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Identification and Quantitation of Pesticides in Tea and Herbal Extracts

Identification and Quantitation of Pesticides in Tea and Herbal Extracts

Identification and Quantitation of pesticides in Tea and Herbal Extracts

iFunnel and triggered MRM - new tools for unmatched sensitivity and unequivocal confirmation Stefan Kittlaus, Dr. Günther Kempe LUA Dresden Dr. Thomas Glauner EMEA LC/MS Food Segment Scientist

Joint Workshop for Pesticides in Food and Feed October 6, 2011 Agilent G6490A QQQ system New developments for utmost sensitivity • Ionization and Ion Transfer Technology – Agilent Jet Stream Ion Generation – Hexabore capillary – Dual ion funnel (iFunnel) technology • Two stages for ion focusing and gas removal • Improvements for wide m/z range transmission • Low capacitance • Collision Cell – Hexapole field axial focusing curved collision cell • Tapered cell structure for increased ion acceptance at entrance • Reduced noise • Improved Quad Drive Electronics – Improved Quad DC frequency response – Higher RF power capability – Quad drive frequency increased to 1.4 MHz

Joint Workshop for Pesticides in Food and Feed 2 Freiburg, October 6, 2011 Agilent iFunnel technology Captures 6 to 10 times more ions Agilent Jet Stream Hexabore Capillary Dual Ion Funnel

• Thermal confinement of • Six bores and half the length • Removes the gas but ESI plume to create ion means much less restriction captures the ions rich zone • Samples 6 to 10 times more • Makes skimmer and one • Efficient desolvation to ion rich gas from the source compound dependant create gas phase ions with 6 capillaries parameter obsolete • Effective ionization across • Captures the majority of the • Removes neutral noise broad range of analyte gas from the source region • Low capacitance design classes including many allows for fast polarity APCI compounds switching

Joint Workshop for Pesticides in Food and Feed 3 Freiburg, October 6, 2011 Agilent iFunnel technology Two stage ion funnel manages the gas load High Low Pressure Pressure Stage 1 Stage 2

Line of Sight

Stage 2 Stage 1 1-3 Torr 8-12 Torr

Offset ion funnels to prevent neutrals from going straight through to MS

Joint Workshop for Pesticides in Food and Feed 4 Freiburg, October 6, 2011 Agilent triple quadrupole instruments Instrument configurations

6410 / 6430 / 6460 LC/MS (Skimmer Configuration)

Ion Detector Cap RF Ion Q1 Coll Q3 Guide Cell Ion Detector

Q3

6490 LC/MS (iFunnel Configuration)

Curved Collision cell

HB Cap Dual Ion RF ion Q1 Funnels guide

Joint Workshop for Pesticides in Food and Feed 5 Freiburg, October 6, 2011 Multi residue pesticide method Compounds included in method

Acephat Butocarboxim-sulfoxid Desmedipham -sulfon Flusilazol Metamitron Oxadiazon Proquinazid Terbutylazin Buturon DMSA Fenamiphos-sulfoxid Flutolanil Metazachlor Oxadixyl Prosulfocarb Tetraconazol Cadusaphos Dichlofluanid Fenarimol Flutriafol Metconazol Pymetrozin Thiabendazol Fenazaquin Foramsulfuron Methabenzthiazuron Oxasulfuron Pyraclostrobin Aldicarb-sulfon Diclofop-methyl Fenbuconazol Formetanat-Hydrochlorid Methacrifos Oxydemeton-sulfon Pyrazophos Aldicarb-sulfoxid 3 hydroxy Fenbutatinoxid Paclobutrazol Pyridaben Thifensulfuron-methyl Alloxydim Carbofuran Diethofencarb Fenhexamid Fosthiazat -methyl Pyridaphenthion Thiodicarb Amidosulfuron Difenoconazol Fuberidazol Penconazol Pyridat sulfon Aminopyralid Carfentrazone-ethyl Difenoxuron Fenoxaprop free acid Furathiocarb Mthiocarb-sulfon Pencycuron Pyrifenox Thiofanox sulfoxid Chlorantraniliprol Halosulfuron-methyl Methiocarb-sulfoxid Pendimethalin Pyrimethanil Thiofanox Asulam Diflufenican Fenpiclonil Haloxyfop free acid Pethoxamid Pyriproxifen Thiophanat Atrazin Chlorfluazuron Dimefuron Fenpropimorph Hexaconazol Methoxyfenozid Phenmedipham Pyroxsulam Thiophanat-methyl Avermectin B1a Metabolite Chloridazon (Pyrazon) Dimethenamid Fenpyroximat Hexaflumuron Metobromuron Phenthoat Tolclophos-methyl Avermectin B1a Chlorimuron-ethyl Dimethoat Hexythiazox Metolachlor Phorat Quinmerac Tolylfluanid Avermectin B1b Chloroxuron Dimethomorph Fenthion- Imazalil Metosulam Phosalon Quinoxyfen Topramezone Azimsulfuron Chlorsulfuron Dimoxystrobin Fenthion-oxon-sulfon Metoxuron Quizalfop free acid Tralkoxydim Azinphos-ethyl Chlozolinat Diniconazol Fenthion-oxon-sulfoxid Metrafenon Phosmet-oxon Quizalofop-ethyl Triadimefon Azinphos-methyl Chromafenozid Fenthion-sulfon Ipconazol Metribuzin Rimsulfuron Triadimenol Azoxystrobin Clethodim Diuron Fenthion-sulfoxid Iprodion Metsulfuron-methyl Rotenone Triasulfuron Beflubutamid Clofentezin EPN Flazasulfuron Iprovalicarb Picoxystrobin Sethoxydim Benalaxyl Clomazon Epoxyconazol Flonicamid Isoproturon Molinat Pinoxaden Siduron Tribenuron-methyl Benfuracarb Clopyralid Ethaboxam Florasulam Isoxaflutol Piperonyl butoxid Silthiopham Trichlorfon Bensulfuron-methyl Fluazifop free acid Kresoxim methyl Monolinuron Simeconazol Tricyclazol Benthiavalicarb Cyazofamid Ethiofencarb-sulfon Fluazifop-P-butyl Lenacil Monuron Pirimiphos-methyl Trifloxystrobin Bifenazat Cycloxydim Ethiofencarb-sulfoxid Flufenacet Linuron Myclobutanil Pirmicarb-desmethyl Spirotetramat Triflumizol Bifenox Cyhexatin Napropamid Prochloraz Spiroxamin Triflumuron Bispyribac Cymoxanil Ethiprol Flumetsulam Neburon Profenophos Sulfosulfuron Triflusulfuron-methyl Bitertanol Cyproconazol Ethirimol Fluometuron Mandipropamid Nicosulfuron Tebuconazol Triforin Bromacil Cyprodinil Ethofumesat Fluopicolid Mecarbam Tebufenozid Trinexapac-ethyl Bromuconazol Cyromazin Fluoroglycofen-ethyl Mepanipyrim Novaluron Propaquizafop Triticonazol Bupirimat Daminozid Fluoxastrobin (E) Mesosulfuron-methyl Nuarimol Propargite Teflubenzuron Tritosulfuron DEET Famoxadon Fluquinconazol Mesotrione Ofurace Propiconazol Tembotrion Zoxamid Butocarboxim -S-methyl Fenamidon Fluroxypyr Omethoat Tepraloxydim Butoxycarboxim Demeton-S-methyl-sulfon Fenamiphos Flurtamon Metalaxyl Orthosulfamuron Propyzamid Terbutryn 313 compounds including 9 isomers

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 6 Freiburg, October 6, 2011 Multi residue pesticide method HPLC method Agilent 1290 Infinity LC system consisting of: - binary pump - wellplate sampler - column compartment - diode array detector (not used) HPLC method Separation column: ZORBAX Eclipse Plus C-18 RRHD column, 100 x 2.1 mm, 1.8 µm @ 25°C Mobile phase: A: 5 mM ammonium formate B: methanol + 5 mM ammonium formate Flow: 0.6 ml/min Gradient: 0.00 min 5 % B 0.20 min 5 % B 2.20 min 50 % B 10.50 min 100 % B 13.00 min 100 % B 13.10 min 5 % B 15.00 min 5 % B Inj.Vol.: 2 µl

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 7 Freiburg, October 6, 2011 Multi residue pesticide method MS method

Spray chamber conditions: Gas temp.: 120°C Dry gas: 15 l/min Nebulizer: 35 psi Sheath gas temp: 375°C Sheath gas flow: 12 l/min

Positive Negative CapVoltage: 3500 V 3000 V Nozzle voltage 300 V 500 V

Automatic setup of MRM tables based on selected cycle time, retention times and retention time windows for the individual compounds • Cycle time 600 ms • Interscan delay 3.5 ms • Total No. of MRMs 635 • Maximum No. Of concurrent MRMs 74 • Minimum Dwell time 4.61 ms • Maximum Dwell time 296.5 ms

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 8 Freiburg, October 6, 2011 Multi residue pesticide method Comparison of MRM and Dynamic MRM

Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4 Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

MRM

Compounds (10/block) 50 80 100 70 Cycle Time (sec) 0.5 0.8 1 0.7

Dynamic MRM

Max Coincident 20 40 40 30 Cycle Time (sec) 0.4 0.4 0.4 0.4

• 2 x shorter cycle times supports narrow chromatographic peaks, more analytes or longer dwell per analyte.

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 9 Freiburg, October 6, 2011 Multi residue pesticide method MRM traces for tomato extract spiked at 10 µg/kg

more than 160 MRM transitions in 1 min

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 10 Freiburg, October 6, 2011 Results for black tea Coverage of method for black tea

number of compounds dectected in spiked black tea

350 312 312 312 312 298 302 300 262

250

200

150

100

50

0 0.5 2.5 5 25 50 250 500 concentration in µg/kg in black tea All compounds found in black tea

MRL

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 11 Freiburg, October 6, 2011 Results for black tea Oxamyl and Carbendazim (High in “Check Your Scope” ranking)

Oxamyl

0.5 µg/kg 2.5 µg/kg 5 µg/kg 25 µg/kg 50 µg/kg

Carbendazim

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 12 Freiburg, October 6, 2011 Results for black tea Avermectine B1a and B1b

Avermectine B1a

Avermectine B1b

blank 2.5 µg/kg 5 µg/kg 25 µg/kg 50 µg/kg 250 µg/kg

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 13 Freiburg, October 6, 2011 Results for black tea Method performance characteristics for black tea extracts

• Replicate injection of black tea extract spiked at 5 µg/kg (n =8)

200 180 160 140 120 100 80 60

40 Target compounds 20 0 0 to 2 2 to 5 5 to 10 10 to 20 > 20 RSD in %

• At 50% of the MRL 303 of 305 pesticides have RSD values within the SANCO criteria of 20% with most of the compounds (n = 274) showing RSDs below 10%

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 14 Freiburg, October 6, 2011 Dilution of black tea extract Results for monocrotophos

1:50

Monocrotophos 1:10

1:5 good S/N 5 µg/kg even in 1:50 1:2 dilution

40 fg o.c. 200 fg o.c. 400 fg o.c. 1 pg o.c. 2 pg o.c.

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 15 Freiburg, October 6, 2011 Dilution of black tea extract Results for fuberidazole

1:50

Fuberidazole 1:10

1:5 1:2 good S/N 5 µg/kg even in 1:50 increased dilution signal

40 fg o.c. 200 fg o.c. 400 fg o.c. 1 pg o.c. 2 pg o.c.

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 16 Freiburg, October 6, 2011 Dilution of black tea extract spiked at 1 pg/ul Equivalent to 5 ug/kg – only compounds with recovery less than 70% are shown

recovery after dilution for pesticides with recoveries less then 70% in undiluted extract

35 recovery range according to SANCO document 30 (70% - 120%)

25

20 undiluted 1:2 15 1:10

10

5

0 < 30% 30% - 40% - 70% - 100% - 120% - 150% - > 200% 50% 70% 100% 120% 150% 200%  significantly better recoveries after dilution for compounds with strong ion suppression in the undiluted tea extract

Joint Workshop for PesticidesConfidentiality in Food and LabelFeed 17 Freiburg, October 6, 2011 Multi residue pesticide method Results of dilution experiments of tea extracts

• Dilution is a possible way to overcome matrix effects like ion suppression

• This allows solvent calibration and still have a method which can be validated according to SANCO recovery requirements (> 70%)

• With any dilution an improved robustness of the method can be expected due to the lower matrix amount which is introduced into the ionization source

Joint Workshop for Pesticides in Food and Feed 18 Freiburg, October 6, 2011 Multi residue pesticide method Other conclusions

• These results show that the 1290/6490 can analyse a range of food types for well over 300 priority pesticides and can detect all compounds down to their MRL achieving excellent precision even for challenging matrix types. • The unique package that enables this cabability is: 1. 1290 UHPLC. 2. Dynamic MRM. 3. Jet Stream/Ion Funnel Ion sampling. 4. Curved Collision cell with ion focussing and Linear acceleration for speed and sensitivty in detection.

Joint Workshop for Pesticides in Food and Feed 19 Freiburg, October 6, 2011 Is it Possible to Simultaneously Obtain Qualitative and Quantitative Results?

Unknown Screening Qual/Quan: Quantitation – how – what’s in the Screening and much compound(s) is sample? Quantitation? in the sample?

Non-Selective Selective

Joint Workshop for Pesticides in Food and Feed Page 20 Freiburg, October 6, 2011 Features of Successful Quantitative Analysis

Fast Cycle Time (QQQ ~5 ms)

Many Data Points Across Peak Quantitative Analysis

Sensitive

Accurate and Reproducible

Joint Workshop for Pesticides in Food and Feed Page 21 Freiburg, October 6, 2011 Features of Conventional Qualitative Analysis

Full Scan Product Ion (Trap, TOF, Scanning or QQQ)

Qualitative Analysis

Cycle Time Library Searching ~200 ms/scan

Joint Workshop for Pesticides in Food and Feed Page 22 Freiburg, October 6, 2011 The Ideal Analytical Solution

tMRM

Cost Quantitative Qualitative Effective

Fast POINTCycle Time High POINTThroughput ProductPOINT Ion Spectrum

ShortPOINTRun Time SinglePOINT Instrument LibraryPOINT Searching

Accurate Confirmation

Sensitive

Joint Workshop for Pesticides in Food and Feed Page 23 Freiburg, October 6, 2011 Triggered MRM Parameters

Primary transitions (A) with defined threshold (B) trigger additional transitions for given number of repeats (C).

(A) (B)

(C)

Joint Workshop for Pesticides in Food and Feed Page 24 Freiburg, October 6, 2011 Triggered MRM (tMRM)

Triggered cycle (above threshold)

Additional MRMs used for compound confirmation Threshold

Primary cycle (below threshold)

Joint Workshop for Pesticides in Food and Feed Page 25 Freiburg, October 6, 2011 tMRM Product Ion Spectrum

334 > 105 334 > 117 334 > 119 x103 334 > 76 x103 334 > 91 x103 x103 x103 8 8 8 8 117.0 8 7 7 7 7 7 6 6 6 6 6 5 5 5 5 5 4 76.0 4 4 4 4 119.0 3 3 91.1 3 105.1 3 3 2 2 2 2 2 1 80 1 1 1 1 80 100 80 100 120 80 100 120 80 100 120 0 0 0 0 0

145.0 x103 334 > 132 x103 334 > 145 x103 334 > 147 x103 334 > 171 8 8 8 8 7 7 7 7 6 6 6 147.0 6 5 5 5 5 4 132.1 4 4 4 3 3 3 3 171.1 2 2 2 2 1 1 1 1 100 140 100 140 180 100 140 180 80 100 120 140 160 180 0 0 0 0 x103 145.0 tMRM Product Ion 117.0 8 Spectrum 7 6 5 4 3 76.0 91.1 132.1 2 105.1 171.1 1 334.2 0 80 100 120 140 160 180 200 220 240 260 280 300 320 340

Joint Workshop for Pesticides in Food and Feed Page 26 Freiburg, October 6, 2011 MRM and tMRM Signal

Primary transitions (integrated), triggered MRM transitions (5 repeats), and resulting MRM spectrum for aflatoxin G1.

1) Quant based on MRM

3) Composite 2) tMRM signal “MS/MS” Spectrum

Joint Workshop for Pesticides in Food and Feed Page 27 Freiburg, October 6, 2011 Automatic tMRM library creation

Joint Workshop for Pesticides in Food and Feed Page 28 Freiburg, October 6, 2011 MH Quant Library editor

Joint Workshop for Pesticides in Food and Feed Page 29 Freiburg, October 6, 2011 Triggered MRM (tMRM) Analysis

tMRM Analysis Workflow

Additional MRMs Report Generated Inject Sample Automatically with Compound Using tMRM Triggered When Concentration Acquisition Compounds and Library Match Method Elute Score

Joint Workshop for Pesticides in Food and Feed Page 30 Freiburg, October 6, 2011 Background information Chamomile

• Chamomile (Matricaria chamomilla) • Typically the flowers are used as medicine or tea • Main production areas are Argentina, Egypt, Bulgaria, Hungary, Spain and Germany • In Germany no use of herbicides is allowed for chamomile production • In 2009 a contract laboratory has identified a compound in chamomile as tebuthiuron in high concentrations based on the two major transitions and a similar qualifier ratio • But: • retention time was slightly different to standard (due to matrix?) • use of tebuthiuron on chamomille production is not likely

Joint Workshop for Pesticides in Food and Feed Page 31 Freiburg, October 6, 2011 Quantitative analysis of pesticides MRM traces of tebuthiuron in QC sample and chamomile extract

QC 50 ppb Chamomile extract RT 6.169 min RT 6.365 min (+ 3.18 %) Qual/Quant ratio 22.5% Calc. Conc. 12.6 µg/kg Qual/Quant ratio 42.7% (189.9%)

Joint Workshop for Pesticides in Food and Feed Page 32 Freiburg, October 6, 2011 Quantitative analysis of pesticides MRM traces of tebuthiuron in spiked chamomile extract

Spiked chamomile extract (+ 100 ppb Tebuthiuron) RT 6.162 and 6.362 min (+ 3.25%) Qual/Quant ratio 21.8 and 44.3% (95.2 and 195.5%)

• In non-spiked chamomile extract both MRM traces for tebuthiuron show up with higher RT than in reference standard (slightly above criteria of SANCO guidelines) • For less resolving HPLC separations co-elution of compounds is very likely • Qualifier/quantifier ratio in non-spiked ginger extract is significantly higher than tolerance allowed in SANCO guidelines • Estimated concentration exceeds default MRL of 0.01 mg/kg)

Joint Workshop for Pesticides in Food and Feed Page 33 Freiburg, October 6, 2011 Quantitative analysis of pesticides Triggered MRM traces of tebuthiuron in chamomile extract

Library match score: 69.57

Joint Workshop for Pesticides in Food and Feed Page 34 Freiburg, October 6, 2011 Quantitative analysis of pesticides Library Searchable Spectrum Confirms ID

Linear Quant Curve

Joint Workshop for Pesticides in Food and Feed Page 35 Freiburg, October 6, 2011 Quantitative analysis of pesticides Library match for tebuthiuron in spiked chamomile extract

Joint Workshop for Pesticides in Food and Feed Page 36 Freiburg, October 6, 2011 Quantitative analysis of pesticides Library match for matrix compound in spiked chamomile extract

Joint Workshop for Pesticides in Food and Feed Page 37 Freiburg, October 6, 2011 Value of tMRM Acquisition tMRM provides fast, accurate quantitation and rigorous qualitative MS/MS confirmation in a single injection

Joint Workshop for Pesticides in Food and Feed Page 38 Freiburg, October 6, 2011 Triggered MRM data acquisition New acquisition mode for enhanced confirmation • Triggered MRM is new data dependant acquisition mode for the confirmation of target compounds • When the signal heights of the primary MRMs exceed a given threshold up to 9 additional MRM transitions are triggered • Triggered MRM makes use of the well established Dynamic MRM functionality • Triggered MRM has advantages over data dependant product ion scans because: • It is more sensitive due to longer dwell times per transition and due to the ideal collision energies for each transition • Peak shapes of the primary transitions are not compromised due to constant cycle times • In spectra fragment ratios are very constant due to relatively long dwell times per transition • Triggered MRM spectra can be saved in an user defined library and can be searched from the qualitative and quantitative software.

Joint Workshop for Pesticides in Food and Feed Page 39 Freiburg, October 6, 2011 Questions ..... ?

Dr. Thomas Glauner Agilent Technologies Sales & Service GmbH & Co KG Hewlett-Packard-Str.8 D-76337 Waldbronn

Tel: +49 (7243) 602-2719 [email protected]

Joint Workshop for Pesticides in Food and Feed 40 Freiburg, October 6, 2011

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