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How Can Orbitrap Technology Help My Food Safety Analysis? Larry Burchfield Mass Spectrometry Product Manager Latin America Region August 28, 2012.

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Presentation on theme: "How Can Orbitrap Technology Help My Food Safety Analysis? Larry Burchfield Mass Spectrometry Product Manager Latin America Region August 28, 2012."— Presentation transcript:

1 How Can Orbitrap Technology Help My Food Safety Analysis? Larry Burchfield Mass Spectrometry Product Manager Latin America Region August 28, 2012

2 2 EFS Target or Non-target analysis workflow SAMPLE Target Analysis Non-Target Analysis Quantitation Target screening ? Profiling Fingerprinting Authenticity Quantitation Food Safety Environmental

3 3 Exactive & Q-Exactive (Orbitrap) Which LCMS Analyzer Do I Choose? Pure Qualitative Pure Quantitative Detect & Quantify Targeted screening Targeted & Unknowns screening Targeted & Unknowns screening 40 – 600 cpds 1 – >40 cpds Multiple cpds Determine structure Structural ID Compound Confirmation Reaction Monitoring Process Monitoring Metabolism Proteomics Metabolomics Structural ID Compound Confirmation Reaction Monitoring Process Monitoring Metabolism Proteomics Metabolomics LTQ ORBITRAP Q-TOF Triple Quads Ion Traps QTRAPs

4 4 Triple Quadrupoles for Target Quan : Traditional Workhorse and still 75% of EFS Instruments Sold in 2011 TSQ Quantum Access TSQ Quantum UltraTSQ Vantage TSQ Quantum Access Max Ultimate ValueUltimate Performance

5 5 However, conventional thinking is evolving… Food Safety: International Requirements and Innovative Analytical Solutions Innovation Applied

6 6 Thermo LCMS EFS Workflow Screen Targeted or Non-targeted. Identify Putative ID web or local DB search PCA for unknowns Confirm ID via MS 2 Quantify Quantify contaminants – either targeted list, or newly detected set. Trace Finder Exactive Plus Q Exactive TSQ ExactFinder UHPLC EQuanMAX Turboflow

7 The benefits of high resolution MS in high throughput screening of mycotoxins in food Michal Godula, PhD Thermo Fisher Scientific Food Safety Group

8 8 Food analysis challenges: Matrix complexity

9 9 Food analysis challenges: Range of analytes  Pesticides (insecticides, herbicides...)  Mycotoxins (Aflatoxins, OTA, fumonisins, trichothecenes..)  Veterinary drug residues (Chloramphenicol, MG..)  Environmental pollutants (PCB, PBDE, Dioxins, PAH..)  Food processing contaminants (Acrylamide, furan, MCPD)  Packaging contaminants (BPA, 4-benzophenone...)  others (melamine)

10 10 Analytical challenges in residue analysis  Matrix complexity  Number of analytes  Number of samples  Performance (LOD)  Speed The complexity of samples requires a more selective way of analyses The complexity of samples requires a more selective way of analyses  Extraction  Clean-up  Determination

11 11 Use of mass spectrometry in screening analysis Simple sample preparation: QuEChERS, extract-n-shoot Target screening Single and Triple quadrupoles (SIM, SRM) Ion traps TOFs, Q-TOFs Non target screening Single and Triple quadrupoles (SCAN) Ion traps (SCAN, Auto MS/MS) TOFs, Q-TOFs (accurate mass)

12 12 MS/MS approach limitations and benefits Limitations: only target compounds screened no post acquisition data mining limited number of compounds per analysis no possibility to search for unknown compounds Benefits: Low detection limits achieved Very good quantitative characteristics Robustness

13 13 HRAM (TOF) approach limitations and benefits Limitations: Limited dynamic range Limited resolution Mass Stability Slow positive/negative switching Detection limits Benefits: Speed Acceptable mass accuracy for many assays

14 14 “Saddle of Mass Accuracy” for DETECTED Ions

15 15 Resolution for various mass separation techniques TOF-TOF 1 Q-TOF 1 Q-TOF 2

16 16 288.0441C9H21O2P1S3Terbufos 288.0949C13H21O3P1S1Iprobenfos 288.1142C15H17N4Cl1Myclobutanil 288.1256C11H20N4O3S1Epronaz 288.1351C11H21N4O3P1Pirimethaphos 288.1474C16H20N2O3Imazamethabenz 0.1033 amu Is a simultaneous measurement possible? Element Exact Mass H 1.007825 C 12.000000 N 14.003074 O 15.994915 Exact Mass and Isobaric Compounds 289.00289.05289.10289.15289.20 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 289.1314 289.0513 289.1022 289.1215 289.1329 289.1424 R = 10,000 289.00289.05289.10289.15289.20 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 289.1390 289.1021 289.0514 289.1215 289.1329 289.1424 R = 20,000 289.00289.05289.10289.15289.20 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 289.1424 289.1547 289.1329 289.1022 289.0514 289.1215 R = 100,000 Yes, at high resolution !

17 17 Analytical Challenges for Screening using MS Detection and identification of isobaric compounds High resolution mass separation Mass accuracy Predictable Isolation of compounds from background matrix components Unknown or uncharacterized species What resolving power is required? Unpredictable! Qualitative Quantitative R = 15,000 > 50 ppm R = 50,000 0.7 ppm 328.95329.00329.05329.10 m/z 0 20 40 60 80 100 0 20 40 60 80 100 Relative Abundance 329.00647 329.00510 329.02475 C 14 H O 5 ClS 0.76388 ppm 328.98993

18 18 EFS LCMS Options for Target or Non-target Analysis Triple Quadrupole TSQ Target Analysis Quantitative SRM confirmation Exactive Plus Target and Non-target Analysis Qualitative & Quantitative AIF Confirmation Q Exactive Target and Non-target Analysis Qualitative & Quantitative AIF and MS/MS Confirmation

19 19 Exactive Plus™ Bench-Top LC-MS “The Exactive LC-MS has the speed, accuracy and precision to routinely give the most confident analysis of both simple and complex samples.”

20 20 Orbitrap – Priciple of Operation z φ r Makarov A. Anal. Chem. 2000, 72, 1156-1162.

21 21 Exactive Plus Benchtop LC-MS Resolution up to 140,000 < 2ppm routine mass accuracy Sub pg sensitivity >10,000 dynamic range Up to 12 scans per second Fast polarity switching One setting: full scan! Easy-to-use!

22 22 Presentation of Dr. Hans Mol

23 23 Resolution (FWHM) Thiamethoxam [M+H] + = 292.02656 Parathion [M+H] + = 292.04031  m 0.0138 amu 292.00292.05 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 292.04031 292.02656  m (Parathion and Thiamethoxam) is 0.0138 amu R = m /  m R = 292 / 0.0138 R = 21,160  m (Parathion and Thiamethoxam) is 0.0138 amu R = m /  m R = 292 / 0.0138 R = 21,160 Mix 1:1

24 24 Resolution (FWHM) 292.00292.05 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 292.04031 292.02656 Thiamethoxam [M+H] + = 292.02656 Parathion [M+H] + = 292.04031  m 0.0138 amu  m (Parathion and Thiamethoxam) is 0.0138 amu R = m /  m R = 292 / 0.0138 R = 21,160  m (Parathion and Thiamethoxam) is 0.0138 amu R = m /  m R = 292 / 0.0138 R = 21,160 Mix 1:3

25 25 Resolution (FWHM) Thiamethoxam [M+H] + = 292.02656 Parathion [M+H] + = 292.04031  m 0.0138 amu 292.00292.05 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 292.04031 292.02656  m (Parathion and Thiamethoxam) is 0.0138 amu R = m /  m R = 292 / 0.0138 R = 21,160 Measured at R = 50,000  m (Parathion and Thiamethoxam) is 0.0138 amu R = m /  m R = 292 / 0.0138 R = 21,160 Measured at R = 50,000 Mix 1:1

26 26 Standard Pesticide Mixture in Horse Feed Matrix XIC with 5 ppm window of selected pesticides Total of about 110 pesticides (each ~ 250 ug/L) in horse feed matrix Unusually high error of 6.5 ppm Reanalyze @ higher resolution 1.20298.2741C18H35NO2 Spiroxamine 1.70297.0556C14H14Cl2N2O Imazalil 0.20296.1161C14H18ClN3O2 Triadimenol 2.00295.0222C10H15O4PS2 Fenthion- sulfoxide 1.40294.1368C15H20ClN3O Paclobutrazol 0.30294.1004C14H16ClN3O2 Triadimefon 0.70253.0309C10H9ClN4S Thiacloprid 1.20249.0192C9H10Cl2N2O2 Linuron 0.90241.0963C9H13ClN6 Cyanazine 0.10240.0786C12H14ClNO2 Clomazone 6.50239.1503C11H18N4O2 Pirimicarb 1.20237.1234C12H16N2O3 Carbetamide 2.00233.0243C9H10Cl2N2O Diuron 2.50216.1011C8H14ClN5 Atrazine 2.00215.0961C8H14N4OS Metribuzin 1.20213.0789C10H13ClN2O Chlortoluron 1.10210.1125C11H15NO3 Propoxur Error [ppm] [M+H]Elemental CompsitionComponent 6.50

27 27 239.00239.05239.10239.15239.20239.25 m/z 0 20 40 60 80 100 Relative Abundance 239.15181 C 11 H 19 O 2 N 4 6.50 ppm 239.00239.05239.10239.15239.20239.25 m/z 0 20 40 60 80 100 Relative Abundance 239.15033 C 11 H 19 O 2 N 4 0.32 ppm R = 15,000 Error = 6.50 ppm R = 80,000 Error = 0.32 ppm Standard Pesticide Mixture in Horse Feed Matrix Unusually high error of 6.5 ppm is suspicious Repeated analysis at 80,000 resolving power Pirimicarb C 11 H 19 O 2 N 4 m/z = 239.15025

28 28 Background Interferences in Pesticide Analysis Expanded view of the pesticide mixture at different resolution settings (top: 15,000 and bottom: 50,000). Pesticide Sulcotrion (m/z 328.02475) is masked under background ions at a resolution of 15,000 but is easily detected at 50,000 resolution (see also mass chromatogram inset)

29 29 Pesticide Analysis at different Resolution Settings Time [min] Overlaid extracted ion chromatograms from a mixture of 116 pesticides and mycotoxins at a 100ppb level. Extraction was done with 3 ppm mass window. The inset chart shows the number of detected compounds at different concentrations (in matrix) at two different resolution settings

30 The benefits of high resolution MS in high throughput screening of mycotoxins in food

31 31 Health Impacts on Human and Livestock Mycotoxins Most potent known carcinogens  Immediate toxic response  Immunosupression  Necrosis of liver cells  Sickness, vomiting  Abdominal pain  Teratogenic  Estrogenic  Hepatotoxic  Nephrotoxic  Carcinogenic effects Acute Chronic

32 32 Outbreak of Aflatoxicosis in Kenya in 2004 Maize products Concentrations 125 deaths reported

33 33 Typical mycotoxin method workflow ExtractCleanupDerivatizeDetect ACN/Water Methanol HAc Hexane Mycosep C18 Silica, Florisil IAC GPC (oil) TLC LC-FL, UV LC-MS/MS LC-HRMS GC-FID, ECD, MS GC-MS/MS Immunoassay: ELISA Lateral flow assay Fumonisins Trichothecenes Aflatoxins B1, G1 KobraTM cell PHRED cell* *http://www.aura-inc.com/phred.html

34 34 AOAC Method 2008.02 Aflatoxins B1, B2, G1, G2 and Ochratoxin A in Ginger and ginseng Homogenized sample 5g Extraction Centrifugation Filtration 25ml of extract load to immunoaffinity column LC-Fluorescence Evaporate to dryness 1g NaCl 25 ml MeOH + 0.5% Na 2 CO 3 (700:300) 7ml of extract dilute with 28ml 0.1M phosphate buffer pH 7.4, 1% Tween 20 AflaOchraTest column Elute with 2x 1ml of MeOH Post-column derivatization for AF

35 35 Multi-mycotoxin screening approaches Simple sample preparation Quantitation Target screening Non-target screening MS/MS: SRM or H-SRM Accurate mass and high resolution MS

36 36 Quick Method for Mycotoxins in Food and Feed Homogenized sample 25g Extraction 2 h Filtration Dilution LC/MS/MS 100 ml ACN:Water (80:20%) 400 ml Water TMO AppNote 377 Accela/TSQ Access Max

37 37 Typical Triple Quad MS Technology in Action Q1 Q2 Q3 408,4 408,2 0.7 Da 256,2 256,3

38 38 Thermo Scientific Quantum and TSQ Series - H-SRM Means Higher Selectivity and Sensitivity Q1 Q2 Q3 408.8 408.4 0.7 Da 256.3 0.4 Da

39 39 H-SRM benefits for mycotoxin analysis Diacetoxyscripenol in wheat, 23 ug/kg TMO AppNote 377 SRM H-SRM

40 40 Multi-mycotoxin screening approaches Simple sample preparation Quantitation Target screening Non-target screening MS/MS: SRM or H-SRM Accurate mass and high resolution MS

41 41 Fusarium mycotoxins  Trichothecenes Type A: T-2, HT-2, diacetoxyscirpenol Type B: deoxynivalenol, nivalenol, fusarenon-X, acetyl-deoxynivalenol  Zearalenone

42 42 Quick screening method for Fusarium mycotoxins EXTRACTION 20 mL beer + 80 mL ACN CENTRIFUGATION RECONSTITUTION in 1 ml MeOH/Water EXACTIVE TM LC-MS ANALYSIS IN: Beer, barley, malt, mush, germs EVAPORATION EXTRACTION 12.5 g + 50 mL ACN/H 2 0 EXTRACTION 12.5 g + 50 mL ACN/H 2 0 CENTRIFUGATION RECONSTITUTION in 1 ml MeOH/Water EXACTIVE TM LC-MS ANALYSIS EVAPORATION

43 43 Exactive MS – High Resolution Benchtop MS Accela UHPLC + Exactive MS, HESI II and APCI probes Column - HSS T3 2.1 x 100mm, 1.8 µm Mobile phase: (A) 5mM ammonium formate, (B) methanol Column temperature – 40°C Flow rate – 0.5 mL/min Injection volume – 5 µL

44 44 Multi-mycotoxin method Standard mix, 5 µg/L Detection capabilities

45 45 Multi-mycotoxin method Real beer sample, 5 µg/L, 25.000 resolution Real life chromatogram

46 46 High Resolution (100,000 FWHM) benefits Beer extract, T-2 toxin 10 µg/L Selectivity ± 100 ppm ± 50 ppm ± 10 ppm ± 2 ppm

47 47 DON APCI HESI Extraction window 5 ppm [M+HCOO]- HESI II vs. APCI Beer matrix matched standard, 25 µg/L

48 48 Performance characteristics of the method Linearity, barley matrix 5-1000 µg/L R2 in range 0.9922 – 0.9993 Linearity: 5 – 1000 ng/mL R 2 : 0.9946 – 0.9997

49 49 Performance characteristics of the method Barley matrix Linearity in range 5 – 500 ng/mL R2 in range 0.9922 – 0.9993  Limit of Quantitation:  Repeatability of injection on the LOQ level n=12 NIVDOND3GFUS-XADONHT-2T-2ZEA LOQ ng/g55551055 NIVDOND3GFUS-XADONsHT-2T-2ZEA RSD (%)7.312.3118.314.913.36.114.2

50 50 Performance characteristics of the method Beer matrix Linearity in range 5 – 500 ng/mL R2 in range 0.9922 – 0.9993 NIVDOND3GFUS-XADONHT-2T-2ZEA LOQ (µg/L)52.5 510 1  Limit of Quantitation:  Repeatability of injection on the LOQ level n=12 NIVDOND3GFUS-XADONHT-2T-2ZEA RSD (%)7.312.3118.314.913.36.116.2

51 51 TOF-MS vs. Exactive MS DON in beer, 5, 10 and 100 ug/L Exactive, 2 ppm mass window TOF, 20 ppm 100 ug/L 10 ug/L 5 ug/L

52 52 Exactive benefits for mycotoxin analysis Mass accuracy and resolving power Dynamic range Positive and negative acquisition in one run Quantitation and screening capabilities Post acquisition data mining Excellent stability and robustness Simple instrument setup and tuning

53 53 Food Safety Seminar Tour Dublin, IrelandSept 21 st London, UK Sept 22 nd Copenhagen, Denmark Sept 23 rd Antwerp, Brussels Sept 24 th Berlin, GermanySept 28 th Munich, Germany Sept 29 th Prague, Czech Rep.Sept 30 th Vienna, AustriaOct 1 st Zurich, SwitzerlandOct 2 nd Bangkok, ThailandOct 5 th Kuala Lumpur, Malay.Oct 6 th Singapore, SingaporeOct 7 th Hanoi, VietnamOct 9 th Ho Chi Minh City, Viet.Oct 12 th Taipei, TaiwanOct 14 th Seoul, S. KoreaOct 16 th White Plains NY, USAOct 26 th Washington DC, USAOct 27 th Atlanta, GA, USAOct 28 th Dallas, TX, USAOct 29 th Chicago, IL, USAOct 30 th Minneapolis, MN, USANov 9 th Cincinnati OH, USANov 10 th N. California, USANov 11 th Los Angeles, CA, USANov 12 th Vancouver, CanadaNov 16 th Toronto, CanadaNov 18 th Ottawa, CanadaNov 19 th Oslo, NorwayDec 7 th Uppsala, SwedenDec 8 th Paris, FranceDec 9 th Rome, ItalyDec 10 th Bologna, ItalyDec 11 th

54 54 Q Exactive EFS Data CDFA, SacramentoCDFA, Sacramento US FDA, DCUS FDA, DC

55 55 Q Exactive for EFS Targeted Screening & GUS High Confidence Confirmation Targeted screening and GUS Goal How Q Exactive addresses the needs Mode/s of operation Software Data collected in HRAM MS and MS/MS Software capable of identifying all unique components in the chromatographic data Database searches for HRAM MS and library searches with MS/MS spectra Highest specificity with 140K resolution Software that supports multiple orthogonal ways of confirmation including elemental composition determination, isotope matching, DB search, spectral library matching. Xcalibur; ExactFinder Highest confidence in Targeted screening & GUS Full Scan MS w AIF Precursor ion selection w HCD MS/MS

56 56 Q Exactive Quan capabilities for EFS Quantitation Performance Similar to High End QqQ Goal How Q Exactive addresses the needs Mode/s of operation Software Unit mass resolution precursor ion selection like QqQ Full scan HRAM MS/MS with stable fragment ion ratio Higher specificity than QqQ due to HRAM fragmment ions Ability to confirm with many fragment ions seen in full scan MS/MS Four to five orders of dynamic range UHPLC compatible Targeted MS/MS similar to SRM; Data dependent MS/MS where quant done using precursor ion in full scan MS and confirmation using HRAM fragment ions in MS/MS; Full scan MS; HRAM SIM Quant capability of high-end QqQ ExactFinder or Xcalibur

57 57 Q Exactive TM Hardware Innovations Hardware DemonstrationDemonstration

58 58 Q Exactive RSLC < 1.5 m

59 59 California Dept of Food & Ag Sample : 60+ pesticides in crop extracts In neat In Green Bell Peppers In avocado Calibration levels vary depending on analytes 500 ppb – 1 ppt Mass tolerance set at 5ppm Sample injection size = 5uL Column = Hypersil Gold aQ 100 x 2.1mm 1.9u

60 60 Q Exactive Workflows for EFS : 1.Full Scan : Screening (target and non-target) Quan 2.Full scan Targeted Data Dependent MS/MS : Full scan screening Targeted precursor ion selection DD MS/MS of precursor ions 3.UHPLC

61 61 Full Scan Targeted DD MS/MS 1)Full Scan a)Quadrupole scans from a mass range of 100- 1000 b)Sends the ions to Ctrap, compress and send into orbitrap for analysis 2)From a targeted list (Data Dependent MS2 a)Quadrupole selects the ion of interest from list b)Pass it to Ctrap c)Then pass it to HCD for fragmentation d)Then sends data packet back to Ctrap and injects to Orbitrap to analyze for MS2 1)Full Scan a)Quadrupole scans from a mass range of 100- 1000 b)Sends the ions to Ctrap, compress and send into orbitrap for analysis 2)From a targeted list (Data Dependent MS2 a)Quadrupole selects the ion of interest from list b)Pass it to Ctrap c)Then pass it to HCD for fragmentation d)Then sends data packet back to Ctrap and injects to Orbitrap to analyze for MS2

62 62 Parent selection list of 62 pesticide analyzed by CDFA of Sacramento Method transferred from TSQ Ultra CDFA samples (targeted list)

63 63 Q Exactive Full Scan Quan (Azoxystrobin at 1.5ppb) Direct comparison of neat vs matrix

64 64 Q Exactive Quantitation Full Scan Targeted DD MS/MS (Azoxystrobin at 1.5ppb) Direct comparison of neat vs matrix Quan IP = 2

65 65 Quanfirmation Boscalid 1.5ppb Boscalid Diuron Azoxystrobin Methomyl Indoxacarb Spinosad D Boscalid Diuron Azoxystrobin Methomyl Indoxacarb Spinosad D Boscalid – MS/MS Accurate Mass Fragmentation Quantitation Confirmation Number of scans across a peak doing MS/MS HR MS(n) IP = 2.0 HRAM IP = 2

66 66 Number of scans across a peak doing MS/MS Azoxystrobin MH+ = 404.1242 Quantitation Confirmation Quanfirmation Azoxystrobin 1.5ppb Boscalid Diuron Azoxystrobin Methomyl Indoxacarb Spinosad D

67 67 Q Exactive Quan performance : Repeat 3x injections at LOD/LOQ in Matrix (Green Bell Pepper) 0.1 – 1.1 ppb

68 68 Comparison of CDFA pesticides in matrix : Q Exactive vs TSQ Vantage LOD/LOQ (ppb)

69 69 Accela 1250 + Q Exactive R = 70,000 All compound elute in 2mins, with peak width of 0.04mins = 9scans under the peak UHPLC compatibility : 60+ pesticides in 2 mins, 4s wide peaks, Full scan

70 70 Target Screen Method

71 71 Unknown Screen Method

72 72 Library/Database Searching Capabilities- Spectral Library Included List selected based on customer feedback and from the major worldwide regulated lists Over 1000 compounds Over 6000 spectra HCD CID Positive Negative Multiple CE Pesticides, hormones, POPs, PPCP Created on LTQ Orbitrap Velos HRAM library courtesy : Eric Genin, Les Ulis

73 73 Q Exactive Quanfirmation High performance HRAM Quantitation and Confirmation bench top LCMS system, capable of : Multi-residue quan performance similar to mid-high end Triples Ideal for targeted and general unknown screening Highest confidence confirmation with R = 140K, and MS/MS UHPLC compatible Best of both worlds! Now, you can have your cake and eat it too!

74 74 Resources : EFS LCMS compendium http://info1.thermoscientific.com/forms/MS527_2010open_Kempel_EFS- Compendium_MultiTactic?gp=NME- Newsletter%20Elec&ce=E.12CMD.MS204.G065390.02http://info1.thermoscientific.com/forms/MS527_2010open_Kempel_EFS- Compendium_MultiTactic?gp=NME- Newsletter%20Elec&ce=E.12CMD.MS204.G065390.02

75 75 Application based Comprehensive Solutions Application protocol Step by step instructions for sample preparation, instrument setup and analysis Guidelines for acceptance criteria Instrument methods Simple method upload into TraceFinder software Enables rapid instrument setup and processing Application specific consumables Eliminates guesswork One central solutions provider

76 76 Applications Kits Hormones in drinking water /EPA 539 PFCs / EPA 537 Pharmaceuticals and personal care products / EPA 1694 Pesticides Antibiotics Allergens Marine toxins β-agonists Mycotoxins Comprehensive LCMS Apps Compendium – updated on a quarterly basis, covering key methods from all geo centers http://info1.thermoscientific.com/forms/MS527_2010open_Kempel_EFS- Compendium_MultiTactic?gp=NME-Newsletter%20Elec&ce=E.12CMD.MS204.G065390.02http://info1.thermoscientific.com/forms/MS527_2010open_Kempel_EFS- Compendium_MultiTactic?gp=NME-Newsletter%20Elec&ce=E.12CMD.MS204.G065390.02

77 77 The End Thank You!


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