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

Transcription

1 Comparison of Two-Dimensional Gas Chromatography and Tandem Mass Spectrometry for Pesticide Analysis of Herbal Teas Michelle Misselwitz, Julie Kowalski, Jack Cochran

2 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 (it s 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.

3 Plant Growth Regulator Organonitrogen Pyrethroid Insecticide Herbicide Fungicide Synergist Organophosphorus Acaricide Organochlorine Triazole

4 Herbal Teas Targeted Pesticide Analysis 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 of black raspberries, strawberries, blueberries, red raspberries, cranberries and cherries 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)

5 Mix A Acephate Diazinon Dichlorvos Dimethoate Malathion Methamidophos Mevinphos Omethoate Chlorpyrifos Coumaphos Fenthion Pirimiphos methyl Azinphos methyl QuEChERS Performance Standards 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 Mix C Carbaryl Methiocarb Iprodione Metalaxyl Vinclozolin Captan Myclobutanil Deltamethrin Bifenthrin cis-permethrin Imazalil trans-permethrin Cyprodinil Fenpropathrin Folpet Thiabendazole Fenhexamid

6 QuEChERS Extraction 1 g ground tea 10 ml H 2 O Vortex 30 min Shake 1 min Soak 30 min Centrifuge 5 min 10 ml Acetonitrile QuEChERS GC/MS Internal Standard Pesticide spike mix Add EN Buffer Salts 4 g magnesium sulfate 1 g sodium chloride 1 g trisodium citrate dihydrate 0.5 g disodium hydrogencitrate sesquihydrate

7 Complex Samples 500 pg ISTD

8 % 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 Omethoate 120 ND ND ND Diazinone Chlorothalonil Vinclozoline Carbaryl Metalaxyl Methiocarb Malathion Fenthion Chlorpyrifos Cyprodinil Thiabendazole ND Captan Imazalil Endosulfan sulfate Propargite ND Iprodione Bifenthrin Fenpropathrin cis-permethrin ND Deltamethrin

9 Cartridge SPE Cleanup 1 ml extract Combo SPE Cartridge, 6mL/500mg Carboprep, 90/500mg PSA Add MgSO 4 to tube Elute 15mL acetone: toluene (3:1)

10 Cartridge SPE Cleanup Elute 15 ml 3:1 Acetone:Toluene 1 ml extract MgSO mg GCB 500 mg PSA Concentrate to1 ml

11 Dispersive SPE (dspe cleanup) 1 ml extract Guide to Sorbents Q213 Sorbent Q252 Primary secondary amine (PSA) Octadecyl (C18) Graphitized carbon black (GCB) Removes Sugars and fatty acids Lipids Pigments and sterols Shake 2 min Centrifuge 5 min dspe Cleanup 150 mg MgSO 4, 50 mg PSA, 50 mg C18, 7.5 mg GCB

12 GCxGC vs GC-MS/MS Chromatographic Separation Resolution! Full Spectral Information! Mass Spectrometer Separation Selectivity! Sensitivity!

13 GCxGC Schematic From Injector Modulator Focuses effluent from primary column (cold jet) and injects this effluent (hot jet) to secondary column To Detector 1 st Dimension 2 nd Dimension Fast detector to record ms wide peaks Orthogonal GC column stationary phases means two independent separation mechanisms

14 2 nd Column Fundamentals of GCxGC Linear Chromatogram Inlet Modulator Detector 1 st Column 2 nd Column Surface Plot Contour Plot

15 Triple Quadrupole Q1 Q2 Q Fragment Ions time m/z m/z

16 Intensity MS n for Signal-to-Noise Improvement Sensitivity Signal Noise S/N Stages of Analysis

17 GCxGC-TOFMS Conditions GC X GC Conditions Injection Temp: 250 C Injection:1 µl 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 µm Rtx m x 0.25 mm x 0.25 µm Column connector: SGE SilTite µ-union Conditions Oven: 70 C (1 min), 8 C/min to 330 C (6.5 min) Secondary Oven Offset: 5 C Carrier: He, constant flow 1.4 ml/min Modulator Temperature Offset: 20 C Modulation Time: 2 sec LECO Pegasus TOFMS Conditions Transfer Line Temperature: 320 C Source Temperature: 225 C Electron Ionization: 70 ev Stored Mass Range: 45 to 550 u Acquisition Rate: 100 spectra/sec

18 GC-MS/MS Conditions GC Conditions Injection Temp: 250 C Injection:1 µl fast, splitless (1.0 min) Liner: Sky Single Taper with wool Column Rxi-5ms 30 m x 0.25 mm x 0.25 µm Conditions Oven: 70 C (1 min), 8.7 C/min to 330 C (5 min) Carrier: He, constant flow 1.4 ml/min Thermo TSQ 8000 GC-MS/MS Transfer Line Temperature: 280 C Source Temperature: 250 C Electron Ionization: 70 ev Acquisition: SRM, 3 per analyte Total Scan Time: sec

19 Liner choice : wool or no wool? Wool No Wool Protects GC column from non-volatile residue Better response for more active pesticides Controlled sample introduction Better response for thermal labile pesticides

20 Complex Samples After 28 tea injections! Use wool in your splitless liner to protect your GC column from non-volatile dirt

21 No Wool - Retention Gap - better solvent focusing No retention gap Methamidophos Dichlorvos 1 m retention gap

22 1.0 m x 0.25 mm x 0.25 µm Rtx-200 Pau d Arco A Traditional South American Herbal Tea Contains: Pau d Arco bark 1.0 m x 0.25 mm Rxi Guard 30 m x 0.25 mm x 0.25 µm Rxi-5ms

23 Pau d Arco A Traditional South American Herbal Tea Contains: Pau d Arco bark N m m [4 P N m m [1 P N m m [1 P N m m [1 P N m m [1 P N m m [2 P N m m [1 P Time (min) m x 0.25 mm x 0.25 µm Rxi-5ms N T P

24 Pesticide % Recoveries of Spiked Pesticides in Pau d Arco Tea (n=2) 1000 ng/g 100 ng/g GC-MS/MS GCxGC-TOFMS GC-MS/MS GCxGC-TOFMS Acephate o-phenylphenol Omethoate Diazinon Chlorothalonil Vinclozolin Carbaryl Metalaxyl Methiocarb Malathion Fenthion Chlorpyrifos Cyprodinil Thiabendazole Captan Imazalil Endosulfan sulfate Propargite Iprodione Bifenthrin Fenpropathrin cis-permethrin Deltamethrin

25 GCxGC - Spectral Deconvolution Caliper - sample "Pau d'arco Tea extract:1", 786, sec, sec to 786, sec, sec Caliper Peak True - sample "Pau d'arco Tea extract:1", peak 292, at 786, sec, sec Library Hit - similarity 948, "o-phenylphenol" Peak True Library Hit o-phenylphenol

26 unknown o-phenylphenol 7000 GCxGC - Spectral Deconvolution Caliper - sample "Pau d'arco Tea extract:1", 786, sec, sec Caliper Peak True - sample "Pau d'arco Tea extract:1", peak 292, at Reference Spectrum - Calibration "QuEChERS Perf AB 500", Analyte Peak True Reference Spectrum st Time (s) 2nd Time (s)

27 MS/MS Selectivity 141 > m/z > m/z > m/z 100 o-phenylphenol 170 OH (mainlib) o-hydroxybiphenyl

28 Pau d Arco A Traditional South American Herbal Tea Contains: Pau d Arco 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 Incurred Pesticides (ng/g): GC-MS/MS Thiabendazole (16) Imazalil (13) GCxGC-TOFMS Thiabendazole (130) Fenpropathrin (43)

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

30 % Recoveries of Spiked Pesticides in Wild Berry Zinger Tea Pesticide 1000 ng/g 100 ng/g GC-MS/MS GCxGC-TOFMS GC-MS/MS GCxGC-TOFMS Acephate o-phenylphenol Omethoate Diazinon Chlorothalonil Vinclozolin Carbaryl Metalaxyl Methiocarb Malathion Fenthion Chlorpyrifos Cyprodinil Thiabendazole Captan Imazalil Endosulfan sulfate Propargite Iprodione Bifenthrin Fenpropathrin cis-permethrin Deltamethrin

31 Wild Berry Zinger Ingredients: Hibiscus, rosehips, roasted chicory, orange peel, blackberry leaves, natural flavors Average % Recovery for ALL target pesticides Spike GC-MS/MS GCxGC-TOFMS 1000 ng/g 79% (± 10) 90% (± 11) 100 ng/g 83% (± 12) 94% (± 33) 10 ng/g 88% (± 19) NA GC-MS/MS Carbaryl (23) Chlorpyrifos (18) Imazalil (470) Thiabendazole (240) Fenhexamid (2) 4 4-DDT (1) Incurred Pesticides (ng/g): GCxGC-TOFMS Carbaryl (10) Chlorpyrifos (16) Imazalil (727) Thiabendazole (429) o-phenylphenol (22)

32 Red Tea Cinnamon Orange Ingredients: Rooibos, cinnamon, orange peel, natural cinnamon and orange flavors

33 % Recoveries of Spiked Pesticides in Red Tea Pesticide 1000 ng/g 100 ng/g GC-MS/MS GCxGC-TOFMS GC-MS/MS GCxGC-TOFMS Acephate o-phenylphenol Omethoate Diazinon Chlorothalonil Vinclozolin Carbaryl Metalaxyl Methiocarb Malathion Fenthion Chlorpyrifos Cyprodinil Thiabendazole Captan Imazalil Endosulfan sulfate Propargite Iprodione Bifenthrin Fenpropathrin cis-permethrin Deltamethrin

34 Red Tea Cinnamon Orange Rooibos, cinnamon, orange peel, natural cinnamon and orange flavors Spike GC-MS/MS GCxGC-TOFMS 1000 ng/g 83% (± 8) 110% (± 13) 100 ng/g 87% (± 12) 91% (± 14)* 10 ng/g 90% (± 15) NA Incurred Pesticides (ng/g): GC-MS/MS o-phenylphenol (20) Endosulfan sulfate (2) Iprodione (2) Imazalil (2) Thiabendazole (8) GCxGC-TOFMS o-phenylphenol (25)

35 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

36 Everyday Detox Ingredients: roasted chicory root, dandelion root, schisandra fruit, lycium fruit, licorice root, ginger rhizome, star anise fruit, kukicha twig

37 GCxGC Resolution Acephate m/z 136

38 Acephate MS/MS Selectivity Quant peak 136 > 42.1 m/z Confirming ions 94 > 64 m/z 136 > 94 m/z 100 pg on-column 10 pg on-column Corruption of peak from interferences

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

40 Pesticide % Recoveries of Spiked Pesticides in Everyday Detox Tea 1000 ng/g 100 ng/g GC-MS/MS GCxGC-TOFMS GC-MS/MS GCxGC-TOFMS Acephate o-phenylphenol Omethoate Diazinon Chlorothalonil Vinclozolin Carbaryl Metalaxyl Methiocarb Malathion Fenthion Chlorpyrifos Cyprodinil Thiabendazole Captan Imazalil Endosulfan sulfate Propargite Iprodione Bifenthrin Fenpropathrin cis-permethrin Deltamethrin

41 Everyday Detox roasted chicory root, dandelion root, schisandra fruit, lycium fruit, licorice root, ginger rhizome, star anise fruit, kukicha twig Spike GC-MS/MS GCxGC-TOFMS 1000 ng/g 85% (± 28) 89% (± 10) 100 ng/g 79% (± 13) 97% (± 24) 10 ng/g 87% (± 19) NA Incurred Pesticides (ng/g): GC-MS/MS gamma-bhc (1) Methiocarb (6) GCxGC-TOFMS None Found

42 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

43 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

44 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.

45 Non-Target Analysis Maté Lemon Ingredients: Yerba maté, 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

46 Maté Lemon Ingredients: Yerba maté, lemon myrtle, green tea

47 Peak Find, Spectral Deconvolution, Library Search Set S/N, automatic deconvolution

48 Sort by Similarity for Qualitative Review

49 Maté Lemon Ingredients: Yerba maté, lemon myrtle, green tea 1425 peaks S/N 100

50 Qualitative Review Caliper - sample "Mate Lemon Tea extract:1", 1206, sec, sec to 1206, sec, sec - 400, sec, sec to 400, sec, sec Some names are easily recognized Peak True - sample "Mate Lemon Tea extract:1", pea k 931, at 1206, sec, sec Caffeine Library Hit - similarity 941, "Caffeine"

51 Qualitative Review Caliper - sample "Mate Lemon Tea extract:1", 610, sec, sec to 610, sec, sec - 400, sec, sec to 400, sec, se Others may need further investigation Peak True - sample "Mate Lemon Tea extract:1", peak 559, at 610, sec, sec 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

52 Qualitative Review 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.

53 Qualitative Review Caliper - sample "Mate Lemon Tea extract:1", 514, sec, sec to 514, sec, sec - 400, sec, sec to 400, sec, sec With less spectral information it is difficult to assign a positive match Peak True - sample "Mate Lemon Tea extract:1", peak 462, at 514, sec, sec Pyroxyfur is a pyridine fungicide Library Hit - similarity 910, "Pyroxyfur"

54 Peak True matches Library Hit at low m/z values Peak True - sample "Mate Lemon Tea extract:1", peak 462, at 514, sec, sec Peak True Library Hit - similarity 910, "Pyridine, 2-chloro-6-(2-furanylmethoxy)-4-(trichloromethyl)-" Library Hit

55 Qualitative Review Caliper - sample "Mate Lemon Tea extract:1", 514, sec, sec to 514, sec, sec - 400, sec, sec to 400, sec, sec With less spectral information it is difficult to assign a positive match Peak True - sample "Mate Lemon Tea extract:1", peak 462, at 514, sec, sec Library Hit - similarity 910, "Pyroxyfur"

56 Peak True - sample "Mate Lemon Tea extract:1", peak 462, at 514, sec, sec No molecular weight ion in Peak True spectrum Pyroxyfur MW = 325 Peak True Library Hit - similarity 910, "Pyridine, 2-chloro-6-(2-furanylmethoxy)-4-(trichloromethyl)-" 20 Library Hit

57 Structured Chromatograms Sceletium & Honeybush Tea Flavonoids polyphenolic compounds that may reduce the risk of certain cancers.

58 Structured Chromatograms Sceletium & Honeybush Tea Phytosterols may help reduce blood cholesterol levels.

59 Sceletium & Honeybush Tea Structured Chromatograms Phytosterols Campesterol Stigmasterol β-sitosterol

60 The South African Sceletium tortuosum herb has been extracted for the medicinally relevant mesembrine-type alkaloids. Mesembranol Mesembrenol Mesembrine Mesembrenone The mesebrine-type alkaloids have shown to elevate mood, reduce stress and promote a sense of calm

61 Mesembrine-type alkaloids elev8 TM elevates mood, assists in coping with stress Sceletium and Honeybush Tea The Cup that Cheers m/z 204, 218, 289 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.