High-Throughput Multiresidue Pesticides Analysis by GC-MS/MS

• Proven and recommended, Rxi-5ms columns reliably separate pesticides with shared ion transitions.
• Exceptionally stable, low-bleed column ensures accurate results at trace levels.
• Pro EZGC chromatogram modeling software dramatically reduces method development time.

Using GC-MS/MS for multiresidue pesticides analysis makes it possible to analyze large numbers of pesticides at once due to the superior secondary separation of selected reaction monitoring (SRM). GC-MS/MS analysis also provides excellent sensitivity, allowing for limits of detection below 10 ppb, which is the typical maximum residue limit. However, comprehensive multiresidue pesticides methods still require reliable chromatographic separation of compounds that share ion transitions because those analytes cannot be distinguished by the MS/MS detector. Rxi-5ms columns contain an ideal stationary phase for this, one that reliably achieves essential chromatographic separations. In addition, these columns are ultra-low bleed, which can improve signal-to-noise ratios and mass spectral integrity. As shown in Figure 1, the combination of GC-MS/MS, and an Rxi-5ms column provides efficient separation, identification, and quantification of more than 200 pesticides. 

Figure 1: Analysis of 200+ Pesticides on an Rxi-5ms Column by GC-MS/MS

	Peaks	tR (min)	Conc. (µg/mL)	Mix
1.	Allidochlor	6.83	10	4
2.	Dichlobenil	7.72	10	3
3.	Biphenyl	8.24	10	3
4.	Mevinphos	8.92	10	9
5.	3,4'-Dichloroaniline	8.95	10	3
6.	Etridiazole	9.31	10	5
7.	Pebulate	9.37	10	4
8.	N-(2,4-Dimethylphenyl)formamide	9.63	10	4
9.	Tetrahydrophthalimide	9.64	10	3
10.	Methacrifos	10.00	10	8
11.	Chloroneb	10.10	10	2
12.	2-Phenylphenol	10.17	10	7
13.	Pentachlorobenzene	10.31	10	2
14.	Tecnazene	11.42	10	3
15.	Propachlor	11.51	10	4
16.	Diphenylamine	11.63	10	3
17.	2,3,5,6-Tetrachloroaniline	11.70	10	3
18.	Cycloate	11.8	10	4
19.	Chlorpropham	12.02	10	7
20.	Ethalfluralin	12.18	10	3
21.	Trifluralin	12.40	10	3
22.	Benfluralin	12.45	10	3
23.	Sulfotepp	12.49	10	8
24.	Phorate	12.59	10	9
25.	Diallate 1	12.6	10	4
26.	BHC, alpha-	12.70	10	2
27.	Diallate 2	12.8	10	4
28.	Hexachlorobenzene	12.90	10	2
29.	Dicloran	12.99	10	3
30.	Pentachloroanisole	13.02	10	2
31.	Atrazine	13.29	10	5
32.	BHC, beta-	13.36	10	2
33.	Clomazone	13.36	10	4
34.	BHC, gamma-	13.52	10	2
35.	Terbutylazine	13.64	10	5
36.	Quintozene	13.65	10	3
37.	Terbufos	13.66	10	8
38.	Pentachlorobenzonitrile	13.69	10	3
39.	Fonofos	13.73	10	9
40.	Propyzamide	13.73	10	4
41.	Profluralin	13.76	10	3
42.	Pyrimethanil	13.85	10	5
43.	Diazinon	13.97	10	1
44.	Disulfoton	14.05	10	9
45.	Fluchloralin	14.06	10	3
46.	BHC, delta-	14.09	10	2
47.	Terbacil	14.11	10	5
48.	Chlorothalonil	14.21	10	3
49.	Tefluthrin	14.23	10	6
50.	Isazophos	14.27	10	1
51.	Triallate	14.28	10	4
52.	Endosulfan ether	14.55	10	2
53.	Pentachloroaniline	14.64	10	3
54.	Propanil	14.82	10	4
55.	Dimethachlor	14.87	10	4
56.	Acetochlor	15.03	10	4
57.	Methyl parathion	15.06	10	9
58.	Vinclozolin	15.06	10	5
59.	Chlorpyrifos-methyl	15.07	10	1
60.	Tolclofos-methyl	15.17	10	8
61.	Transfluthrin	15.18	10	6
62.	Heptachlor	15.25	10	2
63.	Alachlor	15.26	10	4
64.	Propisochlor	15.35	10	4
65.	Metalaxyl	15.38	10	7
66.	Fenchlorphos	15.41	10	8
67.	Fenitrothion	15.72	10	1
68.	Pirimiphos-methyl	15.76	10	1
69.	Prodiamine	15.78	10	3
70.	Linuron	15.79	10	4
71.	Pentachlorothioanisole	15.83	10	2
72.	Dichlofluanid	15.90	10	3
73.	Malathion	15.96	10	8

	Peaks	tR (min)	Conc. (µg/mL)	Mix
74.	Anthraquinone	16.02	10	6
75.	Aldrin	16.06	10	2
76.	Metolachlor	16.1	10	4
77.	Fenthion	16.15	10	8
78.	Chlorpyrifos	16.21	10	1
79.	Parathion	16.22	10	9
80.	Dichlorobenzophenone, 4,4'-	16.26	10	2
81.	Triadimefon	16.28	10	5
82.	Chlorthal-dimethyl	16.33	10	7
83.	Fenson	16.45	10	2
84.	MGK 264 1	16.56	10	5
85.	Bromophos-methyl	16.60	10	8
86.	Diphenamid	16.61	10	4
87.	Pirimiphos-ethyl	16.71	10	1
88.	Isodrin	16.72	10	2
89.	Cyprodinil	16.77	10	5
90.	Isopropalin	16.77	10	3
91.	MGK 264 2	16.82	10	5
92.	Chlorfenvinphos 1	16.87	10	8
93.	Metazachlor	16.91	10	4
94.	Pendimethalin	16.95	10	3
95.	Heptachlor epoxide	16.97	10	2
96.	Penconazole	16.97	10	5
97.	Chlozolinate	17.05	10	7
98.	Tolylfluanid	17.07	10	3
99.	Bromfenvinphos-methyl	17.13	10	8
100.	Captan	17.13	10	5
101.	Chlorfenvinphos 2	17.13	10	8
102.	Bioallethrin	17.17	10	6
103.	Fipronil	17.17	10	5
104.	Quinalphos	17.21	10	1
105.	Triadimenol	17.21	10	5
106.	Folpet	17.28	10	5
107.	Procymidone	17.34	10	5
108.	Chlorbenside	17.37	10	2
109.	Triflumizole	17.41	10	5
110.	Chlordane, trans-	17.51	10	2
111.	Bromophos-ethyl	17.58	10	8
112.	DDE, o,p'-	17.62	10	2
113.	Paclobutrazol	17.63	10	5
114.	Tetrachlorvinphos	17.74	10	8
115.	Endosulfan I	17.79	10	2
116.	Chlordane, cis-	17.85	10	2
117.	Flutriafol	17.90	10	5
118.	Nonachlor, trans-	17.96	10	2
119.	Chlorfenson	17.97	10	2
120.	Fenamiphos	17.98	10	8
121.	Bromfenvinphos	18.03	10	8
122.	Flutolanil	18.07	10	4
123.	Iodofenfos	18.10	10	8
124.	Prothiofos	18.15	10	8
125.	Tricyclazole	18.21	10	5
126.	Profenofos	18.22	10	8
127.	Fludioxonil	18.24	10	5
128.	Pretilachlor	18.29	10	4
129.	DDE, p,p'-	18.31	10	2
130.	Dieldrin	18.37	10	2
131.	Oxadiazon	18.4	10	4
132.	Myclobutanil	18.46	10	5
133.	DDD, o,p'-	18.50	10	2
134.	Oxyfluorfen	18.51	10	3
135.	Flusilazole	18.53	10	5
136.	Bupirimate	18.59	10	5
137.	Nitrofen	18.77	10	3
138.	Fluazifop-P-butyl	18.84	10	7
139.	Endrin	18.85	10	2
140.	Ethylan	18.86	10	2
141.	Chlorfenapyr	18.88	10	5
142.	Chlorthiophos 1	18.94	10	8
143.	Chlorobenzilate	19.02	10	7
144.	Endosulfan II	19.04	10	2
145.	Chlorthiophos 2	19.12	10	8
146.	DDD, p,p'-	19.23	10	2

	Peaks	tR (min)	Conc. (µg/mL)	Mix
147.	DDT, o,p'-	19.31	10	2
148.	Ethion	19.32	10	8
149.	Nonachlor, cis-	19.33	10	2
150.	Chlorthiophos 3	19.40	10	8
151.	Endrin aldehyde	19.45	10	2
152.	Sulprofos	19.58	10	8
153.	Triazophos	19.59	10	9
154.	Carbophenothion	19.78	10	8
155.	4,4'-Methoxychlor olefin	19.84	10	2
156.	Carfentrazone ethyl	19.84	10	7
157.	Edifenphos	19.88	10	8
158.	Norflurazon	19.93	10	4
159.	Lenacil	19.94	10	5
160.	Endosulfan sulfate	19.97	10	2
161.	DDT, p,p'-	20.04	10	2
162.	Hexazinone	20.24	10	5
163.	2,4'-Methoxychlor	20.27	10	2
164.	Tebuconazole	20.31	10	5
165.	Propargite	20.42	10	5
166.	Resmethrin 1	20.43	10	6
167.	Captafol	20.48	10	5
168.	Piperonyl butoxide	20.50	10	9
169.	Resmethrin 2	20.55	10	6
170.	Nitralin	20.69	10	3
171.	Iprodione	20.88	10	5
172.	Endrin ketone	20.97	10	2
173.	Pyridaphenthion	20.99	10	1
174.	Tetramethrin 1	21.00	10	6
175.	Phosmet	21.06	10	1
176.	Bromopropylate	21.12	10	7
177.	EPN	21.14	10	1
178.	Tetramethrin 2	21.14	10	6
179.	Bifenthrin	21.15	10	6
180.	Methoxychlor	21.25	10	4
181.	Fenpropathrin	21.3	10	4
182.	Tebufenpyrad	21.37	10	4
183.	Phenothrin 1	21.59	10	6
184.	Tetradifon	21.68	10	2
185.	Phenothrin 2	21.71	10	6
186.	Phosalone	21.89	10	1
187.	Azinphos-methyl	21.90	10	1
188.	Leptophos	21.94	10	8
189.	Pyriproxyfen	21.97	10	5
190.	Mirex	22.16	10	2
191.	Cyhalothrin, lambda-	22.30	10	6
192.	Fenarimol	22.47	10	5
193.	Acrinathrin	22.51	10	6
194.	Pyrazophos	22.58	10	1
195.	Azinphos-ethyl	22.63	10	1
196.	Pyraclofos	22.73	10	1
197.	Permethrin, cis-	23.14	10	6
198.	Permethrin, trans-	23.29	10	6
199.	Pyridaben	23.3	10	4
200.	Coumaphos	23.40	10	8
201.	Fluquinconazole	23.41	10	4
202.	Prochloraz	23.48	10	4
203.	Cyfluthrin 1	23.83	10	6
204.	Cyfluthrin 2	23.93	10	6
205.	Cyfluthrin 3	24.02	10	6
206.	Cyfluthrin 4	24.06	10	6
207.	Cypermethrin 1	24.19	10	6
208.	Cypermethrin 2	24.30	10	6
209.	Cypermethrin 3	24.39	10	6
210.	Cypermethrin 4	24.43	10	6
211.	Flucythrinate 1	24.43	10	6
212.	Etofenprox	24.53	10	5
213.	Acequinocyl	24.54	10	7
214.	Flucythrinate 2	24.66	10	6
215.	Fluridone	24.92	10	5
216.	Fenvalerate 1	25.25	10	6
217.	tau-Fluvalinate 1	25.47	10	6
218.	Fenvalerate 2	25.48	10	6
219.	tau-Fluvalinate 2	25.53	10	6
220.	Deltamethrin	26.09	10	6

Column	Rxi-5ms, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13423)
Standard/Sample	GC multiresidue pesticide standard #1 (cat.# 32563)
	GC multiresidue pesticide standard #2 (cat.# 32564)
	GC multiresidue pesticide standard #3 (cat.# 32565)
	GC multiresidue pesticide standard #4 (cat.# 32566)
	GC multiresidue pesticide standard #5 (cat.# 32567)
	GC multiresidue pesticide standard #6 (cat.# 32568)
	GC multiresidue pesticide standard #7 (cat.# 32569)
	GC multiresidue pesticide standard #8 (cat.# 32570)
	GC multiresidue pesticide standard #9 (cat.# 32571)
Diluent:	Toluene
Conc.:	10 µg/mL
Injection
Inj. Vol.:	1 µL splitless (hold 0.5 min)
Liner:	Topaz, single taper inlet liner w/wool, 4.0 mm x 6.5 x 78.5 (cat.# 23447)
Inj. Temp.:	250 °C
Oven
Oven Temp.:	90 °C (hold 1 min) to 330 °C at 8.5 °C/min (hold 5 min)
Carrier Gas	He, constant flow
Flow Rate:	1.4 mL/min

Detector	TSQ8000
SIM Program:	50-550 m/z
Transfer Line Temp.:	280 °C
Source Temp.:	325 °C
Solvent Delay Time:	5 min
Tune Type:	PFTBA
Ionization Mode:	EI
Instrument	Thermo Trace GC
Sample Preparation	Individual standards were diluted with toluene to 10 ppm and analyzed in a short-cap, screw-thread vial (cat.# 21143) and capped with a short-cap, screw-vial closure (cat.# 24495).
Notes	Standards were analyzed individually and were overlaid for this chromatogram. Standards are also available as a kit: GC multiresidue pesticide standards kit (cat.# 32562).

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Some of the most challenging pesticides are closely eluting isomers and other compounds that share ions or ion transitions (Table I). Poor separation can affect automated integration, which can skew results and require time-consuming manual integrations. Further, if these compounds coelute completely, then identification and quantitation become impossible by MS/MS. To demonstrate the resolving power of Rxi-5ms columns for multiresidue pesticides in disparate matrices, samples were prepared in brown rice flour and oranges as follows and then analyzed on a Thermo TRACE GC with a TSQ8000 detector.

Brown Rice Flour Extracts

Five grams of brown rice flour was mixed with 10 mL of water. After a 60 second vortex, the brown rice flour was fortified at 10 ng/g with all residues from the GC multiresidue pesticides kit (cat.# 32562). The fortified brown rice flour suspension was shaken for 30 minutes. Extraction was performed using 10 mL of acetonitrile and the original unbuffered Q-sep QuEChERS salts (cat.# 25848). After centrifugation, the supernatant was removed and further cleaned up with pre-filled Q-sep QuEChERS dSPE tubes containing 150 mg MgSO4, 50 mg PSA, and 50 mg C18-EC (cat.# 26125).

Orange Extracts

Ten grams of homogenized orange was fortified at 10 ng/g with all residues from the GC multiresidue pesticide kit (cat.# 32562), mixed with 10 mL acetonitrile and two steel ball bearings (1/16''), and shaken for 10 minutes. Q-sep QuEChERS extraction salts for EN 15662 (cat.# 25849) were added, and the sample was shaken for 1 minute. The sample was centrifuged for 5 minutes, and the supernatant was removed and further cleaned up with prefilled Q-sep QuEChERS dSPE tubes containing 150 mg MgSO4, 50 mg PSA, and 50 mg C18-EC (cat.# 26125).

As shown in Figure 2, the Rxi-5ms column provides excellent resolution of several difficult pesticides. While benfluralin and trifluralin can be separated by MS/MS using different transitions, chromatographic resolution is advantageous here because it allows a shared main ion (m/z 264) to be used (Figure 2A). This is also true for the cyfluthrins and cypermethrins, where baseline chromatographic separation between the two groups of isomers was achieved (Figure 2D). In the case of compounds that share the same ion transitions, the selectivity of the Rxi-5ms column provided excellent chromatographic resolution of 4,4’-DDD and 2,4’-DDT (Figure 2B), as well as the permethrins (Figure 2C) and BHC isomers (Figure 2E). 

Table I: SRM and Shared Ions for the Pesticides in Figure 2

*The window for these transitions was one minute.
Prec – Precursor ion
Prod – Product ion
CE – Collision energy    

Figure 2: Selected Transitions of Isomers and Compounds Sharing Ions

A) Trifluralin and Benfluralin at 10 ppb in Brown Rice Flour Extract B) 4,4’-DDD and 2,4’-DDT at 10 ppb in Brown Rice Flour Extract C) Permethrins at 10 ppb in Brown Rice Flour Extract D) Cyfluthrins and Cypermethrins at 10 ppb in Orange Extract E) BHC Isomers at 10 ppb in Brown Rice Flour Extract

 

	Peaks	tR (min)	Conc. (ng/g)	Precursor Ion	Product Ion	Collision Energy	Mix
1.	Trifluralin	12.35	10	306	264	8	3
2.	Benfluralin	12.43	10	292	264	8	3
3.	α-BHC	12.70	10	181	145	13	2
4.	β-BHC	13.33	10	181	145	15	2
5.	γ-BHC	13.49	10	181	145	15	2
6.	δ-BHC	14.05	10	181	145	13	2
7.	DDD, p,p'-	19.17	10	235	165	24	2
8.	DDT, o,p'-	19.25	10	235	165	24	2
9.	Permethrin, cis-	23.04	10	183	168	10	6
10.	Permethrin, trans-	23.19	10	183	153	14	6
11.	Cyfluthrins	24.00-24.35	10	226	206	10	6
12.	Cypermethrins	24.35-24.70	10	163	127	6	6

Column	Rxi-5ms, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13423)
Standard/Sample	GC multiresidue pesticide standards kit (cat.# 32562)
Diluent:	Toluene
Injection
Inj. Vol.:	1 µL splitless (hold 0.5 min)
Liner:	Topaz, single taper inlet liner w/wool, 4.0 mm x 6.5 x 78.5 (cat.# 23447)
Inj. Temp.:	250 °C
Oven
Oven Temp.:	90 °C (hold 1 min) to 330 °C at 8.5 °C/min (hold 5 min)
Carrier Gas	He, constant flow
Flow Rate:	1.4 mL/min

Detector	TSQ8000
Transfer Line Temp.:	280 °C
Source Temp.:	325 °C
Solvent Delay Time:	5 min
Tune Type:	PFTBA
Ionization Mode:	EI
Instrument	Thermo Trace GC
Sample Preparation	Brown Rice Extract (shown in A, B, C, and E): 5 g of brown rice flour was mixed with 10 mL of water. After a 60 second vortex, the brown rice flour was fortified at 10 ng/g with all residues from the GC multiresidue pesticides kit (cat.# 32562). The fortified brown rice flour suspension was shaken for 30 minutes. Extraction was performed using 10 mL of acetonitrile and the original unbuffered Q-sep QuEChERS salts (cat.# 25848). After centrifugation, the supernatant was removed and further cleaned up with pre-filled Q-sep QuEChERS dSPE tubes containing 150 mg MgSO4, 50 mg PSA, and 50 mg C18-EC (cat.# 26125). Orange Extract (shown in D): 10 g of homogenized orange was fortified at 10 ng/g with all residues from the GC multiresidue pesticide kit (cat.# 32562), mixed with 10 mL acetonitrile and two steel ball bearings (1/16''), and shaken for 10 minutes. Q-sep QuEChERS extraction salts for EN 15662 (cat.# 25849) were added, and the sample was shaken for 1 minute. The sample was centrifuged for 5 minutes, and the supernatant was removed and further cleaned up with prefilled Q-sep QuEChERS dSPE tubes containing 150 mg MgSO4, 50 mg PSA, and 50 mg C18-EC (cat.# 26125). The extracts were analyzed in a short-cap, screw-thread vial (cat.# 21143) and capped with a short-cap, screw-vial closure (cat.# 24495).

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As demonstrated above, using an Rxi-5ms column together with GC-MS/MS is an effective combination in achieving high-quality qualitative and quantitative pesticides analysis. Critical separations are achieved through both the power of the MS/MS and—when ions or ion transitions are shared—through the high efficiency and chromatographic selectivity of the Rxi-5ms column. In addition to improving data accuracy, good chromatographic separation reduces manual integration, which saves time and allows increased sample throughput. Labs interested in additional time savings can also explore virtual method development and optimization using Restek’s Pro EZGC chromatogram modeling software (www.restek.com/EZGC). With this free online tool, methods for multiresidue pesticides analysis can be developed or modified in minutes on a computer instead of taking days of instrument time in the laboratory.