Forensic

Science International, 51 (1991) 89-93 Elsevier Scientific Publishers Ireland Ltd.

89

SOLID PHASE EXTRACTION METHOD FOR RAPID CLEAN-UP OF SOME SYNTHETIC PYRETHROID FROM HUMAN URINE AND PLASMA

ISOLATION AND INSECTICIDES

L. JUNTING and F. CHUICHANG Department of Forensic Chemistry, Shenyang 110001 (China)

Faculty

of Forensic

Medicine,

China Medical University,

(Received February 13th, 1991) (Revision received May 14th, 1991) (Accepted July 17th, 1991)

Summary A simple and rapid method for the isolation of seven synthetic pyrethroid insecticides (methothrin, fenpropathrin, cyhalothrin, permethrin, cypermethrin, fenvalerate, deltamethrin) with a solid phase extraction (SPE), utilizing Sep-Pak C1s cartridges, from human urine and plasma is presented. The detection of the insecticides was performed using a wide bore capillary gas chromatograph (GC) with flame ionization detection (FID). The insecticide-containing samples mixed with 70% methanol were directly applied to the cartridges and eluted with 2 ml chloroform. The recoveries using the cartridges were between 90 - 102% for urine and 81- 93% for plasma. Mixing samples with 70% methanol prior to extraction, seems very useful for the screening of synthetic pyrethroid insecticides. Key words: Pyrethroid;

Pyrethrin;

Sep-Pak Cis cartridges;

Solid phase extraction;

Capillary gas

chromatography

Introduction Pyrethroids represent a relatively new group of synthetic insecticides. Their applications have increased substantially in recent years. Suicides or accidents due to synthetic pyrethroid insecticide intake are rarely encountered in forensic practice. The commonly used adsorbent in the solid phase extraction of pyrethroids is Florisil. In 1984, Spittler [l] used Sep-Pak Florisil cartridges for the extraction of fenvalerate from processed tomato products and by-products. The application of Sep-Pak Silica as a clean-up procedure for permethrin in lettuce has also been reported by Papadopoulou [2]. In this paper, we present a simple and rapid isolation method of seven pyrethroids from human samples using Sep-Pak Cis cartridges and their analysis by wide bore capillary gas chromatography (GC). 0379-0738/91/$03.50 0 1991 Elsevier Scientific Publishers Printed and Published in Ireland

Ireland Ltd.

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Experimental Materials Seven synthetic pyrethroid insecticides were used in our experiments. Fenpropathrin and fenvalerate were obtained from Sumitomo Chemical Co. (Japan); cyhalothrin was obtained from ICI (England); cypermethrin from FMC (America); deltamethrin from Roussel-Uclaf Group (France) and methothrin and permethrin were kindly provided by China Insecticide Examination Center (China). Sep-Pak C is cartridges were purchased from Waters Associates (Milford, Massachusetts, America) and fused silica wide bore capillary columns (HP-l, 5 m x 0.53 mm i.d., film thickness 2.65 pm) were obtained from Hewlett Packard Co. (America). Other common chemicals used were of the highest purity commercially available. Urine and plasma were obtained from healthy subjects. Isolation with Sep-Pak Cl8 cartridges Pretreatment of a Sep-Pak Cis cartridge involved passing 5 ml chloroform, 5 ml methanol, 5 ml methanol/water (1:l) and 5 ml water through it. When the cartridge was reused, the above passage procedure, excluding the 5 ml chloroform, was repeated once. Human samples (1 ml) containing the pyrethroids were mixed with 10 ml 70% methanol and poured into the pretreated Sep-Pak Cis cartridge at a flow rate not greater than 5 ml/min. The cartridge was washed with 5 ml water and finally 2 ml chloroform was passed through it to elute the pyrethroids which were then collected in a vial. The eluate consisted of a large amount of an organic layer (lower phase) and a small amount of an aqueous layer (upper phase). The eluate was evaporated almost to dryness under a stream of nitrogen at room temperature; it took less than 10 min. The residue was dissolved in 100 ~1 pure ethanol and l-cl1 aliquots of this were then subjected to GC analysis. Gas chromatographic conditions Gas chromatography (GC) was carried out on a Hewlett Packard 5890A gas chromatograph connected to a Hewlett Packard 33928 integrator, equipped with a fused silica wide bore capillary column (methyl silicone gum) and flame ionization detector (FID). The conditions were: detector temperature 240°C, injection temperature 240°C and column temperature 190 - 235°C (lO”C/min) with nitrogen flow rate 20 ml/min. Results Figure 1 shows gas chromatograms obtained with a non-polar HP-l wide bore capillary column for seven pyrethroids (10 pg of each) which had been added to urine and plasma and simply extracted using the Sep-Pak Cl8 cartridges. Separation of most of the compounds was generally satisfactory except deltamethrin (5.8 min) which had lower chromatographic sensitivity. The recoveries were between 90 - 102% for urine and 81- 93% for plasma. The plasma extract gave a large peak at 6.35 min. Since three chromatograms

91 Authentic

Urine

Plasma

extract

extract

0

I

I

190

210

I

235

1

235 ‘C

I

190

,

210

I

I

235

235’C

I

190

I

210

I

235

I

235 ‘C

Fig. 1. Wide bore capillary chromatograms for synthetic pyrethroid insecticides isolated from human urine and plasma using Sep-Pak C,s cartridges. The insecticides and their retention times were: methothrin, 1.2 min; fenpropathrin, 2.6 min; cyhalothrin, 3.2 min; permethrin, 3.8 min; cypermethrin, 4.5 min; fenvalerate, 5.1 min and deltamethrin, 5.8 min. Gas chromatography was carried out with a fused silica wide bore capillary column (HP-l, 5 m x 0.53 mm i.d., film thickness 2.65 pm). Chromatographic conditions were: column temperature 195-235°C (10”Clmin) and nitrogen flow rate 30 mllmin. The mixture of 7 pyrethroids (10 fig each) was added to 1 ml urine or plasma.

were obtained with one Sep-Pak Cls cartridge, this large peak was from plasma itself. Figure 2 shows the curve of recoveries of fenpropathrin relative to the concentrations of methanol. The other pyrethroids presented exactly the same curve as fenpropathrin . The maximum recovery occurred at 70% methanol. The plasma samples diluted by 70% methanol scarcely brought about any protein precipitation. It could be poured directly into the cartridge without any difficulty. Instead of 70% methanol, 0.001 N HCl was used for the mixing of urine and plasma samples containing pyrethroids. The same recoveries were obtained as in the case of 70% methanol for the urine extract, but they were only 10 - 20% for the plasma extract (unpublished observation). To check the sensitivity of the present wide bore capillary GC with FID, a calibration curve for fenpropathrin was prepared. Linearity was found up to 100 ng and the detection limit was 2 ng in 1 ~1 injected volume. Discussion In this paper, we have presented

a simple and rapid isolation procedure

for

92

(rec.

1

100%

(cont.) Fig. 2. The elution recoveries from human plasma samples for fenpropathrin with different concentrations of methanol. The elution results at 90% and 100% methanol were obtained from the supernatant after centrifuging at 3000 rev./min for 5 min.

synthetic pyrethroid insecticides using Sep-Pak Cis cartridges. In most previous reports, the insecticides were isolated from biological or environmental samples by repeated extraction with organic solvents and centrifugation, which were much more complicated and time consuming [3 - 41. In addition, the solvent extraction followed by centrifugation sometimes presented the problem of emulsion formation. This study is the first in the trial use of Sep-Pak Cis cartridges for the isolation and clean-up of pyrethroids in human samples although Sep-Pak Florisil clean-up procedure had been used for the analysis of pyrethroid insecticides in some agricultural sample extracts [5]. In 1989, Debon and Segalen [6] used SepPak Cis cartridges for the extraction of pyrethroids in water. To our knowledge, this is the only reference in the literature of the use of Sep-Pak Cis cartridges for the isolation of pyrethroid insecticides. Debon and Segalen [6] eluted the compounds with pure methanol. According to the manufacturer’s manual, various concentrations of methanol or acetonitrile are usually used for the elution of compounds from Sep-Pak Cis cartridges. In the present study, we used only chloroform for the elution. It made the evaporation time much shorter, (less than 10 min) and the recoveries much better than with the use of methanol or acetonitrile.

93

Since Sep-Pak Cis cartridges were used for the extraction of internal constituents in human samples, the existence of some impurities was to be expected, such as the component at 6.35 min (Fig. 1, right panel). We consider it important to mix urine or plasma samples with 10 ml of 70% methanol, prior to extraction through Sep-Pak Cis cartridges. It has been reported that human samples containing organophosphate or carbamate insecticides were mixed with pure water [7,8]. However with pure water, pyrethroid insecticides could not be adsorbed onto the surface of Cls cartridges as shown in Fig. 2. Thus, using 70% methanol seems very useful for separating pyrethroids from other organophosphate or carbamate insecticides. Acknowledgement Technical Hamamatsu

advice from Dr. Osamu Suzuki (Department of Legal Medicine, University School of Medicine, Japan) is gratefully acknowledged.

References T.D. Spittler, R.J. Argauer, D.J. Lisk, R.O. Mumma, G. Winnett and D.N. Ferro, Gas chromatographic determination of fenvalerate insecticide residues in processed tomato products and by-products. J. Assoc. Ojjj Anal. Chem., 6’7 (1984) 824-826. E. Papadopoulou-Mourkidou, Y. Iwata and F.A. Gunther, Application of a high performance liquid chromatographic system with an on-line infrared detector to the residue analysis of permethrin. J. Agric. Food C&m., 31 (1983) 629 - 633. Cao Xiupeng and Wang Youmei, Determination of synthetic pyrethroid insecticides in biological materials by GUECD Chin. J. Forensic Med., 4 (1989) 156- 161. E. Papadopoulou-Mourkidou, Recent advances in pyrethroid determination. Anal. Methods Pestic. Plant Growth Regul., 16 (1988) 179-206. Paul R. Haddad, John G. Brayan, Gerard J. Sharp and Sergio Dilli, Determination of pyrethroid J. residues on paddy rice by reversed phase high performance liquid chromatography. Chromatogr., 461 (1989) 337-346. A. Debon and J.L. SegaIen, Trace analysis of pyrethrins and piperonyl butoxide in water by high performance liquid chromatography. Pyrethrum Post, 17 (1989) 43 - 46. J. Liu, 0. Suzuki, T. Kumazawa and H. Seno, Rapid isolation with Sep-Pak C,s cartridges and wide bore capillary gas chromatography of organophosphate pesticides. Forensic Sci. Int., 41 (1989) 67 - 72. 0. Suzuki, H. Rattori, J. Liu, H. Seno and T. Kumazawa, Positive and negative ion mass spectrometry and rapid clean-up of some carbamate pesticides. Forensic Sci. Znt., 46 (1990) 169- 180.

Solid phase extraction method for rapid isolation and clean-up of some synthetic pyrethroid insecticides from human urine and plasma.

A simple and rapid method for the isolation of seven synthetic pyrethroid insecticides (methothrin, fenpropathrin, cyhalothrin, permethrin, cypermethr...
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