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 aSanchez Departamento de Qu•mica Anal•tica, Facultad de Ciencias, Universidad de Malaga, 29071-Malaga, 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 Haen, 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.