A liquid chromatography-electrospray tandem mass spectrometry method for the determination of multiple pesticide residues involved in suspected poisoning of non-target vertebrate wildlife, livestock and pets

Abstract

A liquid chromatography tandem mass spectrometry (LC/MS/MS – triple quadrupole) method that facilitates the qualitative and quantitative determination of multiple pesticide residues that could be present in wild and domestic vertebrate animals has been developed. The method involves the direct analysis of crude extracts from a variety of test specimens taken from (non-target) birds and mammals suspected as being victims of accidental or deliberate exposure to pesticides. Performance of the method was validated following replicate analysis of pseudo-matrices (chicken muscle and chicken liver tissue) that had been fortified with 102 different pesticides and their degradation products at two different concentration levels (0.1 mg kg⁻¹ and 1.0 mg kg⁻¹). Residues detected were quantified following interpolation against external calibration curves obtained using matrix-matched standards that covered a residue concentration range between 0.025 μg ml⁻¹ and 0.5 μg ml⁻¹. Application of the method is demonstrated through inclusion of a few examples that show screening and confirmation results of pesticide residues detected in animals involved in suspected poisoning incidents.

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Introduction

The United Kingdom's Wildlife Incident Investigation Scheme (WIIS) is operated in Scotland by SASA, a scientific division of the Scottish Government's Agriculture, Food and Rural Communities Directorate. The WIIS^1,2 investigates poisoning of non-target vertebrate wildlife, beneficial insects such as honeybees, livestock and pets whenever accidental or deliberate exposure to pesticides is suspected. The scheme provides a means of post-registration surveillance of pesticide use and a measure of the success of the pesticide registration process. Incidents resulting from approved use or misuse can highlight problems with the conditions of approval or label instructions and provide valuable feedback into the regulatory process. In contrast, operation of the scheme can also reveal the sinister practice of deliberate and illegal attempts to poison animals. Evidence from such incidents can be used by the Scottish Government and police to enforce a variety of legislation relating to the safe use of pesticides and the protection of the environment and animals.^3–5

The chemical diagnosis of suspected pesticide exposure or poisoning incidents involving wildlife, livestock, domestic animals, beneficial insects (e.g. honeybees) or baits i.e. animal carcases deliberately laced with pesticides, is extremely challenging primarily because:

• Hundreds of professional and amateur plant protection and biocidal products are commercially available and currently approved for use in the UK.

• Products withdrawn from use in the UK are still accessible.

• New products are regularly introduced to the market.

• Analytical methods, techniques and instrumentation used should be capable of the detection, identification and quantitation of multiple pesticide residues in a variety of sample types and complex mixtures even if they are present at ultra-low levels.

• Analytical methods, data and records must comply with rigorous quality assurance procedures and data can be presented, scrutinised and challenged in associated enforcement actions or legal proceedings.

• Targeted chemicals must be extracted from a variety of test specimens such as liver tissue, stomach contents or viscera.

There are several examples of how Liquid Chromatography-Electrospray Tandem Mass Spectrometry (LC/MS/MS) has been successfully applied to determine the presence of multiple pesticide residues that remain in or on food of animal origin including various animal tissues e.g. red/white meat or offal. In 2009, Pang et al.⁶ combined gas chromatography mass spectrometry (GC/MS) and LC/MS/MS techniques following gel permeation chromatography (GPC) clean up, for the quantitative determination of several hundred pesticides in animal muscles (cow, sheep, chicken and rabbit). They concluded that LC/MS/MS was more sensitive than GC/MS for the determination of over 200 pesticides. Mol et al. utilised Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC/MS/MS) in the development of a generic extraction method for the simultaneous determination of pesticides, mycotoxins, plant toxins and veterinary drugs in feed and food matrices which included meat.⁷ Advanced LC/MS based residue methods for the determination of multiple pesticides and their degradation products in foodstuffs which included animal tissues were reviewed by Fernandez-Alba et al. in 2008. They described the advantages and pitfalls of these methods, particularly relating to large-scale screening, identification and quantitation of multiple pesticide residues.⁸

However, examples of the use of LC/MS/MS for routine monitoring of multiple pesticide residues that may remain in or on specimens taken from vertebrate wildlife or pets suspected of being accidentally or deliberately exposed to pesticides are rare. In 1996, Brown et al. published details of analytical methods used for the determination of multiple pesticide residues and single pesticide residues in wildlife specimens submitted to the WIIS operating in England and Wales. The different methods involved a variety of sample clean-up and derivatisation techniques, combined with GC, GC/MS and/or High Performance Liquid Chromatography (HPLC) and covered a wide range of pesticides and their metabolites.⁹ The same author updated this review in 2005 and LC/MS and LC/MS/MS were included as front-line techniques for the determination of single pesticide residues (imidacloprid, paraquat and diquat), multiple (carbamate) pesticide residues and anticoagulant rodenticides.¹⁰ Hunter et al. published details of a single residue method in 2004 that used LC/MS/MS (negative ion mode) for the quantitative determination of chloralose in specimens collected from suspected poisoned birds of prey.¹¹ Wang et al. (2006) carried out a 6-year retrospective review of pesticide poisoning in domestic animals and livestock in Austria whereby pesticides were characterised by non-MS based chromatographic methods only.¹²

Most recently (2011), Novotný et al. published details of case studies involving carbamate intoxication of animals in the Czech Republic.¹³ The authors only noted the use of ‘chromatographic techniques’ for the analysis of tissue (liver), solids (bait, unidentified material) or liquid samples (gastric content).

At SASA, before the method reported here was developed in 2010, we depended almost exclusively on GC/MS/MS multiple pesticide residue methods to screen biological specimens for organochlorine, organophosphate and pyrethroid pesticides or to confirm the presence of a few carbamate pesticides such as carbofuran and aldicarb (initially determined by HPLC-UV). This situation combined with the dearth of relevant multiple pesticide residue methods based on LC/MS/MS highlighted the need for us to significantly and quickly extend our WIIS-Scotland ‘target’ pesticide inventory to include pesticides that were amenable to LC/MS/MS. In an effort to optimise the efficiency of sample preparation procedures, it was decided to employ the same extraction solvent (ethyl acetate) procedures for LC/MS/MS as that used to extract multiple pesticides from specimens prepared for existing GC/MS/MS methods. The ambition was to be able to simply split a common sample extract prior to LC/MS/MS (no clean-up) and GC/MS/MS (GPC clean-up) experimentation. The advantages of using a common sample extract for subsequent LC/MS/MS and GC/MS/MS analyses have been demonstrated by Pihlström¹⁴ and Lehotay.¹⁵ Both authors utilised this approach for the determination of multiple pesticide residues in various foodstuffs following extraction by ethyl acetate and QuEChERS, respectively.

Experimental

High performance liquid chromatography

HPLC gradient separation was achieved using an 1100 Series Liquid Chromatograph system comprising a column heater module, vacuum degasser, quaternary pump and auto injector/sampler (Agilent Technologies, Santa Clara, USA). A 3 μm Hypersil Gold C₁₈ BDS column 100 × 4.6 mm (Thermo Fisher Scientific, Loughborough, UK) fitted with a Security Guard™ guard cartridge (Phenomenex, Macclesfield, UK) was operated at 35 °C and at a flow rate of 0.5 ml min⁻¹. This flowrate proved optimum for the LCMS system used, eliminated the need for post-column flow splitting and did not compromise peak resolution. The overall run-time and solvent consumption would be reduced through the use of e.g. 2.1 mm internal diameter columns. However, narrow-bore columns were not readily compatible with this particular LCMS system. The gradient elution timetable using A: methanol (5 mM ammonium acetate) and B: water, methanol (5 mM ammonium acetate) 95/5, v/v was:

Time (min)	%A	%B
0.00	30.0	70.0
1.00	30.0	70.0
2.00	60.0	40.0
5.00	60.0	40.0
22.00	85.0	15.0
34.00	85.0	15.0
35.00	30.0	70.0
40.00	30.0	70.0

An injection volume of 10 μl was used throughout.

An isocratic HPLC separation utilising the above instrument configuration, general operating parameters and mobile phase solutions was used for confirmation purposes i.e. 80% A: 20% B; flow rate: 0.5 ml min⁻¹; stop time: 12 minutes; injection volume: 10 μl.

Mass spectrometry

Electrospray ionisation was achieved using a Quattro Ultima Tandem (triple quadrupole) Mass Spectrometer (Waters Corporation (Micromass), Manchester, UK). The instrument was operated in positive ionisation mode and 102 different pesticides, metabolites and degradation products were monitored during the chromatographic separation using multiple reaction monitoring (MRM) combined with time-scheduled data acquisition sequences. An interchannel delay of 0.02 s, an interscan time of 0.1 s, dwell times between 0.05 and 0.25 seconds and span corresponding to 0.2 Da were used. Argon of 99.9% purity (BOC, Manchester, UK) was used as collision gas (1.4 × 10⁻³ mbar gas cell pressure).

Optimum cone voltage and collision energy values were determined for each analyte following direct infusion (Harvard syringe pump) of individual pesticide solutions. The molecular ion species was identified i.e. [M + H]⁺, [M + Na]⁺, [M + NH₄]⁺ or in the case of 3-hydroxy carbofuran [M-H₂O + H]⁺and selected as the precursor ion. The precursor ion → product ion transitions listed in Table 1 were used for screening, confirmation and construction of associated calibration curves. However, the data acquisition capability of the LCMS system employed limited the number of MRM transitions that could be adequately monitored per data acquisition function/channel, especially when time-scheduled data acquisition windows overlapped. Consequently, it was not possible to effectively combine screen and confirmation MRMs in a single experiment for all 102 pesticides sought. Therefore, whenever a positive result was indicated following the initial screen a separate confirmation experiment was performed whereby the original screen and an alternative precursor ion → product ion transitions were monitored (where possible) in combination with the much faster isocratic HPLC separation. In addition, our strategy was to prepare a fresh set of matrix-matched standards and re-analyse the sample extract as soon as possible in order to minimise any instability of analytes in sample matrix solution (ideally within 24 h). The quantitative results obtained from the two separate MRMs monitored in the confirmation experiment had to agree within ±20% in order to satisfy in-house quality control procedures. Analytical method performance criteria and guidelines for confirmation of organic residues or contaminants present in animal products are specified in Commission Decision 2002/657/EC¹⁶ whereby the number of (chromatographic/mass spectrometric) identification points (IPs) is specified i.e. at least 3 IPs depending upon the pesticide. We believe that this confirmatory approach accumulates a comparable (minimum) number of IPs.

Table 1 LC/MS/MS parameters (positive ion mode)^a

Pesticide [precursor ion assignment]	Rt min	MRM screen	CV V	CE eV	MRM confirmation
a Rt = retention time (minutes); CV = cone voltage; CE = collision energy.
Acetamiprid [M + H]	6.31	223 > 126	25	15	223 > 90
Aldicarb [M + Na]	7.20	213 > 89	8	8	213 > 116
Aldicarb sulphone [M + NH4]	3.28	240 > 86	13	22	240 > 148
Aldicarb sulphoxide [M + H]	3.49	207 > 89	16	15	207 > 143
Atrazine [M + H]	9.44	216.1 > 74	30	20	216.1 > 104
Azoxystrobin [M + H]	10.98	404 > 372	21	16	404 > 344
Bendiocarb [M + H]	7.90	224.2 > 109	20	20	224 > 167
Benfuracarb [M + H]	20.19	411 > 194.9	20	25	411 > 251.9
Bitertanol [M + H]	17.86	338 > 269	25	8	338 > 70
Boscalid [M + H]	11.74	343 > 307	25	25	343 > 140
Bromuconazole [M + H]	15.68	378.1 > 158.8	30	20	378.1 > 69.8
Bupirimate [M + H]	15.23	317 > 272	25	25	317 > 166
Carbaryl [M + H]	8.32	202 > 145	20	15	202 > 127
Carbendazim [M + H]	6.77	192 > 160	35	15	192 > 132
Carbofuran [M + H]	7.95	222 > 165	20	10	222 > 123
Carbofuran-3 hydroxy [M-H2O + H]	6.24	220 > 162.9	26	12	220 > 181
Carbosulfan [M + H]	27.43	381 > 118	25	25	381 > 160
Chlorotoluron [M + H]	9.12	213.1 > 72.1	30	20	213.1 > 140
Clofentizene [M + H]	17.33	303 > 138	25	14	303 > 102
Clothianadin [M + H]	5.93	250 > 168.8	20	15	250 > 131.9
Cyazofamid [M + H]	14.41	325 > 108	20	10	325 > 261
Cymoxanil [M + H]	6.62	199.1 > 128	15	10	199.1 > 111
Cyproconazole [M + H]	13.56	292.3 > 125	25	30	294 > 127
Cyprodinil [M + H]	16.04	226 > 108	25	25	226 > 93
Demeton-S-methyl [M + H]	6.31	231 > 89	15	10	231 > 199.8
Demeton-S-methyl sulphone [M + H]	4.02	263 > 169	26	18	263 > 109
Difenoconazole [M + H]	18.91	406.2 > 250.9	30	25	408 > 252.9
Diflubenzuron [M + H]	15.05	311 > 158	25	10	311 > 141
Dimethoate [M + H]	6.31	230 > 125	16	22	230 > 199
Dimethomorph [M + H]	12.44	388 > 301	25	15	388 > 165
Dimoxystrobin [M + H]	15.50	349 > 260	25	18	349 > 228
Disulfoton [M + H]	17.81	275 > 89	25	18	349 > 61.1
Disulfoton sulphoxide [M + H]	9.08	291.1 > 184.9	25	10	291.1 > 213
Diuron [M + H]	10.07	233 > 159.9	30	25	235 > 161.9
Epoxiconazole [M + H]	14.28	330.2 > 121	20	25	330.2 > 101
Famoxadone [M + NH4]	17.15	392.2 > 331	15	10	392.2 > 238
Fenamidone [M + H]	11.46	312.1 > 236	25	15	312.1 > 264
Fenarimol [M + H]	14.01	331 > 268	25	25	331 > 81
Fenbuconazole [M + H]	14.87	337 > 125	25	25	337 > 70
Fenhexamid [M + H]	13.56	302 > 97	36	26	302 > 55
Fenpropimorph [M + H]	26.94	304 > 147	25	30	304 > 116
Fenpyroximate [M + H]	24.66	422 > 138	25	25	422 > 366
Fenthion [M + H]	16.18	279.1 > 168.9	25	15	279.1 > 246.9
Fenthion sulphone [M + H]	8.44	311.1 > 278.9	40	15	310.8 > 124.9
Fenthion sulphoxide [M + H]	8.23	294.9 > 279.9	35	20	294.9 > 264
Fludioxonil [M + H]	12.51	266 > 158	25	25	266 > 180
Flufenacet [M + H]	13.83	364.1 > 194	20	10	364.1 > 152
Fluopicolide [M + H]	12.37	385 > 174.8	20	25	385 > 194
Fluoxastrobin [M + H]	13.47	459.1 > 426.8	30	20	461.1 > 428.9
Fluquinconazole [M + H]	13.23	376.2 > 349	25	20	376.2 > 306.9
Flusilazole [M + H]	15.32	316 > 165	20	25	316 > 247
Flutriafol [M + H]	9.23	302.2 > 69.8	20	25	302.2 > 122.8
Imazalil [M + H]	16.98	297 > 159	32	25	297 > 201
Imidacloprid [M + H]	5.60	256 > 175	25	25	256 > 209
Indoxacarb [M + H]	19.26	528 > 203	25	35	528 > 150
Isofenphos [M + H]	17.55	346 > 245	20	10	346 > 217
Isoproturon [M + H]	9.75	207 > 165	20	12	207 > 72
Kresoxim-methyl [M + H]	15.50	314 > 222	25	10	314 > 116
Linuron [M + H]	11.46	249.7 > 160	26	20	249.7 > 182
Malathion [M + H]	12.30	331 > 127	30	20	331 > 285
Mepanipyrim [M + H]	13.37	224 > 106	25	30	224 > 77
Metconazole [M + H]	17.37	320.3 > 69.8	25	25	320.3 > 124.7
Methiocarb [M + H]	11.81	226 > 121	25	15	226 > 169
Methomyl [M + H]	4.13	185 > 128	22	10	163 > 88
Metolcarb [M + H]	7.66	166 > 109	25	9	166 > 194
Metrafenone [M + H]	17.64	409.1 > 209	25	15	409.1 > 229
Monocrotophos [M + H]	4.67	241 > 127	18	18	224 > 127
Myclobutanil [M + H]	13.09	289 > 70	25	20	289 > 125
Ofurace [M + H]	7.90	282 > 254	25	10	284 > 160
Omethoate [M + H]	3.23	214 > 155	20	12	214 > 125
Oxamyl [M + NH4]	3.56	237 > 72	25	12	237 > 90
Oxydemeton methyl [M + H]	3.82	247 > 109	18	15	247 > 169
Penconazole [M + H]	16.31	284 > 159	30	30	284 > 70
Pencycuron [M + H]	18.08	329 > 218	30	15	329 > 125
Phorate [M + H]	17.24	261 > 75	25	25	261 > 171
Phorate sulphone [M + H]	9.18	293 > 115	25	25	293 > 171
Picoxystrobin [M + H]	15.41	368 > 145	25	20	368 > 205
Pirimicarb [M + H]	9.02	239 > 72	28	21	239 > 181.9
Prochloraz [M + H]	17.94	376 > 308	25	10	376 > 266
Propamocarb [M + H]	6.77	189 > 102	30	15	189 > 144
Propiconazole [M + H]	16.98	342 > 159	25	30	342 > 69
Pymetrozine [M + H]	4.01	218 > 105	25	30	218 > 79
Pyraclostrobin [M + H]	17.15	388.1 > 194	25	10	388.1 > 163.9
Pyrethrins [M + H] summed	24.26	361 > 149	25	8	361 > 107
		329 > 161			329 > 133
		317 > 149			317 > 121
		373 > 161			373 > 133
		375 > 163			375 > 121
		331 > 163			331 > 121
Pyrifenox [M + H]	14.91	295.1 > 93	30	25	297 > 93
Pyrimethanil [M + H]	11.46	200 > 107	40	28	200 > 82
Quinoxyfen [M + H]	22.21	308 > 197	20	30	308 > 162
Simazine [M + H]	8.00	202.1 > 131.9	30	20	202.1 > 124
Spinosad [M + H] summed	27.69	732.5 > 142	25	25	732.5 > 98.5
		746 > 142			746 > 95
Spiromesifen [M + H]	23.34	371 > 273	25	22	371 > 255
Spiroxamine [M + H]	22.48	298.2 > 143.9	30	22	298.2 > 100
Tebuconazole [M + H]	16.31	308 > 70	30	24	308 > 125
Tebufenpyrad [M + H]	20.98	334 > 145	25	25	334 > 117
Tetraconazole [M + H]	14.32	372 > 70	25	15	374 > 161
Thiabendazole [M + H]	7.60	202 > 175	30	25	202 > 131
Thiacloprid [M + H]	6.70	253 > 126	25	20	253 > 186
Thiamethoxam [M + H]	4.38	292.1 > 210.9	20	10	292.1 > 181
Thiodicarb [M + H]	8.63	355 > 88	25	20	355 > 108
Triazamate [M + H] summed	6.62	287 > 198, 315 > 224	25	15	315 > 72
Trichlorfon [M + H]	7.90	256.9 > 108.7	25	25	256.9 > 220.9
Trifloxystrobin [M + H]	19.26	409 > 186	20	25	409 > 206
Triticonazole [M + H]	13.83	318 > 69.8	30	35	318 > 124.8

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A nitrogen generator (Peak Scientific, Renfrew, UK) and compressor system (Atlas Copco, Cumbernauld, UK) were used to supply nitrogen as the desolvation, cone and nebuliser gas. These were set at universally applied values of approximately 500 l h⁻¹ (desolvation gas flow rate) and 80 l h⁻¹ (cone gas flow rate). The ion source was operated at 150 °C, the desolvation temperature held at 350 °C and the capillary voltage was maintained at 3 kV. The LC/MS/MS instrument was controlled and data processed using MassLynx 4.0 and QuanLynx Application Manager software (Waters Corporation (Micromass), Manchester, UK).

Materials and apparatus

Certified pesticide reference standards (purity ≥98%) were purchased from Qm_x (Thaxted UK) and pure solvent standard solutions were prepared individually then as mixtures using methanol (HPLC grade, Rathburn, Walkerburn, UK). High purity laboratory water used for HPLC mobile phase was generated in-house and AnalaR grade ammonium acetate (VWR, Lutterworth, UK) was used to prepare aqueous mobile phase ‘buffer’ solution. Ethyl acetate (glass distilled grade) was the extraction solvent (Rathburn, Walkerburn, UK) and anhydrous sodium sulphate used to absorb any moisture released during sample processing was purchased from Sigma-Aldrich (Gillingham, UK). HPLC (0.45 μm PTFE) syringe filters (Chromacol Ltd, Welwyn Garden City, UK) were used to filter the final extract.

Sample extraction

Liver tissue was chopped and a portion (≤5 g) weighed into a beaker (100 ml). Sufficient anhydrous sodium sulphate was added to the sample in order to absorb any moisture. The mixture was allowed to dry for 20–30 minutes until friable then transferred into a conical flask (250 ml) and 100 ml of ethyl acetate added. Digestive tract material (≤10 g of a representative subsample) was weighed directly into a 150 ml or 250 ml conical flask, sodium sulphate added and left for 20–30 minutes then 100 ml of ethyl acetate added. The flask was securely stoppered and tumbled for approximately 1 hour. The resulting extract was filtered through a Whatman no. 1 filter paper (18.5 cm) into a round-bottomed flask (150 ml). The extract was evaporated to low volume (<1 ml) by rotary evaporation (bath temperature was kept below 30 °C). The extract was quantitatively transferred to a 5 ml volumetric flask containing 2.5 ml of cyclohexane and made to volume with ethyl acetate. 1 ml of this solution was then transferred into a round-bottomed (25 ml) flask along with 10 ml methanol (the remaining ethyl acetate–cyclohexane solution was retained for additional GCMS sample preparation). The methanolic solution was evaporated to low volume (approximately 1 ml) before it was quantitatively transferred into a 2 ml volumetric flask and made up to volume with methanol containing 5 mM ammonium acetate. An appropriate aliquot of the final solution was simply filtered into an auto sampler vial using a 1 ml disposable syringe fitted with a 0.45 μm PTFE syringe filter. The sample was then ready for LC/MS/MS analysis.

Suspected bait materials were either dried with sodium sulphate and the whole sample was soaked in ethyl acetate or a representative subsample was taken and extracted in the same way as described for digestive tract material. When sample amounts were limited the extraction method was the same but reagent amounts were adjusted proportionately.

Preparation of pure solvent standards, matrix-matched standards and fortified samples

Stock solutions of individual pesticides were prepared from certified reference materials into methanol (≈400 μg ml⁻¹) and aliquots taken to compose standard mixtures (5 μg ml⁻¹). Serial dilution with methanol generated a range of mixture solutions which were subsequently used to prepare matrix-matched calibration standards and for the fortification of ‘spiked’ samples. Chicken muscle and liver tissue ideally described as organic, were purchased from local retail outlets in order to generate ‘blank’ pseudo-matrices using the same extraction/solvent exchange procedures described above. The matrix solutions were prepared so that 1 ml ≡ 10 g of tissue in methanol. A range of matrix-matched calibration standards were subsequently prepared by admixing aliquots of pure solvent standard mixture and matrix solution to ensure a matrix concentration of 0.5 g ml⁻¹. The matrix-matched pesticide concentrations used were 0.025 μg ml⁻¹; 0.1 μg ml⁻¹; 0.2 μg ml⁻¹; 0.5 μg ml⁻¹. Matrix solution ‘blanks’ were screened during the validation process in order to ensure that they were free from pesticide residues. Even though none of the pesticides included in this method were found, it is recommended that matrix is similarly screened whenever matrix supplies are replenished.

Recovery and repeatability were determined following analysis of ‘spiked’ samples that were fortified with known amounts of each pesticide. Mixed standards were used to fortify chicken muscle tissue or chicken liver with amounts consistent with the majority of lower residue levels found in liver tissue from animals known to have been accidentally or deliberately exposed to pesticides i.e. 0.4–1 mg kg⁻¹. Two fortification levels of 0.1 mg kg⁻¹ and 1.0 mg kg⁻¹ were selected for chicken muscle tissue and chicken liver.

Results and discussion

The pesticides chosen to extend the WIIS-Scotland target inventory were readily available from an existing and comprehensive target inventory (several hundred pesticides) assembled to facilitate SASA's participation in UK and EU ‘Pesticide Residues in Food’ annual surveillance programs.¹⁷ It was extremely important that the selection criteria presented the most efficient and cost-effective provision of an enhanced monitoring service. This was achieved by filtering the ‘foodstuffs’ inventory so that, in the first instance, the new and extended WIIS-Scotland target inventory reflected past and present pesticide usage patterns in Scotland.¹⁸ It was also deemed vital to include some pesticides that were no longer or had never been approved for use in the UK because ‘illegal’ compounds such as carbofuran, aldicarb and isofenphos were known to be accessible and were being used to deliberately poison animals. Some other pesticides that showed no appreciable use in Scotland but had been detected in non-target animals elsewhere in the UK were also included e.g. benfuracarb, fenthion, indoxacarb, methomyl or myclobutanil.¹⁹ Finally all pesticides selected were amenable to LC/MS/MS. This process allowed us to quickly identify candidate pesticides and improve our capability to ‘capture’ pesticides known to have been used throughout Scotland in particular.

Multi-point (n = 4) calibration curves were generated from (MRM) ion-chromatogram peak area measurements for each analyte present in matrix-matched standards (chicken muscle or liver). Matrix-matched standards were chosen in order to compensate for any signal suppression/enhancement compared to their relative response in pure solvent.¹¹ The calibration acceptance criteria were that determination coefficients (r²) obtained were ≥0.96 for each analyte and that the signal:noise (S:N) value of the lowest calibration level (LCL) was >5 : 1. All pesticides that yielded r² values ≥0.96 and S:N values ≥5 : 1 for the LCL were subsequently considered for generic extraction and method optimisation. Pesticides that failed repeatedly to satisfy these criteria (i.e. despite additional optimisation of general experimental parameters such as preparation of fresh standards and/or minor changes to universally applied instrument parameters) were deferred in order to minimise the method development effort and avoid any compromise in the response of those pesticides that passed.

Further assessment of the utility of the generic ethyl acetate extraction process was achieved following analysis of muscle and liver tissue that had been fortified at two different fortification levels i.e. 0.1 mg kg⁻¹ and 1.0 mg kg⁻¹ of each pesticide prior to sample extraction. The efficiency of the ethyl acetate extraction was considered acceptable if mean recoveries following replicate analysis (n = 5 or n = 6) of the target analyte in each matrix fell within the range 60–140% and yielded a relative standard deviation (RSD) ≤20%. These analytical performance measures were comparable with European Union guidelines for method validation and quality control procedures for the determination of pesticide residues in food and feed²⁰ where mean recoveries should fall within the range 70–120% with a RSD of ≤20%. The guidelines also specify that a generalised mean recovery range of 60–140% for routine multi-residue analysis may be used (the guidelines used to establish in-house performance criteria during method development were superseded in 2011). The majority of pesticides listed in Table 1, complied with the above performance criteria with some minor exceptions. These are highlighted in italics in Tables 2 and 3 which contain the analytical performance data for chicken muscle tissue and chicken liver tissue fortified at each level, respectively.

Table 2 LC/MS/MS method performance data – fortified chicken muscle tissue^a

Pesticide	Fortification level 0.1 mg kg^−1					Fortification level 1 mg kg^−1
	Min	Max	n	Mean	CV (%)	Min	Max	n	Mean	CV (%)
a Mean recoveries <60% or >140% and RSDs >20% are shown in italics.
Acetamiprid	73	84	6	80	4.7	75	89	6	81	6.3
Aldicarb	77	96	6	90	7.7	66	92	6	81	12.3
Aldicarb sulphone	82	92	6	88	5.0	77	90	6	84	6.1
Aldicarb sulphoxide	66	86	6	77	10.3	83	92	6	87	3.7
Atrazine	79	87	6	82	4.0	71	81	6	75	4.7
Azoxystrobin	73	79	6	76	2.9	70	84	6	77	5.9
Bendiocarb	78	92	6	85	6.9	80	86	6	84	3.0
Benfuracarb	72	79	6	76	3.3	68	77	6	73	5.7
Bitertanol	75	83	6	79	3.9	70	81	6	76	6.1
Boscalid	64	81	6	72	8.8	75	85	6	80	5.1
Bromuconazole	80	92	6	85	5.2	68	84	6	73	7.8
Bupirimate	60	86	6	74	14.6	77	98	6	86	9.2
Carbaryl	71	86	6	80	7.3	79	93	6	85	5.9
Carbendazim	75	87	6	82	5.6	81	88	6	85	3.6
Carbofuran	91	101	6	94	3.9	88	118	6	101	10.2
Carbofuran (3 hydroxy)	71	91	6	84	8.7	77	92	6	84	6.4
Carbosulfan	66	75	6	69	5.8	61	72	6	67	6.0
Chlorotoluron	79	95	6	85	6.4	74	81	6	77	3.4
Clofentizene	72	84	6	80	5.6	71	81	6	76	5.4
Clothianadin	68	90	6	79	11.4	64	91	6	74	12.4
Cyazofamid	78	88	6	82	5.1	72	83	6	78	5.0
Cymoxanil	75	86	6	80	5.9	80	92	6	86	5.1
Cyproconazole	62	93	6	83	13.3	71	83	6	77	5.0
Cyprodinil	75	93	6	84	8.3	66	77	6	70	6.1
Demeton-S-Methyl	54	153	6	84	44.6	45	90	6	79	21.7
Demeton-S-methyl sulphone	76	81	6	78	2.7	78	91	6	86	5.2
Difenoconazole	72	86	6	80	7.1	72	80	6	75	4.9
Diflubenzuron	71	85	6	77	7.3	71	82	6	76	5.2
Dimethoate	71	88	6	80	8.1	74	89	6	81	6.6
Dimethomorph	78	87	6	81	4.2	65	76	6	72	5.6
Dimoxystrobin	75	92	6	82	8.4	71	93	6	79	10.4
Disulfoton	59	79	6	68	11.3	58	85	6	72	13.2
Disulfoton sulphoxide	75	94	6	82	7.9	81	89	6	84	3.8
Diuron	62	95	6	76	15.6	66	99	6	77	16.0
Epoxiconazole	73	89	6	83	7.5	72	84	6	78	5.8
Famoxadone	71	78	6	76	3.6	68	80	6	73	6.2
Fenamidone	74	83	6	78	5.0	69	78	6	74	4.1
Fenarimol	62	100	6	79	16.3	70	90	6	80	9.0
Fenbuconazole	71	90	6	79	9.5	73	84	6	78	5.3
Fenhexamid	81	91	6	85	4.2	74	82	6	78	4.0
Fenpropimorph	94	107	6	100	5.1	68	79	6	73	5.5
Fenpyroximate	75	84	6	80	4.6	66	84	6	74	8.4
Fenthion	74	89	6	79	6.7	59	75	6	71	8.7
Fenthion sulphone	53	85	6	69	18.3	69	104	6	89	16.4
Fenthion sulphoxide	74	84	6	79	6.1	77	89	6	82	6.2
Fludioxonil	86	108	6	97	7.2	75	92	6	83	8.3
Flufenacet	76	86	6	81	4.6	72	81	6	76	4.6
Fluopicolide	76	87	6	83	5.6	74	79	6	77	2.7
Fluoxastrobin	65	76	6	71	5.3	71	80	6	75	5.0
Fluquinconazole	63	109	6	83	22.1	97	115	6	104	6.6
Flusilazole	82	96	6	88	5.6	69	84	6	76	6.5
Flutriafol	74	85	6	80	4.8	49	83	6	71	17.6
Imazalil	64	82	6	77	8.7	74	86	6	78	6.1
Imidacloprid	71	87	6	77	7.6	67	85	6	76	9.6
Indoxacarb	65	88	6	72	12.0	64	81	6	72	10.5
Isofenphos	42	75	6	59	18.2	56	121	6	81	29.4
Isoproturon	74	90	6	81	7.3	68	79	6	76	5.4
Kresoxim methyl	77	94	6	86	8.0	72	100	6	81	12.8
Linuron	70	124	6	93	23.6	77	89	6	83	5.6
Malathion	73	82	6	78	3.9	74	78	6	76	2.3
Mepanipyrim	77	86	6	82	3.9	70	82	6	77	6.3
Metconazole	91	98	6	93	2.8	57	86	6	74	14.7
Methiocarb	75	90	6	81	6.8	75	84	6	80	4.2
Methomyl	97	117	6	102	7.6	94	119	6	107	8.3
Metolcarb	67	82	6	73	8.2	75	90	6	80	6.9
Metrafenone	74	89	6	81	7.1	74	80	6	76	3.5
Monocrotophos	79	87	6	83	3.3	76	91	6	83	7.0
Myclobutanil	77	88	6	84	4.8	74	86	6	80	5.4
Ofurace	66	83	6	77	8.5	77	90	6	85	5.0
Omethoate	67	75	6	72	5.5	85	96	6	91	5.3
Oxamyl	80	87	6	85	3.0	78	89	6	84	5.4
Oxydemeton methyl	73	85	6	79	6.8	79	89	6	84	4.9
Penconazole	68	86	6	79	9.4	74	84	6	78	4.9
Penycycuron	77	90	6	86	5.6	68	80	6	75	5.5
Phorate	74	83	6	78	4.0	68	78	6	74	5.5
Phorate sulphone	75	87	6	82	5.9	77	91	6	83	6.4
Picoxystrobin	77	92	6	87	6.6	74	86	6	79	6.1
Pirimicarb	83	91	6	87	3.5	72	81	6	78	4.2
Prochloraz	72	82	6	76	5.3	73	80	6	77	3.7
Propamocarb	33	43	6	38	9.5	51	60	6	55	10.1
Propiconazole	77	81	6	79	1.9	77	87	6	83	5.3
Pymetrozine	63	74	6	70	5.4	72	80	6	76	4.3
Pyraclostrobin	75	87	6	80	6.0	72	83	6	77	5.4
Pyrethrins	77	87	6	81	5.1	72	81	6	76	4.5
Pyrifenox	80	88	6	84	3.7	72	81	6	77	5.0
Pyrimethanil	73	96	6	87	9.2	74	85	6	78	5.0
Quinoxyfen	68	81	6	76	6.1	65	80	6	72	8.7
Simazine	67	87	6	80	9.0	75	84	6	79	5.5
Spinosad	56	64	6	59	5.1	71	98	6	86	10.8
Spiromesifen	75	97	6	83	9.6	74	94	6	82	9.3
Spiroxamine	78	86	6	81	3.7	70	80	6	74	4.9
Tebuconazole	78	89	6	85	5.2	71	83	6	78	5.7
Tebufenpyrad	76	90	6	82	7.3	67	78	6	74	6.7
Tetraconazole	78	101	6	85	10.1	68	96	6	80	11.7
Thiabendazole	72	85	6	78	7.3	75	85	6	80	4.6
Thiacloprid	74	82	6	78	3.8	77	89	6	83	6.0
Thiamethoxam	78	88	6	83	4.8	74	84	6	81	5.0
Thiodicarb	42	49	6	46	6.6	35	49	6	40	11.8
Triazamate	63	73	6	67	6.7	60	70	6	65	5.4
Trichlorfon	56	101	6	77	22.3	72	128	6	93	21.9
Trifloxystrobin	75	86	6	80	5.0	73	83	6	76	4.5
Triticonazole	84	95	6	91	4.3	51	88	6	70	17.8

---

Table 3 LC/MS/MS method performance data – fortified liver tissue^a

Pesticide	Fortification level 0.1 mg kg^−1					Fortification level 1 mg kg^−1
	Min	Max	n	Mean	CV (%)	Min	Max	n	Mean	CV (%)
a Mean recoveries <60% or >140% and RSDs >20% are shown in italics.
Acetamiprid	68	84	5	76	7.7	81	108	6	91	11.0
Aldicarb	76	86	5	81	5.9	71	107	6	92	14.1
Aldicarb sulphone	37	50	5	47	11.9	62	94	6	73	16.2
Aldicarb sulphoxide	34	47	5	40	13.1	58	79	6	68	11.5
Atrazine	69	81	5	74	6.6	77	100	6	84	10.3
Azoxystrobin	71	79	5	76	4.2	82	100	6	86	8.0
Bendiocarb	79	94	5	84	7.7	87	110	6	92	9.8
Benfuracarb	56	68	5	63	7.3	75	97	6	81	10.4
Bitertanol	73	77	5	75	2.0	80	99	6	85	8.3
Boscalid	65	77	5	70	6.6	81	98	6	87	6.9
Bromuconazole	74	99	5	86	10.6	77	116	6	89	16.6
Bupirimate	70	80	5	75	6.6	76	94	6	85	7.3
Carbaryl	70	79	5	75	4.1	83	109	6	92	10.8
Carbendazim	70	79	5	74	5.6	82	115	6	93	13.4
Carbofuran	87	95	5	91	3.4	93	115	6	100	8.1
Carbofuran (3 hydroxy)	78	85	5	81	3.7	79	111	6	91	12.3
Carbosulfan	29	34	5	31	6.2	58	65	6	62	3.8
Chlorotoluron	74	88	5	81	6.4	75	107	6	87	14.3
Clofentizene	69	77	5	73	4.3	81	101	6	86	9.1
Clothianadin	61	83	5	74	10.9	69	106	6	88	13.5
Cyazofamid	76	84	5	80	4.6	75	100	6	83	10.7
Cymoxanil	67	79	5	74	6.5	83	118	6	92	13.9
Cyproconazole	66	86	5	75	9.9	76	104	6	91	14.2
Cyprodinil	67	86	5	78	9.3	68	100	6	82	17.3
Demeton-S-methyl	33	89	5	63	38.2	93	106	6	99	5.6
Demeton-S-methyl sulphone	71	87	5	80	7.4	83	112	6	92	11.3
Difenoconazole	70	77	5	75	3.9	77	95	6	82	8.6
Diflubenzuron	66	83	5	74	11.6	75	100	6	84	10.2
Dimethoate	79	83	5	81	2.5	79	105	6	87	10.7
Dimethomorph	75	81	5	78	3.3	74	110	6	86	15.7
Dimoxystrobin	55	87	5	74	18.2	80	107	6	92	11.7
Disulfoton	64	77	5	70	7.1	66	107	6	83	17.7
Disulfoton sulfoxide	73	80	5	77	3.6	82	106	6	91	9.1
Diuron	64	96	5	76	17.0	80	109	6	92	11.2
Epoxiconazole	93	106	5	99	4.7	81	109	6	89	11.9
Famoxadone	69	79	5	75	5.4	77	93	6	84	6.6
Fenamidone	69	83	5	74	7.6	76	98	6	83	9.8
Fenarimol	67	88	5	78	12.0	81	99	6	87	7.7
Fenbuconazole	64	73	5	68	4.9	80	107	6	88	11.6
Fenhexamid	72	85	5	77	7.9	74	112	6	87	16.6
Fenpropimorph	81	90	5	84	4.5	82	116	6	94	13.4
Fenpyroximate	54	69	5	61	9.8	72	102	6	82	14.1
Fenthion	67	93	5	75	14.2	71	103	6	84	12.8
Fenthion sulphone	63	79	5	68	10.1	81	115	6	94	14.6
Fenthion sulphoxide	73	80	5	77	3.5	87	110	6	92	9.8
Fludioxinil	64	125	5	89	26.4	67	116	6	87	21.3
Flufenacet	75	83	5	78	5.1	79	102	6	86	9.8
Fluopicolide	72	85	5	79	6.1	79	106	6	86	12.0
Fluoxastrobin	69	78	5	74	4.8	77	90	6	81	5.8
Fluquinconazole	67	91	5	79	11.9	84	121	6	109	12.3
Flusilazole	71	91	5	80	9.4	81	106	6	90	10.9
Flutriafol	66	92	5	77	13.8	54	150	6	89	41.7
Imazalil	69	82	5	75	6.7	80	98	6	86	7.3
Imidacloprid	55	125	5	80	34.3	77	111	6	90	12.9
Indoxacarb	67	90	5	77	11.1	73	92	6	83	9.9
Isofenphos	30	46	5	38	16.9	46	71	5	58	20.0
Isoproturon	78	88	5	81	5.2	71	104	6	84	14.5
Kresoxim methyl	76	91	5	82	7.1	83	109	6	89	11.3
Linuron	76	95	5	87	9.1	75	87	6	82	7.1
Malathion	68	84	5	77	8.4	79	96	6	86	6.4
Mepanipyrim	73	87	5	78	6.7	73	104	6	85	13.2
Metconazole	73	85	5	79	5.4	56	131	6	89	35.6
Methiocarb	73	81	5	76	4.3	81	105	6	88	10.2
Methomyl	111	131	5	123	7.1	110	123	6	113	9.0
Metolcarb	83	115	6	93	13.4	78	86	6	81	4.1
Metrafenone	73	82	5	78	4.9	80	100	6	84	9.1
Monocrotophos	77	108	6	89	12.6	66	87	6	75	11.0
Myclobutanil	74	83	5	77	4.8	75	112	6	89	15.1
Ofurace	74	79	5	77	2.5	84	119	6	96	12.8
Omethoate	38	48	5	44	8.4	62	78	6	68	8.4
Oxamyl	43	57	5	52	10.1	65	95	6	76	14.7
Oxydemeton methyl	57	73	5	67	9.5	74	111	6	88	14.6
Penconazole	60	77	5	71	9.6	80	104	6	88	10.2
Penycycuron	74	87	5	78	7.0	72	113	6	86	17.1
Phorate	67	80	5	72	7.3	75	95	6	83	9.0
Phorate sulfone	72	80	5	76	4.7	84	101	6	92	6.2
Picoxystrobin	76	80	5	78	1.9	77	104	6	88	11.8
Pirimicarb	70	88	5	78	8.3	73	110	6	87	15.5
Prochloraz	72	80	5	75	4.3	83	98	6	87	6.4
Propamocarb	35	40	5	38	5.4	76	110	6	89	14.3
Propiconazole	69	82	5	77	6.4	85	104	6	91	7.3
Pymetrozine	62	79	5	72	8.7	74	108	6	85	15.1
Pyraclostrobin	74	81	5	77	3.6	78	97	6	84	7.9
Pyrethrins	82	89	5	86	3.5	75	96	6	82	9.3
Pyrifenox	76	81	5	78	2.7	74	113	6	88	16.7
Pyrimethanil	66	79	5	71	8.0	72	109	6	87	16.7
Quinoxyfen	57	84	5	68	16.5	74	91	6	82	7.2
Simazine	71	86	5	77	7.8	74	115	6	90	17.1
Spinosad	43	46	5	45	3.0	47	57	6	51	9.0
Spiromesifen	79	90	5	84	4.8	81	116	6	91	14.0
Spiroxamine	73	85	5	77	6.1	76	108	6	87	13.9
Tebuconazole	72	83	5	77	5.7	73	115	6	89	17.6
Tebufenpyrad	79	98	5	87	8.8	77	99	6	85	10.4
Tetraconazole	72	75	5	74	1.5	66	121	6	85	25.3
Thiabendazole	74	82	5	78	5.0	85	113	6	94	11.5
Thiacloprid	65	76	5	72	6.1	80	112	6	90	12.7
Thiamethoxam	70	74	5	72	2.1	77	102	6	86	11.5
Thiodicarb	54	59	5	56	3.7	54	66	6	61	7.3
Triazamate	69	78	5	73	4.4	67	94	6	76	12.5
Trichlorfon	42	87	5	70	28.2	67	141	6	106	34.6
Trifloxystrobin	72	79	5	76	4.3	79	97	6	84	8.0
Triticonazole	72	85	5	78	6.8	60	116	6	86	24.5

---

In chicken muscle tissue, target performance criteria were achieved at both fortification levels for 95 compounds. Seven compounds yielded mean recovery or RSD values outwith the target ranges. In chicken liver tissue, target performance criteria were achieved for 85 compounds at both fortification levels. Seventeen compounds yielded mean recovery or RSD values, respectively outwith the target ranges. The magnitude of these failures was such that they were either just below or just above the recovery and/or RSD values set by us. There were a couple of exceptions but even the lowest mean recovery values (30–50%) showed acceptable RSDs. Therefore, these anomalies were not considered to be prohibitive for screening purposes or to present a significant risk of generating false positives or negatives. Consequently, all 102 pesticides listed in Table 1 were incorporated into the method.

LC/MS/MS ion chromatograms of analytes selected from throughout the experimental mass and retention time ranges for the lowest calibration level (LCL) matrix-matched standard (chicken muscle) are shown in Fig. 1. The MRM peaks yielded by the matrix-matched LCL are annotated with retention time (Rt) and peak area (PA) values and serve to illustrate the selectivity and sensitivity of the method. There is an occasional requirement to re-extract and re-analyse samples e.g. in cases where results are disputed. However, this approach is not possible with ‘single-shot’ samples due to finite sample availability. Such samples may have to be retained for corroboration by third parties involved in e.g. enforcement investigations or actions. Consequently, it was important to assess the stability of the selected pesticides in crude extract. This was achieved by simply comparing the analyte peak retention time and peak area measurement following analysis of a LCL matrix-matched (chicken muscle) standard at 0 days and following storage at 5 °C for 7 days. Table 4 shows the results obtained for the 8 analytes presented in Fig. 1. A 46% reduction in the peak area response of carbosulfan between 0 and 7 days was most dramatic. The data were extremely limited from this basic exercise and must be interpreted with caution. However, the study serves to illustrate that quantitation of certain pesticides may be significantly compromised and should be considered if and when a sample extract needs to be re-analysed.

Fig. 1 MRM ion chromatograms (gradient separation) selected throughout the experimental mass range and retention time range. Peaks of the LCL (chicken muscle matrix – 0.025 μg ml⁻¹) are annotated with retention time (Rt – minutes) and peak area values.

Table 4 Simple stability study of pesticides in crude extract at 0-days and 7-days evaluated using the LCL matrix-matched standard (chicken muscle 0.025 μg ml⁻¹)

Analyte	Rt (min)	Rt (min)	Peak area	Peak area	Ratio
	(0-days)	(7-days)	(0-days)	(7-days)
Methomyl	4.23	4.19	4934	6191	0.80
Carbofuran – 3OH	6.27	6.27	17 488	25 391	0.69
Carbofuran	7.95	7.95	86 558	87 781	0.99
Azoxystrobin	11.18	11.25	414 354	450 930	0.92
Imazalil	17.59	17.68	107 385	106 967	1.00
Indoxacarb	19.90	19.99	7078	6173	1.15
Tebufenpyrad	21.72	21.81	30 581	29 473	1.04
Carbosulfan	28.59	28.68	98 075	45 479	2.16

---

Application of the method has established the utility of LC/MS/MS for the determination of multiple pesticide residues in investigations into suspected poisoning. Residues ranging from 0.01 to several thousand mg kg⁻¹ (not corrected for recovery) have been detected in specimens from a variety of wild and domestic animals. It is important to ensure that any dilution factors are applied whenever extracts have been diluted to fall within the narrow but linear experimental concentration range. Results obtained indicated that exposure to pesticides was responsible for the death or illness of the animal since other potential causes of death such as disease, trauma or starvation were ruled out following post mortem examination.

A detailed presentation of typical results obtained is shown in Fig. 2. This figure contains the total ion chromatogram (TIC), screen, confirmation and proximate standard MRM chromatograms generated following detection of a residue of carbofuran in the crude extract of liver taken from a golden eagle (Aquila chrysaetos) found dead in the Grampian region of Scotland (March 2011). Carbofuran is the active ingredient of insecticidal products used to control ‘soil dwelling’ and ‘foliar feeding’ insects. Carbofuran (pure chemical), classified by the World Health Organisation as ‘highly hazardous’, is extremely toxic to birds and mammals²¹ particularly when used incorrectly. Furthermore, products containing carbofuran have not been approved for use in the United Kingdom since December 2001 (although they are approved for use in other countries). Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate) yielded an intense [M + H]⁺ molecular ion at m/z 222 and a product-ion mass spectrum rich in structural detail, when subject to positive ion mode MS/MS. The MRM transition used for screening (m/z 222 → m/z 165) and transitions used for confirmation (m/z 222 → m/z 165 and m/z 222 → m/z 123) were used in conjunction with gradient and isocratic HPLC separation, respectively. In this particular example, it was necessary to dilute the original extract for the confirmation experiment, as the response from the screening experiment indicated that it was outwith the linear range.

Fig. 2 Total ion chromatograms (TIC), screen, confirmation and proximate standard ion chromatograms obtained following analysis of crude extract from the liver of a golden eagle (Aquila chrysaetos) found dead in Grampian, Scotland (March 2011) and confirmed to be a victim of carbofuran poisoning.

It is important to recognise that application of the method can refute or confirm any suspicion that a dead or sick animal has been exposed to any of the pesticides included in the LC/MS/MS target inventory. Consequently, results presented in this paper represent a select and small proportion of the numerous animal species and specimens that have been and continue to be analysed using this method. It would be straightforward to assess the incorporation of additional pesticides, e.g. identified from annual pesticide usage information, into the method.

Conclusions

The chemical diagnosis of suspected animal poisonings or environmental contamination following accidental or deliberate exposure to pesticides has been significantly enhanced following the development and routine application of a relatively simple and quick LC/MS/MS method. It is possible to examine crude extract directly i.e. without laborious sample clean-up, for the presence of multiple pesticide residues due to the high selectivity and sensitivity associated with LC/MS/MS. We have also found the method to be suitable for the analysis of digestive tract content and also blood, vomit and faecal specimens e.g. from sick animals believed to be victims of sub-lethal exposure. In addition, the method is routinely and successfully applied to the characterisation of suspicious chemicals/substances and analysis of suspected baits i.e. carcases, meat or eggs that may have been deliberately laced with toxic pesticides.

Ultimately, the results obtained provide robust scientific evidence that confirms or refutes any suspicion that non-target vertebrate animals have been exposed to any of the pesticides included following their approved use, misuse, abuse or due to the persistence of some of these chemicals in the environment. The evidence collected can be used to initiate investigations into how and why the exposure occurred and to identify any violation of legislation designed to protect animals and the environment.

References

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