Analysis of Pesticide Residues in Pistachios –
Using QuEChERS Extraction and Cleanup with Supel™ QuE Z-Sep+

Kathy Stenerson, Principle R&D Scientist; Megan Wesley, 2016 R&D Summer Intern

Introduction

PistachioPistachios are popular and enjoyed for both taste and health benefits such as decreased cholesterol, weight management, protection against diabetes and hypertension, and improved digestion.1 These nuts are grown in the United States (specifically, California), Italy, and countries in Central Asia like Iran, Turkey, Afghanistan and Syria. Pesticide tolerances set by the US EPA for pistachios range from 0.01 - 0.7 μg/g before harvest to 3 - 200 μg/g after harvest, depending on the pesticide.2 Testing for pesticide residues then requires a method which will allow for low level and accurate determination. The "quick, easy, cheap, effective, rugged and safe" (QuEChERS) approach has been used to analyze multiple pesticide residues found in pistachios.3 Pistachios contain approximately 45% fat, which can result in a significant amount of co- extracted matrix in the acetonitrile extract generated using the QuEChERS procedure. The use of a cleanup sorbent which can reduce this fat is essential to prevent fouling of LC-MS/MS and GC-MS/MS systems, and minimize ion suppression, thus allowing low level detection. In this application, Supel™ QuE Z-Sep+ sorbent was used as part of the QuEChERS method in the analysis of pesticide residues in pistachios. Z-Sep+ is a zirconia and C18 functionalized silica sorbent which acts to retain fatty constituents through both Lewis acid/base and hydrophobic interactions. The selectivity of the zirconia present in Z-Sep+ offers retention of a wider range of fats than C18 alone. In this application, QuEChERS extraction and cleanup using Z-Sep+ sorbent were used before the LC-MS/MS and GC-MS/ MS analysis of pesticide residues in pistachios. The targeted analyte list included pesticides relevant to pistachios.4,5

 

Experimental

Pistachios were purchased from a local grocery store. They were frozen with liquid nitrogen (shells on), ground, and spiked at 10 ng/g with the pesticides listed in Tables 2 and 4, and allowed to equilibrate for 1 hour. Samples were then subjected to QuEChERS extraction and cleanup with Z-Sep+ following the procedure in Figure 1. A 100 μL aliquot of the final extract was diluted to 1 mL with 5 mM ammonium formate/0.1% formic acid in water, and analyzed by LC-MS/MS using the conditions shown in Table 1.

The remaining acetonitrile extract was analyzed directly by GC-MS/MS using the conditions shown in Table 3. Spiked samples were quantitated against 5-point matrix-matched calibration curves prepared in unspiked pistachio matrix blanks (after cleanup). No internal standard was used.

Figure 1. QuEChERS Extraction and Cleanup Procedure Used for Pistachios.

Table 1. LC-MS/MS analysis conditions.

column: Ascentis® Express RP-Amide, 10 cm × 2.1 mm I.D., 2 μm (51576-U)
mobile phase: [A] 5 mM ammonium formate, 0.1% formic acid in water;
[B] 5 mM ammonium formate, 0.1% formic acid in 95:5 acetonitrile:water
gradient: 5% B held for 1 min; 5 to 100% B in 12 min;
held at 100% B for 1.5 min; 100 to 5% B in 0.5 min; held at 5% B for 1.5 min
flow rate: 0.4 mL/min
column temp.: 30 °C
detector: MS, ESI (+), MRM (see Table 2)
injection: 5 μL

Table 2. MRMs used for quantitation, LC-MS/MS.

Compound CAS no. MRM Frag (V) CE
Aclonifen 74070-46-5 265/182.1 115 28
Aldicarb 116-06-3 208.1/89.1   70 12
Aldicarb-sulfone 1646-88-4 223.1/86.1   80   8
Bifenazate 149877-41-8 301.1/170.1   95 16
Butocarboximsulfoxide 34681-24-8 207.1/132   65   0
Carbendazim 10605-21-7 192.1/160.1 105 16
Carbofuran 1563-66-2 222.1/165.1   80 20
Chlorantraniliprole 500008-45-7 483.9/452.9 105 16
Etrimfos 38260-54-7 293.1/125 120 28
Flufenoxuron 101463-69-8 489.1/158 100 20
Isoxathion 18854-01-8 314.1/105 135 12
Malathion 121-75-5 331/126.9   80   5
Methabenzthiazuron 18691-97-9 222.1/165.1   90 12
Methomyl 16752-77-5 163.1/106   50   4
Neburon 555-37-3 275.07/57.1 100 20
Omethoate 1113-02-6 214/109   80 24
Pyraflufen-ethyl 129630-19-9 413/339 120 25
Quinalphos 13593-03-8 299/163   90 20
Rotenone 83-79-4 395/213.1 145 20
Spinetoram 187166-40-1 748.5/142.2 206 32
Spiromesifen 283594-90-1 388/273 110 10
Thiacloprid 111988-49-9 253/126 100 16
Thiophanate-methyl 23564-05-8 343/151   90 20
Triazophos 24017-47-8 314.1/162.1 110 16
Trichlorfon 52-68-6 256.9/109   80 12

Table 3. GC-MS/MS analysis conditions.

column: SLB®-5ms, 20 m × 0.18 mm I.D., 0.18 μm (28564-U)
oven: 50 °C (2 min), 15 °C/min to 320 °C (5 min)
inj. temp.: 250 °C
carrier gas: helium, 1.2 mL/min constant flow
detector: MSD, scan and MRM (see Table 4)
MSD interface: 325 °C
injection: 1 μL, splitless (0.75 min)
liner: 4 mm I.D. FocusLiner™ with taper

Table 4. MRMs Used for quantitation; GC-MS/MS.

Compound CAS # MRM CE
Chlorpyrifos-methyl 5598-13-0 286/93 20
Tolclofos-methyl 57018-04-9 265/250 15
Fenthion 55-38-9 278/169 15
MGK-264 18691-97-9 164/98 10
Endosulfan sulfate 1031-07-8 274/239 15
Etoxazole 153233-91-1 141/63 30

Results and discussion

Background

Initially, cleanup using Z-Sep+ sorbent was compared to PSA/C18, a common QuEChERS cleanup sorbent for fat-rich samples. A visual comparison of the QuEChERS extracts (in acetonitrile) is shown in Figure 2. Both cleanups removed some green color, resulting in similar light yellow extracts. GC-MS-scan comparisons (Figure 3) show lower background after Z-Sep+ cleanup compared to PSA/C18. The predominant peaks present in the uncleaned extract are fatty acids and monoglycerides. While PSA/C18 only reduced the levels of these compounds, almost none were detected after Z-Sep+ cleanup.

Pesticide recovery

Table 5 shows the average %Recovery and %RSD for n=3 replicates of spiked pistachio samples. The majority of the pesticides were analyzed by LC-MS/MS; and those without sufficient response were analyzed by GC-MS/MS. Out of the 30 pesticides analyzed, 22 had recoveries within the generally accepted range of 70-120 %. Reproducibility was good, with RSD values < 20% for all 30 pesticides, and < 10% for many. Two pesticides, etoxazole and trichlorfon, had recoveries < 50%. Trichlorfon was most likely retained by the Z-Sep+ sorbent during the cleanup step. This could be due to the Lewis base character of the phosphate group present in its structure. Etoxazole, on the other hand, does not contain a phosphate group. It is a very lipophilic pesticide, indicated by its log P value of 5.6. Extraction efficiency of this compound from the fatty pistachio matrix was probably very poor using acetonitrile. Spinetoram, with a log P of 6.3, also showed lower recovery (56%) than a majority of the pesticides studied. This trend of decreased recovery for high log P pesticides has been observed by others for high fat matrices.6 Recovery of both of these compounds may be increased by addition of a less polar solvent such as ethyl acetate for the extraction; however, an increase in the level of co-extracted background can be expected.

Figure 2. Comparison of Pistachio Extracts; Before and After Cleanup.

Figure 3. GC-MS-Scan Comparison of Pistachio Extracts With (a) No Cleanup, (b) PSA/C18 Cleanup, and (c) Z-Sep+ Cleanup; All the Same Y-scale.

Table 5. Pesticide recoveries from pistachios Using Z-Sep+ cleanup, spike level of 10 ng/g.

Pesticide Avg. % recovery (n=3) % RSD Analysis
Aldicarb 102%   3% LC-MS/MS
Aldicarb-sulfone 108%   1% LC-MS/MS
Bifenazate   88%   4% LC-MS/MS
Butocarboximsulfoxide   83%   5% LC-MS/MS
Carbendazim   71%   4% LC-MS/MS
Carbofuran 104%   4% LC-MS/MS
Chlorantraniliprole   90%   5% LC-MS/MS
Chlorpyrifos-methyl   66% 10% GC-MS/MS
Endosulfan sulfate   58%   6% GC-MS/MS
Etoxazole   45%   9% GC-MS/MS
Etrimfos   90%   7% LC-MS/MS
Fenthion   72%   9% GC-MS/MS
Flufenoxuron   62% 15% LC-MS/MS
Isoxathion   92%   3% LC-MS/MS
Malathion 102%   4% LC-MS/MS
Methabenzthiazuron   84%   3% LC-MS/MS
Methomyl 106%   5% LC-MS/MS
MGK-264 (avg. 2 isomers)   57%   17% GC-MS/MS
Neburon   92%   7% LC-MS/MS
Omethoate   66%   2% LC-MS/MS
Pyraflufen-ethyl   97% 18% LC-MS/MS
Quinalphos 104%   7% LC-MS/MS
Rotenone 100%   3% LC-MS/MS
Spinetoram   56% 10% LC-MS/MS
Spiromesifen   83%   4% LC-MS/MS
Thiacloprid 100%   2% LC-MS/MS
Thiophanate-methyl 100%   3% LC-MS/MS
Tolclofos-methyl   71% 10% GC-MS/MS
Triazophos (avg. 2 isomers)   89%   3% LC-MS/MS
Trichlorfon   14% 13% LC-MS/MS

Conclusions

Pistachios, which contain 45% fat, present a challenging matrix when doing pesticide residue analysis. If using QuEChERS extraction, some fat will be co-extracted with the analytes of interest. Thus, the cleanup step must be able to reduce this background. In this application, the use of Supel™ QuE Z-Sep+ was demonstrated for the effective cleanup of these extracts prior to LC-MS/MS and GC-MS/MS analysis. Fatty acid and monoglyceride background were significantly reduced using Z-Sep+, and compared to PSA/C18 cleanup, the resulting extract had lower background; as evidenced by GC-MS-scan data.

Pesticide recovery was within the acceptable range of 70-120% for 22 out of 30 targeted pesticides, with excellent reproducibility demonstrated for spiked replicates.

References

  1. Organic Facts. Health Benefits of Pistachios. www.organicfacts.net/health-benefits/seed-and-nut/health-benefits-of-pistachio.html (accessed June 2016)
  2. Index to Pesticide Chemical Names, Part 180 Tolerance Information, and Food and Feed Commodities (by Commodity); US Environmental Protection Agency Office of Pesticide Programs, U.S. Government Publishing Office: Washington, DC, 2012.
  3. Emami, A.; Rastegar, H.; Amirahmadi, M.; Shoeibi, S.; Mousavi, Z. Multi-Residue analysis of pesticides in pistachio using gas chromatography-mass sSpectrometry (GC/MS). Iranian J. of Tox. 2015, 8 (27), 1174-1181.
  4. California Pistachio Research Board. Pistachio Pesticides (as of May 2012). calpistachioresarch.org, (accessed May 2016)
  5. PAN Pesticide Database. Pesticide use in California, Pesticide Use on Pistachios 2012. www.pesticideinfo.org (accessed May 2016)
  6. Rajski, L.; Lozano, A.; Uclés, A.; Ferrer, C.; Fernández-Alba, A.R. Determination of pesticide residues in high oil vegetal commodities by using various multi-residue methods and clean-ups followed by liquid chromatography tandem mass spectrometry. J. Chrom. A, 2013, 1304, 109-120.

 

Materials