Analyst, January 1996, Vol. 121 (55-59) 55 Microbore Liquid Chromatography-Electrospray Mass Spectrometry of Selected Synthetic Pyrethroid Insecticides Ian A. Fleeta, John J. Monaghanajb, Derek B. GordonQ'c and Gwyn A. Lorda,= a Michael Barber Centre for Mass Spectrometry, Department of Chemistry, UMIST, Sackville Street, Manchester, UK M60 I QD b Department of Chemistry, The University of Edinburgh, West Mains Road, Edinburgh, UK EH9 3JJ c Department of Biological Sciences, Manchester Metropolitan University, Chester Street, Manchester, UK MI 5GD The pyrethroid insecticides kadethrin, a-cypermethrin, flucythrinate and SSI-116 have been studied by positive-ion electrospray mass spectrometry (+ESMS) in the presence of ammonium acetate and formic acid. Ammoniated molecule base peak ions [M + NH4]+ were observed for all the insecticides studied at low electrospray source sampling cone voltages.The effect of increasing the cone voltage (40-120 V) and its influence on the extent of fragmentation experienced by each insecticide were studied. A number of these key fragment ions found in +ESMS spectra of a-cypermethrin have been examined by MS-MS under low-energy collisional activation (CA) conditions. On-line microbore reversed-phase liquid chromatographic separations were performed on mixed pyrethroid standards. The eluates were analysed by +ESMS to establish the lower limits of detection using full-scan and selective-ion recording (SIR) modes. Limits of detection (signal-to-noise ratio better than 3 : 1) for each component of the pyrethroid mixture, injected on column, were in the range 120-300 pg (0.30-0.77 pmol) using full-scan mode and 12-60 pg (0.03-0.15 pmol) by SIR.Keywords: Microbore liquid chromatography; electrospray ionization mass spectrometry; electrospray source sampling cone voltage; tandem mass spectrometry; collisional activation; selective-ion recording; ester and non-ester pyrethroid insecticide Introduction Pesticide research over the last thirty years has yielded many structurally diverse insecticides which are classed as synthetic pyrethroids. This research effort was instigated by the in- secticidal properties and low mammalian toxicities of the natural pyrethrins' and a desire to produce synthetic analogues having improved photo-stability and potency, while undergoing faster biodegradation and photodegradation, than the more persistent chlorinated pesticides such as DDT.Research by Elliott2 and co-workers, involving the correlation of stereo- chemical structure with insecticidal activity, led to the discov- ery of permethrin, cypermethrin and many other important pyrethroids which are insecticidal analogues of cyclopropane- carboxylic acid esters. Further efforts by various research groups worldwide, involving a sequence of isosteric replacements of groups originally present in pyrethrin I, have yielded a number of new pyrethroids of commercial interest.3-7 These new pyrethroids lack the cyclopropanecarboxylic acid ester bond and are mainly achiral. They are broadly insecticidal, have low mammalian toxicities and show markedly lower toxicities towards fish than pyrethroids containing the cyclopropanecarboxylic acid ester bond.These structurally diverse pyrethroids are applied to crops, forests, soils, animal feeds and in household use. The resulting loss of these compounds as the intact molecules themselves, their degradation products and/or metabolites to the environ- ment requires the detection of these compounds at the microgram and sub-microgram levels. While the combination of positive-ion electron ionization (EI) and positive/negative-ion chemical ionization (+/-CI) mass spectrometry offers the analytical chemist specificity in the detection of certain synthetic pyrethroids,*99 no single ionization method provides the desired sensitivity for the detection of all members of this structurally diverse insecticide class at the trace level.The fissile nature of the bond between the cyclopropanecarboxylic acid ester oxygen and the alpha- carbon of the benzylphenoxy-containing pyrethroids lo leads to low abundance or non-existent molecular ions in EIMS. Because the central linkages of the non-ester-type pyreth- roides' 1312 show similar facile cleavage, these compounds also yield molecular ions of low abundance. Cyclopropanecarboxylic acid ester pyrethroids'3 and non- ester types yield predominently protonated and/or ammoniated molecule ions when studied by +CI (ammonia). Using -CI (methane) the mass spectra of ester-type pyrethr~ids'~ show almost exclusive formation of the corresponding carboxylate anion. Under similar conditions, the non-ester pyrethroid types cannot form stable carboxylate anions, and yield poor spectra.The development of electrospray ionization14 has revolu- tionized the mass spectrometric analysis of high relative molecular mass compounds. More recently electrospray ioniza- tion has been successfully ultilized to analyse many compounds of lower molecular mass including pharmaceuticals, drugs and sulfonylurea herbicides.15 In this work we have studied a number of structurally diverse pyrethroids using positive-ion electrospray mass spectrometry (+ESMS) in the presence of ammonium acetate and formic acid at low electrospray source sampling cone voltages. The structures of each insecticide are shown in Scheme 1. Kadethnn and a-cypermethrin are representative of the cyclopropanecarboxylic acid ester-type pyrethroids, flucythrinate is an ester type, lacking a cyclopro- pane ring, and SSI- 1 16 is a silicon-containing non-ester-type pyrethroid.The effect of increasing the cone voltage and its influence on the extent of fragmentation experienced by each56 Analyst, January 1996, Vol. 121 insecticide were investigated. A number of key fragment ions found in +ESMS spectra of a-cypermethrin, at higher cone voltages, have also been examined by electrospray tandem mass spectrometry, (+ESMS-MS) under low-energy collisional acti- vation (CA) conditions. Additionally we have investigated the use of on-line microbore reversed-phase HPLC coupled with +ESMS at low cone voltages to separate and detect these structurally diverse pyrethroid insecticides at the trace level.Microbore reversed- phase HPLC offers higher column efficiencies than conven- tional packed columns, drastically reduced flow rates (compati- bility with mass spectrometric techniques) and the ability to work with a smaller sample volume, thus allowing scope for sample enrichment by concentration.16 Experimental A VG Quattro tandem quadrupole mass spectrometer (VG Organic, Altrincham, Cheshire, UK) fitted with an electrospray ionization source, interfaced to a tri-axial probe held at 4.4 kV, was used to carry out various +ESMS and +ESMS-MS experiments. The electrospray source high voltage lens was held at 0.55 kV, for all experiments, and the sampling cone voltage was varied between 40 and 120 V. Mass spectrometry and MS-MS experiments were carried out with the resolution set such that the peak width at half height of the ammoniated molecule ion (a-cypermethrin) was <0.45 u.The mass spectrometer was calibrated over the desired mass range, using poly(ethy1ene glycol). The first quadrupole analyser (MS) was used to study +ES sampling cone voltage induced fragmentations while tandem Kadeth ri n a-Cypermethrin v Flucythyrinate Y FN Scheme 1 Pyrethroid structures. experiments (MS-MS) were performed on selected key ions observed following cone voltage induced fragmentation studies. Individual 25 pg ml-l pyrethroid standards were infused through the tri-axial probe, using a 100 pl syringe and syringe infusion pump at a rate of 5 pl min-1 (Model 22, Harvard Apparatus South Natick, MA, USA). A 'make-up' flow consisting of 70 + 30 propan-2-ol-H20 containing 10 mmol l-1 CH3COONH4 and 22 mmol 1-l HCOOH at a flow rate of 5 pl min-1 was pumped via a micro-gradient syringe pump (Brownlee Labs., Santa Clara, CA, USA) to the tri-axial probe as shown in Fig.1. The tri-axial probe comprises of two concentric stainless-steel capillaries and an inner 700 mm X 75 pm id (375 pm od) uncoated (inner wall) fused-silica capillary (Polymicro Technologies, AZ, USA) which terminates approx- imately 0.5 mm beyond the inner steel capillary. On-line isocratic reversed-phase HPLC experiments were performed by injecting, via a Valco C14W injector with a 0.06 pl rotor volume (Valco, Houston, TX, USA), mixed pyrethroid standards (1-250 pg ml-l) using a 25 cm X 0.5 mm id Spherisorb ODs2 microbore poly(etherether ketone) (PEEK) column.A micro-gradient syringe pump, Fig. 1, was used to pump a mobile phase of 70 + 30 propan-2-ol-H20 containing 10 mmol l-1 CH3COONH4 and 22 mmol l-1 HCOOH at a flow rate of 10 p1 min-1. All MS-MS experiments were obtained using argon gas in the radiofrequency only, hexapole collision cell. The gas pressure was adjusted within the cell so that 50% suppression of the selected ion was obtained. The collision energy was 25 eV in the laboratory frame of reference for each experiment. The mobile phase solvents, propan-2-01 and water, were HPLC grade (Rathburn Chemicals, Walkerburn, Scotland, UK). Ammonium acetate and formic acid were of ACS quality (Sigma-Aldrich, Poole, Dorset, UK). Individual and mixed pyrethroid standards were freshly prepared in the range 1-250 pg ml-1, by serial dilution of their respective stock standards, using 70 + 30 propan-2-ol-H20 containing 10 mmol 1-1 CH3COONH4 mobile phase.All standards were stored in amber 1 cm3 glass vials at 5 "C. All pyrethroid samples used in this study were of research grade. Common names or research codes of each pyrethroid have been used throughout this paper: kadethrin, CAS Registry voltage \Analyst, January 1996, Vol. 121 57 No. 58769-20-3, 5-benzyl-3-furylmethyl(E)-( lR)-cis-2,2-di- methyl-3 -(2-oxothiolan-3-ylidenemethyl)cyclopropanecar- boxylate; a-cypermethrin, CAS Registry No. 67375-30-8, a racemate comprising (R ,S)-a-cyano-3 -phenoxybenzyl-( 1 S&)- cis-3-( 2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxy- late; SSI- 1 16, CAS Registry No.99503- 10-3, (4-ethoxyphe- ny1)-dimethyl- { [ (3-phenoxyphenyl)methoxy]methyl} -silane; flucy-thrinate, CAS Registry No. 70 124-77-5, (+)-a-cyano(3- phenoxyphenyl)methyl(+)-4-(difluoromethoxy)-a-( 1 -methyl- ethy1)benzeneacetate. Flucythrinate (Cybolt) and SSI- 1 16 were donated by Cyanamid, Gosport, Hampshire, UK and Shionogi & Co. Shiga, Japan, respectively. Samples of kadethrin and a- cypermethn were purchased from Promochem (Welwyn Garden City, Hertfordshire, UK). Results and Discussion We have studied a number of structurally diverse pyrethroids using +ESMS in the presence of ammonium acetate and formic acid at low-electrospray-source sampling cone voltages. The structures of kadethrin, a-cypermethrin, flucythrinate and SSI- 116 are shown in Scheme 1.The effect of increasing the cone voltage and the influence on the extent of fragmentation experienced by each insecticide were investigated. A number of key fragment ions found in +ESMS spectra of a-cypermethrin have been examined by +ESMS-MS under low-energy CA. We have also investigated the use of on-line microbore reversed- phase HPLC coupled with +ESMS at low cone voltages to separate and detect these structurally diverse pyrethroid in- secticides at the trace level. Sampling Cone Voltage-induced Fragmentation Experiments The positive-ion electrospray spectra of cypermethrin, at sampling cone voltages between 40 and 120 V (in increments of 20 V), were acquired by scanning the mass range, m/z 50-500, at a rate of 8 s per scan.The resulting spectral data for each scan were stored as an averaged spectrum. At lower cone voltages the positive-ion electrospray spec- trum of a-cypermethrin consists of predominantly ammoniated molecule ions [M + N&]+, in their predicted isotopic abun- dances of 9 : 6 : 1 for a species containing two chlorine atoms, Fig. 2. Progressively higher cone voltages resulted in a simultaneous diminution of the [M + NH4]+ population, with a concomitant increase in the abundance of the protonated molecule ion [M + HI+ and of diagnostically useful fragment ions, Fig. 3. Additionally, the electrospray spectra of a- cypermethrin show that the [M + NH4]+ and [M + HI+ ions (for cone voltages up to 100 V) both yield the correct isotopic abundances for a species containing two chlorine atoms. Many fragment ions observed in the +ES spectra of a-cypermethrin are commonly observed in the corresponding +EIMSl0 and/or +CI (ammonia) MS spectra.l3 Kadethrin shows a similar behaviour to a-cypermethrin, yielding predominantly [M + NH4]+ ions (m/z 414), at lower cone voltages (cone voltage 40 V), in its +ES spectra. Progressively higher cone voltages produce a simultaneous diminution of the [M + NH4]+ population, with a concomitant increase in the abundance of [M + HI+ (m/z 397) and the fragment ions. The +ES spectra of flucythrinate and SSI-116 (cone voltage 40 V), also show the same trends in fragment ion production at progressively higher cone voltages. However, these two pyrethroids do not yield protonated molecule ions [M + HI+ when undergoing transition from low to higher cone voltages.A positive-ion electrospray spectrum of the mobile phase in the absence of analyte, at a cone voltage of 40 V, is dominated by an intense protonated propan-2-01 solvent dimer ion at m/z 12 1 (base peak). At higher cone voltages the protonated propan- 2-01 solvent dimer ion diminishes in abundance. Low cone voltage +ES spectra of the mobile phase also show a minor artifact ion at m/z 391 which has been attributed to the protonated molecule ion of bis(2-ethylhexyl) phthalate. At higher cone voltages, the bis(2-ethylhexyl) phthalate ion undergoes fragmentation with subsequent loss of the side chains to form the ions at m/z 279 and m/z 149 (protonated phthalic anhydride). MS-MS of Some Key Ions Observed Following Sampling Cone Voltage-induced Fragmentation of a 25 pg ml-1 a-Cypermethrin Standard Many fragment ions observed in the +ES spectra of each pyrethroid, at higher sampling cone voltages, are identical to fragment ions observed in either their +EIMS and/or +CI (ammonia) MS spectra.We have investigated at a source sampling cone voltage of 70 V a number of key fragment ions observed in the +ES of a-cypermethrin. Individual +ES product-ion spectra of the ammoniated molecule ions of a-cypermethrin, m/z 433 (2 X 373) and m/z 435 (1 X W I , 1 X 37Cl), show direct,connectivity between these ions and the product ions m/z 4 16 [M + HI+ (2 X 3Tl); m/z 418 [M + HI+ (1 X 3T1, 1 x 37C1), and m/z 191 (2 X T 1 ) ; m/z 193 (1 X 3Tl, 1 X 37Cl), respectively. Product-ion spectra of the ions at m/z 416 and 418 also show connectivity between these ions and the product ions m/z 191 and 193, respectively. The fragment ions m/z 191 and 193 are observed in the +CI (ammonia) spectra of a-cypermethrin but not in their +EI spectra.The formation of protonated molecule ions at higher sampling cone voltages from their ammoniated molecule ions reflects the dissociation of [M + NH4]+ to [M + HI+ + NH3, Scheme 2. Confirmation that NH3 has been lost directly as a neutral species from the [M + NH4]+ ion is provided by a constant neutral loss scan of 17 Da. A product-ion scan of the 359 m/z Fig. 2 Positive-ion electrospray spectrum of Cypermethrin (cone voltage 40 V). 193 I m/z Fig. 3 Positive-ion electrospray spectrum of Cypermethrin (cone voltage 100 V).58 Analyst, January 1996, Vol.121 m/z lBylBWl95 Scheme 2 Formation of a dichlorovinyldimethylcyclopropane acylium ion, [C8HgCl20]+, following homolytic cleavage of the carbon-oxygen bond alpha to the ester carbonyl group. m/z 191 ion (2 X 35C1), obtained at the same cone voltage, shows connectivity between this ion and the fragment ions at m/z 163,127 and 91. The ions, m/z 191,193 and 195, have been attributed to the formation of a dichlorovinyldimethylcyclo- propane ac ylium ion, following homolytic cleavage of the carbon-oxygen bond alpha to the C=O group, Scheme 2. Subsequent loss of CO from the m/z 191 ion yields a dichlorovinyldimethylcyclopropane carbenium ion [C7H935C12]+ at m/z 163, which undergoes further successive loss of molecules of HC1 to form m/z 127 and 91 ions.1° The ions, m/z 163,127 and 91, are also observed in the +EI spectrum of a-cypermethrin.Tandem electrospray mass spectrometry experiments at higher sampling cone voltages on key ions indicate that a- cypermethrin undergoes similar fragmentations to those ob- served under EIMS (70 eV) and/or +CI (ammonia) ionization conditions. This suggests that the fragment ions produced in each ionization process have similar internal energies. Low Sampling Cone Voltage, Microbore Isocratic Reversed-phase HPLC Separation of Mixed Pyrethroid Standards On-line reversed-phase liquid chromatography positive elec- trospray ionization experiments were performed on mixed pyrethroid standards to establish the lower limits of detection. Individual mixed standards were chromatographed through a 25 cm X 0.5 mm id Spherisorb ODs2 microbore PEEK column, Fig.1, and the resulting eluates were subjected to +ES ionization. Mass spectral data were acquired by scanning either v la*] Kndethrin .- 5 Tl<MS,Ps+).4 s 0-1 ’ I . ’ - ’ ‘ ’ ’ ” I . ’ . I ’ ” ‘ ‘ Time/min Fig. 4 standard, and narrow scan range (m/z 380-480). LC-(+ESMS) reconstructed mass chromatograms of individual pyrethroid ammoniated molecule ions, using a 10 pg ml-l mixed pyrethroid 18Q 541M ES+), 418 S414C ES+, - 4 4 h lea! Kadethrin 43 Time/min LC-(+ESMS) single-ion chromatograms of individual pyrethroid ammoniated molecule ions, using a 1 pg ml-l mixed pyrethroid standard. Fig. 5Analyst, January 1996, Vol. 121 59 ~~ Table 1 Limits of detection* for individual components of a pyrethroid mixture using full scant and SIR$ modes Pyrethroid M, Full scan/pg SIR/pg SSI-116 392 300 30 (4 10)s Kadethrin 396 120 12 (414)s a-Cypermethrin 415 300 60 (433)s) Flucythrinate 451 240 18 (469)s * Signal-to-noise ratio better than 3 : 1 for individual components of a pyrethroid mixture injected on column.t Using 10 pg ml-1 mixed standard of individual pyrethroids. * Using 1 pg ml-l mixed standard of individual pyrethroids. Monoisotopic mass of ammoniated molecule ion, [M + N&]+. a narrow mass range between m/z 380 and 480 (full-scan mode) at a rate of 3 s per scan or by monitoring single ions (selective- ion recording, SIR) characteristic of the ammoniated molecule ions for each pyrethroid (dwell time 0.2 s, inter-scan delay 0.02 s and 1 Da span).Full-scan experiments were performed using mixed pyrethroid standards in the range 10-250 yg ml-l and mixed pyrethroid standards in the range 1-10 yg ml-l were used for SIR experiments. Loss of chromatographic resolution (band broadening) is observed, Figs. 4 and 5, compared with off-line experiments using isocratic reversed-phase HPLC with a standard size analytical column, 25 cm X 4.6 mm id Shandon ODS column, and UV detection (not shown). The band broadening is attributed largely to post-column dispersion in the length of unpacked capillary necessary for interfacing to the mass spectrometer. ‘Memory’ effects in the electrospray source region may also contribute. Although band broadening is observed, the specificity of mass spectrometry obviates the need to chromatographically resolve every single component by detection of unique ammoniated molecule ions and/or fragment ions (higher cone voltages). However, this does not apply in the case of isobaric compounds, including diastereoisomers, where chromatographic separation is necessary.Using full-scan mode, limits of detection at a signal-to-noise ratio better than 3 : 1 were calculated from the LC-(+ESMS) reconstructed mass chromatograms for each ammoniated mole- cule ion, Fig. 4, and found to be in the range 120-300 pg (0.30-0.77 pmol) injected on column, Table 1. Limits of detection at a signal-to-noise ratio better than 3 : 1 were calculated from the LC-(+ESMS) single-ion chromatograms of each ammoniated molecule ion, Fig. 5, and found to be in the range 12-60 pg (0.03-0.15 pmol) injected on-column, Table 1.Conclusion This study provides evidence that microbore reversed-phase LC-(+ESMS) at lower sampling cone voltages, in the presence of ammonium acetate and formic acid, is a sensitive analytical technique for the separation and unambiguous detection of structurally diverse pyrethroid insecticides at the trace level. Ammoniated molecule base peak ions were observed for all the insecticides studied at low cone voltage. Progressively higher cone voltages resulted in a diminution of the [M + NH4]+ population, with a concomitant increase in the abundance of diagnostically useful fragment ions. At higher sampling cone voltages the +ES spectrum of a- cypermethrin was found to yield fragment ions in common with the compound’s +EIMS and/or +CIMS spectra.Tandem electrospray mass spectrometry experiments, performed on key ions observed in the spectra of a-cypermethrin, have provided evidence that the fragment ions produced in each ionization process have similar internal energies. Limits of detection at a signal-to-noise ratio better than 3 : 1 for each individual component of a pyrethroid mixture injected on column were in the range 120-300 pg using full-scan mode and 12-60 pg by SIR. Where tandem mass spectrometry is available further improvements may be made to achieve ultimate sensitivity, especially in the presence of high chemical noise, by monitoring in selective-reaction monitoring mode (SRM) key ions which have a product/precursor ion relationship. The authors thank Dr.T. Takahashi of Shionogi & Co. and R. E. Hyson of Cyanamid for the donation of research-grade samples and provision of technical help. We also thank Dr. P. Myers of Phase Separations, for the kind donation of the microbore HPLC column and Professor S. Gaskell and colleagues at the Michael Barber Centre for Mass Spectrometry, UMIST, for their help and support. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Pyrethrum-The Natural Insecticide, ed. Casida, J. E., Academic Press, New York, 1973. Elliott, M., in Recent Advances in the Chemistry of Insect Control, Special Publication No. 53, ed. Janes, N. F., The Royal Society of Chemistry, London, 1985, pp. 73-102. Udagawa, T., Numata, S., Oda, K., Shiraishi, S., Kodaka, K., and Nakatani, K., in Recent Advances in the Chemistry of insect Control, Special Publication No.53, ed. Janes, N. F., The Royal Society of Chemistry, London, 1985, pp. 192-204. Bushell, M. J., in Recent Advances in the Chemistry ofinsect Control, Special Publication No. 79, ed. 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A., Tetler, L. W., and Monaghan, J. J., J . Mass Spectrom., 1995,30, 617. Fleet, I. A., and Monaghan, J. J., unpublished work. Lidgard, R. O., Duffield, A. M., and Wells, R. J., Biomed. Environ. Mass Spectrom., 1986, 13, 677. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M., Mass Spectrom. Rev., 1990, 9, 37. Volmer, D., Wilkes, J. G., and Levsen, K., Rapid Commun. Mass Spectrom., 1995, 9, 767. Ahuja, S., Trace and Ultratrace Analysis by HPLC, Wiley, New York, 1992. Paper 51051 77K Received August 3, 1995 Accepted September 20, I995