flow rate (ml min~1) W ater Acetonitrile Methanol

Gradient elution: A 0É0 245 1É0 303040 3É0 230 1É0 303040 10É0 230 1É0 303040 20É0 230 1É0 202060 Variable Ñow: B 0É0 245 1É0 303040 3É0 230 1É0 303040 5É0 230 1É2 303040 10É0 230 1É4 303040 20É0 230 1É2 303040 25É0 245 1É0 303040 rate was used. In conditions B, a variable Ñow rate with contaminants. The Ðrst step applied to such samples is a constant mobile phase composition was used. The the extraction of the lipid material from the homoge- wavelength program used in conditions A and B is nized sample matrix. Isolation of the lipid-soluble con- described in Table 1. In each case a good separation is taminants from the fatty extract for subsequent achieved. We chose conditions A for the chromatog- determination is the second step. raphy of pesticides. The clean-up step can be carried out in di†erent ways.16 Gel permeation columns commonly used for separating high-molecular-weight polymers, have been 3.2 Recovery assays adapted to lipid sample clean-up and are applied to the clean-up of pesticides in fats and oils of both plant and In the absence of sample matrix, the e†ect of all the animal origin.17 This clean-up system was evaluated for pesticides on the quantitation of each other was evalu- the pesticides studied in this work. After the gel per- ated. Synthetic mixtures were prepared using a Ðxed meation column step, further clean-up was advisable for quantity of the pesticide to be recovered, namely pirimi- multi-residue analysis to remove interference and carb 5, azinphos-ethyl 5, fenitrothion 10, chlorfenson 10, improve sensitivity. We selected silica Sep-Pak car- chlorobenzilate 10, tetradifon 10 and dicofol 20 ng, and tridges because the animal tissue extracts, after gel per- adding the other six at levels between 10 and 200 ng meation column, were in a polar solvent. (Table 3). In general, recoveries were consistently good, In Fig. 2, the chromatograms of a liver and a brain giving mean values around 100% at the levels assayed extract (spiked and control) are shown. Percentage for pirimicarb and azinphos-ethyl. Only tetradifon gave recoveries were determined in three di†erent liver and mean values of 85% for the 150 ng level. Dicofol, feni- brain samples which were each spiked prior to extrac- trothion, chlorfenson and chlorobenzilate gave mean tion at three levels, 0É25, 1É00 and 2É50 mg kg~1, after values slightly high for the studied levels. checking the sample mix for the absence of the pesti- Samples of high fat content are of particular concern cides under study. After extraction the samples were because of their propensity to accumulate many of these cleaned-up by the gel-permeation-column Sep-Pak pro-

TABLE 2 E†ect of Di†erent Conditions in Separation Achieved by Liquid Chromatography

Gradient elution: A V ariable Ñow: B

Compounds tR(min)k atR(min)k a Pirimicarb 2É40 1É32 2É24 1É24 Fenitrothion 3É72 2É81 2É13 3É62 2É63 2É12 Azinphos-ethyl 4É20 3É22 1É15 4É08 3É08 1É17 Chlorfenson 6É75 6É09 1É89 6É32 5É32 1É73 Chlorobenzilate 8É24 7É57 1É24 7É46 6É46 1É21 Tetradifon 11É50 11É10 1É46 9É81 8É81 1É36 Dicofol 18É40 17É12 1É54 15É07 14É07 1É60 60 Aurora N. D•az, Angeles G. Paveja, Francisco G. Sa nchez

TABLE 3 Interference Study

Recovery of the pesticides in synthetic mixtures (%) Amount of each other pesticide (ng) Pirimicarb Azinphosethyl Fenitrothion Chlorfenson Chlorobenzilate T etradifon Dicofol

10 100 100 100 100 100 100 101 50 100 98 103 103 104 98 105 100 104 102 105 101 107 92 104 150 96 104 106 108 109 85 104 200 94 92 105 107 108 85 104

Fig. 2. Liquid chromatographic separation of (1) pirimicarb, (2) fenitrothion, (3) azinphos-ethyl, (4) chlorfenson, (5) chlorobenzilate, (6) tetradifon and (7) dicofol (1 mg kg~1 each) of (A) liver and (C) brain extracts. (B) control liver chromatogram and (D) control brain chromatogram. Multi-residue pesticide determination in animal tissues 61

TABLE 4 Recoveries of Pesticides by Liquid Chromatography from Spiked Liver and Brain Samples

Recovery (%)

L iver Brain

Added Sample Sample Sample Sample Sample Sample Compounds (mg kg~1) 1 2 3 Mean 1 2 3 Mean

Pirimicarb 0É25 114É095É398É2 102É599É693É596É796É6 1É094É597É193É294É999É795É097É597É4 2É599É5 100É698É099É499É790É895É195É2 Fenitrothion 0É25 89É3 103É592É0 100É397É095É0 100É297É4 1É099É597É999É298É198É497É095É897É1 2É598É4 107É7 101É3 102É899É696É799É798É7 Azinphos-ethyl 0É25 102É7 100É597É794É999É895É093É196É0 1É095É199É499É798É899É395É098É897É7 2É598É4 105É4 104É6 102É598É998É497É396É0 Chlorfenson 0É25 96É095É4 106É299É299É298É498É898É8 1É092É5 108É7 108É5 103É297É095É096É096É0 2É595É996É0 101É597É896É3 101É998É598É9 Chlorobenzilate 0É25 106É797É0 113É9 105É898É7 101É6 100É0 100É1 1É0 108É7 111É097É6 105É898É1 106É0 102É2 102É1 2É597É093É297É395É899É995É497É597É6 Tetradifon 0É25 103É392É293É396É398É293É795É895É9 1É098É798É1 100É098É994É792É593É693É6 2É598É896É997É497É798É996É797É897É8 Dicofol 0É25 106É7 103É099É0 102É996É195É095É695É6 1É097É7 104É4 107É2 103É195É0 105É0 100É0 100É0 2É597É299É8 100É599É299É394É596É996É9

cedure and the results are shown in Table 4. The recov- ranged from 0É10 mg kg~1 for tetradifon to eries obtained from spiked brain and liver samples are 0É23 mg kg~1 for chlorobenzilate in spiked liver excellent, giving mean values around 100% with recov- samples and 0É05 mg kg~1 for chlorfenson to eries ranging from 89É3to114É0%. 0É23 mg kg~1 for chlorobenzilate in spiked brain Since GC is the most commonly used technique in samples. In the GC method the detection limits ranged pesticide residue analysis, the results obtained by the from 0É05 mg kg~1 for chlorfenson to 2É03 mg kg~1 for LC method were compared to those from an optimized azinphos-ethyl in spiked liver samples and GC method. Although the selective detectors used in 0É04 mg kg~1 for tetradifon to 1É93 mg kg~1 for gas chromatography permit very sensitive determi- azinphos-ethyl in spiked brain samples. These results nations, the methodology for the analysis of di†erent indicate that the detection limits of pesticides for the classes of pesticides is laborious. On the other hand, an liquid and gas chromatographic methods were compa- electron-capture detector (ECD) permits easy measuring rable, although the LC method was more sensitive than of the seven compounds with an adequate sensitivity. In the GC method for pirimicarb and azinphos-ethyl. Fig. 3, the gas chromatograms of a liver and brain extract are shown. By using the ECD and the described extraction procedure, recoveries of pesticides in brain and liver samples were consistently good giving mean CONCLUSIONS values around 100% with recoveries ranging from 87É9 to 107É0% (Table 5). Low levels of pirimicarb and Liquid chromatography is a useful technique for the azinphos-ethyl were not detected, because the detection quantitation of pesticide residues in animal tissues. The limit was greater than 0É25 mg kg~1 for pirimicarb and results compare well with those obtained by a gas chro- 1mgkg~1 for azinphos-ethyl. matographic method. Recoveries of pesticides from In Table 6, the detection limit values for all the pesti- spiked liver and brain samples are consistently good in cides are shown. In the LC method the detection limits both methods. So, the HPLC method is an adequate 62 Aurora N. D•az, Angeles G. Paveja, Francisco G. Sa nchez

Fig. 3. Gas chromatographic separation of (1) pirimicarb (1 mg kg~1), (2) fenitrothion (1 mg kg~1), (3) dicofol (1 mg kg~1), (4) chlorfenson (1 mg kg~1), (5) chlorobenzilate (1 mg kg~1), (6) tetradifon (1 mg kg~1) and (7) azinphos-ethyl (2É5mgkg~1)of(A) liver and (C) brain extracts. (B) control liver chromatogram and (D) control brain chromatogram. alternative to GC for the analysis of di†erent classes of 2. Cabras, P., Spanedda, L. & Gennari, M., Separation of pesticide residues. pirimicarb and its metabolites by high-performance liquid chromatography. J. Chromatogr., 478 (1989) 250È4. 3. De Kok, A., Geerdink, R. B. & Brinkman, U. A. T., The determination of polychlorinated naphthalenes in soil ACKNOWLEDGEMENT samples by means of various gas and liquid chromato- graphic methods. Anal. Chem. Symp. Ser., 13 (1983) 203È This work was supported by DGICYT (Proyectos 16. PB93-1006 and BIO94-0548). 4. Priebe, S. R. & Howell, J. A., Post-column reaction detec- tion system for the determination of organophosphorus compounds by liquid chromatography. J. Chromatogr., 324 (1985) 53È63. REFERENCES 5. Opelanio, L. R., Rack, E. P., Blotcky, A. J. & Crow, F. W., Determination of chlorinated pesticides in urine by 1. Blaicher, G., Pfannhauser, W. & Woidich, H., Problems Molecular Neutron Activation Analysis. Anal. Chem., 55 encountered with the routine application of h.p.l.c. to the (1983) 677È81. analysis of carbamate pesticides. Chromatographia, 13 6. Ding, X.-D. & Krull, I. S., Trace analysis for (1980) 438È46. organothiophosphate agricultural chemicals by high- Multi-residue pesticide determination in animal tissues 63

TABLE 5 Recoveries of Pesticides by Gas Chromatography from Spiked Liver and Brain Samples

Recovery (%)

L iver Brain

Added Sample Sample Sample Sample Sample Sample Compounds (mg kg~1) 1 2 3 Mean 1 2 3 Mean

Pirimicarb 1É0 101É5 107É099É595É291É299É595É0 102É6 2É598É092É394É793É694É296É789É795É0 Fenitrothion 0É25 101É895É687É998É699É0 101É595É396É1 1É095É788É588É7 101É299É5 106É597É791É0 2É596É096É6 102É199É199É4 105É592É298É2 Azinphos-ethyl 2É598É62 97É899É097É698É497É896É698É5 Chlorfenson 0É25 101É18 97É294É594É692É995É895É197É6 1É099É93 99É998É897É498É898É295É099É5 2É5 100É87 98É598É795É693É0 101É892É299É4 Chlorobenzilate 0É25 98É1 100É099É197É499É596É396É299É1 1É098É0 100É499É197É299É297É595É099É2 2É595É2 100É499É994É996É694É793É598É5 Tetradifon 0É25 105É095É999É796É498É996É295É0 100É2 1É094É0 102É495É999É2 100É399É298É15 97É4 2É591É798É598É695É696É297É093É596É2 Dicofol 0É25 95É288É386É296É394É4 100É194É489É9 1É097É090É595É3 100É5 100É0 103É498É094É3 2É598É398É998É595É097É398É389É498É6

TABLE 6 Detection Limits of Pesticides in Liver and Brain Extracts by Liquid and Gas Chromatography

L iquid chromatography Gas chromatography

L iver extract Brain extract L iver extract Brain extract a Compounds DL (mg kg~1) DL (mg kg~1) DL (mg kg~1) DL (mg kg~1) Pirimicarb 0É17 0É17 0É46 0É53 Fenitrothion 0É15 0É18 0É17 0É17 Azinphos-ethyl 0É14 0É11 2É03 1É93 Chlorfenson 0É16 0É05 0É05 0É08 Chlorobenzilate 0É23 0É23 0É23 0É26 Tetradifon 0É10 0É08 0É11 0É04 Dicofol 0É18 0É20 0É19 0É25

a D (Detection limit) deÐned as the amount that gave a signal-to-noise ratio of three. L

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