330 Analyst, June, 1974, Vol. 99, PP. 330-337 Quantitative Determination of the Enantiomeric Purity of Synthetic Pyrethroids Part II.* S-Bioallethrin BY F. E. RICKETT AND P. B. HENRY (Wellcome Research Laboratories (Berkharnsted), Berkhamsted Hill, Berkhamsted, Hertfordshire) S-Bioallethrin consists primarily of ( + )-allethronyl-( + )-trans-chrysanthe- mate, but technical samples contain small amounts of other allethrin isomers. The ratio of diastereoisomers can be measured directly from the nuclear magnetic resonance spectrum after using a europium shift reagent, when many of the resonances split into two distinct diastereoisomer signals. If the enantiomeric purity of the chrysanthemate moiety is determined indepen- dently, then the absolute enantiomeric purity of the allethrin sample can be calculated.Standard deviations for the measurement of laboratory or technical samples were between 0.3 and 0.7 per cent. The accuracy of the method was verified by independently determining the enantiomer ratios of various allethrolone samples by gas chromato- graphy of their diastereoisomeric ( - )-a-methoxy-a-trifluoromethylphenyl- acetic esters, esterifying with natural ( + )-trans-chrysanthemic acid and re- determining the enantiomeric purity by nuclear magnetic resonance. Good agreement was achieved between the two determinations. The nuclear magnetic resonance method should also be suitable for measuring the diastereoisomer ratio of cis-allethrins. THE chrysanthemate allethrin, which is an insecticide, can occur in eight isomeric forms; the chrysanthemic acid moiety exhibits both optical and geometrical isomerism and the allethrolone moiety is optically active.This insecticide was introduced in 1949l as the racemic mixture of cis- and tyans- chrysanthemates, in the approximate ratio 25 : 75. Greater insecticidal activity has since been obtained by resolving the acid into the (+)-tram form (bi0allethrint)~-4 and the com- pound is now becoming commercially available with the allethrolone also resolved into the (+)- or S-enantiomer, under the name S-bioallethrin or Esbiol: [( +)-allethronyl-( +)-trans- chry sant hemat el. Polarimetry is a useful method for checking the composition of commercial S-bioallethrin samples as the isomer with the highest insecticidal activity also exhibits the highest negative rotation5 (the values originally published by LaForge, Green and Schechters9' have proved to be inaccurate).However, the method can give only an indication of the minimum amount of (+)-allethronyl-( +)-trans-chrysanthemate present in the sample without defining the other constituents. A more specific method of determining the enantiomeric purity is therefore desirable. An earlier paper8 described a method for determining the enantiomeric purity of cis or trans forms of the chrysanthemic acid moiety by hydrolysis of the allethrin and analysis by gas chromatography of the diastereoisomers formed by reaction of the (+)- and (-)-acids with (+)-or-methylbenzylamine. Analogous methods cannot be used for the allethrolone moiety because it is not possible to recover the free alcohol by hydrolysis of allethrin unless the semicarbazone is first ~repared.~ The method described here provides a means of directly measuring the ratio of diastereoisomers in laboratory and technical S-bioallethrin samples, by use of nuclear magnetic resonance spectroscopy, from which the enantiomeric purity of the allethrolone can readily be calculated.The nuclear magnetic resonance spectra of some natural cyclopropanes with lanthanide shift reagents has been described by Crombie, Findley and Whiting.lo * For details of Part I of this series, see reference list, p. 337. $ Registered trade name, Roussel Uclaf S.A., Romainville, France. @ SAC and the authors. Proposed B.S.I. approved name.RICKETT AND HENRY EXPERIMENTAL REAGENTS- 331 Bioallethrin- ( -J- ) -Allet hron yl- ( + ) -trans-chrysanthemat e, technical grade.S-Bioallethrin- ( + ) -Allet hronyl- ( + ) -trans-chrysant hemat e. Both labor at ory prepared and technical samples were used, the latter supplied by Roussel Uclaf S.A. under the trade name Esbiol. a-( &)-trans-Allethrin-A crystalline racemate of (-)-allethronyl-( +)-trans-chrysanthe- mate and (+)-allethronyl-( -)-trans-chrysanthemate, melting-point 50 to 51 "C, prepared by the method of Schechter, LaForge, Zimmerli and Thomas.11 S-Allethrolone-One sample was kindly supplied by Dr. M. Elliott of the Rothamsted Experimental Station, Harpenden, Herts. Other samples were prepared by hydrolysis of S-bioallethrin semicarbazones by using established procedure~l~s~~ and were blended with (&)-allethrolone (Benzol Products Inc., Edison, New Jersey).(+)-Pyrethrolone-This was supplied by Dr. M. Elliott of Rothamsted Experimental Stat ion. (-)-trans-Chrysanthemoyl chloride-Boiling-point 82 "C (0.5 mm Hg) ; [a]i2 - 24.6" (14.5 per cent. in 2,2,4-trimethylpentane). The chloride was prepared from (+)-trans- chrysanthemic acid that had been obtained by hydrolysis of pyrethrum extract.14 Shift reagent, Eu(fod-d,) ,-Tris- (1 ,l ,l ,2,2,3,3-heptafluoro-7,7-dimethy1-d6-octane-4,6- dioned,) europium(II1) (from Nuclear Magnetic Resonance Ltd., High Wycombe, Bucks). Carbon tetrachloride (nuclear magnetic resonance grade)-Uvasol (E. Merck), dried over type 3A molecular sieve. (-)-cc-Methoxy-cc-trij7aoromethyl~henylacetyl chloride [( -)-MTPA ~hloride]-[a]~~ - 129" (5.0 per cent.in carbon tetrachloride) ; boiling-point 99 "C (12 mm Hg) ; prepared from (-)-MTPA, [a]:' - 72" (neat) (Ralph N. Emanuel Ltd., Wembley, Middlesex) using published methods.15 (-)-Menthol. Nuclear magnetic resonance spectra were recorded on a Vanan T-60 spectrometer by use of high-resolution tubes with tetramethylsilane as internal standard. NUCLEAR MAGNETIC RESONANCE MEASUREMENT OF THE DIASTEREOISOMER RATIO OF The sample (50 mg, 96 to 100 per cent. in total esters) and Eu(fod-d,), (200 mg) were dissolved in the carbon tetrachloride (0.4 d) and filtered into a nuclear magnetic resonance spectrometer tube. Signals for the a- and /3-diastereoisomers of the cyclopropane "CH, (see below) were found at 3.57 and 3.10 p.p.m. downfield of tetramethylsilane, respectively.Peak areas were recorded on a sweep width of 250 Hz and the average of five integrams (each of 50 s sweep time) was taken. S-BIOALLETHRIN- GAS - LIQUID CHROMATOGRAPHIC DETERMINATION OF ALLETHROLONE ENANTIOMERS- (-)-MTPA chloride (22 mg) in 2 ml of dry benzene was added slowly (over 5 minutes) at room temperature to a stirred solution of allethrolone (10 mg) and dry pyridine (5 pl), also in 2 ml of dry benzene. The mixture was stirred at room temperature for 1 hour, then refluxed for 2 hours and evaporated in vacuo at 30 "C to a volume of approximately 1 ml. This product was purified by chromatography on 1 g of Woelm neutral aluminium oxide (Brockmann activity 111) contained in a Pasteur pipette and eluted with 15 ml of benzene. The solvent was evaporated in vacuo and the residue made up to 10 ml with ethyl acetate in preparation for gas - liquid chromatographic analysis, which was performed on a Pye, Series 104, gas chromatograph fitted with a flame-ionisation detector and using a 20 foot x Q inch glass column packed with 6 per cent.LSX-3 on 100 to 120 mesh Gas-Chrom Q. The column oven temperature was 210 "C and the carrier gas (nitrogen) flow-rate 8.5 ml min-l. Retention times were: for (-)-allethronyl-( -)-MTPA, 70.5 minutes; and for (+)- allethronyl-( -)-MTPA, 74.1 minutes. ENANTIOMERIC PURITY OF (-)-MTPA- A solution of 8 mg of (-)-menthol, 10 mg of (-)-MTPA chloride and 50 pl of dry pyridine in 3 ml of dry benzene was refluxed for 2 hours, evaporated in vacuo and made up332 RICKETT AND HENRY: QUANTITATIVE DETERMINATION OF THE [Analyst, Vol.99 to 5 ml with ethyl acetate. The menthyl esters were analysed on a 50 m x 0.2 mm glass capillary column coated with free fatty acid phase (FFAP), operated under the following conditions : column temperature, 163 "C ; injector temperature, 209 "C; carrier gas, nitrogen at 14 p.s.i. inlet pressure. The solution (1 pl) was injected, using an inlet split ratio of approximately 100: 1. The preparation of the column and the equipment used have been described previously.8 Retention times were : (-)-menthyl-( -)-MTPA, 36.4 minutes; and for (-)-menthyl-( +)- MTPA, 37.1 minutes. RECONSTITUTION OF S-BIOALLETHRINS- (-)-trans-Chrysanthemoyl chloride (230 mg), dissolved in 5 ml of dry benzene, was added dropwise over 15 minutes to the S-allethrolone (150 mg) and 0.1 ml of dry pyridine in 5 ml of dry benzene, stirred at 0 "C.The mixture was stirred at room temperature over- night, diluted with 20 ml of diethyl ether, washed with 10-ml portions of 2 M hydrochloric acid, water, saturated sodium hydrogen carbonate solution, then water again and dried over anhydrous calcium sulphate. The solvent was evaporated in vacuo to approximately 1 ml and the product purified by chromatography on Woelm neutral aluminium oxide (Brockmann activity 111), being eluted with 25 per cent. ether in n-hexane. RESULTS AND DISCUSSION Diastereoisomers often exhibit chemical shift differences in their nuclear magnetic resonance spectra, particularly where the protons are close to the asymmetric centres.16117 It has previously been observed18 that synthetic pyrethroids prepared from racemic alcohols show diastereoisomeric non-equivalence for one of the cyclopropane methyl groups.The spectrum of bioallethrin recorded at 60 MHz in solution in carbon tetrachloride shows evidence of diastereoisomeric non-equivalence for three signals [Fig. 1 (a) and 1 ( b ) ] , at 1.25 p.p.m. (cyclopropane "CH,), 1.98 p.p.m. (cyclopentenolone jCH,) and 2.25 p.p.m. (cyclopentenolone 'H) but the separations are not sufficient to allow quantitative determina- tion of the diastereoisomer ratio. With the addition of a europium shift reagent the diastereo- isomers are differentially shifted to lower field, causing many of the resonances to split into two separate diastereoisomer signals [Fig. 2 (a)].The nuclear magnetic resonance spectrum of allethrin, obtained by using a shift reagent, has recently been published by Sugiyama et aZ.l9 The optimum conditions for the quantitative measurement of the diastereoisomer ratio are obtained by using 50 mg of allethrin sample and 200 mg of Eu(fod-d,), dissolved in 0.4 ml of carbon tetrachloride, thus giving a mole ratio of pyrethroid to shift reagent of 0.88: 1. With these concentrations the cyclopropane "CH, gives two signals, at 3.57 p.p.m. and 3.10 p.p.m. for the a- and /3-diastereoisomers, respectively. The #I-diastereoisomer peak consists of the two enantiomers, (+)-allethronyl-( +)-trans-chrysanthemate and (-)- allethronyl-( -)-trans-chrysanthemate, while the a-diastereoisomer comprises the (+) (-) and (-) (+) forms [cf., a-(&)-trans-allethrin].The "CH, peaks are ideal for quantitative measurement, being intense singlets well separated from interfering resonances. The assignments were confirmed by comparing the spectra of bioallethrin [Fig. 2 (a)], S-bio- allethrin [Fig. 2 (b)] and a-(-J-)-trans-allethrin [Fig. 2 (c)]. Measurement of the diastereoisomer ratio does not, of course, give a direct result for the enantiomeric purity of the sample. The (+) to (-) ratio of the chrysanthemic acid moiety can be determined independentlys and, with the nuclear magnetic resonance measurements described above. the enantiomeric Duritv of the allethrolone can be calculated by applying L d the following equation: S-allethrolone, per cent. = and hence S-bioallethrin, per cent.100 (P + x - 100) 2x - 100 x (P + x - 100) - 2x - 100June, 19741 ENANTIOMERIC PURITY OF SYNTHETIC PYRETHROIDS. PART I 333 where P is the observed percentage of the P-diastereoisomer peak and x is the percentage of (+)-enantiomer in the trans-chrysanthemic acid. * In practice this calculation may not be necessary, for if the chrysanthemic acid is of high enantiomeric purity and the allethrolone is predominantly in the (+) form, then the amount of (-)-allethronyl- (-)-trans-chrysanthemate present must be low and the P-diastereo- isomer peak will give an approximate measure of the S-bioallethrin content. d m ? n n H h i 8-0 7 .O 6.0 5.0 4.0 3-0 24 b a TR c 14 0 p.p.m. Fig. 1. Nuclear magnetic resonance spectra of (a) bioallethrin, and (b) S-bioallethrin recorded in solution in carbon tetrachloride without shift reagent.Assignments according to Bramwell et aL1* An optically pure sample of S-bioallethrin was not available for use as a standard to check the accuracy of the nuclear magnetic resonance method. Instead, samples of allethrolone were independently analysed for (+) to (-) ratio, esterified with natural (+)- trans-chrysanthemic acid and used as secondary nuclear magnetic resonance standards. (-)--a-Methoxy-a-trifluoromethylphenylacetic acid [( -)-MTPA] is an established reagent for determining the enantiomeric purity of alcohols by nuclear magnetic resonance spectros~opy.~5~~~ The (-)-MTPA esters of ( j-)-allethrolone give two signals, separated by 8.5 Hz, for the diastereoisomeric cyclopentenolone JCH, [Fig.3 ( a ) ] ; no shift reagents were required in this instance. The lower field resonance at 2-03 p.p.m. was assigned to the (+)-allethronyl ester by comparison with the (-)-MTPA ester of natural (+)-pyrethrolone [Fig. 3 ( b ) ] ; the configurations of the groups around the cyclopentenolone ring are identical in the two compounds,21 the only difference being in the lengths of the side chains. * In the derivation of this equation it is assumed that the enantiomers of acid and alcohol are ran- domly distributed, i.e., that no kinetic resolution has taken place during the esterification stage.334 RICKETT AND HENRY: QUANTITATIVE DETERMINATION OF THE [Analyst, VOl. 99 Although the nuclear magnetic resonance signals were sufficiently well separated for quantitative measurement, an alternative and preferred method for measuring the diastereoisomer ratio was by gas chromatography of the (-)-MTPA esters on a 20-foot glass column packed with 6 per cent. LSX-3 on Gas-Chrom Q support.The resolution obtained 1.. s 8 .O 7.0 6.0 5 .O 4.0 3.0 2.0 1.0 0 p.p.m. Fig. 2. Nuclear magnetic resonance spectra of (u) bioallethrin, (b) S- bioallethrin, and (c) E-( j-)-trans-allethrin with shift reagentJune, 19741 ENANTIOMERIC PURITY OF SYNTHETIC PYRETHROIDS. PART I 335 between the diastereoisomers was 1-45, only marginally above the base-line. Duplicate samples of allethrolone containing 50 to 91 per cent. of (+)-enantiomer were esterified with (-)-MTPA chloride and each was analysed twice by this procedure. Peak areas were measured by use of a disc integrator and the standard deviations calculated from the four analyses of each sample were 0.3 per cent. 0 i" J ~ ~ ~ ~ ~ I ~ ~ ~ ~ ~ 1 1 1 1 ~ ~ 1 ~ 1 1 ~ 1 1 I 1 ~ l l I I 8 .O 7 -0 6.0 5.0 4.0 3.0 2 .o 1 .o 0 p.p.m.Fig. 3. Nuclear magnetic resonance spectra of the ( -)-a-methoxy-a-trifluoro- methylphenylacetic esters of (a) ( f )-allethrolone and ( b ) (+)-pyrethrolone As the (-)-MTPA is a synthetic rather than naturally derived chemical it was considered pertinent to check the enantiomeric purity of the batch used for these determinations. This was accomplished by analysing the diastereoisomeric (-)-menthy1 esters on a 50 m x 0.25 mm glass capillary column coated with FFAP. A base-line separation between the diastereo- isomers was obtained and from four determinations the ratio of (-)-MTPA to (+)-MTPA was found to be 99.3: 0.7 with a standard deviation of 0.2.The enantiomer ratios of the allethrolone samples were corrected for the small amount of (+)-MTPA present in the acid forming the derivatives by using equation (1) where, in this instance, P was the observed percentage of the (+)-allethronyl-( -)-MTPA gas - liquid chromatographic peak and x the percentage of (-)-MTPA in the acid. The corrected values are given in Table I. S-Bioallethrin samples were reconstituted from the allethrolones by use of natural (+)-trans-chrysanthemic acid and the (+) to (-) ratios determined by the nuclear magnetic336 RICKETT AND HENRY: QUANTITATIVE DETERMINATION OF THE [Ana&Sf, VOl. 99 TABLE I PERCENTAGE OF ( +)-ENANTIOMER IN ALLETHROLONE SAMPLES DETERMINED BY GAS - LIQUID CHROMATOGRAPHY AND NUCLEAR MAGNETIC RESONANCE METHODS (+)-Allethrolone by esters, per cent.50.1 76.5 87.7 91-5 91.9 g.1.C. of (-)-MTPA (+)-Allethrolone by n.m.r. of (+)-trans- chrysanthemates, per cent. 50.0 77.0 87-5 91-4 92.3 resonance method. (As, in this instance, the acid is enantiomerically pure, the diastereo- isomer ratio is equal to the enantiomer ratio of the alcohol.) The results given in Table I are the average of five integrations; standard deviations were between 0-3 and 0.7 per cent. The enantiomer ratios of the allethrolone samples determined by the nuclear magnetic resonance method thus compared well with the gas - liquid chromatographic values, all of the differences being less than the combined standard deviations.n H aa I aG I w L Fig. 4. Nuclear magnetic resonance spectra of cis-allethrins with shift reagent. (a). ( j-)-Allethronyl-( +)-cis-chrysanthemate and (b), (+)- allethronyl- ( + ) -cis-chrysanthemateJune, 19741 ENANTIOMERIC PURITY OF SYNTHETIC PYRETHROIDS. PART I 337 Other constituents of technical S-bioallethrin are esters of cis-chrysanthemic acid and free chrysanthemic acid (each comprising generally less than 2 per cent. of the total). When using equivalent amounts of shift reagent, neither compound gave peaks lying within the area from 3.0 to 3.6 p.p.m. that was used for these measurements. No increases in the standard deviations were observed for measurements of technical S-bioallethrin samples ; multiple scan accumulation did not significantly improve precision.The method would also appear to be suitable for determining the diastereoisomer ratio of cis-allethrins. Fig. 4 (a) and (b) shows the nuclear magnetic resonance spectra, after treatment with the shift reagent, of (&)-allethronyl-( +)-cis-chrysanthemate and (+)- allethronyl-( +)-cis-chrysanthemate, respectively (the latter being contaminated with approximately 13 per cent. of the (-)-allethronyl isomer). The cyclopropane aCH3 peaks at 2.88 p.p.m. and 2.27 p.p.m. cannot be used in this instance because the latter overlaps with the methyl peaks of the isobutenyl groups. However, signals from both 'CH, and bCH3 are fully resolved and either should be suitable for quantitative measurement. gift of 1. 2. 3. 4. 5.6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. The authors are grateful to Dr. M. Elliott of Rothamsted Experimental Station for the valuable materials. REFERENCES Stoddard, R. B., and Dove, W. E., Soap Sanit. Chem., 1949, 25, 118. Fales, J. H., Bodenstein, 0. F., Waters, R. M., and Fields, E. S., Soap Chem. Spec., 1970, 46 (12), Chadwick,'P. R.,' Pest. Scz., 1971, 2, 161. Procida S.A., Puteaux, France, technical information. LaForge, F. B., Green, N., and Schechter, M. S., J . Org. Chern., 1954, 19, 457. --- , Ibid., 1956, 21, 455. Rickktt, F.' E., Analyst, 1973, 98, 687. LaForge, F. B., Green, N., and Schechter, M. S., J . Amer. Chem. SOC., 1952, 74, 5392. Crombie, L., Findley, D. A. R., and Whiting, D. A., Tetrahedron Lett., 1972, 39, 4027. Schechter, M. S., LaForge, F. B., Zimmerli, A., and Thomas, J. M., J . Amer. Chem. Soc., 1961, Maciver, D. R., Pyrethrum Post, 1968, 9, 41. Elliott, M., J . Chem. SOC., 1964, 5225. Sawicki, R. M., Elliott, M., Gower, J. C., Snarey, M., and Thain, E. M., J . Sci. Fd Agric., 1962, Dale, J. A., Dull, D. L., and Mosher, H. S., J . Org. Chem., 1969, 34, 2543. Mislow, K., and Raban, M., Topics Stereochem., 1966, 1, 22. Raban, M., and Mislow, K., Ibid., 1967, 2, 199. Bramwell, A. F., Crombie, L., Memesby, P., Pattenden, G., Elliott, M., and Janes, N. F., Telra- Sugiyama, T., Kobayashi, A., and Yamashita, K., Agric. Biol. Chem., 1973, 37, 1497. Koreeda, M., Weiss, G., and Nakanishi, K., J . Amer. Chem. SOC., 1973, 93, 239. Begley, M. S., Crombie, L., Simmons, D. J., and Whiting, D. A., Chem. Commun., 1972, 1276. NOTE-Reference 8 is to Part I of this series. 78. , Ibid., 1971, 47 (l), 64. 73, 3541. 13, 172. hedron, 1969, 25, 1727. Received October 24th, 1973 Accepted November 28th, 1973