An Optimized QuEChERS Workflow to Analyze Over 200 Pesticides in Honey

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
Analyte	RT	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