Analysis of Pesticides in Turmeric Powder by LC/MS/MS and GC/MS/MS After Cleanup with a Novel Dual-Layer SPE Cartridge

By: Katherine K Stenerson, Principal R&D Scientist, Reporter US Volume 34.3

Introduction

Turmeric is a plant indigenous to south Asia, with a majority of its production coming from India. The rhizome of the plant is used to produce powdered turmeric, which is used in foods, cosmetics, and some medicines. It is also an essential constituent of curry, which is a mixture of spices used extensively in Indian cooking. Turmeric has also been used in traditional medicines for thousands of years, and recently has garnered attention for studies showing its potential antioxidant, anti-inflammatory, antimutagenic, antimicrobial and anticancer properties.1

Pesticide residue testing of turmeric and other spices is required by many countries. For example, Canada has set maximum residue limits for 42 different pesticides in turmeric root.2 The US EPA has set tolerance limits for a variety of pesticides in root and tuberous vegetables, of which turmeric is included.2,3

Turmeric contains more than 100 different components, with two of the main constituents being curcumin and volatile oils. Curcumin gives turmeric its distinctive yellow/orange color, while the volatile oils consist primarily of terpenes. Turmeric also contains some fats; specifically, sterols and fatty acids.4 This complex composition makes extracts produced from turmeric a challenge in the chromatographic analysis of pesticides, as residual pigments and oils can contaminate both GC/MS and LC/MS systems.

When dealing with very high background samples such as turmeric, standard QuEChERS cleanup may not offer enough capacity. For better cleanup, solid phase extraction (SPE), including dual-layer cartridges, can be used. These cartridges often contain graphitized carbon black (GCB) in the top bed and primary-secondary amine (PSA) in the bottom bed. PSA retains acidic interferences such as fatty acids. GCB removes planar molecules such as pigments and sterols. Common GCBs, however, will retain all molecules with planar structures, including some pesticide analytes such as hexachlorobenzene. To increase recoveries of these pesticides, toluene is normally added to the elution solvent. However, there are issues associated with the use of toluene. It can affect the ability of the PSA to retain fatty acids, and its presence in the final extract is problematic for HPLC analysis.5

In this application, a different dual-layer SPE cartridge was used in the cleanup of extracts of turmeric powder prior to pesticide analysis by GC/MS/MS and LC/MS/MS. This cartridge, the Supelclean™ Ultra2400, was designed for the cleanup of acetonitrile extracts made from difficult matrices such as dry commodities (spices, tea, etc.) prior to pesticide residue analysis. The top bed consists of a mixture of PSA, C18 and a graphitized, spherical carbon known as Graphsphere™ 2031. This carbon was engineered to remove sufficient pigmentation while allowing for better recoveries of planar compounds, without the need for toluene in the elution solvent. The bottom layer of the cartridge contains Z-Sep, a zirconia-coated silica. Z-Sep removes oily residues and provides additional retention of some pigments. The combination of these sorbents in an SPE format offers more capacity than QuEChERS cleanup, and compared to traditional GCB/PSA dual layer cartridges, does not require the use of toluene in the elution solvent to recover planar pesticides.

Experimental

Turmeric powder was obtained from a local grocery store. Samples were spiked at 100 ng/g with the pesticides listed in Tables 1 and 2. Sample extracts were prepared and cleaned following the procedures in Figure 1. A set of 3 spiked samples and 1 unspiked (blank) were prepared and analyzed for each set of pesticides. Analysis was done by GC/MS/MS and LC/MS/MS using the conditions listed in Tables 3 and 4 (with MS/MS transitions shown in Tables 1 and 2). Quantitation was performed against multi-point calibration curves prepared in unspiked turmeric extract (after cleanup). Recoveries were calculated as the average of the three spiked replicates, less anything found in the unspiked extract. No internal standards were used, thus the values reported represent absolute recoveries.

Table 1. Pesticides Studied in Turmeric Powder by GC/MS/MS Analysis

  MRM 1 CE MRM 2 CE
Alachlor
188/160 10 188/130 40
Aldrin 263/193 35 263/191 35
γ-BHC 183/147 15 181/145 5
Azinphos-methyl 160/77 15 132/77 15
Chloropyrifos 314/286 5 314/258 15
Chloropyrifos-methyl 286/93 20 288/93 20
Cypermthrins 165/91 10 163/91 10
4,4'-DDT 235/199 15 235/165 25
Diazinon 199/135 15 137/84 10
Dichlorvos 185/93 25 145/109 25
Dimethoate 125/79 20 93/63 10
Disulfoton 88/60 5 88/59
15
Endosulfan β 241/206 15
241/170 30
Endosulfan-α 241/206 15 241/170 30
Ethion 231/129
20 121/65 10
Fenitrothion 277/125 20 277/109 20
Heptachlor 274/239 15 272/237 15
Hexachlorobenzene 284/249 20 284/214 35
Iprodione I 314/56 35 187/124 25
Iprodione II 316/56 35 187/124 25
Malathion 173/99 15 158/125 5
Metalaxyl 234/174 10 234/146 20
Methoxychlor 227/169 30 227/141
30
Mevinphos 192/127 25 192/109 25
Parathion-methyl 233/109 10 124/47 10
Permethrins 183/168 10 183/165 10
Phenthoate 274/125 15 274/121 10
Phorate 260/75 5 231/129 25
Phosalone 182/102 15 182/75 30
Pirimiphos-methyl 290/151 20 290/125 25
Profenophos 339/269 15 339/188 15
Quintozene 295/237 20 237/143 30
Vinclozolin 212/145 30 187/124 20

 

Table 2. Pesticides Studied in Turmeric Powder by LC/MS/MS Analysis

  MRM  
Frag (V) CE (V) Cell Acc (V)
Acephate 184/143 70 0 5
Acetamiprid 223.1/126 80 27 2
Boscalid (Nicobifen) 343/307.1 145 16 6
Carbendazim (Azole) 192.1/160.1 105 16 2
Chlorbufam 224/172.02 120 5 3
Cycluron 199.2/72 120 20 2
Diflubenzuron 311/158 80 8 2
Fenoxanil 329.08/189 80 30 3
Fosthiazate 284/61 90 60 2
Methabenzthiazuron
222.1/165.1 90 12 2
Methamidophos 142/125 85 10 2
Methomyl 163.1/106 50 4 2
Monocrotophos (Azodrin) 224.1/193 65
0
5
Nitralin 346.11/304 100 10 3
Oxamyl 237.1/72 60 12 2
Pirimicarb 239.15/72.1 100 20 2
Procymidon 301/284* 70 8 2
Propaquizafop 444.12/100.1 125 16 2
Tetraconazole 372/159 130 36 2
Uniconazole-P 292.1/125 135 40 2

* Ammonium adduct

 

Extraction and cleanup procedure used for turmeric powder

Figure 1. Extraction and Cleanup Procedure Used for Turmeric Powder, GC and LC


Table 3.
GC/MS/MS Run Conditions for the Analysis of Pesticides in Turmeric

column: SLB®-5ms, 30 m × 0.25 mm ID, 0.25 μm (28471-U)
oven: 50°C (2 min), 8°C/min to 320°C (5 min)
inj. temp.: 250°C
carrier gas: helium, 1.4 mL/min, constant
detector: MRM (see table 3)
MSD interface: 320°C
injection : 1μL, splitless (splitter open at 0.75 min)
liner: 4 mm I.D., split/splitless type, single taper wool packed FocusLiner™ design (2879901-U)

 

Table 4. LC/MS/MS Run Conditions for the Analysis of Pesticides in Turmeric

column: Ascentis® Express C18, 10 cm × 2.1 mm ID, 2 μm
mobile phase: [A] 5 mM ammonium formate, 0.1% formic acid in water;
[B] 5 mM ammonium formate, 0.1% formic acid in methanol
gradient: 95% A, 5% B held for 1 min; to 50% A in 3 min; to 100%
B in 8 min; held for 1 min;, to 95% A in 1.5 min; held at 95%
A for 1.5 min
flow rate: 0.4 mL/min
detector: MRM (see table 2)
injection: 5 μL

 

Results and Discussion

Background

Prior to cleanup, the extract appeared orange-brown in color with a yellow oily residue (Figure 2). After cleanup for both LC and GC, the extracts appeared substantially lighter and clearer. Figures 3 and 4 show a comparison between extracts at the same level of dilution with and without cleanup. The LC extract (in 80% aqueous) was almost devoid of color, with very little cloudiness. The extract for GC analysis was a pale yellow color, with substantially less oily residue. Full scan GC/MS analyses of GC extracts are shown in Figure 5 as total ion chromatograms (TICs). The peak pattern is similar between the two, with the main peaks consisting primarily of terpenes. These compounds are easily volatilized in the GC inlet, and do not pose issues with system contamination, however they can interfere with mass spectral detection, requiring the use of MS/MS for selectivity. The overall amplitude of the peaks was less after cleanup, as is shown by a 21% reduction in the peak area sums for each in Figure 5.

Undiluted Acetonitrile Extract of Turmeric Powder Before Cleanup
Figure 2. Undiluted Acetonitrile Extract of Turmeric Powder Before Cleanup

Turmeric Extracts at the Same Dilution (167X total); Without Cleanup, and After Cleanup for LC/MS/MS Analysis
Figure 3. Turmeric Extracts at the Same Dilution (167X total); Without Cleanup, and After Cleanup for LC/MS/MS Analysis

Turmeric Extracts at the Same Dilution (5X); Without Cleanup, and After Cleanup for GC/MS/MS Analysis
Figure 4.
Turmeric Extracts at the Same Dilution (5X); Without Cleanup, and After Cleanup for GC/MS/MS Analysis

 

GC/MS Scan Analyses of Turmeric Extracts Before and After Cleanup with Supelclean™ Ultra 2400

Figure 5. GC/MS Scan Analyses of Turmeric Extracts Before and After Cleanup with Supelclean™ Ultra 2400 Shown with same Y-scale. Sum of area counts for all peaks is indicated with each.

Pesticide Recovery and Reproducibility

The average recoveries obtained from spiked turmeric samples (n=3) are presented in Table 5. Of the 51 pesticides spiked, all except hexachlorobenzene had recovery of greater than 70%. Hexachlorobenzene, a pesticide with a planar structure, was recovered at 67% after cleanup. It should be noted that this was without using toluene in the elution solvent, as is necessary to obtain good recoveries from dual-layer cartridges containing graphitized carbon black.5 Although not shown here, higher recovery of hexachlorobenzene has been obtained by loading more turmeric extract (300 μL) on the Supelclean™ Ultra 2400 cartridge. This indicates that the presence of more matrix displaced the hexachlorobenzene, thus reducing its retention on the carbon. However the higher sample loading produced an extract with more color, a sign that the cleanup capacity of the cartridge had been reached or exceeded for this matrix. Reproducibility, calculated as %RSD for the sets of spiked replicates, was less than 20% for 44 of the 51 pesticides. As is indicated in Figure 6, many compounds had RSD values of less than 10%. Pesticides with RSD values greater than 20% were attributed to those showing low response in the MS/MS method.

 

Table 5. Pesticide Recoveries and % RSD Values (n=3) for Spiked Replicates; Turmeric Spiked at 100 ng/g

Pesticide Avg. Recovery RSD Analysis
Alachlor
99% 23% GC/MS/MS
Aldrin 85% 10% GC/MS/MS
Azinphos-methyl 89% 11% GC/MS/MS
γ-BHC 83% 8% GC/MS/MS
Chloropyrifos 96% 12% GC/MS/MS
Chloropyrifos-Methyl 113% 6% GC/MS/MS
Cypermthrin (isomer 1) 99% 15% GC/MS/MS
4,4'-DDT 95% 8% GC/MS/MS
Diazinon 92% 14% GC/MS/MS
Dichlorvos 78% 31% GC/MS/MS
DIsulfoton 86% 7% GC/MS/MS
Endosulfan β 86% 35% GC/MS/MS
Endosulfan-α 92% 23% GC/MS/MS
Ethion 97% 7% GC/MS/MS
Fenitrothion 63% 5% GC/MS/MS
Heptachlor 81% 7% GC/MS/MS
Hexachlorobenzene 67% 9% GC/MS/MS
Iprodione (isomer 1) 103% 5% GC/MS/MS
Malathion 90% 10% GC/MS/MS
Metalaxyl 86% 21% GC/MS/MS
Methoxychlor 78% 12% GC/MS/MS
Mevinphos 73% 7% GC/MS/MS
Parathion-Methyl 88% 8% GC/MS/MS
Permethrin (isomer 1) 104% 24% GC/MS/MS
Phenthoate 89% 7% GC/MS/MS
Phorate 82% 10% GC/MS/MS
Phosalone
90%
7% GC/MS/MS
Pirimiphos-methyl 74% 3% GC/MS/MS
Profenophos 88% 7% GC/MS/MS
Quintozene 75% 8% GC/MS/MS
Vinclozolin 90% 6% GC/MS/MS
Acephate 89% 6% LC/MS/MS
Acetamiprid 102% 4% LC/MS/MS
Boscalid (Nicobifen) 86% 7% LC/MS/MS
Carbendazim (Azole) 106% 7% LC/MS/MS
Chlorbufam 92% 18% LC/MS/MS
Cycluron 103% 5% LC/MS/MS
Diflubenzuron 101% 5% LC/MS/MS
Fenoxanil 91% 10% LC/MS/MS
Fosthiazate 95% 4% LC/MS/MS
Methabenzthiazuron 96% 4% LC/MS/MS
Methamidophos 85% 5% LC/MS/MS
Methomyl 106%
6% LC/MS/MS
Monocrotophos (Azodrin) 97% 3% LC/MS/MS
Nitralin 124% 55% LC/MS/MS
Oxamyl 104% 3% LC/MS/MS
Pirimicarb 97% 3% LC/MS/MS
Procymidon
91% 13% LC/MS/MS
Propaquizafop 97% 1% LC/MS/MS
Tetraconazole 98% 2% LC/MS/MS
Uniconazole-P 103% 19% LC/MS/MS


 Number of Pesticides with Average Recoveries Within Indicated Percent Relative Standard Deviation (%RSD) Ranges After Cleanup

Figure 6. Number of Pesticides with Average Recoveries Within Indicated Percent Relative Standard Deviation (%RSD) Ranges After Cleanup with Supelclean Ultra 2400

Conclusion

Ultra 2400 dual-layer SPE cartridge. The selection of sorbents in this cartridge allows for cleanup of acetonitrile extracts of very difficult samples such as spices and other dry commodities. The Graphsphere™ 2031 carbon used in the upper layer removes/reduces pigmentation while still allowing for recovery of planar pesticides without the use of toluene in the elution solvent. Z-Sep sorbent in the bottom layer of the cartridge removes oils and some pigments, as was indicated in the cleanup of turmeric extracts for both GC and HPLC analysis. Suitable recoveries for a wide range of pesticides of different polarities and classes were obtained from turmeric extract, and minimal background interference was noted. In this work, a 1 mL Supelclean Ultra 2400 cartridge was used. A larger 3 mL version of the cartridge is also available which can accommodate a higher sample loading.

Trademarks

Ascentis and SLB are registered trademarks of Sigma-Aldrich Co. LLC.
Graphsphere, Supel, Supelclean and Visiprep are trademarks of Sigma-Aldrich Co. LLC.
FocusLiner is a trademark of SGE Analytical Science Pty Ltd.

Materials

     

 References

  1. Prasa, S.; Aggarwal, B. Turmeric, the Golden Spice. Herbal Medicine: Biomolecular and Clinical Aspects, 2nd ed.; CRC Press/Taylor&Francis: Boca Raton (FL), 2011; Chapter 13.
  2. Health Canada MRL database. www.hc-sc.gc.ca (accessed 1/27/2016).
  3. Electronic Code of Federal Regulations (eCFR), Title 40, Chapter 1, Subchapter E, Part 180, updated 1/26/2016.
  4. Li, S.; Yuan, W.; Deng, G.; Wang, P.; Yang, P. “Chemical composition and product quality control of turmeric (Curcuma longa L.)” (2011). Faculty Publications. Paper 1. http://scholarworks.sfasu.edu/agriculture_facultypubs/1 (accessed 1/27/2016)
  5. Shimelis, O.; Yang, Y.; Stenerson, K.; Kaneko, T.; Ye, M. Evaluation of Solid-Phase Extraction Dual-layer Carbon/Primary Secondary Amine for Clean-up of Fatty Acid Matrix Components From Food Extracts in Multiresidue Pesticide Analysis. J. Chrom. A, 2007, 1165, 18-25.