Multi-Residue Pesticide Analysis in Herbal Teas Using the QuEChERS Method and Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry

Michelle Misselwitz, Jack Cochran, Julie Kowalski; Restek Corporation, 110 Benner Circle; Bellefonte, PA, USA

Herbal tea, a non-caffeinated drink made from plants, herbs, or spices has been used throughout history for its potential medicinal benefit.

Used frequently in Traditional Chinese Medicine (TCM) different blends of herbal material will be formulated depending on the desired medicinal properties. As with any plant based commodity, there is the potential for pesticide residues to remain in the final product.

Dried plant material found in herbal tea poses a significant challenge to the analytical chemist to detect trace levels of pesticide residues.

The extract, even after sample cleanup, can contain a large amount of coextractive material that can completely overwhelm the target pesticides, making trace detection very difficult.

Nonvolatile material not removed during extract cleanup deposit onto the inlet and column requiring more frequent maintenance to be performed.

We employed the QuEChERS methodology for a quick extraction and cleanup of store bought herbal teas.

The percent recoveries of spiked pesticide standards were determined using both one dimensional GC and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS).

Introduction

LECO Pegasus 4D GCxGC-TOFMS 1’ Column: 1 m x 0.25 mm Rxi Guard column connected to a 30m x 0.25mm x 0.25µm Rxi-5ms 2’ Column: 1 m x 0.25 mm x 0.25 µm Rtx-200 Column connector: SGE SilTite µ-Union Inlet: 250°C, 1µL splitless (1 min), Sky Single Taper Liner Oven: 70°C (1 min) to 330°C (6.5 min) @ 8°C/min, secondary oven + 5°C offset Carrier: He, corrected constant flow, 1.4 mL/min Modulation: 2 sec Acquisition: 45 – 550 u @ 100 spectra/sec

GC-TOFMS analyses were performed using a 1 m x 0.25 mm Rxi Guard column connected to a 15m x 0.25 mm x 0.25 µm Rxi-5ms and a fast GC oven temperature program.

Table I: Percent recoveries of a subset of spiked target pesticides with relevance to the herbal teas analyzed. Results of both 1D GC-TOFMS and GCxGC-TOFMS analysis. Pau d'Arco Tea Wild Berry Zinger Tea Red Tea Everyday Detox Tea

GC 1 µg/g (n=2)

GCxGC 1 µg/g (n=2)

GCxGC 0.1 µg/g (n=2)

GC 1 µg/g

GCxGC 1 µg/g

GCxGC 0.1 µg/g

GC 1 µg/g

GCxGC 1 µg/g

GCxGC 0.1 µg/g

GC 1 µg/g

GCxGC 1 µg/g

GCxGC 0.1 µg/g

Acephate ND 90 65 ND 88 81 ND 110 80 ND 88 89 o-Phenylphenol 86 87 100 84 92 95 87 98 96 89 88 100 Omethoate 120 96 58 ND 78 88 ND 120 100 ND 85 93 Hexachlorobenzene 59 63 71 53 62 62 66 74 76 61 61 71 Dimethoate 120 97 84 102 94 96 100 110 110 103 92 110 Simazine 88 94 86 80 90 110 90 110 110 88 90 96 Atrazine 89 96 90 78 92 96 92 91 91 ND 98 99 Diazinone 93 99 99 79 93 95 91 99 88 94 88 96 Chlorothalonil 62 71 58 72 79 100 82 110 71 79 74 99 Vinclozoline 88 90 84 81 99 91 87 100 83 87 93 110 Carbaryl 94 98 65 80 94 91 91 110 85 95 97 100 Metalaxyl 84 100 97 84 96 90 86 110 87 91 100 88 PCB 52 ISTD ISTD ISTD ISTD ISTD ISTD ISTD ISTD ISTD ISTD ISTD ISTD Methiocarb 91 100 73 79 90 89 92 120 102 96 96 120 Malathion 100 100 91 80 95 94 89 110 77 93 98 67 Fenthion 87 100 90 81 88 98 89 110 91 89 98 100 Chlorpyrifos 88 91 87 76 93 88 90 100 90 88 88 110 Cyprodinil 80 86 66 59 71 73 82 97 79 80 83 81 Thiabendazole 44 58 39 54 62 77 48 87 75 ND 75 86 Captan 97 110 72 86 99 180 100 140 160 120 100 100 Imazalil 81 80 80 82 91 91 84 120 88 100 75 72 Endosulfan sulfate 110 95 82 76 95 100 81 120 84 93 99 100 Propargite 94 99 70 73 96 110 101 110 92 ND 90 83 Iprodione 89 120 27 76 99 74 94 120 92 100 90 100 Bifenthrin 86 91 77 68 86 82 83 98 85 87 85 84 Fenpropathrin 100 96 85 76 93 93 90 110 76 89 90 100 Azinphos-methyl 92 110 60 69 85 100 84 140 87 92 98 81 cis-Permethrin 87 100 78 67 88 70 82 99 92 ND 87 97 Deltamethrin 92 94 45 71 88 78 95 120 120 110 90 93

500 pg ISTD

1 µg/mL = 92% (± 14) 0.1 µg/g = 81% (± 17)

Incurred Pesticides: Hexachlorobenzene 7.6 ng/g;

Thiabendazole 59 ng/g; Fenpropathrin 57 ng/g

1 µg/mL = 90% (± 11) 0.1 µg/g = 94% (± 33)

Incurred Pesticides: o-Phenylphenol 17 ng/g;

Carbaryl 5.1 ng/g; Chlorpyrifos 16 ng/g; Thiabendazole 190 ng/g; Imazalil 280 ng/g

1 µg/mL = 110% (± 13) 0.1 µg/g = 91% (± 14)*

*Dichlofluanid excluded from calculation due to high bias (460%)

Incurred Pesticides: o-Phenylphenol 24 ng/g

1 µg/mL = 89% (± 10) 0.1 µg/g = 97% (± 24)

Incurred Pesticides: None found, 85% Organic Ingredients

A variety of herbal teas were purchased at a local grocery store and grinded into a powder. 1 g of tea was weighed into 50 mL centrifuge tubes and 10 mL of water was added to the samples.

The water was allowed to hydrate the tea for 30 min. A 100 µL spike of a mixed pesticide standard (10 ng/µL and 1 ng/µL) was added for the spiked

samples. An internal standard was added to every sample. 10 mL of acetonitrile was added to the centrifuge tube and vortexed for 30 min. EN QuEChERS Method extraction salt formulation packets were added to each sample and shaken

for 1 min and subsequently centrifuged for 5 min. A 1 mL aliquot of the supernatant was used for dispersive solid phase extraction (dSPE) containing

150 mg MgSO4, 50 mg PSA, 50 mg C18, 7.5 mg GCB. The dispersive tube was gently shaken for 2 min and centrifuged for 5 min. 5 µL of a 5% formic acid solution in acetonitrile was immediately added for every 0.5 mL of extract.

Experimental

Results and Discussion

Pau d’Arco A Traditional South American Herbal Tea

Contains: Pau d’Arco bark

Wild Berry Zinger Ingredients: Hibiscus, rosehips, roasted chicory, orange peel, blackberry leaves, natural flavors…

Red Tea – Cinnamon Orange Ingredients: Rooibos, cinnamon, orange peel,

natural cinnamon and orange flavors

Everyday Detox Ingredients: roasted chicory root, dandelion

root, schisandra fruit, lycium fruit, licorice root, ginger rhizome, star anise fruit, kukicha twig

The herbal tea that was purchased varied in complexity and ingredients. Target pesticides were determined based on MRL information available for the ingredients.

The pesticide mix evaluated contained 53 pesticides ranging in volatility and covered many of the GC amenable pesticides found in tea or the other ingredients included in the herbal teas.

The sample extracts were first evaluated by one dimensional GC-TOFMS, however the samples were very complex and made pesticide recovery determination difficult ( Figure 1).

Modulation, a quick cryogenic trapping and injection of analyte bands onto the secondary column in the GCxGC system, focuses peaks near the detector and increases the signal-to-noise.

The 0.1 µg/g fortification level equates to 10 pg on-column for 100% recovery, the analysis would not be possible with a 1D GC-MS analysis.

The extra resolution provided by GCxGC-TOFMS allowed the use of the less solvent and labor intensive dispersive SPE cleanup (Figure 2). The EN QuEChERS extraction with the universal dSPE cleanup and GCxGC-TOFMS determination provided good recoveries for both

fortification levels, 1 µg/g and 0.1 µg/g (Table I). A decrease in recovery was observed for the planar pesticides hexachlorobenzene and chlorothalonil when compared to a dSPE

formulation without GCB. The use of the universal dSPE formulation containing 7.5 mg of GCB was important to reduce pigment that can quickly degrade GC performance.

The 1 m x 0.25 mm guard column (retention gap) protected the analytical column from the nonvolatile residue remaining in the samples. The inlet liner becomes very dirty after only 28 sample injections (Figure 3).

Peak shape of early eluting pesticides were also improved with the use of the retention gap (Figure 4).

Figure 2: GCxGC-TOFMS contour

plots of the herbal tea samples after

QuEChERS extraction and dSPE cleanup highlights the complexity of

the samples.

GCxGC-TOFMS overall average % recoveries for all 53 pesticides analyzed and quantified incurred

pesticides

Figure 1: GC-TOFMS analysis of Everyday Detox tea. The 500 pg internal standard, PCB 52, illustrates the overwhelming amount of

matrix and the difficulty to analyze pesticides at even 1 µg/g ( 100 pg on-column).

Figure 3: After 28 injections of tea samples, nonvolatile residue was

present on the liner. After changing the liner GC performance was

restored.

Methamidophos

Dichlorvos

No retention

gap

1 m retention

gap

Figure 4: The use of the retention gap allows for better

solvent focusing and improves the peak shape of the early eluting pesticides

while protecting the analytical column.