Is Your Sample Preparation Good Enough? Sample Preparation Strategies for
Is Your Sample Preparation Good Enough? Sample Preparation Strategies for Multiresidue and Compound/Class Specific Analysis ©2012 Waters Corporation 1 Sample Preparation Is This Good Enough? ©2012 Waters Corporation 2 Method Requirements – Detector Type What Further cleanup/enrichment is required? – LC-MS/MS minimal or none – LC-MS/Tof minimal enrichment – GC-MS enrichment and removal of chlorophyll – LC/UV significant cleanup/enrichment by SPE ©2012 Waters Corporation 3 Method Requirements – Analysis Type Multi-residue vs. Compound-specific analysis Multi-Residue/ Multi-Class Compound or Class Specific Entire procedure (sample prep & analytical method) Generic to a diverse set of analytes Specific for one compound or class of compounds Sample Preparation Protocol Simple (one or two steps) Multi-step Speed Maximizing recovery & matrix cleanup Goal of Sample Cleanup Recovery & cleanup are compromised for a large number of analytes Level of Sample Cleanup Minimal Significant Tandem MS, Time-of-Flight Single quad MS, UV, FLR, ELS, GC/MS, GC Detection Techniques ©2012 Waters Corporation Minimizing interference/ion suppression 4 Multi-residue Test Methods Why are they attractive? –More information per analytical run –Streamlined (fewer) workflows –Better asset utilization What are the technical challenges? –“Universal” sample extraction –Performance demands on separation and detection ©2012 Waters Corporation 5 Method Development Goals Rapid, cost effective sample preparation – Wide variety of analyte/commodity combinations Rapid and efficient separation – Complex extracts, many residues Highly selective, sensitive detection – Demanding regulatory limits (low ppb) – Demanding QC criteria Simple set-up and operation of complex methods ©2012 Waters Corporation 6 Major Areas of Multi-Class and Multi-Residue Screening Pesticides • Major Classes • • • • • Organophosphate Carbamate Organochlorine Pyrethroid Glyphosate • Human Health Effects • Neurological damage • Cancers • Hormone, endocrine system disruption ©2012 Waters Corporation Veterinary Drugs • Major Classes • Topical antiseptics, bactericides, fungicides • Steriods, peptides • Antiparasitic drugs • Antibiotics • Human Health Effects • Drug residue allergies • Cancer, reproductive, developmental effects • Hormone disruption • Antibiotic resistance • Drug misuse 7 Sample Preparation Multi-Residue Pesticide Screening QuEChERS “Quick, Easy, Cheap, Effective, Rugged, Safe” Popular approach to performing sample cleanup – Lower material costs – Rapid and straightforward sample preparation – Minimal use of organic solvent and buffer additives High throughput sample preparation – Multi-class, multi-residue method – High quality results with a minimal number of steps Combination of salting-out LLE and liquid partition cleanup – Analytes are partitioned into acetonitrile for further clean up (if required) ©2012 Waters Corporation 8 DisQuE Products Liquid-extraction and partition cleanup Dispersive SPE cleanup ©2012 Waters Corporation 9 Basic QuEChERS Procedure CEN Method AOAC Method 1.5 g sodium acetate 6 g MgSO4 150 mg MgSO4 50 mg PSA ©2012 Waters Corporation 1 g trisodium citrate dihydrate 0.5 g disodium hydrogencitrate sesquihydrate 1 g NaCl 4 g MgSO4 150 mg MgSO4 25 mg PSA 25 mg C18 10 Extraction/Partitioning AOAC Method Transfer 15 g of sample to a 50 mL extraction tube. Add 15 mL of 1% acetic acid in acetonitrile. Shake vigorously for 1 minute. Add Contents of DisQuE Pouch Centrifuge, take aliquot of top layer for analysis or further cleanup by dSPE ©2012 Waters Corporation 11 First Extraction: 50 mL Tube (Tube 1) Water is IMPORTANT!! – Produces strong liquid/liquid partition cleanup after DisQuE salts are added o Removes unwanted matrix o Acetonitrile extraction more effective – If water not present in matrix, then add it Acetonitrile Layer (ANALYTES) Homogenized sample Aqueous Layer (Saturated buffer salts + ionic/polar analytes) Undissolved buffer salts) ©2012 Waters Corporation 12 Recommended Water Content for QuEChERS by Matrix Sample weight Water added Fruits & vegetables > 80% water content 10 g - Fruits & vegetables 2580% water content 10 g Xg Cereals 5g 10 g Dried fruits 5g 7.5 g Honey 5g 10 g Spices 2g 10 g Sample type Note X = 10 g – water amount in 10 g sample Water can be added during comminution step Note 1: Recommended for CEN QuEChERS by M. Anastassiades Note 2: The water added should be kept at low temperature (< 4 C) ©2012 Waters Corporation 13 Cleanup Tube 2 Dispersive SPE Provides additional cleanup – A 1 mL aliquot of the top layer extract from Tube 1 is transferred to a 2 mL dispersive SPE tube – Most appropriate for cleanup prior to GC-MS Sorbent choices – MgSO4 (standard): removes residual water, important for GC analysis – PSA (standard): removes acids/sugars – Graphitized Carbon Black (GCB): removes chlorophylls/pigments (AND ANALYTES !) – C18: removes fats, oils, non-polar compounds Acetonitrile Layer (ANALYTES ARE HERE) Sorbent (INTERFERENCES) ©2012 Waters Corporation 14 Example #1 – Pesticides in Red Pepper ©2012 Waters Corporation 15 Example of Multi-Residue Screening Pesticides in Red Pepper Instrument: UPLC-QTof Cleanup Required: Minimal – QuEChERS sample preparation – No further cleanup Enrichment Required: Minimal – Sample enriched 5:1 by evaporation ©2012 Waters Corporation 16 UPLC-QTof Conditions Mass Spectrometer Xevo G2-S QTof™ ACQUITY UPLC™BEH C18 50 mm x 2.1 mm, 1.7 µm Ionization Mode ESI + (0.8 kV) Column temperature 45oC Cone voltage 25V Flow 0.45 mL/min Desolvation temperature 550˚C Mobile phase (A) H2O (0.01M Amm. Acetate) Acquisition Range 50-1200 m/z Acquisition Rate 10 spectra/s Low collision energy 4 eV Ramp collision energy 15-35 eV System ACQUITY UPLC™ I-Class Column (B) MeOH (0.01M Amm. Acetate) Injection volume Gradient ©2012 Waters Corporation 5µL Time (min) %age A %age B 0.00 98 2 0.25 98 2 12.25 1 99 13.00 1 99 13.01 98 2 17.00 98 2 17 213 Pesticides in Red Pepper Xevo G2-S QTof 5.82 (retention time) 202.0488 (exact mass) ©2012 Waters Corporation Data provided by Dominic Roberts, Waters 18 Extracted Ion Chromatograms 3 Pesticides from the 213 Compound TIC Atrazine Diuron Metolachlor ©2012 Waters Corporation Data provided by Dominic Roberts, Waters 19 Example #2 – Vet Drugs in Milk ©2012 Waters Corporation 20 Example of Multi-Residue Screening Veterinary Drugs in Milk Instrument: UPLC-MS/MS (Xevo TQ) Cleanup Required: Minimal – Multi-residue sample preparation – C18 pass-thru cleanup Enrichment Required: Minimal – Sample enriched 3:1 by evaporation ©2012 Waters Corporation 21 Veterinary Drug Classes Analyzed by Multiresidue Methods Tetracycline Fluoroquinolone tetracycline enrofloxacin Sulfonamide sulfamethazine Beta Lactam NSAID Steroid Macrolide erythromycin Beta-adrenergic salbutamol oxacillin ©2012 Waters Corporation phenylbutazone dexamethasone 22 Multiresidue Method Milk – 2 mL Sample Initial Extraction/Precipitation Pipet 2 mL sample into centrifuge tube Add 2 mL acetonitrile (ACN) Centrifuge @ 8000 x g Take 2 mL supernatant Protein Precipitation Add 3 mL acetonitrile(0.2% formic acid) Centrifuge @ 8000 x g Take 1 mL supernatant provides good recovery of most compounds minimal extraction of fat much protein in extract secondary protein precipitation step removes most residual protein without significant loss of polar analytes SPE Cleanup ? ©2012 Waters Corporation 23 SPE Cleanup Sep-Pak C18 (pass-thru mode) Condition 1 mL 80:20 acetonitrile/water install collection tubes 1 cc 100 mg Pass-Thru/Collect 1 mL protein ppt sample Rinse/Collect 0.5 mL 80:20 acetonitrile/water 40 mg/well add 0.25 mL 200 mM ammonium formate in 50:50 ACN/methanol* Evaporate/Reconstitute 0.2 mL 25:75 acetonitrile/buffer (25 mM ammonium formate buffer @ pH 4.5) * buffers sample to protect acid labile analytes ©2012 Waters Corporation 24 Results: Milk 0.67 x MRL Level Compound Carbadox Chloramphenicol(P) Chlorotetracyline (T) Ciprofloxacin (F) Dexamethasone (St) Enrofloxacin (F) Erythromycin (M) Lincomycin(M) Oxacillin (B-L) Oxytetracycline (T) Penicillin (B-L) Phenylbutazone (NSAID) Ractopamine Salbutamol Sulfamerazine (S) Sulfamethazine (S) Sulfanilamide (S) Tetracycline (T) Spike Level (ng/g) 67.0 67.0 67.0 67.0 67.0 134.0 6.7 33.0 67.0 67.0 33.0 67.0 200.0 67.0 67.0 67.0 67.0 67.0 %REC (%RSD) %Suppression* (n=3) 27 (27) -43.0 94 (16) 10.0 22 (20) 7.0 67 (20) 32.0 87 (6) -8.0 76 (11) 26.0 59 (10) 5.0 102 (9) 25.0 79 (12) -9.0 24 (16) -9.0 73 (8) -8.0 67 (18) 20.0 65 (14) 0.0 80.4 (3) 96.0 71 (4) -16.0 71 (6) -74.0 110 (30)* 60.0 31 (18) -21.0 * negative number signifies enhancement ©2012 Waters Corporation 25 Review Multi-Class Methods Initial extraction must be appropriate for many classes – QuEChERS for Fruits and Vegetables – Multi-residue meat extraction Only Minimal Cleanup Possible – Cleanup must be suitable for many classes of compounds o dSPE o Pass-thru SPE ©2012 Waters Corporation 26 Method Requirements Multi-residue vs. Compound-specific analysis Multi-Residue/ Multi-Class Compound or Class Specific Entire procedure (sample prep & analytical method) Generic to a diverse set of analytes Specific for one compound or class of compounds Sample Preparation Protocol Simple (one or two steps) Multi-step (if necessary) Speed Maximizing recovery & matrix cleanup Goal of Sample Cleanup Recovery & cleanup are compromised for a large number of analytes Level of Sample Cleanup Minimal Significant Tandem MS, Time-of-Flight Single quad MS, UV, FLR, ELS, GC/MS, GC Detection Techniques ©2012 Waters Corporation Minimizing interference/ion suppression 27 Compound (or Class) Specific Analysis Reasons – Lower detection limit required compared with screening method – Confirmation of detected analyte from screening analysis – Screening method is not appropriate (i.e., tetracyclines) – LC-UV method desired Development of compound specific methods – From screening method extract (where appropriate) – Using optimized alternate extraction procedure o Organic extraction procedure is not suitable • i.e., tetracyclines require aqueous extraction pre-treatment ©2012 Waters Corporation 28 Traditional SPE in Food Analysis For sample enrichment – To achieve lower detection limits (concentrate sample 10-100X) using less sensitive detection For sample cleanup – Reduce matrix effects – Reduce interferences for less selective detectors (UV, FLR, ELS) If multiresidue extraction method is not suitable Before SPE clean-up ©2012 Waters Corporation After SPE clean up 29 Is Multi-Residue Extraction Suitable to Develop Class-Specific Method? Sample preparation: PESTICIDES Typical % Recovery Suitability for Compound-Specific Method Organochlorine/ organophosphorous >80 Very good Carbamate/Urea/triazine >80 Very good Nicotinamide >70 Very good Phenoxyacid herbicides >70 Very good Sulfonylurea >60 Good Quaternary ammonium <20 Not suitable Class ©2012 Waters Corporation QuEChERS 30 Is Multi-Residue Extraction Method Suitable for Class-Specific Analysis? Sample preparation: VETERINARY DRUGS % Recovery Suitability for CompoundSpecific Method Sulfonamides, Fluoroquinolones, Betalactams, Steroids >70 Very good NSAIDs >60 Good Beta-andrenergenics >70 Fair (matrix effects) Macrolides >50 Fair Tetracyclines <40 Poor Aminoglycosides <10 Not Suitable Class ©2012 Waters Corporation 80/20 ACN/H2O with 0.2% formic acid 31 Example #1 – Conazole Fungicides in Orange Juice ©2012 Waters Corporation 32 Sample Preparation for Conazole Fungicides in Orange Juice SPE Protocol for Oasis MCX cartridge (3 cc, 60 mg) carbendazim thiabendazole Condition/Equilibrate Is multi-residue extraction method (QuEChERS) suitable? – Yes, recovery > 90% Wash 1 2% formic acid in H 2O Characterize the analytes – Load diluted QuEChERS extract They are bases Wash 2 MeOH Select the appropriate – Oasis® Sorbent Oasis MCX Elute 5% ammonia in MeOH Prepare sample for SPE – Dilute 2 mL QuEChERS extract 4-fold with aqueous formic acid prior to SPE Evaporate/Reconstitute 0.5 mL for UPLC- MS/MS (4X) 0.2 mL for UPLC -UV (10X) ©2012 Waters Corporation 33 Oasis® MCX prior to UPLC-MS/MS Recovery 120 35 % Suppression 100 % Recovery Matrix Effects 40 80 60 40 20 30 25 20 15 10 5 0 0 Carbendazim Thiabendazole Imazalil Difenoconazole Fenbuconazole Carbendazim Thiabendazole Imazalil Difenoconazole Fenbuconazole QuEChERS QuEChERS + SPE Sample enrichment – SPE provides enrichment to improve detection limits – LOD with QuEChERS only : ~ 1-10 ppb – LOD with QuEChERS + SPE: ~ 50-500 ppt Reduction of matrix effects Decrease instrument down time ©2012 Waters Corporation 34 What about UV Detection? Challenges with UV detection – Much more matrix interference o SPE cleanup essential – Need to use UV detection at 220 nm o Phosphate buffers provide much better UV transparency compared with formate or acetate • pH 6.8 is excellent buffer range for phosphate buffers – What about mobile phase pH? o pH 10 (UPLC-MS/MS conditions): carbendazim co-elutes with orange juice components that were not interferences for MS detection o pH 3: carbendazim elutes near void volume with significant interference from early eluting compounds o pH 6.8: separation gives sufficient retention for carbendazim away from void volume and before major orange juice components ©2012 Waters Corporation 35 UPLC-UV 100 ppb Conazole Fungicides Gradient table Instrument: ACQUITY UPLC H-Class with PDA Column: ACQUITY UPLC BEH C18, 3.0 x 100 mm, 1.7 µm Mobile Phase: A: 20 mM potassium phosphate buffer pH 6.8 B: acetonitrile UV detection at 220 nm Conazole_26Jjuly2012_06a Sm (Mn, 2x3) 3.5e-2 3.0e-2 1 2.5e-2 Flow 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 %A 75.0 75.0 35.0 35.0 5.0 5.0 75.0 75.0 %B 25.0 25.0 65.0 65.0 95.0 95.0 25.0 25.0 Curve 6 6 6 6 6 6 6 6 2: Diode Array 220 Range: 1.171 Carbendazim Thiabendazole Imazalil Fenbuconazole Difenoconazole (cis and trans) 2 2.0e-2 AU 1. 2. 3. 4. 5. Time 0.00 2.5 6.7 7.5 8.0 8.9 9.0 12.4 1.5e-2 5 1.0e-2 3 5.0e-3 0.0 4 -5.0e-3 1.00 ©2012 Waters Corporation 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 Time 8.00 36 Analysis of a Commercial Orange Juice with Incurred Carbendazim carbendazim Incurred sample carbendazim 10 ng/g standard thiabendazole Blank OJ sample LC-MS(MS) QuEChERS (no SPE) 13 ppb ©2012 Waters Corporation LC-UV QuEChERS (Oasis MCX SPE) 11 ppb 37 Analysis of an Incurred Residue of Carbendazim using LC-UV inc sample no SPE (1/2) Conazole_15Jun2012_12 2: Diode Array 281 Range: 1.745 1.0e-2 AU 7.5e-3 5.0e-3 No SPE 2.5e-3 0.0 0.00 1.00 2.00 Conazole_15Jun2012_11 Sm (Mn, 1x2) 3.00 carbendazim 1.0e-2 AU 7.5e-3 5.0e-3 1.00 2.00 5.00 2: Diode Array 281 Range: 4.024e-1 Oasis MCX SPE 2.5e-3 0.0 0.00 4.00 3.00 4.00 Time 5.00 Incurred carbendazim was quantified at 11 ng/g (ppb) in a store bought orange juice. ©2012 Waters Corporation 38 Example #2 – Tetracyclines in Milk ©2012 Waters Corporation 39 Sample Preparation for Tetracyclines in Milk pKa ~10 pKa ~3.5 pKa ~8.5 tetracycline Is multi-residue extraction method (2% formic acid in 80/20 ACN/H2O with SepPak C18 pass-through) suitable? – No, recovery < 40% – Need aqueous buffer extraction (which one?) ©2012 Waters Corporation 40 Which Aqueous Buffer to Use for Extraction? Tetracyclines are commonly extracted from meat and milk using McIlvaine buffers (mixed phosphate and citrate) at pH 4-5 – The McIlvaine buffer extracts the tetracyclines very well (>90%) – The McIlvaine buffer precipitates the proteins very well The McIlvaine aqueous buffer system is compatible with reversed-phase or mixed-mode SPE – Some reversed-phase retention is desirable for mixed-mode SPE because of high buffer salt concentration in load step Tetracyclines are strong chlelating agents – EDTA or oxalic acid is added to the McIlvaine buffer to sequester calcium McIlvaine, T.C., J. Biol. Chem. 49 (1921): 183–186. ©2012 Waters Corporation 41 Sample Preparation for Tetracyclines in Milk pKa ~10 pKa ~3.5 pKa ~8.5 tetracycline Is multi-residue extraction method (2% formic acid in 80/20 ACN/H2O with SepPak C18 pass-through) suitable? – No, recovery < 40% – Need aqueous buffer extraction (which one?) Characterize the analytes – They are amphoteric (acid and base) – They are chelators Bases pKa 2-10 Use Oasis® MCX Acids pKa 2-8 Use Oasis® MAX Condition/ Equilibrate Condition/ Equilibrate Load Load Wash 1 2% formic acid in Wash 1 5% ammonia in H2O* H2O* Wash 2 MeOH Wash 2 MeOH Elute 5% ammonia in Elute 2% formic acid in MeOH* MeOH* Evaporate/ Reconstitute Evaporate/ Reconstitute Select the appropriate Oasis® Sorbent – Oasis MCX or MAX – Try both with standard protocols ©2012 Waters Corporation *Prepare these solutions fresh daily 42 Results from Oasis® MAX Tetracyclines UPLC-MS/MS % Recovery at 50 ppb Spike Level (Milk) % Recovery at 200 ppb Spike Level (Milk) 100 100 Multi-Residue Method 80 Oasis MAX (Standard Protocol) % Recovery % Recovery 80 Multi-Residue Method 60 40 20 Oasis MAX (Standard Protocol) 60 40 20 0 0 Oxytetracycline Tetracycline Chlortetracycline Oxytetracycline Tetracycline Chlortetracycline MCX gave similar recovery – Meat samples had higher level of visible particulates (turbidity) Need to optimize recovery for MAX procedure – Tetracyclines are stong chelators and they are unstable – Leads to variable recovery Replace formic acid with oxalic acid in elute 2 o Oxalic acid counteracts the chelation o ©2012 Waters Corporation 43 SPE Method Optimization UPLC-MS/MS Results % Recovery at 200 ppb Spike Level (Milk) 100 Oasis® MAX Optimized protocol % Recovery Condition/Equilibrate 2 mL MeOH, 2 mL water 80 Oasis MAX (Standard Protocol) Oasis MAX (Optimized Protocol) 40 20 Load Sample from pretreatment 0 Oxytetracycline Wash 1 0.5 mL 5% NH4OH / water Tetracycline Chlortetracycline % Recovery at 50 ppb Spike Level (Milk) 100 Wash 2 0.5 mL methanol 80 % Recovery Elute 0.5 mL 45:55 acetonitrile / 75 mM oxalic acid Multi-Residue Method 60 Multi-Residue Method 60 Oasis MAX (Standard Protocol) Oasis MAX (Optimized Protocol) 40 20 0 Oxytetracycline ©2012 Waters Corporation Tetracycline Chlortetracycline 44 Conclusions – Multiresidue Analysis Is Your Sample Preparation Good Enough? Multiresidue extraction protocols (ie QuEChERS) may be good enough (not require subsequent dSPE or other cleanup/enrichment) – If instrument performance is acceptable – If required detection limits are met Multiresidue extraction protocols (ie QuEChERS) may require subsequent dSPE or other cleanup/enrichment – dSPE or pass-thru SPE to remove matrix interferences prior to GC-MS analysis o i.e. fats from meat extract or chlorophyll from plant extracts – Concentrate (enrich) sample prior to analysis by evaporation o Improve detection limits for MS-QTof (red pepper example) ©2012 Waters Corporation 45 Conclusions – Class Specific Analysis Is Your Sample Preparation Good Enough? A multiresidue extraction protocol (ie QuEChERS) may be suitable for a compound or class specific method – If compound of interest is effectively extracted – If instrument sensitivity is acceptable Compound or class specific methods may not be good enough (may require development) – If compound(s) of interest is not effectively extracted with multiresidue protocol o Alternate extraction protocol required – If chosen instrument is not sensitive or selective enough o SPE enrichment and cleanup required ©2012 Waters Corporation 46 Conclusions Examples From this Presentation Compound-specific analysis may require an optimized sample preparation strategy A multi-residue sample prep method may be adapted for a compound specific analysis (i.e., conazole fungicides) – The QuEChERS extract used for multi-residue extraction was employed to develop a compound-specific method – SPE using Oasis MCX was employed for further sample cleanup and enrichment A specific total sample preparation strategy may be needed for a compound class (i.e., tetracyclines) – The acetonitrile based multi-residue screening extraction protocol was not suitable (< 40% recovery) – An aqueous buffer extraction provides higher recovery (> 80%) o An optimized Oasis MAX SPE protocol was developed for further cleanup and sample enrichment ©2012 Waters Corporation 47 More Information Acknowledgements Kenneth J. Fountain John Martin Kathy Coffey Jennifer Burgess Euan Ross Mia Summers Dominic Roberts www.waters.com/food ©2012 Waters Corporation 48
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