Anal. Chem. 2003, 75, 1430-1435
Direct On-Line Continuous Supercritical Fluid Extraction and HPLC of Aqueous Pyrethrins Solutions Jaroslav Po´l† and Bernd W. Wenclawiak*,‡
Department of Analytical Chemistry, University of Siegen, Adolf-Reichwein-Strasse 2, 57068 Siegen, Germany, and Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veverˇ´ı 97, 611 42 Brno, Czech Republic
A new method, employing supercritical fluid extraction (SFE) connected on-line with high-performance liquid chromatography, was developed for the determination of pyrethrins and piperonyl butoxide in aqueous solutions. The principle of the laboratory-made device for SFE is based on the low mutual solubility of water and supercritical (liquid) CO2. This device works in continuous mode that offers extraction of unlimited sample volumes. Different extraction temperatures and pressures were tested to find optimum extraction conditions. The addition of organic modifier and inorganic salt to the water sample to increase extraction recovery was investigated. The method was evaluated, and it was applied for the extraction of aqueous samples spiked with commercial insecticides. The working concentration range of the method was from the limit of quantification (0.1 µg L-1) to the solubility of the analytes in water. Pyrethrins are insecticides that are commercially extracted from flowers (Chrysanthemum cinerariaefolium).1,2 The extract, stripped of the solvent, is called pyrethrum extract. It contains hydrocarbons, terpenes, and pyrethrins. Pyrethrins are the predominant group of compounds, and they have insect impact. Pyrethrins are a mixture of six compounds: three are esters of the chrysanthemum acid (cinerin I, jasmolin I, pyrethrin Is called pyrethrins I) and three are esters of the pyrethrum acid (cinerin II, jasmolin II, pyrethrin iiscalled pyrethrins II). It has been reported that the Romans used chrysanthemum flower powder as an insecticide. Nowadays the pyrethrins have gained popularity again as “green” and natural insecticides. They are used mostly for indoor purposes as a substitution for persistent and mammal-toxic organochlorine and organophosporus insecticides. The number of pyrethrin preparations, which are used in households (garden and house flower insecticides, indoor insecticides or repellents against indoor tiny insects) and even in human care (shampoos against lice and fleas), is increasing. The potential * Corresponding author. Phone: +49-(0)271-7404573. Fax: +49-(0)2717402414. E-mail:
[email protected]. † Academy of Sciences of the Czech Republic. ‡ University of Siegen. (1) Casida, J. E.; Quistad, G. B. Pyrethrum Flowers; Oxford University Press: New York, 1995. (2) Pan, W. H. T.; Chang, C. C.; Su, T. T.; Lee, F.; Fuh, M. R. St. Talanta 1995, 42, 1745-1749.
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harmful effect of those compounds on people, especially on children, has been discussed, because humans come into very close contact with the compounds, and transfer to humans occurs. Therefore, there is much interest in determining those substances in the environment. Piperonyl butoxide is added to most of the preparations. It acts as a synergist and slows down photodegradation by UV light. The analysis by gas chromatography (GC) is complicated because pyrethrins degrade at temperatures above 200 °C. The thermal conversion can be reduced if a short column with a thin film of stationary phase and on-column injection is used.3,4 A derivatization of pyrethrins prior to the GC analysis could be one way to eliminate this problem. Transesterfication before determination of pyrethrins to chrysantemates and pyrethrates in supercritical CO2 in the presence of acidic alumina and methanol has been described.5 The CO2 also played a role as an extraction agent that transferred the reaction products from the extraction chamber into toluene. An analysis by GC/MS followed. In other work, the authors hydrolyzed pyrethrins in subcritical water in the presence of basic alumina.6 Then the product of hydrolyzation, chrysantemic acid, was extracted from the water using solid-phase microextraction (SPME), and it was analyzed by GC-FID or GC-MSD. Real samples were also successfully analyzed. The analysis by high-performance liquid chromatography (HPLC) is not as difficult as GC analysis, because no degradation of the samples during the analysis has been observed. Several papers describe the analysis and successful separation of all six active compounds in pyrethrum extract.7-10 Wintersteiger and Ofner11 developed the method for determining pyrethrins together with piperonyl butoxide in spiked human plasma by solid-phase extraction (SPE) followed by off- and on-line HPLC analysis. Other papers describe solid-phase extraction of pyrethrins and piperonyl (3) Class, T. J. J. High Resolut. Chromatogr. 1991, 14, 48-51. (4) Ko ¨hle, H.; Haberer, K. Vom Wasser 1990, 75, 75-82. (5) Wenclawiak, B. W.; Krappe, M.; Otterbach, A. J. Chromatogr., A 1997, 785, 263-267. (6) Krappe, M.; Hawthorne, S. B.; Wenclawiak, B. W. Fresenius J. Anal. Chem. 1999, 364, 625-630. (7) Mourot, D.; Boisseau, J.; Gayot, G. Anal. Chim. Acta 1978, 97, 191-173. (8) McEldowney, A. M.; Menary, R. C. J. Chromatogr. 1988, 447, 239-243. (9) I-Hsiung, W.; Subramanian, V.; Moorman, R.; Burleson, J.; Ko, J. J. Chromatogr., A 1997, 776, 277-281. (10) Essig, K.; Zhao, Z. J. Chromatogr. Sci. 2001, 39, 473-480. (11) Wintersteiger, R.; Ofner, B. J. Chromatogr., A 1994, 660, 205-210. 10.1021/ac026047f CCC: $25.00
© 2003 American Chemical Society Published on Web 02/15/2003
Figure 1. Schematic of SFE apparatus (all components that make up this independent unit are defined by the large dashed rectangle) connected on-line to HPLC through the interface (details in Figure 2). (1) CO2 cylinder, (2) CO2-phase pump, (3) water-phase pump, (4) packed extraction column, (5) phase separator, (6) water-phase capillary restrictor, (7) water waste collection, (8) water sample reservoir, (9) CO2-phase capillary restrictor, (10) SFE-HPLC interface (trapping column, heater, two valves), (11) CO2 capillary vent, (12) HPLC, (13) data acquisition, and (14) SFE control computer.
butoxide of tap water12 or milk.13 Both authors used HLPC analysis. Zong-Mao and Yun-Hao14 published a review on extraction and analysis of pyrethrins in environmental samples. We describe here a method that employs supercritical fluid extraction (SFE) and HPLC in an on-line arrangement. It is optimized to determine pyrethrins and piperonyl butoxide in water samples. The method is applied to spiked water samples and commercial products containing pyrethrins. EXPERIMENTAL SECTION SFE Apparatus. Design and theoretical background of a laboratory-made apparatus for dynamic continuous extraction of aqueous media by supercritical CO2 have been described before.15 A schematic is given in Figure 1. The principal part of the extraction apparatus is the vertically mounted extraction column packed with inert material and with a phase separator attached to the bottom. In the concurrent mode, two high-pressure pumps deliver separately aqueous sample and liquid CO2 to the top of the column. There, both phases are brought into mutual contact and flow down the extraction column to the phase separator while reaching a steady state in the system water/supercritical carbon dioxide/solute. In the phase separator, both phases get separated because of their different densities and leave the extractor through fused-silica capillary restrictors that are attached at appropriate vertical position. The end of the CO2 restrictor is connected to a trapping device where analytes are collected. The purpose of the apparatus, which is an independent unit, is to transfer analytes (12) Debon, A.; Segalen, J. L. Pyrethrum Post 1989, 17, 43-46. (13) Nijhuis, H.; Heeschen, W.; Hahne, K. H. Pyrethrum Post 1985, 16, 14-17. (14) Zong-Mao, Ch.; Yun-Hao, W. J. Chromatogr., A 1996, 754, 367-395. (15) Kara´sek, P.; Po´l, J.; Planeta, J.; Roth, M.; Vejrosta, J.; Wicˇar, S. Anal. Chem. 2002, 74, 4294-4299.
Figure 2. Schematic of the SFE-HPLC interface in the trapping mode. The dotted lines are silica capillaries and full lines mark stainless steel capillaries. T indicates the heated T-junction.
from the water phase to the CO2 phase. This allows at ambient conditions the concentration of analytes from expanding CO2. A control unit that is connected with a PC to enter and display extraction parameters (flow rate of the liquid phase, pressure, temperature) controls the extraction apparatus. SFE-HPLC Interface. An additional device, the solid-phase trapping interface that connects the CO2 extractor outlet and HPLC, was developed (Figure 2). The idea to collect analytes onto a solid sorbent is not new, and it was already described for offline17,18 and for on-line arrangement with HPLC19 or with microcolumn LC/GC.20 Our arrangement adds some constructive elements. The coupling consisted of a seven-port valve and a sixport valve (Ecom Ltd., Prague, Czech Republic) and a trapping column. The silica capillary restrictor transferred CO2 from the extractor to the end of the trapping column (stainless steel, length 5 cm, i.d. 4.6 mm; Labio Ltd., Prague, Czech Republic). The trapping column was filled with the sorbent (Separon SGX C18, particle size 60 µm; Tessek Ltd., Prague, Czech Republic). It was proved that the sorbent did not influence significantly the back pressure of flow through gaseous CO2. The end of the CO2 capillary has to be heated to avoid plugging. The temperature of the heater was set to 100 °C for flow rates of liquid CO2 up to 1.5 mL min-1 and to 130 °C for higher flow rates to avoid plugging of the restrictor by freezing CO2. The maximum flow rate of liquid CO2 was 2.3 mL min-1. The trapping process itself is simple: the analytes are collected from the passing gaseous CO2 stream by the stationary phase. After collection, the analytes are eluted with the mobile phase directly onto the HPLC column. The coupling can operate in three modes that can be set by switching the valves: mode of trapping, mode of analysis, and mode of the standard solution injection. (16) Po´l, J. Diploma Thesis, Faculty of Chemistry, Technical Univesity of Brno, 1998. (17) Schantz, M. M.; Chesler, S. N. J. Chromatogr. Sci. 1986, 363, 397-401. (18) Mulcahey, L. J.; Hendrick, J. L.; Taylor, L. T. Anal. Chem. 1991, 63, 22252232. (19) Unger, K. K.; Roumeliotis, P. J. Chromatogr. 1983, 282, 519-526. (20) Cortes, H. J.; Green, L. S.; Campbell, R. M. Anal. Chem. 1991, 2719-2724.
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The features of the SFE-HPLC coupling are as follows: The whole bulk of the extract is directly injected into the HPLC system, and therefore, a high sensitivity of the method is achieved. On the other hand, this can lead to interferences between the compounds of interest and undesirable compounds in the chromatogram when a real sample is analyzed. In this case, the extraction conditions (pressure and temperature) must be adjusted to optimum values to minimize those interferences. The extract is injected using the valve, and no operator error can occur. Therefore, the method is characterized by high reproducibility. HPLC. A liquid chromatographic pump Ecom LCP 4000 with gradient programmer GP 4 (Ecom Ltd.) run at a flow rate of 1 mL min-1 with acetonitrile and water as mobile phase. Two columns were tested for separation efficiency: Nucleosil 100-5 C8 (250 cm × 4.6 mm × 5 µm, Watrex Ltd., Prague, Czech Republic) and C18 LiChrospher 100 (250 cm × 4.6 mm × 5 µm, Merck KGaA, Darmstadt, Germany). A Spectra Series UV100 detector (Thermo Separation Products, San Jose, CA) was set to 235 nm. Data acquisition and evaluation were done by CSW 32 software (Data Apex Ltd., Prague, Czech Republic) that allows displaying of the signal of the UV detector only in millivolt units. These units are not common for the UV detector, but it has no negative effect for interpreting and evaluating the signal. Reagents. HPLC grade acetonitrile was supplied by Promochem GmbH and methanol for trace analysis by J. T. Baker. A technical-grade pyrethrum mixture (20.73% pyrethrins) was obtained from Riedel de-Hae¨n GmbH, allethrin standard was from Labor Dr. Ehrenstorfer and piperonyl butoxide (90%) from Fluka Chemie GmbH. Water was twice distilled before it was used for preparation of standard solutions and as mobile phase for HPLC. Real samples used for the analysis and spiked water samples were as follows: Goldgeist Forte, shampoo against fleas and lice (Eduard Gerlach GmbH); Sprutzit, preparation against household flower insect (W. Neudorf GmbH KG). Standard Solution. The technical-grade pyrethrum mixture was weighed into a brown glass volumetric flask, diluted with methanol, and kept in a refrigerator. It contained 1.89 mg mL-1 pyrethrum extract. For experiments, an aliquot of this solution was pipetted into a brown glass vial. If necessary, a designated amount of piperonyl butoxide or allethrin solution was added. This solution was used for calibration and preparation of standard water solutions. The standard solution in water was prepared by adding an aliquot of pyrethrin solution by microsyringe to distilled water. Then it was stirred well and sonicated. In preparing the model samples, the solubilities of pyrethrins, allethrin, and piperonyl butoxide in water1 were considered. Procedure. The first step of the procedure was to start up the extraction apparatus and wait for the set parameters of pressure and temperature. The interface valves were set into a position that CO2 from the extractor could pass the trapping column (identical to the mode of analysis). Distilled water was extracted during this step. The equilibration of the apparatus took 15 min. The capillary heater in the interface was switched on. After this “steady time period”, the interface valves were switched to the mode of extraction. A vessel containing a sample to extract replaced the vessel with distilled water. When most of the sample was extracted, 50 mL of distilled water was added to flush the system. At this time, the HPLC system was started for equilibra1432
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tion. When all the distilled water was consumed, the interface valves were switched to the mode of analysis, and the HPLC data acquisition was started up. While the HPLC analysis was running, the apparatus was brought to the “standby mode”, and it was flushed with 200 mL of distilled water at atmospheric pressure. Because of the interface arrangement, the process of standard injection was slightly distinct from common HPLC injection that uses a calibrated sample loop. A designated volume of standard solution (from 5 to 200 µL) was aspirated either into a 50-µL or a 1-mL microsyringe, depending on the volume. The remaining void volume in either syringe was filled with distilled water, and the mixture was mixed with a small bubble of air. This should lower the solvent strength of the injected bulk and avoid the unwanted elution of the analytes from the trapping column. This mixture containing an exact quantity of analytes was injected into the trapping column through the seven-port valve, when the appropriate interface mode was adjusted. The microsyringe was flushed once with distilled water onto the trapping column. Then the interface was switched to the mode of analysis by switching the seven-port valve and the HPLC analysis was started. Those sequences can be considered similar to solid-phase extraction. RESULTS AND DISCUSSION HPLC Analysis. The C18 column (LiChrospher 100, 250 cm × 4.6 mm × 5 µm) was selected because of better separation of the six Pyrethrins together with allethrin or piperonyl butoxide in standard solution. A sufficient separation of the standard mixture was achieved by isocratic elution conditions with a 70:30 (v/v) acetonitrile/water mixture (Figure 3). Calibration. The six-point calibration plot had a range from 0.2 µg of pyrethrins, 0.4 µg of piperonyl butoxide, and 0.4 µg of allethrin to 8.2 µg of pyrethrin extract, 19.1 µg of piperonyl butoxide, and 7.2 µg of Allethrin, respectively. The coefficients of regression (R2-) for the calibration plots were between 0.992 and 0.995. Concerning pyrethrins, the calibration plot was made for the six individual compounds as well as for the sum of all six pyrethrins. The reason is practical: for example, the German standards for drinking water require only a maximum concentration of 0.1 µg L-1 for all pyrethrins, and producers of commercial pyrethrin insecticides show the pyrethrins sum on the product etiquette. Therefore, determination of the sum of all pyrethrins seems to be sufficient for analyzing real samples. Last but not least, this fact is a suitable solution for some difficulties with the extract analysis, as will be mentioned later. Extraction of the Standard Water Solution. Several extractions of water spiked with pyrethrins were done to find out appropriate extraction conditions (pressure and temperature) and to improve and evaluate the method (standard addition, calibration on internal standard). To enhance the extraction recovery, the addition of an organic modifier or an inorganic salt to the water sample was investigated, too. A 100-mL water standard solution with a pyrethrins concentration of 340 µg L-1 was usually extracted, if not mentioned otherwise. The apparatus arrangement, especially the capillary restrictors, was not changed during the whole series of experiments. The chromatogram of the extracts shows the coelution of cinerin I with pyrethrin I and of cinerin II with pyrethrin II (Figure 4). This is caused by the interface arrangement. The analytes were spread during trapping on the extraction column, and therefore,
Figure 3. Chromatogram of standard mixture of pyrethrum extract and piperonyl butoxide. Injection: 1.6 µg of pyrethrins and 3.8 µg of piperonyl butoxide. Peaks: (1) cinerin II, (2) pyrethrin II, (3) jasmolin II, (4) piperonyl butoxide, (5) cinerin I, (6) pyrethrin I, and (7) jasmolin I.
Figure 4. Chromatogram of extract. Peaks: (1) cinerin II and (2) pyrethrin IIsmajor constituents of the group of pyrethrins II, (3) jasmolin II, (4) piperonyl butoxide, (5) cinerin I, (6) pyrethrin Ismajor constituents of the group of pyrethrins I, and (7) jasmolin I. For details, see Results and Discussion.
the analytes were not sufficiently focused in a small band. However, this seems to be problematic only at first glance; the six pyrethrins are always present as a “native” mixture, and as mentioned before, the sum of pyrethrins is of practical interest. Another difference that can be seen when the chromatograms of an extract and a calibration standard are compared is the positive time shift of the retention times. It is caused by the presence of gaseous CO2 in the interface. This increases the dead time, because the mobile phase needs more time for compression of this gas volume and might also influence the solubility of analytes in the mobile phase.
Different extraction conditions, as pressure and temperature, can have an effect on the equilibrium between supercritical CO2 and the water phase with a solute. Simply, pressure has the biggest effect on CO2 density and also on the diffusion coefficient. This can influence, for example, the solute transfer from a matrix. On the other hand, temperature influences mostly the solubilities of a solute in both phases. The distribution coefficient of a solute between CO2 and the water phase can be increased by appropriate adjustment of pressure and temperature. The behavior of those three-component systems is difficult to predict, and therefore, it is necessary to determine these values experimentally. Analytical Chemistry, Vol. 75, No. 6, March 15, 2003
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Table 1. Extraction Recoveries and Relative Standard Deviation for Pyrethrins and Piperonyl Butoxide (Triplicate Extraction)a
cinerin II + pyrethrin II jasmolin II piperonyl butoxide cinerin I + pyrethrin I jasmolin I sum of pyrethrins a
Figure 5. Extraction recovery and its dependence on temperature. The extraction recovery is recalculated to pertain to the ratio FL/FG ) 3, as described in Results and Discussion. RSD, 0.4-6.8%.
It has to be noted that flow rates of the CO2 and the water phases changed with pressure and temperature, while keeping the capillary restrictors at the same length and inner diameter. Generally, with increasing pressure and temperature both flow rates increase, too. It could seem that changes in flow rates at different extraction conditions could cause some inaccuracies when the data are compared. However, concerning the theory14 based on the calculation of the mass balance on the extraction column, the ratio of water molar flow rate and CO2 molar flow ratesFL/FGsis determinative. It means for example, that the data from two experiments with different flow rates of water and CO2 can be equally compared, as long as the ratio of molar flow rates of water and CO2 phases is the same in both cases. One series of experiments was performed at constant 20 MPa pressure while temperatures were changed in 10 °C steps from 50 to 90 °C. The water flow rate was changing with increasing temperature more than the CO2 flow rate. This is caused by a more rapid change in water viscosity with temperature compared to that of CO2. Therefore, the data cannot be equivalently compared due to the different FL/FG ratios and a recalculation to a constant FL/FG ratio is necessary. The recalculation is based on the fact that the distribution coefficient of an analyte is constant at the same pressure and temperature, and the equations explained in ref 15 were used. For better comparison of the data, the ratio FL/FG was chosen to be 3. Figure 5 shows the extraction recoveries after recalculation. Extraction recoveries increased with temperature, except for jasmolin II where the recovery, considering the experimental deviation, remains constant from 70 °C. The relative standard deviation (RSD) was 0.4-6.8%. The dependence of recovery on pressure was performed at 80 °C for 20, 25, and 30 MPa. The recalculation to a constant FL/ FG ratio was not necessary, because the FL/FG ratio was constant for all three experiments. The extraction recoveries increased with pressure up to 25 MPa and then stayed nearly constant up to 30 MPa; the RSD was within 0.3-6.6%. The values 80 °C and 30 MPa were selected as optimum for further extractions. Higher extraction recoveries were found at 90 °C, but PEEK holders of the silica capillary restrictors were 1434 Analytical Chemistry, Vol. 75, No. 6, March 15, 2003
recovery (%)
RSD (%)
86.7 57.4 95.8 72.6 67.7 78.4
2.3 0.9 0.3 1.9 1.0
Values of practical interest are marked in boldface type.
getting softer and threatened to shoot the restrictor out at a pressure of 30 MPa. The 30 MPa working pressure was chosen due to faster analysis (higher flow rate of water phase) while keeping the same extraction recoveries. Flow rates of water and liquid CO2 were 2.1 and 2.3 mL min-1, respectively. An addition of inorganic salt to the water sample, commonly used in the field of extraction of water samples, was tested in hope to enhance the extraction recovery of nonpolar pyrethrins from water. The ionic strength of the standard solution (100 mL) was increased by addition of NaCl (2 g) or Na2SO4 (2 g). The results were a little bit surprising: the addition of salts had no influence on the extraction recovery. A similar effect was observed when PCBs and OCPs were extracted from surface water.16 The change of phase behavior in the extraction column after salt addition cannot be fully explained here due to the complexity of the multicomponent system. However, what is important is that the salt content has no influence on the extraction efficiency, and therefore, the method can be suitable, for example, for extraction of seawater or hard water. The presence of an organic modifier in the water phase can change the phase equilibrium in the extraction system and influence the distribution coefficient in both directions. The addition of methanol, as an organic modifier, was 2, 5, and 10% into standard water sample and its influence on extraction recovery was observed. The addition of 2 and 5% methanol slightly increased the extraction recovery; however, the addition of 10% methanol already decreased the extraction recovery. Probably too much methanol was dissolved in CO2, in the extraction column, and the methanol then eluted the analytes from the trapping column. It was decided to continue the work without further modifier addition. Method Evaluation. The previous experiments were done to find the optimal extraction conditions. The extraction efficiencies were confirmed by a six-point calibration, obtained at optimum extraction parameters, and they were evaluated using two methods: method of standard addition and calibration on internal standard (allethrin). Both methods proved the accuracy of the experiments performed. The results were found to be in agreement within 3% deviation. The final results for pyrethrins and also for piperonyl butoxide are shown in (Table 1). Because the sum of pyrethrins is of practical interest, the recovery is also shown and it was used for calculating the amounts of analytes in the analyzed samples. Due to the very similar structure of pyrethrins, we suppose nearly the same response factor for the UV detector. This is the presumption for adding the peaks’ areas together. The extraction recovery for the sum of all pyrethrins was not quantita-
Table 2. Extraction of Commercial Insecticides Dissolved in Aqueous Samples addeda Goldgeist Forte (µg L-1) Sprutzit (µg L-1)
analyzeda
PYRb
PBOc
PYRb
PBOc
21 11
194 43
21 10
188 44
a “Added”, the amount of analytes present in water sample (based on pesticide amount shown on product etiquette). “Analyzed”, the results of our analysis. The RSD was 1.2-4.1%. b PYR, sum of pyrethrins. c PBO, piperonyl butoxide.
Table 3. Determination of Samples of Different Volumes Spiked with 0.1 µg L-1 Pyrethrins and 1.0 µg L-1 Piperonyl Butoxide sample vol (mL) anal. time (h)
PYR PBO
400 3.5
600 5.16
1000 8.5
Figure 6. Extraction recoveries of pyrethrins versus pressure at a constant temperature of 80 °C. RSD, 0.3-6.4%.
c (µg L-1)
RSD (%)
c (µg L-1)
RSD (%)
c (µg L-1)
RSD (%)
0.1 1.1
63.2 60.7
0.1 0.6
17.1 16.0
0.1 0.8
0.7 1.4
tive, but it was balanced by a low RSD value. These data were achieved by triplicate extraction of distilled water that was fortified by 232 µg L-1 piperonyl butoxide and 74 µg L-1 pyrethrins. Piperonyl butoxide interfered with jasmolin II in the chromatogram, and this could add minor inaccuracy to the piperonyl butoxide and total pyrethrin results. However, the content of individual pyrethrins in a natural pyrethrin mixture is known, and we can elaborate the data by simple calculation. The content of jasmolin II in the pyrethrin sum is 2% (in the standard used in this work). The content of individual pyrethrins in the pyrethrin sum is similar in other standards.21 From the chromatogram of the extract (Figure 4) we can easily recognize three major peaks that could refer to the presence of insecticide preparation containing pyrethrins together with piperonyl butoxide: pyrethrins II (including pyrethrin II and cinerin II), piperonyl butoxide (refining of the data for quantitative calculation is necessary because of the mentioned coelution with jasmoline II), and pyrethrins I (including pyrethrin I and cinerin I), respectively. The presence and the mutual position of the three major peaks could be used as a “fingerprint” when one is considering the classification of the compounds and only a UV detector is available. This simplification is based on the identification of either pyrethrins I, pyrethrins II, and piperonyl butoxide or pyrethrin I, pyrethrin II, and piperonyl butoxide, but not on individual pyrethrins, as was described in the literature.11-13 In this work, we show better resolution and better method sensitivity of those three peaks and also we can distinguish a peak of jasmoline I. Real Sample Determination. Two insecticide preparations, mentioned in the Experimental Section, were used for spiking tap water samples. The volumes of samples were 100 mL, and the concentrations of pyrethrins and piperonyl butoxide (depending (21) Wenclawiak, B. W.; Otterbach, A. J. Biochem. Biophys. Methods 2000, 43, 197-207.
on sample) were 11-21 and 43-194 µg L-1, respectively (Table 2). We found agreement between added and analyzed amount concerning the RSD. Samples of tap water, spiked with Goldgeist Forte to a concentration of 0.1 µg L-1 pyrethrins (German standard for drinking water) and 1.0 µg L-1 piperonyl butoxide, were prepared. They had volumes of 400, 600, and 1000 mL to be extracted. The results (Table 3) show that the method is able to determine the mentioned concentration of analytes; however, to get outstanding RSD, the extraction of 1000 mL of sample is necessary. Then the method is more time-consuming, but more accurate, and the extraction step with the HPLC analysis takes roughly 8.5 h. However, this long time is not so surprising when such a trace concentration of analytes is being determined. It has to be noted that analysis of pure tap water showed no appearance of pyrethrins or piperonyl butoxide. CONCLUSION The presented method is suitable for analyzing pyrethrins together with piperonyl butoxide in aqueous media. It is characterized by the robustness to inorganic salt content that enlarges the number of possible samples to analyze and by high reproducibility. Another advantage results from the SFE apparatus arrangement: the method is automated and requires minimum care of an operator. The method seems useful for continuous monitoring of environment. ACKNOWLEDGMENT This work was initiated by the NATO Scientific Fellowship Programme that provided funds for the stay of J.P. at the University of Siegen. The financial support by grants from Academy of Sciences of the Czech Republic S4031110, Fo¨rderverein fu¨r wissenschaftliche Weiterbildung an der Universita¨t Siegen, and Fonds der chemischen Industrie are also gratefully acknowledged. Received for review August 14, 2002. Accepted December 18, 2002. AC026047F
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