Residual Pesticides Analysis of Botanical Ingredients Using Gas Chromatography Triple Quadrupole Mass Spectrometry

Originally published by Shimadzu Corporation in collaboration with Restek Corporation.

Introduction

The use of dietary supplements is increasing in the United States. These dietary supplements are made from various dried botanicals, which may contain residual pesticides from agricultural practices, and because of that, they have to be monitored to ensure their quality and prevent exposure. The ingredients are dried which requires the standard QuEChERS methods [1] to be modified to overcome this difficulty. Gas chromatography is the best technique to separate multicomponents including coextracted interferences, and because the triple quadruple mass spectrometer (GC-MS/MS) is highly sensitive this allows analysis of trace level contamination. In this study, we analyzed over 200 compounds simultaneously using a triple quadrupole gas chromatograph mass spectrometer with the modified QuEChERS method.

Materials and Methods

Pesticides standards, internal standards, quality control standards and a QuEChERS kit were obtained from Restek:

• GC Multiresidue Pesticide Kit (cat.# 32562)
• QuEChERS Internal Standard Mix for GC-MS Analysis (cat.# 33267)
• SV Internal Standard Mix (cat.# 31206)
• Q-sep QuEChERS Extraction Kit (Original cat.# 23991 discontinued; see cat.# 25848)

The total number of targets was 232 compounds (220 pesticides, 6 internal standards, and 6 quality control standards). Ginseng, which can be purchased in any store, was used as a matrix. Using this ginseng, matrix-matched calibration standards (1 to 200 ng/mL) and fortified samples (each two 10 and 50 ng/g) were prepared. Calibration curves were generated by an internal standard method, weighted 1/C and the internal standard was PCB52.

An MRM analytical method was created using the Smart Pesticides Database (Shimadzu). This database has retention indices for all registered compounds, and retention times can be predicted by running an n-alkane sample mixture (AART: Automatic Adjustment of Retention Time). According to estimated retention times, Smart MRM creates an optimum data acquisition time program (Figure 1).

Extraction and Clean-up Procedure

• Weigh 1.0 ± 0.05 g ground ginseng powder into 50 mL polypropylene centrifuge tube.
• Add 10 mL HPLC-grade water and vortex the tube vigorously.
• Add 10 mL of the ACN/IS Extraction Solvent.
• Allow the tube to sit for 15 min.
• Add 4 g anhydrous MgSO₄ and 1 g NaCl.
• Shake the tube vigorously on a mechanical shaker for 30 min.
• Centrifuge the 50 mL tubes at 3000–4500 rpm × 5 min.
• Condition the GCB/PSA (0.25 g/0.5 g) SPE columns with ~250 mg anhydrous Na₂SO₄ on top using 3 column volumes of acetone.
• Insert a collection rack consisting of 15 mL disposable glass centrifuge tube on a SPE vacuum manifold.
• Add 1.25 mL of the ACN extract.
• Rinse with 1 mL acetone.
• Elute with 12 mL of 3:1 v/v acetone:toluene.
• Evaporate (50 °C) the eluent to ~100 μL gently.
• Add 500 μL of toluene to the Blank/fortified samples, calibration standard solutions to matrix matched calibration standards.
• Add 20 μL quality control standards (12.5 μg/mL) and ~50 mg of anhydrous MgSO₄ to all samples.
• Vortex for 5 sec.
• Centrifuge the tubes at 3000 g × 5 min.
• Transfer the toluene extract using a Pasteur pipette to ALS GC vials.

Result and Discussion

Matrix Matched Calibration

The chromatogram in Figure 2 shows a 10 ng/mL matrix matched calibration standard. Of the 232 compounds, 230 could be detected in ±0.1 min of estimated retention time by AART. The remaining two compounds, 1,4-Dichlorobenzene-d4 and Naphthalene-d8 of six quality control standards, had eluted before 4 min. Although retention times were shifted, they were with identified within about ±0.2 min of estimated.

Column	Rxi-5ms, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13423)
	using Rxi guard column 5 m, 0.25 mm ID (cat.# 10029)
	with SilTite μ-Union connector kit (cat.# 23885)
Standard/Sample	GC Multiresidue pesticide kit (cat.# 32562)
Diluent:	Toluene
Conc.:	10 ng/mL
Injection
Inj. Vol.:	2 µL pulsed splitless
Liner:	Topaz 3.5 mm ID single taper inlet liner w/wool (cat.# 23336)
Inj. Temp.:	250 °C
Pulse Pressure:	36 psi (248.2kPa)
Pulse Time:	1.5 min
Oven
Oven Temp.:	90 °C (hold 1 min) to 130 °C at 30 °C/min to 330 °C at 10 °C/min (hold 2 min)
Carrier Gas	He, constant linear velocity
Linear Velocity:	55 cm/sec

Detector	Shimadzu GCMS TQ8040
Transfer Line Temp.:	290 °C
Analyzer Type:	Quadrupole
Source Temp.:	230 °C
Electron Energy:	70 eV
Ionization Mode:	EI
Instrument	Shimadzu GCMS-TQ8040
Notes	Matrix-matched calibration standards were prepared in ginseng. Calibration curves were generated by the internal standard method using PCB52 as the internal standard. An MRM analytical method was created using the Shimadzu pesticides database. Although the multiresidue pesticides kit mixes are formulated to ensure maximum long-term stability and reliability as packaged, stability may become an issue when a large number of compounds with different chemical functionalities are combined together into a single mix. This should be taken into consideration for quantitative analysis.
Acknowledgement	Chromatogram provided by Shimadzu. Publication 3655-11615-10ANS (C146-E334), First Edition, December 2016. Residual Pesticides Analysis of Botanical Ingredients Using Gas Chromatography Triple Quadrupole Mass Spectrometry. Riki Kitano, Tairo Ogura, Nicole Lock, Robert Clifford, Julie Kowalski, Jack Cochran, Dan Li

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Calibration curves were generated from matrix matched calibration standards, then back calculation and linearity were evaluated.

Back calculation was performed by calculating the concentration of each calibration point, and if the concentration exceeds ±20% of theoretical value, the calibration point would be interpolated with the nearest two points. Over 93% of the compounds with concentration of 1 ng/mL were within ±20% of theoretical calculations and all compounds of concentration 20 to 200 ng/mL were within ±20% (Figure 3).

This modified QuEChERS method contains dilution steps, and samples will be diluted by quarter. To quantify 10 ng/g concentration, 2 ng/g or lower calibration point are required. Even at low concentrations, the calibration curves show good linearity (Figure 4) and all coefficients of determination (220 pesticides) were greater than 0.99.

Recovery of Fortified Samples

Each two 10 ng/g and 50 ng/g fortified samples were prepared (10 ng/g-1, 10 ng/g-2, 50 ng/g-1, 50 ng/g-2) and these recovery rates were evaluated from the average of three successive data points for each sample.

Of the compounds, 85% showed good recovery within the range of 70 to 120% on 10 ng/g-1 and 50 ng/g-2. As mentioned previously, fortified samples were diluted to 2.5 ng/mL and 12.5 ng/mL. Since calibration curves showed good linearity at low concentration and modified QuEChERS method suppressed interference, good recovery results were achieved. (Some compounds were not quantified correctly because the matrix for the calibration standards originally contained them. Y-intercept were lifted up and this shift might cause incorrect quantification, especially at low concentration. From the standard addition method, 16 pesticides were detected with more than 10 ng/g in matrix.)

In this study, recovery results were rechecked and combined with qualitative information, the relative ion ratio. Ion ratios between the target and reference were compared to that of the 100 ng/g standard and evaluated according to SANCO/12571/2013 [2]). This mentions that the use of relative ratio ±30% as a criterion is recommended.

Table I shows recovery and relative ion ratio for all compounds and Figure 5 shows the combination map of recovery and relative ion ratio, which was generated from this table. Of the compounds, 76% in the 10 ng/g-1 were within ±30% on relative ion ratio with good recovery of between 70 to 120%. For 50 ng/g-2, 83% of the compounds were within a ±30% relative ion ratio. And here, compounds which showed poor recovery and/or over ±30% relative ion ratio were examined.

Table I: Recovery and Relative Ion Ratio; Relative ion ratios were calculated by those of 100 ng/mL standard solution.

Compounds outside the red box (Figure 5) were classified to four groups.

Group A showed low recovery; this group consisted mainly of compounds which have a low boiling point. They may have been lost in the evaporation step. Group B showed high relative ion ratios and this was caused by interference from matrix. Group C showed high recovery; this group consisted of 10 ng/g fortified sample. Some of these were in matrix originally and quantified incorrectly. Others caused by their transitions which had low response and low stability. Group D showed low relative ion ratio. It was necessary to set higher response transitions. By modifying some procedures and parameters, positions of these compounds may improve.

Conclusion

This study shows that the modified QuEChERS method combined with GC-MS/MS achieved consistent pesticides monitoring in botanical ingredients.

Although dried sample could make a heavy and difficult matrix, the modified QuEChERS method, SPE column cleanup, and toluene dilution steps suppressed interference from matrix. The GC-MS/MS detected very low amounts of pesticides even though the sample was diluted. This analytical method takes only 30 minutes in total run time and covers over 200 pesticides. It provides a high throughput solution in laboratories doing this type of analysis.

References

[1] M. Anastassiades, S. J. Lehotay, D. Štajnbaher, F. J. Schenck, Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning and “Dispersive Solid-Phase Extraction” for the Determination of Pesticide Residues in Produce, J. AOAC Int., 86 (2003) 412–431
[2] European Commission, Health & Consumer Protection Directorate-General, Guidance document on analytical quality control and validation procedures for pesticide residues analysis in food and feed, SANCO/12571/2013