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An Optimized QuEChERS Workflow to Analyze Over 200 Pesticides in Honey

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  • QuEChERS can be modified to work with low-moisture, high-sugar commodities.
  • Optimized workflow describes which products and procedures work best for this commodity.
  • Over 98% of the 203 pesticides analyzed exhibit recoveries between 70-120%.

When it comes to the analysis of pesticide residues in low-moisture, high-sugar matrices, QuEChERS might not be the first sample preparation technique that comes to mind. QuEChERS is better known for its use in high-moisture fruits and vegetables, such as strawberries and spinach, with multiple official methods published for these types of commodities. However, QuEChERS is designed to achieve successful results with matrices that official methods may not cover, such as those low in moisture or high in sugars—it just needs to be modified.

Determining which QuEChERS products are most effective for commodities that extend beyond the scope of official methods can be problematic for food safety labs, which may not have experience with trialing different products and procedures. Similarly, the perception of the complexity involved may add an additional deterrent to labs looking to use this technique with other matrices.

In this article, we demonstrate an optimized workflow for the analysis of over 200 pesticides in honey that delivers acceptable results for this low-moisture, high-sugar commodity. We offer best practices for how to modify your sample preparation to optimize your current method and recommend several products that work best for this matrix.

Building on Our Preliminary Experiments

When we previously performed a QuEChERS extraction on low-moisture commodities using brown rice flour, we tested two approaches using the original unbuffered QuEChERS salts (cat.# 25847) to determine which would produce higher recoveries.

In the first approach, we added equal amounts of water and extraction solvent (10 mL) and kept the sample size the same (10 g). In the second approach, we added 10 mL of water and extraction solvent but cut the sample size in half (5 g). The sample matrix and water were shaken for 30 minutes before extraction. The methods were then tested with Restek’s QuEChERS performance mix (cat.# 31152), and the comparison is shown in Figure 1 for selected pesticides.

Figure 1: Comparison of selected pesticide's recoveries in brown rice flour with two different extraction methods.

 

While the raw recoveries (as area/area of internal standard) for most pesticides selected showed very little difference between the methods, DDT,p,p’- showed a significantly higher response with method 2. However, as brown rice flour has a fairly neutral pH and honey is more acidic, we wanted to see how another extraction salt, AOAC salts (cat.# 25851) performed when comparing the two methods for honey (Figure 2).

Figure 2: Method 1 (left tube) shows a much greater amount of sample solids and interferences, resulting in a small amount of extract. Method 2 (right tube), with its lower sample size, shows a reduced amount of sample solids and a greater amount of extract.

 

Given the higher response with method 2, we rehydrated our samples of honey using this method, and then evaluated different extraction salts and dSPE (Figures 3A & 3B).

Figure 3A: The evaluation of extraction salts showed that the more acidic environment of AOAC salts (cat.# 25851) resulted in higher recoveries, especially for dicofol.

 

Figure 3B: Due to the possible presence of sugars and waxes, the tested dSPE had either PSA (cat.# 26124) or C18-EC (cat. #26242) or a combination of both (cat.# 26125 and cat.# 26243) added. The best-performing dSPE tube was cat.# 26124 with 50 mg PSA.

 

It’s important to note that the best-performing dSPE for a given analysis will vary depending on which pesticides are most critical. For the pesticides featured in this article, we determined that the dSPE with 50 mg PSA (cat.# 26124) was most effective.

Applying Our Optimized Combination to 203 Analytes

Given the performance of our optimized combination of extraction salts and dSPE on a small number of analytes, we wanted to test against a larger group of pesticides. In this case, we spiked our honey samples using Restek’s GC multiresidue kit (cat.# 32562), which contains 203 compounds.

Chromatographic Method:

The chromatographic conditions were as follows. Retention times, ion transitions, and collision energies are provided in Appendix I.

Column: Rxi-5ms, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13423)
Instrument: GC-MS/MS
Oven Program: Temperature (°C) Ramp Rate (°C/min) Hold Time (min)
90 - 1
330 @ 8 °C/min 5
Column Flow (constant): 1.4 mL/min
Injection Volume: 1 µL
Injection Type: splitless, 0.5 min hold time
Inlet Temperature: 250 °C
 

Outstanding Recoveries for Almost All Pesticides

When we look at the recoveries for this analysis, we can see that 201 out of the 203 compounds analyzed exhibit recoveries between 70-120% (Figure 3). Only two compounds, captafol (52%) and sulfotepp (120%) fell out of this range.

Figure 4: Almost all compounds had acceptable recoveries.

 

Conclusion

QuEChERS is a versatile technique that can work for an abundance of matrices—including those not covered by official methods. As shown, QuEChERS can easily be modified to be very effective for low-moisture, high-sugar matrices. By testing two methods to rehydrate our samples, then comparing extraction salts and dSPE options, we were able to create a highly successful workflow that achieved excellent recoveries for 201 analytes.

As the benefits of QuEChERS extractions include being quick, easy, and cheap, labs can utilize these advantages to modify their workflows to test against other matrices. While the official methods provide a great starting point, QuEChERS’ flexibility means it can be adaptable for the specific needs of each lab, even those testing more complicated commodities.

Appenidx I: Analyte MS transitions for spiked honey samples.

Analyte

RT

Quant Ion Qual Ions

Precursor Mass

Product Mass

Collision Energy

Confirm Precursor

Confirm Product

Confirm Energy

Confirm Precursor

Confirm Product

Confirm Energy

2,4'-Methoxychlor

20.24

227

121

12

121

78

20

121

91

12

2-Phenylphenol

10.21

170

141

22

141

115

12

170

115

34

4,4'-Methoxychlor olefin

19.75

308

223

30

238

152

35

238

195

20

Acequinocyl

24.40

342

188

14

188

132

10

189

115

22

Acetochlor

14.99

223

132

20

174

146

12

223

147

10

Acrinathrin

22.47

289

93

10

181

152

26

208

181

10

Alachlor

15.22

188

130

32

146

118

8

188

160

10

Aldrin

16.04

298

263

8

263

191

35

263

193

31

Allidochlor

6.90

138

81

8

132

56

6

138

96

8

Anthraquinone

16.00

208

152

23

180

152

12

208

180

10

Atrazine

13.30

215

173

8

200

122

10

200

132

10

Azinphos-ethyl

22.53

160

77

18

132

77

12

160

132

6

Azinphos-methyl

21.87

160

77

16

132

51

28

132

77

12

Benfluralin

12.40

292

160

20

292

206

12

292

264

8

BHC, alpha-

12.68

219

183

8

181

145

13

217

145

18

BHC, beta-

13.33

219

183

8

181

109

27

181

145

15

BHC, delta-

14.06

219

183

8

181

109

28

181

145

13

BHC, gamma-

13.49

219

183

8

181

109

29

181

145

13

Bifenthrin

21.12

181

165

25

165

115

30

181

166

10

Bioallethrin

17.09

136

93

11

123

81

8

136

108

8

Biphenyl

8.29

154

115

25

152

126

23

154

127

32

Bromfenvinphos

18.00

325

269

10

267

159

15

323

267

10

Bromfenvinphos-methyl

17.12

295

93

10

109

79

5

295

109

15

Bromophos methyl

16.57

331

286

26

329

314

13

331

316

13

Bromophos-ethyl

17.55

359

303

17

331

303

8

357

301

15

Bromopropylate

21.08

341

183

15

183

155

12

341

185

13

Bupirimate

18.56

316

208

8

273

108

16

273

193

8

Captafol

20.75

313

197

30

79

51

22

79

77

12

Captan

17.12

149

79

12

107

79

12

149

105

6

Carbophenothion

19.69

342

157

10

125

97

6

199

143

10

Carfentrazone ethyl

19.75

340

312

10

330

141

44

330

310

8

Chlorbenside

17.35

268

125

8

125

89

17

125

99

19

Chlordane, cis-

17.83

377

268

19

272

237

12

375

266

21

Chlordane, trans-

17.48

375

266

18

272

237

13

373

264

18

Chlorfenapyr

18.85

247

227

12

137

75

28

137

102

14

Chlorfenson

17.95

302

175

8

175

111

8

177

113

8

Chlorfenvinphos

17.13

323

267

10

267

159

15

295

267

5

Chlorobenzilate

18.93

251

111

32

139

111

12

251

139

14

Chloroneb

10.13

206

191

10

191

113

13

191

141

10

Chlorothalonil

14.18

266

133

40

264

168

23

266

170

23

Chlorpropham

12.02

213

171

8

127

65

22

171

127

8

Chlorpyrifos

16.18

314

258

12

197

107

36

197

169

14

Chlorpyrifos-methyl

15.04

286

93

22

286

208

12

286

241

24

Chlorthal-dimethyl

16.29

332

301

8

299

221

24

301

223

24

Chlorthiophos

19.37

325

269

14

269

205

15

297

269

8

Chlozolinate

17.01

331

259

8

186

145

16

259

153

26

Clomazone

13.33

204

107

18

125

89

13

125

99

17

Coumaphos

23.28

362

109

17

210

182

10

362

226

13

Cycloate

11.82

215

83

15

154

83

8

215

154

6

Cyfluthrin

23.93

226

206

10

163

91

14

163

127

6

Cyhalothrin, lambda-

22.26

208

181

8

181

152

23

197

141

10

Cypermethrin

24.10

181

152

18

163

91

11

163

127

6

Cyprodinil

16.74

225

210

12

224

118

34

224

208

20

DDD, o,p'-

18.48

235

165

22

235

199

14

235

200

10

DDD, p,p'-

19.20

237

165

22

235

165

24

235

199

12

DDE, o,p'-

17.59

318

176

56

246

176

32

316

246

15

DDE, p,p'-

18.29

318

248

22

246

176

28

316

246

20

DDT, o,p'-

19.29

237

165

22

235

165

21

235

199

16

DDT, p,p'-

20.01

237

165

22

235

165

21

235

199

16

Deltamethrin

25.94

253

93

19

181

152

20

253

174

8

Diallate

12.65

234

150

18

234

192

12

     

Diazinon

13.93

304

179

12

137

84

12

199

93

16

Dichlofluanid

15.87

224

123

12

123

77

16

167

124

10

Dichloroaniline, 3,4'-

9.00

163

90

16

161

99

21

161

126

8

Dichlorobenzophenone, 4,4'-

16.23

139

75

27

111

75

12

139

111

13

Dichlobenil

7.80

171

100

25

136

100

10

171

136

13

Dicloran

12.96

206

148

18

176

148

10

206

176

10

Dieldrin

18.35

279

243

10

263

193

34

277

241

8

Dimethachlor

14.84

197

148

8

134

77

25

134

105

13

Diphenamid

16.58

239

72

10

167

115

40

239

167

8

Diphenylamine

11.64

169

167

25

167

166

17

169

168

12

Disulfoton

14.02

153

97

12

88

60

6

142

109

6

Edifenphos

19.80

310

109

29

109

65

13

173

109

8

Endosulfan ether

14.53

272

237

10

239

204

13

241

206

13

Endosulfan I

17.77

241

206

10

195

125

19

195

159

6

Endosulfan II

18.97

241

206

10

195

125

23

195

159

8

Endosulfan sulfate

19.87

274

239

12

241

206

8

272

237

10

Endrin

18.84

279

243

8

245

173

25

263

193

30

Endrin aldehyde

19.37

347

253

18

345

243

17

345

245

14

Endrin ketone

20.96

317

101

21

315

279

8

317

281

8

EPN

21.10

169

77

22

157

110

14

169

141

8

Ethalfluralin

12.13

316

202

26

276

202

15

316

276

10

Ethion

19.29

231

129

24

153

97

10

231

175

10

Ethylan

18.83

223

167

12

223

179

22

223

193

28

Etofenprox

24.38

163

77

34

163

107

18

163

135

10

Etridiazole

9.28

211

140

22

183

140

16

211

183

10

Fenamiphos

18.00

303

154

15

217

202

9

303

195

8

Fenarimol

22.45

219

107

12

139

75

28

139

111

16

Fenchlorphos

15.40

287

272

11

285

240

23

285

270

11

Fenitrothion

15.68

277

109

16

260

125

12

277

260

8

Fenpropathrin

21.26

265

210

8

181

127

24

181

152

24

Fenson

16.43

268

77

18

141

77

8

268

141

8

Fenthion

16.12

278

109

18

278

125

22

278

169

17

Fenvalerate

25.34

225

119

15

167

125

8

169

127

10

Fipronil

17.13

369

215

30

213

143

28

367

213

30

Fluazifop-P-butyl

18.80

383

254

20

383

268

8

383

282

12

Fluchloralin

14.01

306

160

20

264

160

15

306

264

8

Flucythrinate

24.52

199

107

24

157

107

13

199

157

8

Fludioxonil

18.20

248

127

30

154

127

8

248

154

20

Fluquinconazole

23.28

340

108

42

340

298

14

340

313

12

Fluridone

24.78

328

127

40

328

189

46

328

259

26

Flusilazole

18.50

233

152

14

206

137

20

233

165

18

Flutolanil

18.04

281

173

10

173

95

26

173

145

14

Flutriafol

17.87

219

123

14

123

75

24

123

95

12

Folpet

17.25

260

130

16

104

76

10

260

232

8

Fonofos

13.70

246

109

16

137

109

6

246

137

8

Heptachlor

15.23

272

237

13

100

65

12

270

235

13

Heptachlor epoxide

16.95

353

263

13

263

193

29

351

261

11

Hexachlorobenzene

12.87

284

214

30

249

214

14

284

249

18

Hexazinone

20.21

171

71

16

128

83

10

171

85

16

Iodofenfos

18.07

377

93

34

377

332

30

377

362

15

Iprodione

20.85

314

245

14

187

124

24

314

271

12

Isazophos

14.23

172

130

10

161

119

8

161

146

8

Isodrin

16.71

261

191

28

193

123

30

193

157

19

Isopropalin

16.73

280

118

25

280

180

10

280

238

8

Lenacil

19.91

153

136

14

136

53

18

136

80

14

Leptophos

21.91

377

269

37

171

77

18

171

124

8

Linuron

15.76

248

61

8

160

133

11

187

124

21

Malathion

15.92

173

99

13

127

99

6

173

127

6

Metalaxyl

15.34

220

160

10

160

130

16

160

145

10

Metazachlor

16.88

209

117

35

133

117

25

209

132

15

Methacrifos

10.03

240

180

10

125

79

6

180

93

8

Methoxychlor

21.14

227

115

50

227

141

33

227

169

25

Methyl parathion

15.03

263

79

30

263

109

10

263

136

8

Metolachlor

16.07

238

133

27

162

133

13

238

162

10

Mevinphos

8.97

192

127

10

127

95

16

127

109

8

MGK 264 1

16.53

164

80

26

164

93

12

164

121

8

MGK 264 2

16.76

164

67

10

164

80

24

164

98

14

Mirex

22.15

274

143

40

272

237

15

274

239

15

Myclobutanil

18.43

179

90

28

179

125

14

179

152

8

N-(2,4-Dimethylphenyl)formamide

9.67

121

106

8

106

77

15

120

77

15

Nitralin

20.66

316

274

8

274

216

6

300

258

8

Nitrofen

18.74

283

162

18

202

139

20

283

202

10

Nonachlor, cis-

19.32

409

300

23

272

237

10

409

302

20

Nonachlor, trans-

17.94

409

263

25

409

300

20

409

302

22

Norflurazon

19.90

303

145

17

145

75

25

145

95

18

Oxadiazon

18.36

258

175

8

175

112

13

175

147

8

Oxyfluorfen

18.47

300

223

15

252

146

33

252

170

27

Paclobutrazol

17.60

238

127

10

236

125

12

236

167

10

Parathion

16.19

291

109

10

109

81

8

139

109

8

Pebulate

9.40

203

128

8

128

57

8

161

128

6

Penconazole

16.93

248

157

26

159

89

30

248

192

16

Pendimethalin

16.91

281

252

8

252

162

10

252

191

8

Pentachloroaniline

14.62

265

107

46

263

192

20

265

194

22

Pentachloroanisole

12.99

280

237

23

265

237

12

267

239

10

Pentachlorobenzene

10.35

250

142

30

250

179

30

250

215

20

Pentachlorobenzonitrile

13.66

275

205

30

273

177

25

273

238

17

Pentachlorothioanisole

15.81

298

265

8

296

246

32

296

263

12

Permethrin, cis-

23.01

183

153

12

163

127

6

183

168

10

Permethrin, trans-

23.16

183

153

14

163

91

13

163

127

6

Phenothrin

21.68

183

153

14

123

81

8

183

168

12

Phorate

12.56

260

75

8

121

65

10

121

93

6

Phosalone

21.86

367

111

36

182

75

30

182

111

14

Phosmet

21.03

160

77

24

160

133

10

160

160

8

Piperonyl butoxide

20.46

176

103

26

176

117

20

176

131

14

Pirimiphos-ethyl

16.67

333

168

22

304

168

12

318

166

12

Pirimiphos-methyl

15.72

305

180

8

290

125

22

290

233

14

Pretilachlor

18.26

262

202

8

162

117

30

162

132

18

Prochloraz

23.35

308

70

13

180

69

15

180

138

12

Procymidone

17.31

283

67

28

283

96

10

283

255

12

Prodiamine

15.73

321

216

15

279

203

8

321

279

6

Profenofos

18.19

339

188

30

337

267

13

339

269

13

Profluralin

13.71

330

69

25

318

199

17

318

264

8

Propachlor

11.53

176

57

8

120

77

19

120

92

8

Propanil

14.78

219

163

8

161

99

25

217

161

8

Propargite

20.31

201

81

10

135

77

24

135

107

14

Propisochlor

15.32

223

132

18

162

120

13

162

144

10

Propyzamide

13.70

173

74

46

173

109

27

173

145

13

Prothiofos

18.13

309

239

15

267

221

16

267

239

8

Pyraclofos

22.69

360

139

14

194

138

18

360

194

10

Pyrazophos

22.54

265

210

10

221

193

10

232

204

10

Pyridaben

23.17

147

117

20

147

119

8

147

132

13

Pyridaphenthion

20.95

340

199

8

199

77

26

199

92

14

Pyrimethanil

13.81

198

118

32

198

158

20

198

183

16

Pyriproxyfen

21.94

226

186

16

136

78

20

136

96

12

Quinalphos

17.17

298

156

8

146

118

10

156

155

14

Quintozene

13.62

295

237

15

235

141

26

237

119

22

Resmethrin

20.44

171

128

15

123

81

8

171

143

6

Sulfotepp

12.50

322

146

23

202

146

10

322

202

10

Sulprofos

19.55

322

97

24

156

141

13

322

156

8

Tau-Fluvalinate

25.35

252

55

17

250

55

23

250

200

19

Tebuconazole

20.29

250

125

22

125

89

16

250

153

12

Tebufenpyrad

21.36

333

276

8

276

171

10

318

131

13

Tecnazene

11.45

261

203

13

215

144

22

215

179

8

Tefluthrin

14.19

197

161

6

177

127

15

177

137

15

Terbacil

14.10

161

88

22

160

117

8

161

144

14

Terbufos

13.62

231

129

23

231

175

12

231

203

8

Terbutylazine

13.61

229

138

12

173

138

8

229

173

8

Tetrachloroaniline, 2,3,5,6-

11.72

231

122

30

231

158

20

231

160

22

Tetrachlorvinphos

17.71

333

109

17

331

79

30

331

109

20

Tetradifon

21.66

227

199

10

159

111

20

159

131

10

Tetrahydrophthalimide

9.67

151

77

32

151

80

6

151

122

10

Tetramethrin

21.11

164

77

25

164

107

12

164

135

8

Tolclofos-methyl

15.14

267

252

10

265

93

25

265

250

10

Tolylfluanid

17.04

240

137

10

137

91

17

238

137

10

Transfluthrin

15.14

163

91

12

127

91

8

163

143

13

Triadimefon

16.24

208

111

24

208

127

14

208

181

10

Triadimenol

17.18

168

70

10

112

58

10

128

65

22

Triallate

14.24

270

186

18

268

184

20

268

226

10

Triazophos

19.55

257

162

8

161

106

12

161

134

8

Tricyclazole

18.20

189

118

20

189

135

18

189

162

12

Triflumizole

17.37

206

179

14

179

144

14

206

186

10

Trifluralin

12.35

306

160

23

306

206

12

306

264

8

Triphenylphosphate

20.35

326

169

30

326

215

20

     

Vinclozolin

15.03

285

212

12

212

145

24

212

172

12

FSAR3866A-UNV