LC-MS Optimierung: Eine Kombination von sowohl Hardware-, als auch Software-Weiterentwicklungen

Transcription

1 Langenauer Wasserforum 2009: Workshop Teil 1 (09:40 h - 10:05 h) Zweckverband Landeswasserversorgung Fa. Agilent [Dr. Volker Gnau] LC-MS Optimierung: Eine Kombination von sowohl Hardware-, als auch Software-Weiterentwicklungen 09. November 2009 Zweckverband Landeswasserversorgung

2 LC-MS Optimierung: Eine Kombination von sowohl Hardware-, als auch Software- Weiterentwicklungen Dr. Volker Gnau Agilent Technologies Waldbronn

3 Agenda 1. Vorstellungs der Agilent Konzepte, Ultra High Definition 2. Quantifizierung 1. direkte Injektion 2. online Solid Phase Extraction 3. Screening mit UHD-QTOF

4 Ultra High Definition Die Optimierung aller analytischen Dimensionen Signal Response Sensitivity Dynamic Range Linearity Peak Resolving Power Peak Capacity Separation Speed Mass Spectrum Data Mining Differential Profiling Annotation Ionization Profile Mass Accuracy Isotopic Fidelity Mass Resolving Power Acquisition Rate

5 Agilent 1290 Infinity LC Lowest Delay Volume Pump (w/o) mixer: 10 µl Pump, Fixed Loop 20 µl Pump, Fixed Loop, JetWeaver 55 µl Highest Precision Best Autosampler Performance ALS precision for small volumes: <1.5% from 0.5-1µLm, <0.7% from 1-2µL, 2-20 µl (40 ul) ** Pump Active Damping: RT stability < 0.2 % (1.5 min runs)** <0.002% carry-over with Chlorhexidine Optional needle seat backflushing with FlexCube Fixed Loop or Variable Loop Injections Greatest Productivity 2mL/min for highest resolution per time Reduced Ion & Matrix Suppression HT-Solution for up to 2000 samples/day (ACR) Complete Integration and control from MassHunter Enables method conversion from/to any (U)HPLC

6 Der perfekte Match - Highest Performance LC/MS Systems Agilent 1290 Infinity LC and Agilent 6540 Accurate Mass Q-TOF MS The ultimate Qual solution Agilent 1290 Infinity LC and Agilent 6460 Triple Quad MS The ultimate Quan solution

7 Methoden Entwicklung mit MassHunter Optimizer Principal workflow Setup compounds and rules Setup rules for precursor and fragment selection Setup optimization parameters Execute Optimization Creation of Acquisition Method MassHunter Optimizer is an Add-on software to MassHunter Acquisition It is used for the optimization of the compound dependant QQQ parameters (Fragmentor voltage and collision energy) Depending on the chosen rules MassHunter optimizer will make 1 to 5 individual injections for each compound All other QQQ parameters are optimized by the Autotune functionality in relation to the molecular mass over the whole mass range

8 Direkte Injektion Quantitative Analyse von Pestiziden Compounds included in method (236 compounds in Acephate Acetamiprid total) Acibenzolar-S-methyl Acrinathrin Aldicarb Aldicarb sulfone Aldicarb sulfoxide Amitraz Atrazine Avermectin B1a Azinphos-methyl Azoxystrobin Benalaxyl Bendiocarb Benfuracarb Benthiavalicarb Bifenthrin Bitertanol Boscalid Bromuconazole Bupirimate Buprofezin Butralin Cadusafos Carbaryl Carbendazim Carbofuran Carbofuran-3-hydroxy Carbophenothion Carbosulfan Chlorfenvinphos Chlorotoluron Chlorpyrifos Chlorthiophos Cinerin 1 Cinerin 2 Clofentezine Clothianidin Crufomate Cyanofenphos Cyazofamid Cyflufenamid Cyhalothrin Cymoxanil Cyproconazole Cyprodinil Cyromazine Demeton-S-methyl Demeton-S-methyl sulfone Oxydemethon methyl Dialifos Diazinon Diclobutrazol Dicrotophos Diethofencarb Difenoconazol Diflubenzuron Dimethoate Dimethomorph Dimethylvinphos(E) Dimoxystrobin Disulfoton-Sulfone Disulfoton-Sulfoxide Ditalimfos Diuron Dodine Edifenphos Emamectin B1a EPN Epoxiconazole Ethiofencarb Ethiofencarb sulfone Ethiofencarb sulfoxide Ethion Ethofumesate Ethoprophos Etofenprox Etrimfos Fenamidone Fenamiphos Fenamiphos-Sulfone Fenamiphos-Sulfoxide Fenarimol Fenazaquin Fenbuconazol Fenhexamid Fenoxycarb Fenpropimorph Fenpyroximate Fenthion Fenthion-sulfon Fenthion-Sulfoxide Fipronil Fluazifop acid Fluazifop-P-butyl Fludioxonil Flufenacet Flufenoxuron Fluopicolide Fluoxastrobin Fluquinconazole Flusilazole Flutriafol Fluvalinate Fonofos Formothion Fosthiazate Furalaxyl Furathiocarb Haloxyfop acid Heptenophos Hexaconazole Hexazinone Hexythiazox Imazalil Imidacloprid Indoxacarb Iprovalicarb Isazofos Isoprocarb Isoproturon Jasmolin 1 Jasmolin 2 Kresoxim-methyl Lenacil Linuron Lufenuron Malaoxon Malathion Mecarbam Mepanipyrim Mepronil Metalaxyl Metazachlor Metconazole Methamidophos Methidathion Methiocarb Methiocarb sulfone Methiocarb Sulfoxide Methomyl Methoxifenozide Metolachlor Metolcarb Metrafenone Metribuzin Mevinphos-1 Monocrotophos Myclobutanil Neo-Quassin Nitenpyram Nuarimol Ofurace Omethoate Oxadixyl Oxamyl Paclobutrazol Paraoxon-ethyl Paraoxon-methyl Penconazol Pencycuron Pendimethalin Phenthoate Phorate Sulfone Phorate Sulfoxide Phosalone Phosmet Phosphamidon Phoxim Picoxystrobin, Piperonyl butoxide Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Profenofos Prometryn Propachlor Propamocarb Propanil Propargite Propazine Propetamphos Propham Propiconazole Propoxur Propyzamide Proquinazid Prosulfocarb Pymetrozine Pyraclostrobin Pyrazophos Pyrethrin 1 Pyrethrin 2 Pyridaben Pyridaphenthion Pyrifenox Pyrimethanil Pyriproxifen Quassin Quinalphos Quinoxyfen Resmethrin Rotenone Simazine Spinosad A Spinosad D Spirodiclofen Spiromefesin Spiroxamine Sulfotep Tebuconazole Tebufenozide Tebufenpyrad Teflubenzuron Terbufos Terbufos sulfone Terbufos sulfoxide Tetrachlorvinphos Tetraconazole Tetramethrin Thiabendazole Thiacloprid Thiamethoxam Thiobencarb Thiodicarb Tolylfluanid Triadimefon Triadimenol Triazamate acid Triazophos Trichlorfon Trietazine Trifloxystrobin Triticonazole Zoxamidec

9 Methoden Entwicklung mit MassHunter Optimizer Dynamic MRM database with transitions for > 600 pesticides

10 Rt # Concurrent dmrm = x3 x4 x4 x3 x4 x2 x1 Cycle time Selection of 24 pesticide transitions from previous slide To illustrate efficiency of cycle times using dynamic MRMs.

11 # Concurrent dmrm = x3 x4 x4 x3 x4 x2 x1 Cycle time Selection of 24 pesticide transitions from previous slide To illustrate efficiency of cycle times using dynamic MRMs.

12 Quantitative Analyse von Pestiziden Chromatographic conditions Agilent 1290 Infinity LC system consisting of: HPLC method Separation column: Mobile phase: - binary pump Inj.Vol.: 1 or 10 µl - wellplate sampler - column compartment - diode array detector (not used) ZORBAX Eclipse Plus C-18 RRHD column, 150 x 2.1 mm, C, p max = 1030 bar A: 5 mm ammonium formate B: methanol + 5 mm ammonium formate Flow: 0.4 ml/min Gradient: 0.00 min 10 % B 0.50 min 10 % B 3.50 min 50 % B min 100 % B min 100 % B min 10 % B min 10 % B

13 Quantitative Analyse von Pestiziden Agilent Jet Stream conditions Spray chamber conditions: Gas temp.: 200 C Dry gas: 8 l/min Nebulizer: 30 psi Sheath gas temp: 300 C Sheath gas flow: 11 l/min Positive Negative CapVoltage: 4500 V 3000 V Nozzle voltage 300 V 0 V Automatic setup of MRM tables based on selected cycle time, retention times and retention time windows for the individual compounds Cycle time 500 ms Interscan delay 3.5 ms Total No. of MRMs 484 Maximum No. of concurrent MRMs 68 Minimum Dwell time Maximum Dwell time 3.85 ms ms

14 Quantitative Analyse von Pestiziden MRM traces of 236 pesticides in sample (10 ng/ml) Method includes 236 pesticides with 2 MRM transitions each Variable dwell times, Inter-scan delay 3.5 ms

15 Quantitative Analyse von Pestiziden - Ausschnitt MRM traces of 236 pesticides in sample (10 ng/ml) more than 120 MRM transitions in 1.2 min

16 Quantitative Analyse von Pestiziden Example 1: Cyromazine EIC for Cal_3 (1.0 pg o.c.) Quantifier: Qualifier: Peak width: 12.0 s Cycle time: 500 ms Data points: 24

17 Quantitative Analyse von Pestiziden Example 2: Linuron EIC for Cal_3 (1.0 pg o.c.) Quantifier: Qualifier: Peak width: 12.3 s Cycle time: 500 ms Data points: 25

18 Agilent 1290 Infinity LC Methode für Pestizide MRM traces of Avermectin B1a (10 ng/ml) 2x10 3 5x10 3 8x10 3 RRLC method (25 min gradient) RT Width min min 1290 method (12 min gradient) RT Width 9.70 min min 1290 method (7 min Gradient) RT Width 5.51 min min

19 Online SPE Proben Vorbereitung An Agilent solution for water analysis the AQUA system

20 Agilent AQUA Online-SPE System - Setup System configuration SPE system: Quarternary pump G1329A autosampler with 900 µl head 6 port 2 pos valve 12 port 6 pos valve 12 port stream selection valve (optional) RRLC system: Binary pump SL Well plate sampler SL (not used) Column department SL

21 Das Setup des Agilent AQUA Online-SPE Systems Method implementation in Mass Hunter Acquisition Single 900 µl injection: DRAW def. amount from sample VALVE mainpass WAIT 0.7 min / 1.5 min** REMOTE startpulse ** The delay volume of the online-spe system is approximately 100 µl. If the 900 µl seat extension is used the wait time has to be extended to 1.5 min.

22 Quantitative Bestimmung von Pestiziden MRM traces of pesticides in calibration samples w/wo SPE HPLC separation no SPE Cal_7 (1 µg/l), Inj.Vol. 20 µl HPLC separation SPE cartridge as precolumn Cal_7 (1 µg/l), Inj.Vol. 20 µl All compounds elute from cartridge under gradient conditions, increased RTs and slightly broader peaks (late eluting compounds) due to longer column. HPLC separation + SPE (backflush mode) Cal_5 (100 ng/l), Inj.Vol. 200 µl RTs similar to separation with SPE cartridge mounted as precolumn but decreased peak widths and better chromatographic resolution (result of backflush mode).

23 Quantitative Bestimmung von Pestiziden Example 1: 2-Hydroxyatrazine EIC for Cal_1 (1.0 ng/l) Quantifier: Qualifier: Peak width: 22.7 s Data points: 25 Linear range: < 1 ng/l to 1 µg/l

24 Quantitative Bestimmung von Pestiziden Example 1: 2-Hydroxyatrazine (S/N calc. peak-to-peak) 2-OH-Atrazine Water Blank 2-OH-Atrazine Test Sample 1 2-OH-Atrazine Cal 1 (1.0 ng/l) 2-OH-Atrazine Cal 2 (5.0 ng/l) 2-OH-Atrazine Test Sample 2 2-OH-Atrazine Cal 3 (10.0 ng/l) 2-OH-Atrazine Test Sample 3 2-OH-Atrazine Cal 4 (50.0 ng/l)

25 Quantitative Bestimmung von Pestiziden 20-fold injection of Cal_4 (50 ng/l, same cartridge) TIC-Overlay for 20-fold injection of Cal_4 Compound Average (in ng/l) SD RSD 2OH-Atrazine Atrazine-desisopropyl ,6-Dichlorobenzamide Metamitron Atrazine-desethyl Dimethoate Hexazinone Simazine Cyanazine Bentazone Atrazine Compound Average (in ng/l) SD RSD Isoproturon ,4-D MCPA DNOC Mechlorprop Dichlorprop Terbuthylazin Dinoseb Pendimethalin

26 Zusammenfassung Agilent LC/MS has achieved a new, higher level of performance Combined operation in MassHunter software Agilent 1290 Infinity LC allows fast, hi resolution separations Huge peak capacity for complex separations Excellent injection and RT precision Low delay volume for very fast methods Very low AS carry-over Agilent G6460 QQQ system can keep up! Fast pos/neg switching Fast MRM and Dynamic MRM methods for triple quad Dynamic MRM databases for pesticides and forensics Excellent sensitivity and linear dynamic range

27 6540 UHD Accurate Mass Q-TOF LC/MS The Highest Performing Bench-top Q-TOF - By Far Exceptional Ultra High Definition Performance With No Trade-Offs Up to 40,000 resolution Excellent isotopic fidelity Mass measurement error down to 500 ppb 5 orders of linear dynamic range Femtogram-level sensitivity with Agilent Jet Stream FAST acquisition for UHPLC up to 20 spectra/second Made Possible by Continuing Technology Breakthroughs Ion Beam Compression (IBC) cools & focuses ion beam Extended Flight Tube with Enhanced Mirror Technology (EMT) New Photonis Fast Bipolar Detector The Ultimate Qualitative Analysis System Proteomics/Metabolomics Non-targeted food/environmental screening Impurity analysis Metabolite ID

28 6540 Ultra High Definition Q-TOF Resolving Power Across the Mass Range Resolution is Scan Rate Independent 5 x R= R= R= R=30218 R= R= R= R= R= Counts vs. Mass-to-Charge (m/z)

29 6540 MS Resolution Is Invariant With Acquisition Rate - Pesticides Example Resolution azoxystrobin 404 m/z cyproconazole 292 m/z cyprodinil 226 m/z diazinon 305 m/z linuron 249 m/z paclobutrazol 294 m/z propazine 229 m/z tetraconazole 372 m/z tricyclazole 190 m/z Acquisition Rate (scans/second)

30 6540 Ultra High Definition QTOF Mass Accuracy Repetitive Injections 40pg reserpine on-column, 10 injections +ESI EIC( ) Scan Frag=240.0V Reserpine_40pgms3.d Counts vs. Acquisition Time (min) Error (ppm) Run Mean 0.25 Std. Dev 0.32 x Isotope Obs % Calc % Obs m/z Calc m/z Diff (ppm) Counts vs. Mass-to-Charge (m/z) 250 ppb mass accuracy calibration and very accurate isotopic ratios

31 Agilent MassHunter Software for Mass Spectrometry also available for GC/MS and ICP-MS in 2009 MassHunter Workstation Data Acquisition/Control Qualitative Analysis Quantitative Analysis Application Software MassHunter Profiling MS Optimizer (small mol and peptides Metabolite ID Easy Access for MassHunter MassProfiler Prof. for MassHunter

32 Pesticide Screen 1290 Infinity/6540 Q-TOF 1290/ sec analysis 250 pesticides (125pg on-column) 1-naphthol Mass [M+H] + = Resolution = ppm Peak Width=0.8 sec 23 data points Spinosad A Mass [M+H] + = Resolution = ppm Fenpyroximate E Mass [M+H] + = Resolution = ppm

33 Analysenzeit: 17 Minuten 224 Pesticides 6530 TOF/1290 Infinity UHPLC Daten-Akquisitionsrate: 3 Hz 50pg on-column

34 Analysenzeit: 3 Minuten 224 Pesticides 6530 TOF/1290 Infinity UHPLC Daten-Akquisitionsrate: 10 Hz 50pg on-column

35 Analysenzeit: 90 Sekunden 224 Pesticides 6530 TOF/1290 Infinity UHPLC Daten-Akquisitionsrate: 20 Hz 50pg on-column

36 Pesticide Screen 1290 Infinity/6540 Q-TOF Beispiel: Extrahierte Ionenchromatogramme mit unterschiedlichen Extraktionsbreiten Spinosyn A EIC ± 0.5 Da ± 0.1 Da ± 50 ppm (ca Da) ± 10 ppm (ca Da)

37 Target screening confirmation via MS/MS Beispiel: Compoundspektren für Spinosyn A Oben: MS1-Spektrum Unten: MS/MS-Spektrum, Annotationen zeigen m/z und generierte Summenformel Spinosyn A Referenzmasse

38 Target screening confirmation via MS/MS Beispiel: MS1 bzw. MS/MS-Chromatogramm für Spinosyn A mit vergleichbarem Signal/Rausch-Verhältnis Spinosyn A MS/MS EIC S/N = 96 Spinosyn A MS1 EIC S/N = 84

39 Agilent TOF Screening Solution (PCD) FBF Find by Formula

40 Non-Targeted Screen Summary (QTOF/TOF) Agilent TOF/Q-TOF for unknown screening (low ppm mass accuracy.) Agilent MassHunter PCD utilizes the accurate mass data to provide reliable positives for qualitative screens. Extra Confirmation with Isotope abundance (pattern), spacing and mass matching Pesticide analytes Fully Automated integration with MassHunter Qualitative Analysis SW. Reliable Data mining Algorithms (MFE or FBF.) Low pico-gram on-column Sensitivity. Fully Automated. Faster data acquisition with 1290 Infinity UHPLC.