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 (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.
Plant Growth Regulator Organonitrogen Pyrethroid Insecticide Herbicide Fungicide Synergist Organophosphorus Acaricide Organochlorine Triazole
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)
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
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
Complex Samples 500 pg ISTD
% 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 MgSO 4 to tube Elute 15mL acetone: toluene (3:1)
Cartridge SPE Cleanup Elute 15 ml 3:1 Acetone:Toluene 1 ml extract MgSO 4 500 mg GCB 500 mg PSA Concentrate to1 ml
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
GCxGC vs GC-MS/MS Chromatographic Separation Resolution! Full Spectral Information! Mass Spectrometer Separation Selectivity! Sensitivity!
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 50-500 ms wide peaks Orthogonal GC column stationary phases means two independent separation mechanisms
2 nd Column Fundamentals of GCxGC Linear Chromatogram Inlet Modulator Detector 1 st Column 2 nd Column Surface Plot Contour Plot
Triple Quadrupole Q1 Q2 Q3 89 110 245 68 Fragment Ions 99 133 time m/z m/z
Intensity MS n for Signal-to-Noise Improvement Sensitivity Signal Noise S/N 0 1 2 3 4 5 6 Stages of Analysis
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-200 1.0 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
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: 0.150 sec
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
Complex Samples After 28 tea injections! Use wool in your splitless liner to protect your GC column from non-volatile dirt
No Wool - Retention Gap - better solvent focusing No retention gap Methamidophos Dichlorvos 1 m retention gap
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
- 28.9 11.0 11.2 12.2 12.3 Pau d Arco A Traditional South American Herbal Tea Contains: Pau d Arco bark 16.3 16.2 16.4 17.1 17.0 17.2 17.7 17.9 22.6 22.8 22.9 25.0 25.2 25.3 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 0 5 10 15 20 25 Time (min) 8.0 11.1 12.2 15.8 16.9 30 m x 0.25 mm x 0.25 µm Rxi-5ms 17.7 21.7 18.4 22.4 28.6 N T P
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 100 90 78 65 o-phenylphenol 101 87 77 100 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 Metalaxyl 99 100 83 97 Methiocarb 72 100 94 73 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 Deltamethrin 85 94 94 45
GCxGC - Spectral Deconvolution Caliper - sample "Pau d'arco Tea extract:1", 786, 0.790 sec, sec to 786, 0.790 sec, sec - 1000 135 800 152 Caliper 600 400 200 63 77 107 121 169 231 285 35 50 100 150 200 250 300 350 Peak True - sample "Pau d'arco Tea extract:1", peak 292, at 786, 0.790 sec, sec 65 1000 800 169 600 141 400 115 200 87 47 285 356 50 100 150 200 250 300 350 Library Hit - similarity 948, "o-phenylphenol" 170 1000 800 Peak True Library Hit o-phenylphenol 600 400 200 115 141 63 39 89 155 50 100 150 200 250 300 350
unknown o-phenylphenol 7000 GCxGC - Spectral Deconvolution Caliper - sample "Pau d'arco Tea extract:1", 786, 0.790 sec, sec 135 1000 Caliper 800 152 6000 5000 4000 3000 2000 600 77 400 63 107 200 121 169 231 50 100 150 200 250 Peak True - sample "Pau d'arco Tea extract:1", peak 292, at 786 65 1000 800 600 400 200 87 169 141 115 105 50 100 150 200 250 Reference Spectrum - Calibration "QuEChERS Perf AB 500", Analyte 170 1000 Peak True Reference Spectrum 1000 800 600 115 141 400 63 0 1st Time (s) 2nd Time (s) 786 0.72 786 0.74 786 0.76 786 0.78 170 135 786 0.8 786 0.82 200 89 74 102 155 50 100 150 200 250
MS/MS Selectivity 141 > 115.1 m/z 169.7 > 115.1 m/z 169.7 > 141.3 m/z 100 o-phenylphenol 170 OH 50 115 141 63 39 51 89 62 77 27 52 58 65 70 87 55 91 98 102 126 131 151 155 0 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 (mainlib) o-hydroxybiphenyl
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)
Wild Berry Zinger Ingredients: Hibiscus, rosehips, roasted chicory, orange peel, blackberry leaves, natural flavors
% 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 76 88 88 81 o-phenylphenol 86 92 113 95 Omethoate 81 78 101 88 Diazinon 85 93 86 95 Chlorothalonil 72 79 74 100 Vinclozolin 84 99 89 91 Carbaryl 98 94 89 91 Metalaxyl 86 96 84 90 Methiocarb 84 90 85 89 Malathion 91 95 96 94 Fenthion 84 88 85 98 Chlorpyrifos 84 93 84 88 Cyprodinil 65 71 66 73 Thiabendazole 65 62 86 77 Captan 86 99 102 180 Imazalil 86 91 102 91 Endosulfan sulfate 86 95 87 100 Propargite 85 96 84 110 Iprodione 88 99 85 74 Bifenthrin 76 86 77 82 Fenpropathrin 85 93 97 93 cis-permethrin 78 88 78 70 Deltamethrin 83 88 74 78
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)
Red Tea Cinnamon Orange Ingredients: Rooibos, cinnamon, orange peel, natural cinnamon and orange flavors
% 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 69 110 74 80 o-phenylphenol 86 98 83 96 Omethoate 58 120 66 100 Diazinon 84 99 94 88 Chlorothalonil 69 110 71 71 Vinclozolin 85 100 88 83 Carbaryl 83 110 82 85 Metalaxyl 85 110 84 87 Methiocarb 83 120 90 102 Malathion 85 110 93 77 Fenthion 83 110 84 91 Chlorpyrifos 81 100 88 90 Cyprodinil 75 97 78 79 Thiabendazole 52 87 51 75 Captan 89 140 76 160 Imazalil 73 120 76 88 Endosulfan sulfate 80 120 80 84 Propargite 78 110 89 92 Iprodione 82 120 89 92 Bifenthrin 74 98 78 85 Fenpropathrin 80 110 74 76 cis-permethrin 74 99 76 92 Deltamethrin 77 120 93 120
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)
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 Confirming ions 94 > 64 m/z 136 > 94 m/z 100 pg on-column 10 pg on-column Corruption of peak from interferences
Acephate has a skinny mass spectrum. There is not a lot of high abundance, high m/z values for more selective fragmentation
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 75 88 81 89 o-phenylphenol 85 88 81 100 Omethoate 66 85 95 93 Diazinon 87 88 78 96 Chlorothalonil 73 74 71 99 Vinclozolin 84 93 83 110 Carbaryl 86 97 74 100 Metalaxyl 85 100 76 88 Methiocarb 86 96 78 120 Malathion 86 98 75 67 Fenthion 87 98 81 100 Chlorpyrifos 85 88 84 110 Cyprodinil 75 83 69 81 Thiabendazole 62 75 37 86 Captan 82 100 82 100 Imazalil 84 75 63 72 Endosulfan sulfate 87 99 77 100 Propargite 89 90 83 83 Iprodione 91 90 81 100 Bifenthrin 81 85 75 84 Fenpropathrin 88 90 83 100 cis-permethrin 80 87 67 97 Deltamethrin 86 90 89 93
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
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 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
Maté Lemon Ingredients: Yerba maté, lemon myrtle, green tea
Peak Find, Spectral Deconvolution, Library Search Set S/N, automatic deconvolution
Sort by Similarity for Qualitative Review
Maté Lemon Ingredients: Yerba maté, lemon myrtle, green tea 1425 peaks S/N 100
Qualitative Review 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 1000 67 194 Some names are easily recognized. 100 200 300 400 500 Peak True - sample "Mate Lemon Tea extract:1", pea k 931, at 1206, 1.190 sec, sec 1000 67 194 Caffeine 500 100 200 300 400 500 Library Hit - similarity 941, "Caffeine" 1000 500 55 109 194 100 200 300 400 500
Qualitative Review Caliper - sample "Mate Lemon Tea extract:1", 610, 1.420 sec, sec to 610, 1.420 sec, sec - 400, 0.000 sec, sec to 400, 0.000 sec, se 1000 800 600 400 51 78 122 106 Others may need further investigation. 200 94 50 100 150 200 250 300 350 400 450 500 Peak True - sample "Mate Lemon Tea extract:1", peak 559, at 610, 1.420 sec, sec 1000 800 600 400 200 51 67 78 94 106 122 50 100 150 200 250 300 350 400 450 500 Library Hit - similarity 912, "Niacinamide" 1000 800 600 400 200 51 66 78 94 106 122 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. 50 100 150 200 250 300 350 400 450 500
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.
Qualitative Review Caliper - sample "Mate Lemon Tea extract:1", 514, 1.110 sec, sec to 514, 1.110 sec, sec - 400, 0.000 sec, sec to 400, 0.000 sec, sec 1000 81 800 600 400 200 53 71 107 With less spectral information it is difficult to assign a positive match. 50 100 150 200 250 300 350 400 450 500 550 Peak True - sample "Mate Lemon Tea extract:1", peak 462, at 514, 1.110 sec, sec 1000 81 800 600 400 Pyroxyfur is a pyridine fungicide. 200 53 107 50 100 150 200 250 300 350 400 450 500 550 Library Hit - similarity 910, "Pyroxyfur" 1000 81 800 600 400 200 53 50 100 150 200 250 300 350 400 450 500 550
200 53 71 107 Peak True matches Library Hit at low m/z values 50 100 150 200 250 300 350 400 450 500 550 Peak True - sample "Mate Lemon Tea extract:1", peak 462, at 514, 1.110 sec, sec 81 1000 800 600 400 Peak True 200 53 51 107 55 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 Library Hit - similarity 910, "Pyridine, 2-chloro-6-(2-furanylmethoxy)-4-(trichloromethyl)-" 81 1000 800 600 400 Library Hit 200 51 53 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125
Qualitative Review Caliper - sample "Mate Lemon Tea extract:1", 514, 1.110 sec, sec to 514, 1.110 sec, sec - 400, 0.000 sec, sec to 400, 0.000 sec, sec 1000 81 800 600 400 200 53 71 107 With less spectral information it is difficult to assign a positive match. 50 100 150 200 250 300 350 400 450 500 550 Peak True - sample "Mate Lemon Tea extract:1", peak 462, at 514, 1.110 sec, sec 1000 81 800 600 400 200 53 107 50 100 150 200 250 300 350 400 450 500 550 Library Hit - similarity 910, "Pyroxyfur" 1000 81 800 600 400 200 53 50 100 150 200 250 300 350 400 450 500 550
200 305 310 315 320 325 330 335 340 345 350 Peak True - sample "Mate Lemon Tea extract:1", peak 462, at 514, 1.110 sec, sec 20 15 No molecular weight ion in Peak True spectrum Pyroxyfur MW = 325 Peak True 10 5 305 310 315 320 325 330 335 340 345 350 Library Hit - similarity 910, "Pyridine, 2-chloro-6-(2-furanylmethoxy)-4-(trichloromethyl)-" 20 Library Hit 15 10 5 305 310 315 320 325 330 335 340 345 350
Structured Chromatograms Sceletium & Honeybush Tea Flavonoids polyphenolic compounds that may reduce the risk of certain cancers.
Structured Chromatograms Sceletium & Honeybush Tea Phytosterols may help reduce blood cholesterol levels.
Sceletium & Honeybush Tea Structured Chromatograms Phytosterols Campesterol Stigmasterol β-sitosterol
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
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.