Dr Stephen Lock Manager LCMS Support Northern Europe Global Co-ordinator Food/Beverage Network PSM

Transcription

1 LC/MS/MS & Two Dimensional LC/MS/MS and its s application to the analysis of contaminents in Food and the Environment Dr Stephen Lock Manager LCMS Support Northern Europe Global Co-ordinator Food/Beverage Network PSM

2 Traditional analysis of drinking water for pesticides Normally each pesticide screen is run seperately. Each pesticide suite of compounds requires a separate solid phase extraction step follow by a concentration step (typically large volumes > 100ml of sample are needed per extraction). If GC-MS analysis is used then a derivitisation step is often needed, especially for the more modern pesticides, before analysis. Typically sample preparation costs in excess of EUR 10 / per sample (excluding labour) Applera Corporation and MDS Inc.

3 Why use 2D LC in the analysis of water Allows concentration of a large volume injection with removal of matrix effects. Improves column lifetimes compared to direct injections on column Solid phase extraction is widely used in the water industry for sample preparation and therefore customers are familiar with this sample preparation. Huge reduction in sample preparation costs (both man hours and consumable costs). Faster analyses Applera Corporation and MDS Inc.

4 2DLC Procedure The 2DLC method uses two fluid paths; a high flow trapping path through the trapping column and a slower path through a small analytical column. The Trapping path is shown in blue, the Analytical path in red and is the same as that used for the clinical immunosurpressant analysis. Trapping Column Switching Valve Waste Analytical Column Applera Corporation and MDS Inc.

5 Results from the analysis of Triazine and Organonitrogen pesticides on the API 3200 System using 2D LC/MS/MS

6 HPLC Conditions for analysis of ON and Triazine pesticides on Binary PE Series 200 system Injection Volume = 5000 µl Mobile Phase A = Water, 0.1% Formic acid, 2 mm Ammonium Acetate Mobile Phase B = Methanol, 0.2 % Formic acid, 2 mm Ammonium Acetate Flow Rate = 0.6 ml/min HPLC Column = Agilent SB-C18 C8, 46 x 50mm, 1.8 µm Gradient Time % B Applera Corporation and MDS Inc.

7 HPLC Conditions for analysis of ON and Triazine pesticides loading pump conditions Delivery trapping Solvent Trap On Line SPE Cartridge = Water = Strata C18, 20 x 2 mm Valve Switch program Loading pump program Time Position Time Flow (ml/min) 1.0 To Waste (A) To MS (B) To Waste (A) Applera Corporation and MDS Inc.

8 Source Conditions & Resolution settings Resolution Q1 = Unit (peak width 0.75 at half height) Resolution Q3 = Low (off set = 0.1) Source Type = TURBO V Source (ESMS) IonSpray Voltage = 5500 V (+ve mode) Nebuliser gas = Air Source Temp. = 700 C Applera Corporation and MDS Inc.

9 MRM Transition were selected directly from the Cliquid software with often no further optimisation In this feasability study single MRM transition were used for each compound. A mixture of 27 compounds organonitrogen/triazine and azoles pestides were analysed. The Dwell time was generally 32ms per compound (including pause time) but was higher for weaker compounds e.g. EPTC This gave us >10 data points across a typical HPLC peak Applera Corporation and MDS Inc.

10 MS Conditions used with HPLC retention time observed Compound Q1 Mass Q3 Mass DP Voltage CE Voltage Retention Time (mins) Dwell Time (ms) ATRATON PROMETON TERBUTHYLAZINE DESMETRYN SIMETRYN AMETRYN PROMETRYN FENPROPIDIN TERBUTRYN FENPROPIMORPH EPTC PROPACHLOR SIMAZINE ATRAZINE PROPAZINE TRIETAZINE PROPYZAMIDE TRIALLATE TRIADIMEFON PENDAMETHALIN METAZACHLOR FLUTRIAFOL FLUSILAZOLE PROPICONAZOLE TEBUCONAZOLE EPOXICONAZOLE Applera Corporation and MDS Inc.

11 Example of a calibration line obtained for a organonitrogen pesticide, EPTC, in tap water range ppt Applera Corporation and MDS Inc.

12 Example of a calibration line obtained for a triazine, terbutylazine, in tap water range ppt Applera Corporation and MDS Inc.

13 Chromatograms obtained for the injection of a tap water blank, 10ppt and 100ppt pesticide spike in tap water Tap water blank 100 ppt spike in Tap water 10 ppt spike in Tap water Applera Corporation and MDS Inc.

14 The effect of matrix on the relative response of a 100ppt spike of pesticides in humic acid, distilled and tap water Distilled water Tap water 10ppm humic acid in water Applera Corporation and MDS Inc.

15 10ppt spiked into water of Prometryn (top) with a corresponding tap water blank on the bottom of the window Applera Corporation and MDS Inc.

16 10 ppt spiked into water of Terbutylazine (top), EPTC (middle) and Atrazine (bottom) and on the left with a corresponding tap water blank on the right hand side of the window Applera Corporation and MDS Inc.

17 Signal noise and statistical data from the repeat analysis of triazine and organo nitrogen pesticides spiked into tap water Compound Name Standard Deviation of 10 ppt standard from 6 injections %CV of 6 injections at 100 ppt signal / noise (peak - peak calculation) of 10 ppt in tap water Retention Time R value for calibration line ( ppt) Ametryn Desmetryn Fenpropidin Terbutylazine Pendimethalin Fenpropimorph Prometon Prometryn Simetryn Terbutryn Atraton Atrazine Epoxiconazole EPTC Flusilazole Flutriafol Metazachlor Propachlor Propazine Propiconazole Propyzamide Simazine Tebuconazole Triadimorph Triallate Trietazine Applera Corporation and MDS Inc.

18 Most MRM Transition were selected directly from the Cliquid software with often no further optimisation In this feasability study single MRM transition were used for each compound. A mixture of 21 organophosphate pestides were analysed. The Dwell time was generally 52ms per compound (including pause time) but was higher for weaker compounds e.g. parathion methyl This gave us >10 data points across a typical HPLC peak. In order to improve the signal to noise the run was split into 3 periods Applera Corporation and MDS Inc.

19 MS Conditions used with HPLC retention time observed Compound Q1 Mass Q3 Mass DP Voltage CE Voltage Period Retention Time (mins) Dwell Time (ms) DICHLORVOS MEVINPHOS DEMETON-S-METHYL DIMETHOATE OXADIXYL PROPETAMPHOS PARATHION-METHYL FENCHLORPHOS FENITROTHION MALATHION TRIAZOPHOS BIFENOX AZINPHOS-METHYL DIAZINON CHLORPYRIPHOS-METHYL PIRIMIPHOS-METHYL CHLORPYRIPHOS-ETHYL PARATHION-ETHYL CHLORFENVINPHOS PHOSALONE COUMAPHOS Applera Corporation and MDS Inc.

20 Calibration line for Demeton-S-methyl in tap water range 25 ppt 1000 ppt no internal standard Concentration (ng/l) Applera Corporation and MDS Inc.

21 Calibration line for Coumaphos in tap water range 25 ppt 1000 ppt no internal standard Concentration (ng/l) Applera Corporation and MDS Inc.

22 Calibration line for Parathion-ethyl in tap water range 25 ppt 1000 ppt no internal standard Concentration (ng/l) Applera Corporation and MDS Inc.

23 Calibration line for Parathion methyl in tap water range 25 ppt 1000 ppt no internal standard Concentration (ng/l) Applera Corporation and MDS Inc.

24 25 ppt of Organophosphate pesticides spiked into tap water (top) and a tap water blank (bottom) Applera Corporation and MDS Inc.

25 25ppt spiked into water of oganophosphate pestidices (left hand panes) and the corresponding tap water blank (right hand side panes) analysed in period 1 DEMETON-S-METHYL DIMETHOATE MEVINPHOS OXADIXYL DICHLORVOS Applera Corporation and MDS Inc.

26 Initail data for Signal noise and statistical calculation from the repeat analysis of Organophosphate in tap water Compound Standard Deviation at 25ppt (5 injections) 5 x Standard Deviation 25 ppt sample Signal to Noise of 25 ppt sample Peak to Peak Signal / 1x Std Dev noise CV of 100 ppt sample (3 injections) r Value DICHLORVOS MEVINPHOS DEMETON-S-METHYL DIMETHOATE (Quadratic fit) PROPETAMPHOS DIAZINON CHLORPYRIPHOS-METHYL * PARATHION-METHYL * FENCHLORPHOS FENITROTHION * PIRIMIPHOS-METHYL MALATHION CHLORPYRIPHOS-ETHYL PARATHION-ETHYL CHLORFENVINPHOS OXADIXYL TRIAZOPHOS BIFENOX * AZINPHOS-METHYL PHOSALONE COUMAPHOS Date for the signal-noise on these compounds was calculated after 1 smooth was applied to the raw data Applera Corporation and MDS Inc.

27 Results from the analysis of Uron pesticide standard (single period method) on the API 3200 System

28 10 ppt of Chorturon in water (top) and a water blank (bottom) signal to noise calculated using peak peak script Applera Corporation and MDS Inc.

29 Calibration line for Chorturon transition 1 concentration range 10 ppt 150 ppt no internal standard used only 1mL injections Concentration (ng/l) Applera Corporation and MDS Inc.

30 Statistics for Uron pesticides screened (Default settings from Cliquid software used) Compound Concentration ppt No of injections Mean Std Dev CV% LOD (5 X Std Dev of 20ppt standard) Diuron 20ppt precison 12 of ppt precison 13 of Linuron 20ppt precison 12 of ppt precison 13 of Chloroxuron 20ppt precison 12 of ppt precison 13 of Isoproturon 20ppt precison 12 of ppt precison 13 of Monlinuron 20ppt precison 12 of ppt precison 13 of Chorturon 20ppt precison 12 of ppt precison 13 of Applera Corporation and MDS Inc.

31 onclusions for Initial 2D_LC analysis of pesticides in drinking water All pesticide classes analysed have been detected at <50 ppt on a API 3200 system. 95% of all pesticides have LODs < 10 ppt. The classes tested include acidic herbicides, triazine, urons, organophosphate, azoles and carbamates > 100 pesticides. All analyses have been performed using the same mobile phase and column conditions The trap cartridge, at a cost of 125, has been shown to last over 500 large volume injections making sample preparation costs of less than 25p/sample or 0.4 EUR per sample Applera Corporation and MDS Inc.

32 Results from the quantitative and qualitative analysis of analysis of Organophosphate pesticides in tap water using a 3200 Q TRAP System

33 Can you detect and generate a library searchable spectra when pesticides are at low levels in water? The problem : How Can we detect Accidental or Malicious Contamination of the Water Supply? Applera Corporation and MDS Inc.

34 Location of Linear Ion Trap Aux AC N 2 CAD Gas Exit lens Q0 Q1 Q2 Q3 LINAC Collision Cell linear ion trap 3x10-5 Torr The ion trap is in the Q3 region of a triple quadrupole. Because the trap is now a quadrupole it can hold 70x more ions than a conventional 3D trap which reduces space charge effects. This leads to better spectra, giving you more confidence in the results Applera Corporation and MDS Inc.

35 Radial Trapping simultaneously Ramp EXB Axial Trapping Axial Trapping Auxiliary AC Ramped. Radial Trapping Exit Lens with Grid

36 Combining QQQ and Linear trap scans together - The standard set-up for Qualitative analysis on the Q TRAP Systems Survey Scan (1) Survey Scan (2) Enhanced Resolution Survey scan be EMS, EMC, Neutral Loss, Precursor Scan, MRM or EPI (combinations of 2 surveys) Improve Resolution/Accuracy IDA Criteria Level 1 Two levels of criteria Dependent Dependent Scan Scan (s) (s) Dependent Scan (s) Dependent Scan (s) Multiple dependant scans (EPI, Product Ion and MS3) IDA Criteria Level 2 Second Second Dependent Dependent Scan Scan (s) Second Dependent Scan (s) (s) Add to Exclusion List Inclusion/Exclusion List Second Level Dependant Scan (MS 3 ) Applera Corporation and MDS Inc.

37 Enhanced Product Ion Scanning N 2 CAD Gas Aux AC Exit lens Ion accumulation Q0 Q1 Q2 Q3 Advantages: Precursor ion selection Fragmentation No time required to isolate the precursor ion. No loss for isolation of fragile precursor ions. Reduce ion current into Q3 linear trap. Triple quad. fragmentation patterns. No inherent low mass cut-off. linear ion trap 3x10-5 Torr Applera Corporation and MDS Inc.

38 The effect of collision energy on the frgamentation patterns of two organophosphate pesticides dimethoate (left panes) & Primiphos-methyl (right panes) CE = 20 V CE = 20 V CE = 35 V CE = 35 V CE = 50 V CE = 50 V CE = 35 V CES 15 V CE = 35 V CES 15 V Applera Corporation and MDS Inc.

39 MRM to Product ion analysis of a water sample spiked at 100 ppt spiked with organophophaste pesticides Triazophos Azinphos-methyl Malathion Applera Corporation and MDS Inc.

40 MRM to Enhanced Product ion analysis of a drinking water sample spiked at 100 ppt spiked with organophophaste pesticides Triazophos Azinphos-methyl Malathion Applera Corporation and MDS Inc.

41 Example of calibration line produced from the MRM survey scan for Malathion Applera Corporation and MDS Inc.

42 MRM to Enhanced Product ion analysis of a drinking water sample spiked at 10 ppt spiked with organophophaste pesticides Diazinon Triazophos Applera Corporation and MDS Inc.

43 Application of 2D_LC analysis to the LC/MS/MS analysis of Food Extracts

44 Sulphonamide analysis in Food Extracts Sulphonamides are a family of broad-spectrum synthetic bacteriostatic antibiotics with activity against most gram-positive and many gram-negative organisms, as well as protozoa. They have been and are used as vetinary medicines and their residues have been found in foods such as eggs, milk, honey and meats. The European Union has set maximum residue limits (MRL) of 100 µg/kg for each parent sulphonamide in food and 10 µg/kg for sulphonamides in baby food. The standard high performance liquid chromatography (HPLC) approach to the analysis of sulphonamides usually involves extensive sample pretreatment in the form of liquid/liquid or solid phase extractions Applera Corporation and MDS Inc.

45 The solution is to use 2D_LC for sample preparation? Use a generic approach utilising Dispersive SPE, a technique which has previously been applied as a rapid analytical technique for the extraction of pesticides from fruit and vegetables, in combination with an on line solid phase extraction step for sample preparation. Offers a simple two stage sample clean-up providing samples, which are suitable for analysis by LCMSMS Applera Corporation and MDS Inc.

46 Initial method for using 2D_LC for sample preparation of milk and honey samples Honey Dissolve 5g honey in 5 ml of water. Add 5 ml of a 50:50 mixture of Acetone & acetonitrile. Shake for 2 minutes and add sodium chloride (1 ± 0.1 g) and magnesium sulphate (2 ± 0.1 g), shake well for 2 minutes and centrifuge for 5 mins at 2000 rpm. Take 200 ul of the top layer and dilute with 800ul of water and inject 50ul onto the 2D_LC set up. Meat To 5 g of meat add 5 ml of water and 5 ml of A 50:50 mixture of Acetone & acetonitrile. Shake for 2 minutes and add sodium chloride (1 ± 0.1 g) and anhydrous magnesium sulphate (2 ± 0.1 g) shake well for 2 minutes and centrifuge for 5 mins at 2000 rpm. Take 200 ul of the top layer and dilute with 800ul of water and inject 50ul onto the 2D_LC set up Applera Corporation and MDS Inc.

47 HPLC Conditions for the analysis of Sulphonamides Injection Volume = 50 µl Mobile Phase A = Water, 5 mm Ammonium Acetate Mobile Phase B = Methanol, Flow Rate = 0.6 ml/min HPLC Column = Gemini C18, 4.6 x 100 mm, 3.5 µm Gradient Time % B Applera Corporation and MDS Inc.

48 HPLC Conditions for the analysis of Sulphonamides loading pump conditions Delivery trapping Solvent Trap On Line SPE Cartridge = Water = Strata X, 20 x 2 mm Valve Switch program Loading pump program Time Position Time Flow (ml/min) 2.0 To Waste (A) To MS (B) To Waste (A) Applera Corporation and MDS Inc.

49 Source Conditions & Resolution settings Resolution Q1 & Q3 Source Type IonSpray Voltage Nebuliser gas = Unit (peak width 0.75 ± 0.1 amu at half height) = Turbo V Source (ESMS) = 5500 V (+ve mode) = Air Source Temp. = 650 C Applera Corporation and MDS Inc.

50 LC/MS Conditions for the analysis of Sulphonamides Compound Name Transition Q1 Mass (amu) Q3 Mass (amu) Dwell (ms) DP Voltage (V) CE Voltage (V) Sulfacetamide Sulfacetamide Sulfadiazine Sulfadiazine Sulfamethoxazole Sulfamethoxazole Sulfathiazole Sulfathiazole Sulfamerazine Sulfamerazine Sulfabenzamide Sulfabenzamide Sulfadimidine Sulfadimidine Sulfaquinoxaline Sulfaquinoxaline Sulfadoxin Sulfadoxin Sulfadimethoxine Sulfadimethoxine Applera Corporation and MDS Inc.

51 Example of the analysis of a 5 ppb spike into Meat (top) and Honey (bottom) Applera Corporation and MDS Inc.

52 Example of a calibration line for a sulphonamide, sulfathiazole, range ppb in honey Applera Corporation and MDS Inc.

53 The Cliquid Software for Food Analysis

54 Homepage - showing tasks and already acquired samples Applera Corporation and MDS Inc.

55 Single button to choose from preconfigured tests Applera Corporation and MDS Inc.

56 Admin can use MRM catalogue to generate new methods Applera Corporation and MDS Inc.

57 Easily create project folders Applera Corporation and MDS Inc.

58 Quickly build or import sample list (from Excel, etc.) Applera Corporation and MDS Inc.

59 Choose the report(s) that you want to generate Applera Corporation and MDS Inc.

60 Specify report delivery options (print,.pdf, ..) Applera Corporation and MDS Inc.

61 Review selection, verify HPLC setup and specify equilibration time before submitting samples Applera Corporation and MDS Inc.

62 Acquisition starts automatically Instrument status is graphically displayed Applera Corporation and MDS Inc.

63 Live View for real time TICin browser Applera Corporation and MDS Inc.

64 Report calibration curves and statistics of analytes Applera Corporation and MDS Inc.

65 Report results of unknown samples including chromatograms and confirmation (MRM ratios) Applera Corporation and MDS Inc.

66 Method Documentation (Order information, recommended sample preparation, experimental, references ) Applera Corporation and MDS Inc.

67 Cliquid software The latest version in different languages Applera Corporation and MDS Inc.

68 Cliquid software version Applera Corporation and MDS Inc.

69 Trademark Acknowledgements Applera, Applied Biosystems, Celera and AB (design) are registered trademarks of Applera Corporation or its susidiaries in the US and/or certain other countries. API 3200, Cliquid and TurboV are trademarks and LINAC and Q TRAP are registered trademarks of Applied Biosystems/MDS SCIEX, a joint venture between Applera Corporation and MDS Inc. All other trademarks are the sole property of their respective owners Applera Corporation and MDS Inc. All Rights Reserved Applera Corporation and MDS Inc.

70 For any further questions please contact Dr Stephen Lock at address:-