c14-mis-p1

Comparison of Two-Dimensional Gas Chromatography and Tandem Mass Spectrometry for Pesticide

Analysis of Herbal Teas

Michelle Misselwitz, Julie Kowalski, Jack Cochran

Herbal Teas Ancient Medicine Nettle - Remedies for an assortment of ailments including anemia, high blood

pressure, rheumatism, arthritis, coughs and colds, congestion, urinary tract infections, and kidney and bladder problems.

Chamomile Tea - Soothes the stomach and relieves bloating and indigestion. Calms the mind and helps people relax and deal better with their stresses.

Ginger Tea - Stimulates and soothes the digestive system. Ginger has been known to aid people experiencing nausea.

Peppermint Tea - Helps you digest foods better and also reduces flatulence and digestive issues. Eases nausea and vomiting, especially if you suffer motion sickness.

Hibiscus Flower Tea (Sorrel) - Lower blood pressure, reduce high cholesterol and strengthen the immune system (its rich in Vitamin C.

Lemon Balm Tea - Effective tonic to calm nerves and anxiety.

Green Tea - the wonder herb lowers cancer risk and contains potent antioxidants. Green tea also lowers blood pressure, prevents and fights tooth decay and dental

issues, and inhibits different viruses from causing illnesses.

Organochlorine

Organonitrogen

Pyrethroid

Triazole

Fungicide

Insecticide Herbicide

Synergist Acaricide

Plant Growth Regulator

Organophosphorus

Herbal Teas Targeted Pesticide Analysis

Wild Berry Zinger Ingredients: Hibiscus, rosehips, roasted chicory, orange peel,

blackberry leaves, natural flavors of black raspberries, strawberries, blueberries, red raspberries, cranberries and

cherries

Pau dArco A Traditional South American Herbal Tea

Contains: Pau dArco bark

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, lycium fruit, licorice root, ginger rhizome, star anise

fruit, kukicha twig (85% Certified Organic Ingredients)

QuEChERS Performance Standards Mix A

Acephate Diazinon

Dichlorvos Dimethoate Malathion

Methamidophos Mevinphos Omethoate Chlorpyrifos Coumaphos

Fenthion Pirimiphos methyl Azinphos methyl

Mix C Carbaryl

Methiocarb Iprodione Metalaxyl Vinclozolin

Captan Myclobutanil Deltamethrin

Bifenthrin cis-Permethrin

Imazalil trans-Permethrin

Cyprodinil Fenpropathrin

Folpet Thiabendazole

Fenhexamid

Mix B Chlorothalonil

4,4'-DDT Dichlofluanid

Dicofol Endrin

o-Phenylphenol Propargite

Endosulfan sulfate Phosalone

gamma-BHC

Red = Tea leaves Purple = Hibiscus Teal = Citrus Italics = Chicory Blue = Ginger

QuEChERS Extraction 1 g ground tea

+ 10 mL H2O

Soak 30 min

+ 10 mL Acetonitrile

+ QuEChERS GC/MS Internal Standard

+ Pesticide spike mix

Vortex 30 min

Add EN Buffer Salts 4 g magnesium sulfate

1 g sodium chloride 1 g trisodium citrate dihydrate 0.5 g disodium hydrogencitrate

sesquihydrate

Shake 1 min

Centrifuge 5 min

500 pg ISTD

Complex Samples

% Recoveries of Fortified Tea at 1000 ng/g (100 pg on-column) quantified using GC-MS

Pau d'Arco Tea Zinger Tea Red Tea Detox Tea

Acephate ND ND ND ND

o-Phenylphenol 86 84 87 89

Omethoate 120 ND ND ND Diazinone 93 79 91 94 Chlorothalonil 62 72 82 79

Vinclozoline 88 81 87 87 Carbaryl 94 80 91 95

Metalaxyl 84 84 86 91

Methiocarb 91 79 92 96

Malathion 100 80 89 93

Fenthion 87 81 89 89

Chlorpyrifos 88 76 90 88 Cyprodinil 80 59 82 80

Thiabendazole 44 54 48 ND Captan 97 86 100 120

Imazalil 81 82 84 100

Endosulfan sulfate 110 76 81 93 Propargite 94 73 101 ND

Iprodione 89 76 94 100 Bifenthrin 86 68 83 87

Fenpropathrin 100 76 90 89 cis-Permethrin 87 67 82 ND

Deltamethrin 92 71 95 110

Cartridge SPE Cleanup

1 mL extract

Combo SPE Cartridge, 6mL/500mg Carboprep,

90/500mg PSA

Add MgSO4 to tube

Elute 15mL acetone: toluene (3:1)

Cartridge SPE Cleanup

1 mL extract

MgSO4

500 mg GCB

500 mg PSA

Elute 15 mL 3:1 Acetone:Toluene

Concentrate to1 mL

Dispersive SPE (dSPE cleanup)

1 mL extract

Shake 2 min

Centrifuge 5 min

Q252 Q213

Guide to Sorbents

Removes Sugars and fatty acids Primary secondary amine (PSA)

Sorbent

Octadecyl (C18) Lipids

Pigments and sterols Graphitized carbon black (GCB)

dSPE Cleanup

150 mg MgSO4, 50 mg PSA, 50 mg C18, 7.5 mg GCB

GCxGC vs GC-MS/MS

Chromatographic Separation Resolution!

Full Spectral Information!

Mass Spectrometer Separation Selectivity! Sensitivity!

GCxGC Schematic

Modulator

1st Dimension 2nd Dimension

From Injector

To Detector

Focuses effluent from primary column (cold jet) and injects this effluent

(hot jet) to secondary column

Fast detector to record 50-500 ms wide peaks

Orthogonal GC column stationary phases means two independent separation mechanisms

Modulator

1st Column 2nd

Column

Inlet

Detector

Linear Chromatogram

2n

d C

olu

mn

Fundamentals of GCxGC

Surface Plot Contour Plot

+ +

+

+

+ +

time m/z

110

245 89 +

+

+

+ + + +

+

+ +

+

+

+ +

+

Q1

+ + + + +

+ +

+ + +

m/z

99

133

68 + + +

+

+

+

+

+ + +

Q3 Q2

Fragment Ions

+

Triple Quadrupole

MSn for Signal-to-Noise Improvement

0 1 2 3 4 5 6

Stages of Analysis

Inte

nsi

ty

Signal

Noise

S/N

Sensitivity

GC X GC Conditions Injection

Temp: 250C Injection:1 L fast, splitless (1.0 min) Liner: Sky Single Taper

Column

Rxi Guard 1.0 m x 0.25 mm Rxi-5ms 30 m x 0.25 mm x 0.25 m Rtx-200 1.0 m x 0.25 mm x 0.25 m Column connector: SGE SilTite -Union

Conditions Oven: 70C (1 min), 8C/min to 330C (6.5 min) Secondary Oven Offset: + 5C Carrier: He, constant flow 1.4 mL/min Modulator Temperature Offset: 20C Modulation Time: 2 sec

LECO Pegasus

TOFMS Conditions

Transfer Line Temperature: 320C

Source Temperature: 225C

Electron Ionization: 70 eV

Stored Mass Range: 45 to 550 u

Acquisition Rate: 100 spectra/sec

GCxGC-TOFMS Conditions

GC-MS/MS Conditions GC Conditions

Injection

Temp: 250C Injection:1 L fast, splitless (1.0 min) Liner: Sky Single Taper with wool

Column

Rxi-5ms 30 m x 0.25 mm x 0.25 m

Conditions Oven: 70C (1 min), 8.7C/min to 330C (5 min) Carrier: He, constant flow 1.4 mL/min

Thermo TSQ 8000 GC-MS/MS

Transfer Line Temperature: 280C

Source Temperature: 250C

Electron Ionization: 70 eV

Acquisition: SRM, 3 per analyte

Total Scan Time: 0.150 sec

Liner choice : wool or no wool?

Wool No Wool

Better response for thermal labile pesticides

Better response for more active pesticides

Controlled sample introduction

Protects GC column from non-volatile residue

Complex Samples

After 28 tea injections!

Use wool in your splitless liner to protect

your GC column from non-volatile dirt

Methamidophos

Dichlorvos

No retention

gap

1 m retention

gap

No Wool - Retention Gap - better solvent focusing



1.0

m x

0.2

5 m

m x

0.2

5

m R

tx-2

00

1.0 m x 0.25 mm Rxi Guard + 30 m x 0.25 mm x 0.25 m Rxi-5ms

RT: 0.0 - 28.9

0 5 10 15 20 25Time (min)

0

50

1000

50

1000

50

1000

50

1000

50

100

Relative A

bundance 0

50

1000

50

1000

50

10011.2

11.012.2

12.3

16.3

16.416.217.1

17.0 17.217.7

17.9

22.8

22.922.625.0

25.2

25.3

16.915.8

17.7 21.722.418.412.2 28.611.18.0

NL: 6.94E4m/z= 42.10-286.00 F: + c EI SRM ms2 [email protected] [42.095-42.105] MS Pau_d_Arco_Spike_10ppbDUP_inj7







NL: 1.67E7TIC MS Pau_d_Arco_Spike_10ppbDUP_inj7



30 m x 0.25 mm x 0.25 m Rxi-5ms

% Recoveries of Spiked Pesticides in Pau dArco Tea (n=2)

Pesticide 1000 ng/g 100 ng/g

GC-MS/MS GCxGC-TOFMS GC-MS/MS GCxGC-TOFMS

Acephate 100 90 78 65


Omethoate 88 96 76 58

Diazinon 101 99 94 99

Chlorothalonil 56 71 65 58

Vinclozolin 96 90 92 84

Carbaryl 61 98 80 65



Malathion 102 100 86 91

Fenthion 98 100 88 90

Chlorpyrifos 94 91 81 87

Cyprodinil 85 86 75 66

Thiabendazole 68 58 66 39

Captan 73 110 90 72

Imazalil 101 80 78 80

Endosulfan sulfate 88 95 87 82

Propargite 89 99 84 70

Iprodione 92 120 92 27

Bifenthrin 92 91 83 77

Fenpropathrin 96 96 93 85

cis-Permethrin 93 100 87 78


o-Phenylphenol

GCxGC - Spectral Deconvolution

50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 135

152

77

63

107

121 169 434 285 399 357 231

Caliper - sample "Pau d'Arco Tea extract:1", 786 , 0.790 sec , sec to 786 , 0.790 sec , sec - 150 , 0.000 sec , sec to 150 , 0.000 sec , sec

50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 65

169

141

115

87

47 434 285 356

Peak True - sample "Pau d'Arco Tea extract:1", peak 292, at 786 , 0.790 sec , sec

50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 170

115 141

63 89 39

155

Library Hit - similarity 948, "o-Phenylphenol"

Caliper

Peak True

Library Hit

0.72786

0.74786

0.76786

0.78786

0.8786

0.82786

0

1000

2000

3000

4000

5000

6000

7000

1st Time (s)2nd Time (s)

170 135

GCxGC - Spectral Deconvolution

50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 135

152

77

63

107

121 169 434 285 399 357 231

Caliper - sample "Pau d'Arco Tea extract:1", 786 , 0.790 sec , sec to 786 , 0.790 sec , sec - 150 , 0.000 sec , sec to 150 , 0.000 sec , sec

50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 65

169

141

115

105 87

434 285 356

Peak True - sample "Pau d'Arco Tea extract:1", peak 292, at 786 , 0.790 sec , sec

50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 170

115

141

63

89 74

102 155

Reference Spectrum - Calibration "QuEChERS Perf AB 500", Analyte "o-Phenylphenol"

Caliper

Peak True

Reference Spectrum

o-P

hen

ylp

hen

ol

un

kno

wn

MS/MS Selectivity

141 > 115.1 m/z 169.7 > 115.1 m/z 169.7 > 141.3 m/z

(mainlib) o-Hydroxybiphenyl

20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 1800

50

100

27

39 51

52 5558

62

63

65 7077 87

89

91 98102

115

126 131

141

151 155

170

OH

o-phenylphenol

Pau dArco

A Traditional South American Herbal Tea Contains: Pau dArco bark

Average % Recovery for ALL target pesticides

Spike GC-MS/MS GCxGC-TOFMS

1000 ng/g 93% ( 15) 92% ( 14)

100 ng/g 87% ( 12) 81% ( 17)

10 ng/g 84% ( 24) NA

GCxGC-TOFMS Thiabendazole (130) Fenpropathrin (43)

Incurred Pesticides (ng/g):

GC-MS/MS Thiabendazole (16)

Imazalil (13)

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

% Recoveries of Spiked Pesticides in Wild Berry Zinger Tea



Acephate 76 88 88 81



Diazinon 85 93 86 95



Carbaryl 98 94 89 91




Fenthion 84 88 85 98




Captan 86 99 102 180

Imazalil 86 91 102 91








Ingredients: Hibiscus, rosehips, roasted chicory, orange peel, blackberry leaves, natural flavors

Wild Berry Zinger

Average % Recovery for ALL target pesticides


1000 ng/g 79% ( 10) 90% ( 11)

100 ng/g 83% ( 12) 94% ( 33)

10 ng/g 88% ( 19) NA

GCxGC-TOFMS Carbaryl (10)

Chlorpyrifos (16) Imazalil (727)

Thiabendazole (429) o-Phenylphenol (22)


GC-MS/MS Carbaryl (23)

Chlorpyrifos (18) Imazalil (470)

Thiabendazole (240) Fenhexamid (2)

44-DDT (1)

Red Tea Cinnamon Orange Ingredients: Rooibos, cinnamon, orange

peel, natural cinnamon and orange flavors

% Recoveries of Spiked Pesticides in Red Tea



Acephate 69 110 74 80


Omethoate 58 120 66 100




Carbaryl 83 110 82 85




Fenthion 83 110 84 91




Captan 89 140 76 160

Imazalil 73 120 76 88








Rooibos, cinnamon, orange peel, natural cinnamon and orange flavors


1000 ng/g 83% ( 8) 110% ( 13)

100 ng/g 87% ( 12) 91% ( 14)*

10 ng/g 90% ( 15) NA

GCxGC-TOFMS o-Phenylphenol (25)


GC-MS/MS o-Phenylphenol (20)

Endosulfan sulfate (2) Iprodione (2) Imazalil (2)

Thiabendazole (8)

Red Tea Cinnamon Orange

Red Tea Cinnamon Orange Incurred Imazalil

2 ng/g = 0.2 pg on-column

Quant peak 215 > 173 m/z

Confirming Ion Ratio 20% 3 SRM transitions

Everyday Detox Ingredients: roasted chicory root, dandelion root, schisandra fruit, lycium fruit, licorice root, ginger

rhizome, star anise fruit, kukicha twig

GCxGC Resolution

Acephate m/z 136

Acephate MS/MS Selectivity

Quant peak 136 > 42.1 m/z

100 pg on-column

10 pg on-column

Corruption of peak from

interferences

Confirming ions 94 > 64 m/z

136 > 94 m/z

Acephate has a skinny mass spectrum. There is not a lot of high abundance, high m/z values for more selective fragmentation

% Recoveries of Spiked Pesticides in Everyday Detox Tea



Acephate 75 88 81 89






Carbaryl 86 97 74 100




Fenthion 87 98 81 100




Captan 82 100 82 100

Imazalil 84 75 63 72








roasted chicory root, dandelion root, schisandra fruit, lycium fruit, licorice root, ginger rhizome, star anise

fruit, kukicha twig


1000 ng/g 85% ( 28) 89% ( 10)

100 ng/g 79% ( 13) 97% ( 24)

10 ng/g 87% ( 19) NA

GCxGC-TOFMS None Found


GC-MS/MS gamma-BHC (1) Methiocarb (6)

Everyday Detox

GCxGC TOFMS Benefits/Drawbacks

+ Non- target screening possible + High identification confidence: 2 retention times and full

mass spectrum

+ Interferences: simple reprocessing of data with different quant ion

- More difficult GCxGC hardware setup and method development

- Higher detection limits - Large data files and slower data processing

GC-MS/MS Benefits / Drawbacks

+ Lower Detection Limits most compounds easily detected at 1 pg on-column

+ Faster data processing/smaller file sizes + Easier hardware setup - Targeted screening only - More difficult compound identification (management of

ion ratios and allowable deviation)

- Less identification confidence: one retention time and limited mass spectral information

- Time consuming MS method development

Conclusions

A QuEChERS extraction and dSPE cleanup of herbal teas resulted in good recoveries of target pesticides.

Using GC-MS/MS allowed detection of target pesticides at 10 ng/g.

Non-target screening of active ingredients is possible with GCxGC-TOFMS.

Average percent recoveries were similar for both instrument platforms.

Non-Target Analysis

Mat Lemon Ingredients: Yerba mat, lemon myrtle, green tea

Its stimulating effect evokes a cheerful mental lucidity

Sceletium & Honeybush Tea Ingredients: Sceletium and african honeybush

The cup that cheers

elev8 Ingredients: Zembrin 25 mg, equivalent to 50 mg

Sceletium tortuosum above ground herb

Mat Lemon Ingredients: Yerba mat, lemon

myrtle, green tea

Peak Find, Spectral Deconvolution, Library Search

Set S/N, automatic deconvolution

Sort by Similarity for Qualitative Review

Mat Lemon Ingredients: Yerba mat, lemon

myrtle, green tea

1425 peaks S/N 100

Some names are easily recognized.

Qualitative Review

100 200 300 400 500

1000 67

194

Caliper - sample "Mate Lemon Tea extract:1", 1206 , 1.190 sec , sec to 1206 , 1.190 sec , sec - 400 , 0.000 sec , sec to 400 , 0.000 sec , sec

100 200 300 400 500

500

1000 67

194

Peak True - sample "Mate Lemon Tea extract:1", peak 931, at 1206 , 1.190 sec , sec

100 200 300 400 500

500

1000 194

109

55

Library Hit - similarity 941, "Caffeine"

Caffeine

Others may need further investigation.

Qualitative Review

50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 78

51

122

106

94


50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 78

51

122

106

94 67


50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 122

78

106

51

94 66

Library Hit - similarity 912, "Niacinamide"

Niacinamide is part of the vitamin

B group. It has anti-inflammatory,

anti-anxiety properties.

Be critical with NIST library matches. Is it real? More unique

spectrum with abundant high m/z

ions gives better confidence in match.

Recognizing chemical names can sometimes be difficult. Using the synonym list is helpful. Pyridine, 2-chloro-6-(2-furanylmethoxy)-4-

(trichloromethyl)- is also known as Pyroxyfur, a fungicide.

Qualitative Review

Qualitative Review

50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 81

53

107 71


50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 81

53

107


50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 81

53

Library Hit - similarity 910, "Pyroxyfur"

Pyroxyfur is a pyridine fungicide.

With less spectral information it is difficult to assign a positive match.

50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 81

53

107 71


45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125

200

400

600

800

1000 81

53

51 107 55


45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125

200

400

600

800

1000 81

53

51

Library Hit - similarity 910, "Pyridine, 2-chloro-6-(2-furanylmethoxy)-4-(trichloromethyl)-"

Peak True matches Library Hit at low m/z values

Peak True

Library Hit

Qualitative Review

50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 81

53

107 71


50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 81

53

107


50 100 150 200 250 300 350 400 450 500 550

200

400

600

800

1000 81

53

Library Hit - similarity 910, "Pyroxyfur"

With less spectral information it is difficult to assign a positive match.

305 310 315 320 325 330 335 340 345 350

200

400

600

800

1000


305 310 315 320 325 330 335 340 345 350

5

10

15

20


305 310 315 320 325 330 335 340 345 350

5

10

15

20

Library Hit - similarity 910, "Pyridine, 2-chloro-6-(2-furanylmethoxy)-4-(trichloromethyl)-"

No molecular weight ion in Peak True spectrum

Pyroxyfur MW = 325

Peak True

Library Hit

Flavonoids polyphenolic compounds that may reduce

the risk of certain cancers.

Structured Chromatograms Sceletium & Honeybush

Tea

Phytosterols may help reduce blood cholesterol

levels.

Structured Chromatograms Sceletium & Honeybush

Tea

-Sitosterol

Stigmasterol Campesterol

Structured Chromatograms

Phytosterols Sceletium & Honeybush

Tea

The South African Sceletium tortuosum herb has been extracted for the medicinally relevant mesembrine-type alkaloids.

Mesembranol Mesembrine Mesembrenol Mesembrenone

The mesebrine-type alkaloids have shown to elevate mood, reduce stress

and promote a sense of calm

m/z 204, 218, 289

Mesembrine-type alkaloids elev8TM

elevates mood, assists in coping with stress

Sceletium and Honeybush Tea

The Cup that Cheers

The minor mesembrine alkaloid mesembranol was not detected in the tea sample, however many other beneficial compounds (phytosterols, flavonoids) that were present

in the tea were not in the tablet.

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