Implementation of Modified QuEChERS Techniques for Modern Applications. Derick Lucas, Ph.D. Agilent Technologies

Transcription

1 Implementation of Modified QuEChERS Techniques for Modern Applications Derick Lucas, Ph.D. Agilent Technologies 1

2 Today s Agenda QuEChERS A Brief Overview Preservatives Analysis in Pet Food Pesticide Analysis in Juice Concentrates Pesticide Analysis in Chicken Pesticide Analysis in Herbal Teas Conclusions and Summary 2

3 Today s Agenda QuEChERS A Brief Overview Preservatives Analysis in Pet Food Pesticide Analysis in Juice Concentrates Pesticide Analysis in Chicken Pesticide Analysis in Herbal Teas Conclusions and Summary 3

4 What is QuEChERS (pronounced Catchers ) Quick, Easy, Cheap, Effective, Rugged and Safe Developed jointly by USDA and Food Regulatory Agencies as a sample preparation method for multi-residue analyses Simplified extraction and cleanup approaches that reduce use of expensive and/or dangerous solvents Primarily for preparing fruits and vegetables for pesticide analysis Rapidly being extended to other food types and compound classes

5 What are the Benefits of QuEChERS? QuEChERS Approach: Extract +250 compounds at one time Final extract amenable to GC/MS or LC/MS Reduced solvent, reduced labor, increased lab productivity QuEChERS Approach Advantages 30 minutes to extract multiple samples at once Minimal solvent usage per sample: ml Chlorinated Solvents: None If you can weigh, shake and have a centrifuge, you can perform QuEChERS

6 Why Choose QuEChERS? Lives up to its name Flexibility and adaptability Works with solids and liquids Perfect complement to tandem MS instruments Called out in many regulatory methods and norms 6

7 Sample Homogenization Pre-Preparation Page 7

8 QuEChERS Easy as Page 8

9 Today s Agenda QuEChERS A Brief Overview Preservatives Analysis in Pet Food Pesticide Analysis in Juice Concentrates Pesticide Analysis in Chicken Pesticide Analysis in Herbal Teas Conclusions and Summary 9

10 QuEChERS Application Examples: Preservatives in Pet Food Looked at BHA, BHT and ethoxyquin in pet food Used QuEChERS sample prep approach to preparing pet foods for LC-MS/MS analysis Developed LC-MS/MS method Results: Incurred sample analysis proved suitability of the technique Difficult to find pet food samples that would be free of preservatives Method optimization was required We will get back to the method optimization in a moment 10

11 Extraction Procedure: General QuEChERS Method Add 2 g pet food, either dry or wet, to a 50 ml centrifuge tube Add 2 ceramic homogenizers (to help facilitate extraction) Add 8 ml water, vortex, 1 min at 2500 rpm Add 10 ml ACN, vortex, 1 min at 2500 rpm Add QuEChERS extraction salts Shake vigorously for 1 min, or vertical shake at 1000 rpm Centrifuge at 5000 rpm, 5 min 11

12 Various Pet Foods After QuEChERS Extraction (Step 1) 12

13 d-spe (Step 2) Transfer 6 ml to 15 ml d-spe tube Vortex 1 min at 2500 rpm Centrifuge at 5000 rpm for 5 min Transfer 1 ml to tube for evaporation Reconstitute in 1 ml ACN:water (1:4) Filter sample with 0.45 μm PP Analyze by LC-MS/MS 13

14 LC-MS/MS Conditions LC conditions: Poroshell 120 SB-C18, 2.7 μm, 2.1 x 50 mm Mobile phase A: Water (0.1% FA) Mobile Phase B: ACN (0.1% FA) Time 0 25% B % B % B % B % B MS Settings: Compound Ethoxyquin TS1 EMV 300 BHA TS3 EMV 1000 MRM Channels (m/z) 218 > > > > 138 Dwell Vol Fragmentor (V) CE (V) BHT TS4 EMV > > Propyl Paraben IS* TS2 EMV > >

15 400 ppb Post-Spiked Sample x Ethoxyquin x IS 2 0 x x BHA Counts vs. Acquisition Time (min) BHT 15

16 400 ppb Pre-Spiked Sample x Ethoxyquin x x BHA IS x Counts vs. Acquisition Time (min) BHT 16

17 Incurred Samples x Raw Pet Food: Organic x Product E: high grade, dry x Product M: High grade, wet 2 0 x x Product P: Lower grade, dry Product PP: Lower grade, wet Counts vs. Acquisition Time (min) 17

18 QuEChERS Method Development Approaches Evaluate extraction salt mixtures Evaluate dspe cleanup mixtures We will use the pet food example to highlight method development and optimization approaches 18

19 Optimization of QuEChERS Procedure: Pet Food Extraction Three variations of the QuEChERS extraction salts were investigated Non-buffered: 4 g MgSO 4, 1 g NaCl AOAC: 6 g MgSO 4, 1.5 g NaAc EN: 4 g MgSO 4, 1 g NaCl, 1 g NaCitrate, 0.5 g disodium citrate sesquihydrate TIP! Use one dspe mixture and keep this part the same for the extraction salt optimization 19

20 QuEChERS dspe Method Optimization Summary Use one dspe type with three salt types to identify the best combination for the application Method Development Products Fruits & Vegetables Method Development Process Select Representative Sample Homogenize dspe Mix 1 EN Extraction Salts CH Original CH AOAC CH Weigh Mix with salts, solvent Centrifuge Transfer aliquots of supernatant to each of the dspe tubes Mix Centrifuge Transfer supernatant to vials Analyze Identify best extraction salts 20

21 Optimization of QuEChERS Procedure: Pet Food Cleanup Four variations of the d-spe material were investigated 25 mg PSA, 150 mg MgSO 4 25 mg C18, 150 mg MgSO 4 25 mg PSA, 25 mg C18, 150 mg MgSO 4 25 mg PSA, 2.5 mg GCB, 150 mg MgSO 4 TIP! Use the extraction salt mix identified in the first experiment and keep this part the same 21

22 QuEChERS dspe Method Optimization Summary Use one extraction tube and 4 dspe tubes to identify the best combination for the application Method Development Products Method Development Process Select Representative Sample EN Extraction Salts CH dspe Mix CH dspe Mix CH dspe Mix CH dspe Mix CH Homogenize Weigh Mix with salts, solvent Centrifuge Transfer aliquots of supernatant to each of the dspe tubes Mix Centrifuge Transfer supernatant to vials Analyze Identify best dspe sorbent 22

23 Pet Food QuEChERS Optimization Results Extraction: Use the EN extraction salts: 4 g MgSO 4, 1 g NaCl, 1 g NaCitrate, 0.5 g disodium citrate sesquihydrate (PN CH) Cleanup: d-spe: 25 mg PSA, 2.5 mg GCB, 150 mg MgSO 4 (PN CH) In a minimal number of steps and experiments, the ideal match of extraction and dspe processes can be determined 23

24 Results for Spike Samples Compound 400 ppb Recovery RSD (%) (%) 600 ppb Recovery RSD (%) (%) 800 ppb Recovery RSD (%) (%) Ethoxyquin BHA BHT

25 Incurred Samples x Raw Pet Food: Organic x Product E: high grade, dry x Product M: High grade, wet 2 0 x x Product P: Lower grade, dry Product PP: Lower grade, wet Counts vs. Acquisition Time (min) 25

26 Today s Agenda QuEChERS A Brief Overview Preservatives Analysis in Pet Food Pesticide Analysis in Juice Concentrates Pesticide Analysis in Chicken Pesticide Analysis in Herbal Teas Conclusions and Summary 26

27 QuEChERS Method Optimization Multi-residue Pesticide Analysis in Juice Concentrates A GC/MS/MS multiple reaction monitoring method was developed on the Agilent 7890A/7000 GC Triple Quadrupole Mass Spectrometer system(gc/qqq) for 84 pesticide residues in lemon and orange juice concentrates provided by a world wide beverage company. Sample Preparation process follows the QuEChERS EN extraction with modifications to account for ph and sample volume. Results: Lemon juice concentrate was acidic (ph = 2) and treatment with NaOH gave better results for several pesticides. Analyte protectants (AP) were required to obtain good peak shapes by GC- MS. The method recoveries for the majority of the compounds were between % with RSD below 10%.

28 28 Optimized QuEChERS Sample Preparation for Juice Concentrates Weigh 4 g Juice Concentrate in EN 50 ml centrifuge tube Add QC spike solution as necessary, shake on mechanical shake for 10 min Add 6 ml of H 2 O (and 2 ml 5N NaOH for lemon juice conc.) Add 10 ml ACN and vortex briefly Add 2 ceramic homogenizers and QuEChERS EN salt packet Cap and load extraction tubes on SPEX ShaQer for 60 seconds (or by hand) 4000rpm for 2 min Transfer 1 ml of upper ACN layer to Dispersive SPE 2 ml tube (EN fruits & Vegetables; p/n: ) Vortex 10 s then 4000 rpm for 2 min Add 10 µl of AP, 20 µl of STD mix (ACN for recovery samples), 30 µl of ISTD mix, and 250 µl of supernatant from dspe tubes to autosampler vial. Vortex vial and place in sample tray for GC-MS/MS analysis

29 Improving Peak Shape with ph Piperonyl Butoxide Atrazine 0 ml 5 N NaOH 0.6 ml 5 N NaOH 1 ml 5 N NaOH 2 ml 5 N NaOH 29

30 QuEChERS Optimization AP (Analyte Protectant) with AP without AP Diazinon Hexachlorobenzene Ethion APs are a must in multi-residue pesticide analysis 30

31 Biphenyl Omethoate BHC, alpha- Hexachlorobenzene Demeton S Atrazine Lindane (gamma-bhc) Diazinon Acetochlor Vinclozolin Chlorpyrifos Methyl Parathion Methyl Dicofol,o,p'- Fenthion Fenitrothion Pirimiphos-methyl Dichlofluanid Malathion Diethofencarb Chlorpyrifos Parathion Dicofol, p,p'- Cyprodinil Penconazole Tolyfluanid Captan Isofenphos Mecarbam Quinalphos Phenthoate Folpet Procymidone Methidathion Prothiofos Profenofos DDE-p,p' 2,4-DEHE Buprofezin Oxyfluorfen Bupirimate Kresoxim Methyl Ethion Triazophos Quinoxyfen Carfentrazone-ethyl DDT-p,p' Imazalil Norflurazon TPP Piperonyl Butoxide Pyridaphenthion Bifenthrin Phosalone Lamba, Cyhalothrin I_II Fenarimol Coumaphos Cypermethrin I-IV Esfenvalerate I Precision & Accuracy for Lemon Juice Conc. (CAL range: ppb, n=6 for %REC and %RSD)

32 Biphenyl Omethoate BHC, alpha- Hexachlorobenzene Demeton S Atrazine Lindane (gamma-bhc) Diazinon Acetochlor Vinclozolin Chlorpyrifos Methyl Parathion Methyl Dicofol,o,p'- Fenthion Fenitrothion Pirimiphos-methyl Dichlofluanid Malathion Diethofencarb Chlorpyrifos Parathion Dicofol, p,p'- Cyprodinil Penconazole Tolyfluanid Captan Isofenphos Mecarbam Quinalphos Phenthoate Folpet Procymidone Methidathion Prothiofos Profenofos DDE-p,p' 2,4-DEHE Buprofezin Oxyfluorfen Bupirimate Kresoxim Methyl Ethion Triazophos Quinoxyfen Carfentrazone-ethyl DDT-p,p' Imazalil Norflurazon TPP Piperonyl Butoxide Pyridaphenthion Bifenthrin Phosalone Lamda, Cyhalothrin I -II Fenarimol Coumaphos Cypermethrin I-IV Esfenvalerate I Precision & Accuracy for Orange Juice Conc. (CAL range: ppb, n=6 for %REC and %RSD)

33 Today s Agenda QuEChERS A Brief Overview Preservatives Analysis in Pet Food Pesticide Analysis in Juice Concentrates Pesticide Analysis in Chicken Pesticide Analysis in Herbal Teas Conclusions and Summary 33

34 QuEChERS Method Pesticides in Chicken using GC-Tandem MS A GC/MS/MS multiple reaction monitoring method was developed on the Agilent 7890/7000 GC Triple Quadrupole Mass Spectrometer system(gc/qqq) for 54 pesticide residues in chicken. Sample Preparation process follows the QuEChERS AOAC extraction. Two dispersive cleanup options were evaluated. Results: Chicken samples were analyzed to verify the calibration coefficient, recovery and reproducibility. The detection limit of most compounds is down to 5 µg/kg (5 ppb). The method recoveries for all of the compounds were between % with RSD below 15%.

35 Sample Preparation Weigh 7.5 g chicken muscle sample (±0.1g) in 50 ml centrifuge tube and add 7.5 ml water Add Internal Standard (TPP) solution, and QC spike solution if necessary, vortex 1 min Add 15mL of ACN containing 1% AcOH Add Bond ElutEN QuEChERS Extraction salt packet Cap and shake vigorously for 30 seconds 4000rpm and 4 for 5 min Transfer 8 ml of upper ACN layer to Bond Elut Dispersive-SPE* 15 ml tube Vortex 1min, 4000 rpm and 4 for 5 min Transfer 4 ml extract to tube and dry under nitrogen Reconstitute with 1 ml of ACN and analysis with GC-QQQ * Two different Dispersive-SPE kits were used. Bond Elut QuEChERS Dispersive Kit for other food methods, CH Bond Elut QuEChERs Dispersive Kit for all food types, CH

36 Picture of the Solutions after Sample Preparation Clean up with Bond Elut QuEChERS Dispersive Kit for all food types PSA, C18, GCB, MgSO4 Clean up with Bond Elut QuEChERS Dispersive Kit for other food methods C18, MgSO4

37 TIC of spike 10 ug/kg pesticides in chicken muscle with Bond Elut QuEChERS AOAC kits. Clean up with Bond Elut QuEChERS Dispersive Kit for other food methods. TIC of chicken muscle sample blank with Bond Elut QuEChERS AOAC kits. Clean up with Bond Elut QuEChERS Dispersive Kit for other food methods.

38 TIC of spike 10 ug/kg pesticides in chicken muscle with Bond Elut QuEChERS AOAC kits. Clean up with Bond Elut QuEChERS Dispersive Kit for all food types. TIC of chicken muscle sample blank with Bond Elut QuEChERS AOAC kits. Clean up with Bond Elut QuEChERS Dispersive Kit for all food types.

39 Methamidophos Dichlorvos Acephate Omethoate Monocrotophos BHC-alpha Hexachlorobenzene Dimethoate BHC-beta BHC-gamma Diazinon BHC-delta Vinclozolin Parathion-methyl Chlorpyrifos-methyl Metalaxyl Heptachlor Pirimiphos-methyl Fenitrothion Malathion Fenthion Aldrin Chlorpyrifos Parathion Triadimefon Kelthane Pendimethalin Heptachlor exo-epoxide Chlordane-oxy Isofenphos Triadimenol Quinalphos Methidathion DDE-p,p' Myclobutanil Dieldrin Endrin Chlorfenapyr DDD-p,p' Ethion DDT-o,p' DDT-p,p' Iprodione Acetamiprid Phosmet Bifenthrin Tetradifon Phosalone Cyhalothrin Permethrin I Permethrin II Fenvalerate I Difenoconazole I Deltamethrin Results Recoveries, Continued 120.0% 100.0% 80.0% 60.0% 40.0% 20.0% 0.0% Recoveries for the pesticides of 50 µg/kg spiked in chicken muscle sample with QuEChERS and clean up with Bond Elut Dispersive Kit for other food methods. 39

40 Methamidophos Dichlorvos Acephate Omethoate Monocrotophos BHC-alpha Hexachlorobenzene Dimethoate BHC-beta BHC-gamma Diazinon BHC-delta Vinclozolin Parathion-methyl Chlorpyrifos-methyl Metalaxyl Heptachlor Pirimiphos-methyl Fenitrothion Malathion Fenthion Aldrin Chlorpyrifos Parathion Triadimefon Kelthane Pendimethalin Heptachlor exo-epoxide Chlordane-oxy Isofenphos Triadimenol Quinalphos Methidathion DDE-p,p' Myclobutanil Dieldrin Endrin Chlorfenapyr DDD-p,p' Ethion DDT-o,p' DDT-p,p' Iprodione Acetamiprid Phosmet Bifenthrin Tetradifon Phosalone Cyhalothrin Permethrin I Permethrin II Fenvalerate I Difenoconazole I Deltamethrin Results Recoveries 120.0% 100.0% 80.0% 60.0% 40.0% 20.0% 0.0% Recoveries for the pesticides of 50 µg/kg spiked in chicken muscle sample with QuEChERS and clean up with Bond Elut Dispersive Kit for all food types. 40

41 Today s Agenda QuEChERS A Brief Overview Preservatives Analysis in Pet Food Pesticide Analysis in Juice Concentrates Pesticide Analysis in Chicken Pesticide Analysis in Herbal Teas Conclusions and Summary 41

42 QuEChERS Method Multi-residue analysis of Pesticides in Herbal Tea An LC/MS/MS multiple reaction monitoring method was developed on the Agilent 1290/6410 LC Triple Quadrupole Mass Spectrometer system(lc/qqq) for 83 pesticide residues in herbal green and black teas. Sample Preparation process follows the QuEChERS original 10 g extraction. Several dispersive cleanup sorbent options were evaluated. Results: Tea samples were analyzed to verify the calibration coefficient, recovery and reproducibility. Planar pesticide recoveries are affected by carbon sorbent mass and type. The method recoveries for the majority of the compounds were between % with RSD below 15%.

43 Sample Preparation Weigh 1.00 g dried tea (±0.01g) in 50 ml centrifuge tube and add 10 ml water Add QC spike solution as necessary, vortex 1 min Add 10 ml of ACN containing I.S. (50 ppb dimethoate-d6) Cap and shake vigorously for 60 seconds Add Bond Elut Original QuEChERS Extraction salt packet Cap and shake vigorously for 60 seconds 4000rpm for 5 min Transfer 5 ml of upper ACN layer to Dispersive-SPE, 15 ml tube Vortex 1 min, 4000 rpm for 5 min Transfer 2 ml extract to tube and dry under nitrogen Reconstitute with 1 ml of 70/30 Water/ ACN, syringe filter and analysis by LC-QQQ

44 Chromatogram Chromatogram of 100 ppb spike in black tea.

45 Green and Black Tea dspe Cleanup Black Tea Green Tea Extract A B C Extract A B C A = GCB, B = Alternative Carbon Sorbent, C = Chlorophyll Specific Sorbent

46 % Pesticides % Pesticides Black Tea 10 ppb Spike >120 ND Green Tea 10 ppb Spike 46 Black Tea 100 ppb Spike >120 ND Green Tea 100 ppb Spike >120 ND >120 ND % Recovery GCB, Alternative carbon sorbent, No carbon, Commercially available, chlorophyll specific, polymeric sorbent, High GCB, High alternative carbon

47 %Recovery Comparison of Recoveries for Planar Pesticides Black Tea Green Tea GCB, Alternative carbon sorbent, Commercially available, chlorophyll specific, polymeric sorbent, High GCB, High alternative carbon 47

48 Today s Agenda QuEChERS A Brief Overview Preservatives Analysis in Pet Food Pesticide Analysis in Juice Concentrates Pesticide Analysis in Chicken Pesticide Analysis in Herbal Teas Conclusions and Summary 48

49 QuEChERS Conclusions and Summary QuEChERS is a flexible, simple, yet powerful tool for preparing a wide variety of food products for analysis by GC and LC-MS. A straightforward approach to method development and optimization is easy with pre-packaged kits and bulk sorbents. QuEChERS reproducibility and ruggedness can be improved with products designed for optimal performance. Agilent offers the QuEChERS expertise along with the range of products needed for successful QuEChERS sample preparation. 49

50 Questions 50

51 Acknowledgements Agilent Technologies SPP Team Angelica Riemann Trisa Robarge Mike Chang Andy Zhai Irina Dioumaeva Special thanks to Jon Wong 51

52 Thank you for your time and attention 52

53 Addition of Anhydrous Extraction Salts The extraction addition step is very crucial to recovery of pesticides The addition of the anhydrous extraction salt to the homogenized food sample that has already been mixed with the extraction solvent ACN produces the greatest recoveries and smallest relative standard deviation The sealed foil package easily facilitates this requirement versus the extraction salts being in a 50 ml PP tube, where the extraction salt integrity is questionable Extraction salts are easily poured directly into the matrix extract, minimizing exothermic effects or possible errors in adding the matrix to the salts

54 Exothermic Temperature Tracking Apparatus A 50 ml Polypropylene (PP) tube cap was modified to accommodate a thermocouple The thermocouple (Omega: HSTC-TT-K-24S-36-SMPW-CC) is attached to a digital thermometer (FLUKE 52 K/J Thermometer) recording temperature as a function of time The produce sample and extract are added to the 50 ml PP tube according to experiment 1 or 2 The 50 ml PP is vigorously shaken either by hand or by a mechanical vertical grinder Geno/Grinder The temperature is recorded over a period of time: 60 seconds Averages were taken over several experiments and the results are shown in graphs 1 and 2 Sample and Buffered Extraction Salts The Geno/Grinder is a registered trademark of SPEX Sample Prep

55 Temperature ºC Temperature ºC Temperature Graphs from AOAC and EN Extraction Kits AOAC Add Buffered Salts to Sample AOAC Sample Added to Buffered Salt EN Add Buffer Salts to Sample EN Sample Added to Bufferd Salt Time (seconds) Time (seconds) These graphs represent the thermal data acquired from the AOAC and EN extraction kits. A substantial increase in temperature is observed (increase of approx 25 ºC for the AOAC method and approx 10 ºC for the EN method) when the produce sample was added to the extraction salt in the 50 ml PP tube versus when the extraction salt from an anhydrous pouch was added to the produce sample.

56 Pesticides used to Evaluate the Effect of Temperature on Recovery The following four pesticides were used to evaluate the thermal effects that are prevalent when the produce is added to the extraction salts Isocarbophos Isofenphos Fenthion Nicotine* The 4 pesticides were chosen based on their labile chemical structures, nicotine being stable control Analysis was performed on a Agilent 6410 Triple Quad LC/MS using a Phenyl- Hexyl column 2.1 x 50 mm, 3.5 µm

57 Recovery % Relative Recoveries for the Labile Pesticides Pre-Spiked Extraction Salts Added to Matrix AOAC Method Pre-Spiked Matrix Added to Extraction Salts AOAC Method 10 0 Represents the negative effect adding the produce matrix to the salt has on recovery during the QuEChERS extraction step, AOAC method

58 Improvements to the Extraction of Samples Step 1: Extraction salts with homogenized food sample Consistency in shaking: Everyone shakes differently Variability in QuEChERS applications, recovery and RSDs Bond Elut Ceramic Homogenizers Reduces shaking time from 1 minute to <20 seconds! Consistent extraction of the sample with the salts Breaks up salt agglomerates Facilitates homogenization with angle cut Increase recovery of pesticides from sample 3 different sizes: 50 ml extraction tube and for dispersive tubes (15 and 2 ml) Page 58

59 QuEChERS Extraction: With and Without Ceramic Homogenizers CH no CH CH no CH

60 Recoveries Ceramic Homogenizers 120 Comparison of QuEChERS Method AOAC and EN With and Without Ceramic Homogenizers AOAC Method with Ceramic Homogenizers EN Method with Ceramic Homogenizers AOAC Method without Ceramic Homogenizers EN Method without ceramic Homogenizers 0 Inert ceramic material No loss in pesticide recovery Consistent recoveries Less individual variation Page 60

61 Percent Recovery Average Recovery with 20 Second Shaking 240 Extraction 220 and Ceramic Homogenizers Time in Seconds of Shaking Extract with Ceramic Homogenizers Organophosphates Carbamates Benzimidazoles Anilinopyrimidines Neonicotinoids Triazoles Strobilurins Pesticides used in study: Acephate, Carbaryl, Carbendazim, Cyprodinil, Imidacloprid, Imazalil, Methamidophos, Penconazole, Propoxur, Pymetrozine, Thiabendazole, Thiophanate-methyl, Ethoprophos, Kresoxim-methyl ; Apple matrix

62 Pre-Packaged Kits or Bulk and In-House Preparation? Pre-Packaged Kits Quality is free built into kits Reduced labor costs Increased productivity UV and moisture protection maintain quality of product Consistent results Less flexibility for method development In-House Preparation Increased labor costs Hygroscopic materials left open may reduce effectiveness Inconsistency in measurement variability in results Reduced productivity Good for method development, low sample volumes 62