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Carbon-based quantitation of pyrethrins by supercritical-fluid chromatography
J. Biochem. Biophys. Methods 43 (2000) 197–207 www.elsevier.com / locate / jbbm
Carbon-based quantitation of pyrethrins by supercritical-fluid chromatography B. Wenclawiak*, A. Otterbach Department of Analytical Chemistry, University of Siegen, Adolf-Reichwein-Strasse 2, 57068 Siegen, Germany
Abstract All six insecticide active ingredients in pyrethrum extract were quantified by supercritical fluid chromatography and carbon calibration. Allethrin is a suitable reference compound for carbon calibration and pyrethrins calibrations. Carbon quantification in SFC is also applied to pyrethroids (phenothrin, permethrin, cypermethrin, fenvalerate and deltamethrin) and alkanes. Halogen substitution on pyrethroids requires halogens on the reference calibration compound. The method was applied to commercial extracts. 2000 Elsevier Science B.V. All rights reserved. Keywords: SFC; Pyrethrin; Pyrethroids; Carbon calibration
1. Introduction Pyrethrins are offered as a substitute for organochlorine insecticides like DDT, lindane, aldrin or toxaphene, which are forbidden in many countries, and for organophosphates (E 605 forte), where serious intoxications were often reported. Pyrethrins (Fig. 1) are natural ingredients in flowers like Chrysanthemum cinerariae folium and Chrysanthemum coccineum. The cold solvent extract is called pyrethrum extract. Pyrethrum is one of the oldest known insecticides [1]. The dried plants came from the middle east and they were used by the Romans against lice and fleas. In 1840, powdered flowers were imported to western Europe as ‘Dalmatian insect powder’. Kenya was the main pyrethrum-producing country until 1995. Pyrethrum extract contains six optically active esters of ( 1 )trans-configured chrysanthemum acid and pyrethrum acid with cyclic ketoalcohols ( 1 )-cinerolon, ( 1 )-jasmolon and ( 1 )pyrethrolon [2]. Because of the different acids, two distinct groups (chrysanthemates and pyrethrates) originate. *Corresponding author. Tel.: 149-271-740-4573. 0165-022X / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 00 )00076-2
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Fig. 1. Structures of pyrethrins and allethrin.
Derived from the natural structure and intended to overcome some limitations of the natural products, synthetic compounds with a similar structure and activity were made and these were called pyrethroids (Fig. 2) [3]. Pyrethroids show high insecticide activity, low human toxicity and good plant compatibility. Natural pyrethrum and approximately 15 commercially interesting pyrethroids account for more than 30% of the world trade in insecticides. The active ingredients are used in different formulations for spray, sprinkling powder and as expedients in professional and private plant protection [4]. They are also inserted in tins, sprays and on strips to combat different pests. The use of those agents in dermatology remedies in human and veterinary science for control of ectoparasites has increased dramatically in the last few years. The increasing consumption and the broad application of these insecticides requires a reliable analytical method for the quantitative and qualitative determination of each single compound. The amount of pyrethrins in pyrethrum extracts is analyzed by different methods that are described in the literature [5]. Wenclawiak et al. [6] described a quantification method for the analysis of pyrethrins using in situ transesterfication in supercritical carbon dioxide with GC–MS. Usually, the total amount or the ratio between chrysanthemates and pyrethrates is calculated [7]. The calculation of single amounts of the six active insecticide substances in chrysanthemic flowers is difficult. One reason is the
B. Wenclawiak, A. Otterbach / J. Biochem. Biophys. Methods 43 (2000) 197 – 207
199
Fig. 2. Structure of pyrethroids.
nonexistence of single standards for every pyrethrin because of their fast degradation by UV-irradiation, heat and oxygen [8]. Another reason is the difficulty in quantifying each ingredient in complex mixtures. Jorgensen et al. [9] described the prediction of response factors from molecular structures using flame ionization detection (FID). They stated that their technique allows quantitation of individual components in a complex organic mixture, if structural identification can be made. Scanlon and Willis [10] showed that the response factor calculated from a reference substance can be compared if the structure is similar. Another example is the analysis of engine exhaust gases using GC–FID–MS. Well over 200 single substances cannot not determined individually. All peak areas of the exhaust chromatogram are quantified, based on the carbon calibration response factor of a certified fuel standard with well-known carbon content [11]. In this study, a quantification method for pyrethrins based on carbon calibration and supercritical fluid chromatography (SFC) is presented.
2. Experimental
2.1. Instrumentation A MIPS / 225 (SUPREX, Pittsburgh) was used in this study. This SFC system was equipped with a pneumatically driven four port, timed split injection valve (Valco, Houston, TX, USA). The injection time was 0.5 s. A column (DB5; J&W Scientific, CA, USA) with a length or 10 m and an I.D. of 0.05 mm was used. A laboratory-made
200
B. Wenclawiak, A. Otterbach / J. Biochem. Biophys. Methods 43 (2000) 197 – 207
Guthrie-restrictor was placed at the end of the column. Carbon dioxide with a He headspace (SFE-grade; p, 11 MPa; Messer-Griesheim, Germany) was used as the mobile phase. It is important that the mobile phase flows as a liquid through the sample loop. Therefore, 200 mm of the inlet capillary and the injector were cooled to 188C with water. The temperature of the flame ionization detector was set to 3508C. The flame gases were hydrogen (5.0) and synthetic air (Kl. 2) from Messer-Griesheim (Germany). The H 2 -flow was set at 37 ml / min. The hydrogen-to-air ratio was 1:10. GC-Star chromatographic software (4.0) (Varian Germany) recorded the signals from the FID system.
2.2. Chemicals ¨ Germany) and n-hexane were of high purity (PromoIsopropanol (Riedel de Haen, chem, Germany). A standard solution (A) with known concentrations of different hydrocarbons was made in n-hexane [e.g. n-octadecane (6.57 mg / l), n-eicosane (6.43 ¨ mg / l), n-docosane (7.86 mg / l) and n-tetracosane (6.50 mg / l); 99.9%; Riedel de Haen]. The investigated pyrethroids [phenothrin (91.7%), permethrin (97.0%), cypermethrin (95%), fenvalerate (99.1%) and deltamethrin (99.3%)] were from the laboratory of Dr. Ehrenstorfer (Germany). Solution B contained n-octadecane (57.5 mg / l), n-docosane (57.7 mg / l), n-tetracosane (54.8 mg / l) and solution C contained allethrin (146.5 mg / l), phenothrin (112.9 mg / l), permethrin (92.5 mg / l), cypermethrin (85.5 mg / l), fenvalerate (126.5 mg / l) and deltamethrin (185.6 mg / l) and these were prepared in isopropanol. The analyzed pyrethrum extracts were from Fluka (Switzerland) (1995), Riedel de ¨ (Germany) (1996) and the World Standard Pyrethrum Extract (Bayer, Germany) Haen (1992). The extracts were dissolved in isopropanol. Pyrethrum (Fluka) and allethrin were dissolved in isopropanol (solution D).
2.3. Quantification by carbon calibration Fig. 3a shows a SF-chromatogram of pyrethroids. A linear, positive pressure gradient from 11 MPa up to 23 MPa at a rate of 0.2 MPa / min was used. The oven temperature was kept constant at 1108C. Cypermethrin and fenvalerate exhibit double peaks due to isomers. A chromatogram of allethrin and pyrethrins from solution C is shown in Fig. 3b. All analytes are well (baseline) separated. The elution order in SFC depends on the solubility and the binary diffusion coefficient (D12 ) of the analytes [12]. Besides temperature and polarity, the molecular weight or molecular size contribute to the D12 . Allethrin, which has the lowest molecular weight, elutes first, followed by the chrysanthemates and pyrethrates. The elution order in both groups is the same (cinerin, jasmolin, pyrethrin). This does not exactly follow the elution order with respect to molecular weight: the double-bond in pyrethrins I and II may cause a stronger interaction with the stationary phase (DB5) resulting in longer retention times. For carbon calibration, solution A was used. n-Eicosane was added as a reference substance and internal standard, in order to minimize injection errors. Using an internal
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201
Fig. 3. (a) Chromatogram of pyrethroids with a positive pressure program: from 11.1 MPa at 0.2 MPa / min to 24.3 MPa; temperature, 1108C; detection (FID) temperature, 3258C. (b) Chromatogram of pyrethrins and allethrin with a positive pressure program: from 11.1 MPa at 0.2 MPa / min to 24.3 MPa; temperature, 1108C; detection (FID) temperature, 3258C.
sample loop, a defined sample volume (VI ) was delivered onto the column. Thus, the exact carbon mass (m CR ) of the injected reference can be calculated (Eq. (1)) m CR 5 x CR ? c R ?VI where:
(in ng)
(1)
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202
x CR : cR:
carbon content in the reference substance concentration of the reference substance (in mg / l).
In this case, only carbon atoms cause a FID signal [13,14]. The response factor (Rf ) (Eq. (2)) was calculated from the reference peak area (a R ; in counts) and the carbon mass of n-eicosane (Eq. (1)). aR Rf 5 ]] (in counts / ng) (2) m CR Substance-specific peaks were found following the chromatographic separation. The analyte peak area (aA ) is proportional to the carbon mass. By mathematical combining of the analyte area with the response factor, the carbon content in analyte (x CA ) and injected volume, the concentration of the concerned substance can be obtained using Eq. (3). aA cA 5 ]]] Rf ? x CA ?VI
(in mg / l)
(3)
As an example of the carbon calibration method, n-alkanes were tested (solution A). The calculated, the experimentally determined and the corrected concentrations are shown in Table 1. In addition, the correction factor ( fcorr ) and the value of a t-test [15] are also listed in Table 1. All measurements were repeated five times. The relative standard deviation (R.S.D.) was always less than 2.9%. The deviation between the calculated and experimental concentration was less than 2.64%. The correction factor was between 1.01 and 1.03. A t-test was carried out between the fcorr values of analytes and the reference. The critical value of t (0.95; 8) is 2.34. It can be seen that there was no significant difference between the fcorr values, and between the calculated and experimental concentrations. However, it is important that the reference substance possesses the same basic structure as that of the analytes. For example, it is not possible to determine pyrethroids using n-docosane as the carbon reference. This is shown in Table 2. It can be seen from Table 2 that the quantification of n-octadecane and n-tetracosane with n-docosane as the reference substance is possible. This is supported by the low Table 1 Carbon calibration of paraffin using n-eicosane as the reference substance a Substance
C 18 C 20 C 22 C 24 a
H 38 H 42 H 46 H 50
Concentration (mg / l) Calculated
Experimental
6.48 Reference 7.85 6.54
6.3160.07 6.3960.09 7.7460.02 6.5060.19
Critical value: t (0.95; 8)52.34 [15].
Deviation (%)
Fcorr
t-test
Concentration (mg / l) corrected
22.64 0.0 21.42 20.65
1.03 1.0 1.01 1.01
2.02 0 1.00 0.19
6.48 6.39 7.85 6.54
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Table 2 Carbon calibration of alkanes and pyrethroids using n-docosane as the reference substance a Substance
n-Octadecane n-Docosane n-Tetracosane Allethrin Phenothrin Permethrin Cypermethrin Fenvalerate Deltamethrin a
Concentration (mg / l) Calculated
Experimental
57.5 Reference 54.8 146.5 112.9 92.5 85.5 126.5 185.6
58.060.7 57.561.2 54.261.5 113.862.51 109.663.7 83.562.5 77.361.4 119.263.3 169.062.7
Deviation (%)
Fcorr
t-test
Concentration (mg / l) corrected
1.3 0.0 22.7 222.3 22.8 29.8 29.6 25.8 28.9
0.99 1.0 1.01 1.29 1.03 1.11 1.11 1.06 1.10
0.55 0 1.22 17.54 1.87 6.30 7.89 3.94 7.87
57.5 57.5 54.8 146.5 112.9 92.5 85.5 126.5 185.6
t (0.95; 8)52.34.
t-values of the n-alkanes. The deviation of experimental to theoretical concentration is minimal. However, the experimental content of allethrin is 22 % less with regard to the calculated content. The t-value is 17.54 and, so, it is higher than the critical t-value of t (0.95; 8)52.34. There was a significant difference between the experimental and calculated concentration. Similar results were obtained for the other pyrethroids, with the exception of phenothrin. Therefore, n-docosane cannot be used for the quantitative analysis of pyrethroids. The results of the pyrethroid quantification based on carbon-calibration are illustrated in Table 3. Deltamethrin, a dibromide insecticide ester (Fig. 2), was used as the reference substance. The concentrations of permethrin and cypermethrin were confirmed. The similarity in their molecular structures, both contain two chlorines instead of bromine as found in deltamethrin, is probably responsible for this (Fig. 2). The missing cyano-group in permethrin is of no significance, because it does not contribute to the FID signal. The calculated t-values for permethrin and cypermethrin were similar to the critical value. There was no significant difference in the correlation factor. Phenothrin and fenvalerate (Fig. 2) had significantly higher experimental concentrations. Their t-values were larger than 2.34. Phenothrin contains methyl groups instead of halogens. Table 3 Carbon calibration of pyrethroids using deltamethrin as the reference substance a Substance
Allethrin Phenothrin Permethrin Cypermethrin Fenvalerate Deltamethrin a
Concentration (mg / l) Calculated
Experimental
146.5 112.9 92.5 85.5 126.5 Reference
126.762.8 122.264.1 93.062.7 86.161.5 131.963.7 185.663.0
t (0.95; 8)52.34.
Deviation (%)
Fcorr
t-test
Concentration (mg / l) corrected
213.5 8.3 0.5 0.7 4.9 0.0
1.16 0.92 0.99 0.99 0.95 1.0
10.61 4.59 0.30 0.57 3.09 0
146.5 112.9 92.5 85.5 162.5 185.6
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The acid component in fenvalerate is a-(4-chlorphenyl-)isovalerianic acid instead of chrysanthemic acid. Both acids cause a higher FID response. Allethrin (Fig. 1) had a 13.5 % lower experimental concentration compared to the calculated content. The significant difference in the correction factor was due to the high t-value of 10.61. This demonstrates that carbon calibration only works if the analytes consist of the same or very similar structures as the reference substance. Thus, carbon-based quantitation in SFC is possible for 1. paraffins and phenothrin with n-alkane as the reference substance 2. permethrin and cypermethrin based on a halogenated pyrethroid (deltamethrin). It is not possible for structurally different pyrethroids, such as fenvalerate, phenothrin and allethrin.
3. Results For carbon-based quantitation to work for pyrethrins, a substance with a similar molecular structure is needed. The only available reference substance in our study was allethrin (Fig 1). Carbon calibration was applied to three different pyrethrum standards. With allethrin as the reference substance, the contents of the pyrethrum standards given by the manufacturer were studied. ¨ extracts were measured five times (n55). The contents The Fluka and Riedel de Haen of the carbon calibration were confirmed using SFC (Table 4). The average content of all reference methods in the World Standard Pyrethrum Extract are listed in Table 5 [16]. The experimental value obtained using SFC was 18.78%. The t-values were calculated from the average pyrethrin content. The results obtained depended on the reference method used, with two of them (AOAC and HPLC) giving results that were significantly different from those obtained using SFC. At the time of analysis, the World Standard Pyrethrum Extract was six years old. It can be assumed that part of the pyrethrins had degraded already. After examination of the total content of pyrethrins in pyrethrum extracts, it is also possible to determine the content of each single compound. As an example, the calibration of cinerins I and II is shown in Fig. 4. The linear range extends over two orders of magnitude. Limits of determination (LOD) and quantification (LOQ) were calculated according to DIN [17] (Table 6). At Table 4 ¨ extract using Comparison of the certified and experimental content of pyrethrins in Fluka- and Riedel de Haen carbon calibration with allethrin as the reference substance Sample
Method
w / w (%)
S.D.
Fluka extract
HPLC SFC HPLC SFC
|25 25.5 46 45.5
2 60.7
¨ Riedel de Haen extract
] 62.7
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205
Table 5 Comparison of the certified and experimental content of pyrethrins in World Standard Pyrethrum Extract using carbon calibration with allethrin as the reference substance Sample
Method a
World Standard Pyrethrum Extract
AOAC PBK a HPLC a UV a SFC
w / w (%)
t exp
t
19.5660.23 20.8660.24 20.2860.33 20.8860.03 18.7860.15
4.61 1.03 6.62 0.47 0
t (0.95; 30)52.04 t (0.95; 21)52.08 t (0.95; 26)52.06 t (0.95; 19)52.09
a Contents in the World Standard Pyrethrum Extract were calculated by AOAC, PBK, HPLC and UV methods in 1992. Values are taken from Reference [16].
Fig. 4. Calibration of cinerins I and II.
Table 6 LOD and LOQ of allethrin and 6 pyrethrins Substance
allethrin cinerin I jasmolin I pyrethrin I cinerin II jasmolin II pyrethrin II
DIN LOD (mg / l)
LOQ (mg / l)
13.00 2.91 1.81 11.77 2.05 1.22 8.87
38.96 8.68 5.43 35.27 6.11 3.67 26.64
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Table 7 Contents of single pesticide active substances and ratio (Chr:Pyr) in World Standard Pyrethrum Extract, and in ¨ extract obtained from Fluka and Riedel de Haen Extract substance
Cinerin I Jasmolin I Pyrethrin I Cinerin II Jasmolin II Pyrethrin II Ratio (Chr:Pyr)
¨ Riedel de Haen extract
World Standard Pyrethrum Extract
Fluka extract
%
S.D. (n517)
%
S.D. (n55)
%
S.D. (n55)
11.1 6.3 45.6 7.3 3.7 24.5 1.8
60.5 60.5 62.5 60.7 60.5 60.9 60.09
9.5 5.2 43.0 6.7 3.7 31.9 1.36
60.9 61.0 61.3 61.0 60.7 62.2 60.19
9.6 7.4 40.4 7.9 4.4 30.4 1.34
60.8 61.3 61.3 60.6 60.3 60.3 60.07
first glance, the LODs seem to be a bit high. However, the injection loop had a volume of only 0.1 ml, so, the absolute detected mass of pyrethrins was in the range of 0.1–1.3 ng. Chapman and Simmons [18] published a detection limit of 10–200 pg for some pyrethroid insecticides using gas–liquid chromatography with ECD. Meinen and Kassera [19] described a linear detector response for pyrethrins in the range from 0.15 to 1.1 ng using gas–liquid chromatography with ECD [19]. Table 7 shows the individual quantities of all six pyrethrins in the three different pyrethrum extracts available for this study. The composition of the pyrethrins was very similar in both commercial extracts. Pyrethrin I constitutes about 45.5% and is the main part of the six insecticide active substances. Pyrethrin II is the next largest component, comprising 24.5%. The content of jasmolin I, as well as of cinerins I and II, was between 6 and 11%. The chrysanthemate:pyrethrate ratios were 1.33 (Fluka extract) and ¨ extract). These values are in the range of 1.2–1.5, as reported for 1.38 (Riedel de Haen natural products [20].
4. Conclusion A quantification method for pyrethrins based on carbon calibration in SFC using allethrin as the reference substance is presented. We have shown that carbon calibration in SFC is possible, even when no reference compounds are commercially available, for example, for all six pyrethrins. In this case, allethrin is a suitable reference compound. Each single compound, as well as the total amount, can be analyzed. This method offers a new approach for the analysis of pyrethrin. Significant or non-significant differences between correction factor or contents were supported by calculation of t-values. It was also demonstrated that there must be structural similarity in order to obtain the correct results, e.g., the halogenated deltamethrin is suitable for the determination of cypermethrin and permethrin. The determination of extracts from solid and aqueous samples will be tested in the future.
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¨ ¨ Jager MI, Mischke I, Wollny V. Oko-Mitteilung, 1988, pp. 40–3. ¨ Streit B. In: Lexikon Okotoxikologie, 2. Aufl, Weinheim: VCH, 1994, pp. 611–3. Staudinger H, Ruzicka W. Helv Chim Acta 1924;7:177–458. Roth L. In: Pyrethroide: Eigenschaften, Verwendung, Arbeitsschutz, Umwelt, Lagerung Entsorgung, ¨ Diagnostik, Therapie, ecomed Verlagsgesellschaft, Landsberg: AG & Co. KG, 1996, Sanierung, Toxizitat, pp. 1–7. Perkow W. In: Die Insektizide, Heidelberg: Huthig, 1968, pp. 512–4. Wenclawiak BW, Krappe M, Otterbach A. J Chromatogr A 1997;785:263–7. Maciver DR. In: Casida JE, Quistad GB, editors, Pyrethrum Flowers — Production, Chemistry, Toxicology and Uses, New York, Oxford: Oxford University Press, 1995, pp. 108–22. Schlenger R. Deutsch Apoth Zeitung 1993;30:29–35. Jorgensen A, Picel KC, Stamoudis VC. Anal Chem 1990;62:683–9. Scanlon JT, Willis DE. J Chromatogr Sci 1985;23:333–40. Hammerle RH, Siegl WO, Herrmann HM, Wenclawiak BW. SEA Technical Paper 1995;952353:62. Schneider GM. In: Wenclawiak BW, editor, Analysis with Supercritical Fluids: Extraction and Chromatography, Berlin: Springer, 1992, pp. 9–31. Oehme M. In: Gas-Chromatographische Detektoren, Heidelberg: Huthig, 1982, p. 17. Otto M. In: Analytische Chemie, Weinheim: VCH, 1995, p. 437. Doerffel K. In: Stastistik in der analytischen Chemie, Leipzig: VEB deutscher Verlag, 1982, pp. 121–31. Odinga WA. Pyrethrum Post 1992;18(4):121–5. Frehse H, Thier HP. GIT Fachz Lab 1991;35:258–91. Chapman RA, Simmons HS. J Assoc Off Anal Chem 1977;60(4):977–8. Meinen VJ, Kassera DC. J Assoc Off Anal Chem 1982;62(2):249–55. Maciver DR. In: Casida JE, Quistad GB, editors, Pyrethrum Flowers, New York: Oxford University Press, 1995, pp. 110–2.
Carbon-based quantitation of pyrethrins by supercritical-fluid chromatography B. Wenclawiak*, A. Otterbach Department of Analytical Chemistry, University of Siegen, Adolf-Reichwein-Strasse 2, 57068 Siegen, Germany
Abstract All six insecticide active ingredients in pyrethrum extract were quantified by supercritical fluid chromatography and carbon calibration. Allethrin is a suitable reference compound for carbon calibration and pyrethrins calibrations. Carbon quantification in SFC is also applied to pyrethroids (phenothrin, permethrin, cypermethrin, fenvalerate and deltamethrin) and alkanes. Halogen substitution on pyrethroids requires halogens on the reference calibration compound. The method was applied to commercial extracts. 2000 Elsevier Science B.V. All rights reserved. Keywords: SFC; Pyrethrin; Pyrethroids; Carbon calibration
1. Introduction Pyrethrins are offered as a substitute for organochlorine insecticides like DDT, lindane, aldrin or toxaphene, which are forbidden in many countries, and for organophosphates (E 605 forte), where serious intoxications were often reported. Pyrethrins (Fig. 1) are natural ingredients in flowers like Chrysanthemum cinerariae folium and Chrysanthemum coccineum. The cold solvent extract is called pyrethrum extract. Pyrethrum is one of the oldest known insecticides [1]. The dried plants came from the middle east and they were used by the Romans against lice and fleas. In 1840, powdered flowers were imported to western Europe as ‘Dalmatian insect powder’. Kenya was the main pyrethrum-producing country until 1995. Pyrethrum extract contains six optically active esters of ( 1 )trans-configured chrysanthemum acid and pyrethrum acid with cyclic ketoalcohols ( 1 )-cinerolon, ( 1 )-jasmolon and ( 1 )pyrethrolon [2]. Because of the different acids, two distinct groups (chrysanthemates and pyrethrates) originate. *Corresponding author. Tel.: 149-271-740-4573. 0165-022X / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 00 )00076-2
198
B. Wenclawiak, A. Otterbach / J. Biochem. Biophys. Methods 43 (2000) 197 – 207
Fig. 1. Structures of pyrethrins and allethrin.
Derived from the natural structure and intended to overcome some limitations of the natural products, synthetic compounds with a similar structure and activity were made and these were called pyrethroids (Fig. 2) [3]. Pyrethroids show high insecticide activity, low human toxicity and good plant compatibility. Natural pyrethrum and approximately 15 commercially interesting pyrethroids account for more than 30% of the world trade in insecticides. The active ingredients are used in different formulations for spray, sprinkling powder and as expedients in professional and private plant protection [4]. They are also inserted in tins, sprays and on strips to combat different pests. The use of those agents in dermatology remedies in human and veterinary science for control of ectoparasites has increased dramatically in the last few years. The increasing consumption and the broad application of these insecticides requires a reliable analytical method for the quantitative and qualitative determination of each single compound. The amount of pyrethrins in pyrethrum extracts is analyzed by different methods that are described in the literature [5]. Wenclawiak et al. [6] described a quantification method for the analysis of pyrethrins using in situ transesterfication in supercritical carbon dioxide with GC–MS. Usually, the total amount or the ratio between chrysanthemates and pyrethrates is calculated [7]. The calculation of single amounts of the six active insecticide substances in chrysanthemic flowers is difficult. One reason is the
B. Wenclawiak, A. Otterbach / J. Biochem. Biophys. Methods 43 (2000) 197 – 207
199
Fig. 2. Structure of pyrethroids.
nonexistence of single standards for every pyrethrin because of their fast degradation by UV-irradiation, heat and oxygen [8]. Another reason is the difficulty in quantifying each ingredient in complex mixtures. Jorgensen et al. [9] described the prediction of response factors from molecular structures using flame ionization detection (FID). They stated that their technique allows quantitation of individual components in a complex organic mixture, if structural identification can be made. Scanlon and Willis [10] showed that the response factor calculated from a reference substance can be compared if the structure is similar. Another example is the analysis of engine exhaust gases using GC–FID–MS. Well over 200 single substances cannot not determined individually. All peak areas of the exhaust chromatogram are quantified, based on the carbon calibration response factor of a certified fuel standard with well-known carbon content [11]. In this study, a quantification method for pyrethrins based on carbon calibration and supercritical fluid chromatography (SFC) is presented.
2. Experimental
2.1. Instrumentation A MIPS / 225 (SUPREX, Pittsburgh) was used in this study. This SFC system was equipped with a pneumatically driven four port, timed split injection valve (Valco, Houston, TX, USA). The injection time was 0.5 s. A column (DB5; J&W Scientific, CA, USA) with a length or 10 m and an I.D. of 0.05 mm was used. A laboratory-made
200
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Guthrie-restrictor was placed at the end of the column. Carbon dioxide with a He headspace (SFE-grade; p, 11 MPa; Messer-Griesheim, Germany) was used as the mobile phase. It is important that the mobile phase flows as a liquid through the sample loop. Therefore, 200 mm of the inlet capillary and the injector were cooled to 188C with water. The temperature of the flame ionization detector was set to 3508C. The flame gases were hydrogen (5.0) and synthetic air (Kl. 2) from Messer-Griesheim (Germany). The H 2 -flow was set at 37 ml / min. The hydrogen-to-air ratio was 1:10. GC-Star chromatographic software (4.0) (Varian Germany) recorded the signals from the FID system.
2.2. Chemicals ¨ Germany) and n-hexane were of high purity (PromoIsopropanol (Riedel de Haen, chem, Germany). A standard solution (A) with known concentrations of different hydrocarbons was made in n-hexane [e.g. n-octadecane (6.57 mg / l), n-eicosane (6.43 ¨ mg / l), n-docosane (7.86 mg / l) and n-tetracosane (6.50 mg / l); 99.9%; Riedel de Haen]. The investigated pyrethroids [phenothrin (91.7%), permethrin (97.0%), cypermethrin (95%), fenvalerate (99.1%) and deltamethrin (99.3%)] were from the laboratory of Dr. Ehrenstorfer (Germany). Solution B contained n-octadecane (57.5 mg / l), n-docosane (57.7 mg / l), n-tetracosane (54.8 mg / l) and solution C contained allethrin (146.5 mg / l), phenothrin (112.9 mg / l), permethrin (92.5 mg / l), cypermethrin (85.5 mg / l), fenvalerate (126.5 mg / l) and deltamethrin (185.6 mg / l) and these were prepared in isopropanol. The analyzed pyrethrum extracts were from Fluka (Switzerland) (1995), Riedel de ¨ (Germany) (1996) and the World Standard Pyrethrum Extract (Bayer, Germany) Haen (1992). The extracts were dissolved in isopropanol. Pyrethrum (Fluka) and allethrin were dissolved in isopropanol (solution D).
2.3. Quantification by carbon calibration Fig. 3a shows a SF-chromatogram of pyrethroids. A linear, positive pressure gradient from 11 MPa up to 23 MPa at a rate of 0.2 MPa / min was used. The oven temperature was kept constant at 1108C. Cypermethrin and fenvalerate exhibit double peaks due to isomers. A chromatogram of allethrin and pyrethrins from solution C is shown in Fig. 3b. All analytes are well (baseline) separated. The elution order in SFC depends on the solubility and the binary diffusion coefficient (D12 ) of the analytes [12]. Besides temperature and polarity, the molecular weight or molecular size contribute to the D12 . Allethrin, which has the lowest molecular weight, elutes first, followed by the chrysanthemates and pyrethrates. The elution order in both groups is the same (cinerin, jasmolin, pyrethrin). This does not exactly follow the elution order with respect to molecular weight: the double-bond in pyrethrins I and II may cause a stronger interaction with the stationary phase (DB5) resulting in longer retention times. For carbon calibration, solution A was used. n-Eicosane was added as a reference substance and internal standard, in order to minimize injection errors. Using an internal
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201
Fig. 3. (a) Chromatogram of pyrethroids with a positive pressure program: from 11.1 MPa at 0.2 MPa / min to 24.3 MPa; temperature, 1108C; detection (FID) temperature, 3258C. (b) Chromatogram of pyrethrins and allethrin with a positive pressure program: from 11.1 MPa at 0.2 MPa / min to 24.3 MPa; temperature, 1108C; detection (FID) temperature, 3258C.
sample loop, a defined sample volume (VI ) was delivered onto the column. Thus, the exact carbon mass (m CR ) of the injected reference can be calculated (Eq. (1)) m CR 5 x CR ? c R ?VI where:
(in ng)
(1)
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202
x CR : cR:
carbon content in the reference substance concentration of the reference substance (in mg / l).
In this case, only carbon atoms cause a FID signal [13,14]. The response factor (Rf ) (Eq. (2)) was calculated from the reference peak area (a R ; in counts) and the carbon mass of n-eicosane (Eq. (1)). aR Rf 5 ]] (in counts / ng) (2) m CR Substance-specific peaks were found following the chromatographic separation. The analyte peak area (aA ) is proportional to the carbon mass. By mathematical combining of the analyte area with the response factor, the carbon content in analyte (x CA ) and injected volume, the concentration of the concerned substance can be obtained using Eq. (3). aA cA 5 ]]] Rf ? x CA ?VI
(in mg / l)
(3)
As an example of the carbon calibration method, n-alkanes were tested (solution A). The calculated, the experimentally determined and the corrected concentrations are shown in Table 1. In addition, the correction factor ( fcorr ) and the value of a t-test [15] are also listed in Table 1. All measurements were repeated five times. The relative standard deviation (R.S.D.) was always less than 2.9%. The deviation between the calculated and experimental concentration was less than 2.64%. The correction factor was between 1.01 and 1.03. A t-test was carried out between the fcorr values of analytes and the reference. The critical value of t (0.95; 8) is 2.34. It can be seen that there was no significant difference between the fcorr values, and between the calculated and experimental concentrations. However, it is important that the reference substance possesses the same basic structure as that of the analytes. For example, it is not possible to determine pyrethroids using n-docosane as the carbon reference. This is shown in Table 2. It can be seen from Table 2 that the quantification of n-octadecane and n-tetracosane with n-docosane as the reference substance is possible. This is supported by the low Table 1 Carbon calibration of paraffin using n-eicosane as the reference substance a Substance
C 18 C 20 C 22 C 24 a
H 38 H 42 H 46 H 50
Concentration (mg / l) Calculated
Experimental
6.48 Reference 7.85 6.54
6.3160.07 6.3960.09 7.7460.02 6.5060.19
Critical value: t (0.95; 8)52.34 [15].
Deviation (%)
Fcorr
t-test
Concentration (mg / l) corrected
22.64 0.0 21.42 20.65
1.03 1.0 1.01 1.01
2.02 0 1.00 0.19
6.48 6.39 7.85 6.54
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Table 2 Carbon calibration of alkanes and pyrethroids using n-docosane as the reference substance a Substance
n-Octadecane n-Docosane n-Tetracosane Allethrin Phenothrin Permethrin Cypermethrin Fenvalerate Deltamethrin a
Concentration (mg / l) Calculated
Experimental
57.5 Reference 54.8 146.5 112.9 92.5 85.5 126.5 185.6
58.060.7 57.561.2 54.261.5 113.862.51 109.663.7 83.562.5 77.361.4 119.263.3 169.062.7
Deviation (%)
Fcorr
t-test
Concentration (mg / l) corrected
1.3 0.0 22.7 222.3 22.8 29.8 29.6 25.8 28.9
0.99 1.0 1.01 1.29 1.03 1.11 1.11 1.06 1.10
0.55 0 1.22 17.54 1.87 6.30 7.89 3.94 7.87
57.5 57.5 54.8 146.5 112.9 92.5 85.5 126.5 185.6
t (0.95; 8)52.34.
t-values of the n-alkanes. The deviation of experimental to theoretical concentration is minimal. However, the experimental content of allethrin is 22 % less with regard to the calculated content. The t-value is 17.54 and, so, it is higher than the critical t-value of t (0.95; 8)52.34. There was a significant difference between the experimental and calculated concentration. Similar results were obtained for the other pyrethroids, with the exception of phenothrin. Therefore, n-docosane cannot be used for the quantitative analysis of pyrethroids. The results of the pyrethroid quantification based on carbon-calibration are illustrated in Table 3. Deltamethrin, a dibromide insecticide ester (Fig. 2), was used as the reference substance. The concentrations of permethrin and cypermethrin were confirmed. The similarity in their molecular structures, both contain two chlorines instead of bromine as found in deltamethrin, is probably responsible for this (Fig. 2). The missing cyano-group in permethrin is of no significance, because it does not contribute to the FID signal. The calculated t-values for permethrin and cypermethrin were similar to the critical value. There was no significant difference in the correlation factor. Phenothrin and fenvalerate (Fig. 2) had significantly higher experimental concentrations. Their t-values were larger than 2.34. Phenothrin contains methyl groups instead of halogens. Table 3 Carbon calibration of pyrethroids using deltamethrin as the reference substance a Substance
Allethrin Phenothrin Permethrin Cypermethrin Fenvalerate Deltamethrin a
Concentration (mg / l) Calculated
Experimental
146.5 112.9 92.5 85.5 126.5 Reference
126.762.8 122.264.1 93.062.7 86.161.5 131.963.7 185.663.0
t (0.95; 8)52.34.
Deviation (%)
Fcorr
t-test
Concentration (mg / l) corrected
213.5 8.3 0.5 0.7 4.9 0.0
1.16 0.92 0.99 0.99 0.95 1.0
10.61 4.59 0.30 0.57 3.09 0
146.5 112.9 92.5 85.5 162.5 185.6
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The acid component in fenvalerate is a-(4-chlorphenyl-)isovalerianic acid instead of chrysanthemic acid. Both acids cause a higher FID response. Allethrin (Fig. 1) had a 13.5 % lower experimental concentration compared to the calculated content. The significant difference in the correction factor was due to the high t-value of 10.61. This demonstrates that carbon calibration only works if the analytes consist of the same or very similar structures as the reference substance. Thus, carbon-based quantitation in SFC is possible for 1. paraffins and phenothrin with n-alkane as the reference substance 2. permethrin and cypermethrin based on a halogenated pyrethroid (deltamethrin). It is not possible for structurally different pyrethroids, such as fenvalerate, phenothrin and allethrin.
3. Results For carbon-based quantitation to work for pyrethrins, a substance with a similar molecular structure is needed. The only available reference substance in our study was allethrin (Fig 1). Carbon calibration was applied to three different pyrethrum standards. With allethrin as the reference substance, the contents of the pyrethrum standards given by the manufacturer were studied. ¨ extracts were measured five times (n55). The contents The Fluka and Riedel de Haen of the carbon calibration were confirmed using SFC (Table 4). The average content of all reference methods in the World Standard Pyrethrum Extract are listed in Table 5 [16]. The experimental value obtained using SFC was 18.78%. The t-values were calculated from the average pyrethrin content. The results obtained depended on the reference method used, with two of them (AOAC and HPLC) giving results that were significantly different from those obtained using SFC. At the time of analysis, the World Standard Pyrethrum Extract was six years old. It can be assumed that part of the pyrethrins had degraded already. After examination of the total content of pyrethrins in pyrethrum extracts, it is also possible to determine the content of each single compound. As an example, the calibration of cinerins I and II is shown in Fig. 4. The linear range extends over two orders of magnitude. Limits of determination (LOD) and quantification (LOQ) were calculated according to DIN [17] (Table 6). At Table 4 ¨ extract using Comparison of the certified and experimental content of pyrethrins in Fluka- and Riedel de Haen carbon calibration with allethrin as the reference substance Sample
Method
w / w (%)
S.D.
Fluka extract
HPLC SFC HPLC SFC
|25 25.5 46 45.5
2 60.7
¨ Riedel de Haen extract
] 62.7
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Table 5 Comparison of the certified and experimental content of pyrethrins in World Standard Pyrethrum Extract using carbon calibration with allethrin as the reference substance Sample
Method a
World Standard Pyrethrum Extract
AOAC PBK a HPLC a UV a SFC
w / w (%)
t exp
t
19.5660.23 20.8660.24 20.2860.33 20.8860.03 18.7860.15
4.61 1.03 6.62 0.47 0
t (0.95; 30)52.04 t (0.95; 21)52.08 t (0.95; 26)52.06 t (0.95; 19)52.09
a Contents in the World Standard Pyrethrum Extract were calculated by AOAC, PBK, HPLC and UV methods in 1992. Values are taken from Reference [16].
Fig. 4. Calibration of cinerins I and II.
Table 6 LOD and LOQ of allethrin and 6 pyrethrins Substance
allethrin cinerin I jasmolin I pyrethrin I cinerin II jasmolin II pyrethrin II
DIN LOD (mg / l)
LOQ (mg / l)
13.00 2.91 1.81 11.77 2.05 1.22 8.87
38.96 8.68 5.43 35.27 6.11 3.67 26.64
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Table 7 Contents of single pesticide active substances and ratio (Chr:Pyr) in World Standard Pyrethrum Extract, and in ¨ extract obtained from Fluka and Riedel de Haen Extract substance
Cinerin I Jasmolin I Pyrethrin I Cinerin II Jasmolin II Pyrethrin II Ratio (Chr:Pyr)
¨ Riedel de Haen extract
World Standard Pyrethrum Extract
Fluka extract
%
S.D. (n517)
%
S.D. (n55)
%
S.D. (n55)
11.1 6.3 45.6 7.3 3.7 24.5 1.8
60.5 60.5 62.5 60.7 60.5 60.9 60.09
9.5 5.2 43.0 6.7 3.7 31.9 1.36
60.9 61.0 61.3 61.0 60.7 62.2 60.19
9.6 7.4 40.4 7.9 4.4 30.4 1.34
60.8 61.3 61.3 60.6 60.3 60.3 60.07
first glance, the LODs seem to be a bit high. However, the injection loop had a volume of only 0.1 ml, so, the absolute detected mass of pyrethrins was in the range of 0.1–1.3 ng. Chapman and Simmons [18] published a detection limit of 10–200 pg for some pyrethroid insecticides using gas–liquid chromatography with ECD. Meinen and Kassera [19] described a linear detector response for pyrethrins in the range from 0.15 to 1.1 ng using gas–liquid chromatography with ECD [19]. Table 7 shows the individual quantities of all six pyrethrins in the three different pyrethrum extracts available for this study. The composition of the pyrethrins was very similar in both commercial extracts. Pyrethrin I constitutes about 45.5% and is the main part of the six insecticide active substances. Pyrethrin II is the next largest component, comprising 24.5%. The content of jasmolin I, as well as of cinerins I and II, was between 6 and 11%. The chrysanthemate:pyrethrate ratios were 1.33 (Fluka extract) and ¨ extract). These values are in the range of 1.2–1.5, as reported for 1.38 (Riedel de Haen natural products [20].
4. Conclusion A quantification method for pyrethrins based on carbon calibration in SFC using allethrin as the reference substance is presented. We have shown that carbon calibration in SFC is possible, even when no reference compounds are commercially available, for example, for all six pyrethrins. In this case, allethrin is a suitable reference compound. Each single compound, as well as the total amount, can be analyzed. This method offers a new approach for the analysis of pyrethrin. Significant or non-significant differences between correction factor or contents were supported by calculation of t-values. It was also demonstrated that there must be structural similarity in order to obtain the correct results, e.g., the halogenated deltamethrin is suitable for the determination of cypermethrin and permethrin. The determination of extracts from solid and aqueous samples will be tested in the future.
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