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What is DART?

Direct Analysis in Real Time “DART-MS” Application of Ambient Pressure Desorption Ionization Technology in the Natural Products, Food, and Pharmaceuticals Research Brian Musselman, Ph.D., IonSense, Inc., Saugus, Massachusetts, USA. What is DART?.

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What is DART?

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  1. Direct Analysis in Real Time“DART-MS”Application of Ambient Pressure Desorption Ionization Technology in the Natural Products, Food, and Pharmaceuticals ResearchBrian Musselman, Ph.D., IonSense, Inc., Saugus, Massachusetts, USA

  2. What is DART? • DART is a non-contact surface sampling technique for desorption ionization mass spectrometry where samples are at atmospheric pressure. • DART can be used to analyze gases, liquid, solids and materials on surfaces. • Developed by J. Laramee and R. Cody at JEOL USA, Inc. • Commercialized by JEOL and IonSense Inc.

  3. DART Schematic • Gas flows through DART • Electrical discharge creates a plasma • Lenses remove charged particles • Grid prevents ion-ion recombination at exit, and other functions • No exposed high voltages • Operated at ambient pressure in open air

  4. Operating Principle • Excited-state Helium atoms or nitrogen molecules interact with sample and atmosphere • Different results can be obtained by using different types of mass spectrometers including TOF, ion trap, quadrupole, and triple quadrupole as well as various hybrid mass spectrometers

  5. Basics: The Ionization ProcessPenning Ionization • Metastable atoms or molecules react with analytes that posses ionization potentials less than the metastable energy, M*+ S  S+. + M + electron

  6. Proton Transfer He(23S) + H2O  H2O+• + He(11S) + electron H2O+• + H2O  H3O+ + OH• H3O+ + nH2O  [(H2O)nH]+ [(H2O)nH]+ + M  MH+ + nH2O • Metastable atoms react with atmospheric water to produce ionized water clusters • Dominant reaction mechanism when helium carrier used: He(23S) energy = 19.8 eV • Huge reaction cross section: 100 A2

  7. Ionized Water Clusters [(H2O)nH]+ Produced by DART (He carrier) in Room Air

  8. Negative Ion Formation • Electrons produced by direct or surface Penning ionization are rapidly thermalized • Thermal electrons react with atmospheric oxygen and water to produce ionized clusters • Oxygen/water cluster ions react with analyte molecules to produce analyte ions

  9. Negative Ions Produced by DART from Room Air Note the absence of nitrogen oxide ions that would be produced by electrical discharge in air.

  10. Acetaminophen MH+ Oxycodone MH+ 150 200 250 300 350 “Ecstasy” (MDMA) Mass MH+ Original DART Hold sample, analysis in a few seconds

  11. DOP Ritalin Triethanolamine Cocaine 100 200 300 400 500 600 m/z US One-Dollar Bill • Triethanolamine is a pH-adjuster used in cosmetics • All labeled compounds are detected as MH+ and confirmed by exact mass. • Common on bills: sunscreen, DEET, nicotine, glycerol and polyglycols (from printing process?)

  12. Simple Mass Spectra Make it Easier to Analyze Unknowns • DART never forms sodium or potassium adducts: • No [M+Na]+ or [M+K]+ • DART never forms multiple-charge ions • Solvent clustering and analyte clustering are less likely than in ESI

  13. 20 0 300 310 320 330 340 350 m / z 20 0 20 300 310 320 330 340 350 m / z 0 300 310 320 330 340 350 m / z 20 0 300 310 320 330 340 350 m / z Contamination Resistance [M+H]+ DI Water Chlorpromazine [M+H]+ No alkali metal cation adducts No multiple charging No apparent suppression Saturated NaCl Saturated Phosphate Buffer Raw Urine

  14. Deploying DART • Everyone wants to knows what’s is in their sample • Most methods require significant sample prep to get answers… • DART simplifies the process by generating simple MS data.

  15. Conventional Technology • You can test every components every step of the way • After each step… • Making new methods… • Trying to figure out what you already know… • So you can spend a lot of money, or…

  16. Formulated ProductsUtilize Thermal Desorption Technique • For small tablets you can subsequently load up to 10 tablets then analyze for content by DART-MS in under 2 minutes • Vary the carrier gas temperature to thermally profile the product • Small tablets are held it place by a spring loaded plate pushing the tablets against a template • Large tablets can simply be placed in the tablet tray

  17. DART-SVP with Angle • Insert the Tablet Carrier onto the ET (Experiment Table) • Vary the carrier gas temperature while scanning across the surface to thermally desorb layer after layer of product • Operation at up to 45 degree angle for efficient desorption

  18. Thermal Desorption Method Development Daytime Caplet – 4 DART-ET Temperatures - Search for 3 Active Ingredients 150 ºC A A D 250 ºC D A 350 ºC P D 450 ºC P A

  19. Night time Caplet DART-ET 350 ºC Dextromethorphan HBr 1 Chlorine Isotope Pattern Chlorpheniramine Maleate 0.3%

  20. Exact Mass 527 Mass 483 DFC Powder Blend: Degradation Analysis R1 R1 Previous experiments have shown poor photostability when exposed to light between 280 nm and 450 nm, but colored capsules can be utilized to minimize degradate formation. Purpose: DART used to replicate the original study of colored capsules with benefit of minimal sample preparation and fast analysis. Results Courtesy of R. Helmy, Merck, “Ambient Pressure Desorption Ionization Mass Spectrometry In Support of Preclinical Pharmaceutical Development,” submitted 10/2009

  21. Photostability Results DART-MS Results in Arbitrary Units‡ Data Generated with HPLC-UV ‡Ratios of deg:API normalized to ratio obs.for orange capsule sample after 20 klux hr Results: DART can be used to determine dark green capsule best for reducing degradation. Total Sample Prep Time: ~ 1 min Total Analysis Time: 2 – 5 min 528 528 528 528 Orange Capsule (400 klux hr) Dark Green Capsule (400 klux hr) White Capsule (400 klux hr) Control (no exposure) 484 * 484 *

  22. Detection of API in Tablets at Low %DL Purpose: Evaluate ability to use DART to analyze API in tablets at low %DL Results: DART able to detect low level API in RC platform formulation. Adjusting temperature plays significant role in signal intensity. DL = Drug Load

  23. Counterfeit Analysis Merck Receives a Customer Complaint ? Is this really Merck’s Arcoxia or a counterfeit ?

  24. TLC Spot Identification via DART SM MW = 293 Bis Nitro MW = 383 Desired MW = 338 Results Courtesy of R. Helmy, Merck, “Ambient Pressure Desorption Ionization Mass Spectrometry In Support of Preclinical Pharmaceutical Development,” submitted 10/2009

  25. Rapid Analysis of Liquids • For liquids you can analyze 12 samples per minute at high speed for qualitative analysis • Or slow the presentation speed to deliver more quantitative analysis • Analysis of 64 samples of Virgin Olive Oil in 22 minutes looking for di- and tri- glyceride content

  26. Oleocanthal • Present in fresh extra-virgin olive oil • Reported to have ibuprofen-like activity • Beauchamp, G. K. et. al. Nature2005, 437, 45-46. “Phytochemistry: Ibuprofen-like activity in extra-virgin olive oil”

  27. [M+NH4]+ [M+H]+ 305 310 315 320 325 330 335 m / z 305 310 315 320 325 330 335 m / z Oleocanthal in Olive Oil Fresh-pressed Grocery brand Not present

  28. Oleic Acid Gourmet Olive Oil 200 400 600 800 1000 Oleic Acid m / z Grocery-Store Olive Oil Linoleic Acid 200 400 600 800 1000 m / z Spray-on Oil Triglycerides Linoleic Acid 200 400 600 800 1000 m / z Sesame Oil 200 400 600 800 1000 m / z Comparison of Cooking Oils

  29. Raw Materials • The Tweezer Block permits analysis of irregular shaped objects • Or determine the content of active ingredients in seconds

  30. 100 150 200 250 300 350 400 450 500 m / z 100 150 200 250 300 350 400 450 500 m / z Two Basil-Leaf Chemotypes 1 Vietnamese Restaurant 7 6 5 8 3 1 Grocery Store 5 4 6 7 • C10H16 (pinene, terpinine) • C10H12O (methylchavicol) • C10H16O (citral) • C10H12O2 (eugenol) 5. C15H24 (sesquiterpenes) 6. Hydroxytrimethoxyflavone 7. Dihydroxytrimethoxyflavone 8. Hydroxytetramethoxyflavone

  31. Lachrymator in Freshly Cut Chive Bulb C3H5S+ Propanethial-S-oxide [M+H]+ CHS+ 20 40 60 80 100 120 140 160 m/z

  32. Tomato Skin (Positive ions) Phytoene Lycopene and carotenes 500 550 600 650 m/z 100 200 300 400 500 600 700 800 m/z

  33. Dioctyl adipate Leached from plastic tube Generic Hydrocortisone Cream

  34. Automation of DART Analysis + + • Need automated sampling mechanism • Need efficient, less labor intensive data analysis

  35. It was never separated on the prep-column Applications of DART in Prep-HPLC R1 Which fraction contains the bromo imp ? Results Courtesy of W. Schafer, Merck, “Ambient Pressure Desorption Ionization Mass Spectrometry In Support of Preclinical Pharmaceutical Development,” submitted 10/2009

  36. Degradation solutions sampled directly Mk 1918 Hundreth N HCL_080131185320 # 3 RT: 0.01 AV: 1 NL: 3.69E7 T: ITMS + c NSI Full ms [100.0-1200.00] 352 352 352.14 36000000 34000000 32000000 324 Acid Base Thermal AIBN Control 30000000 28000000 26000000 Peroxide 24000000 22000000 20000000 Intensity 18000000 16000000 14000000 Oxidative Stress 12000000 10000000 8000000 6000000 4000000 334.20 2000000 127.12 380.10 324.24 701.68 168.11 214.36 394.19 480.90 537.13 656.84 757.87 838.27 981.99 1051.68 1107.29 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z Utilization of DART for Automated Force Degradation Workflow

  37. MW=168 MW=210 MW=195 MW=182 MW=181 MW=197 MW=278 MW=246 Common internal standards used by catalysis groups

  38. TIC SM (M+1=134) Prod (SM+16) 96 Well Plate Analysis

  39. 150 DART Linearity on Agilent MSD Sensitive enough for to track product formation Semi-quantitative: allows chemist to quickly identify best hits on plate

  40. Higher Resolution permits separation of Melamine from Hydroxymethyl Furfural thermal degredation product generated at 350C MEL HMF

  41. DART of Milk Powder Extract with stable isotope standard

  42. Pyridoxinecarboxylic acid Hypoxanthine Ascorbic acid Creatinine Phenylacetyl glutamine pGlu Pyruvate Lactate Ranitidine Ranitidine+Cl HR DART MS of Urine

  43. Computer Analysis of SpectrumAccurate Mass Assignments Courtesy of Robert “Chip” Cody, JEOL USA

  44. Ala Val I/L Thr Gly Pro Leu/Ile Ser Phe 80 100 120 140 160 m/z Blood droplet: Amino Acids (Positive Ions) No sample prep, no cleanup, no derivatization, no MS/MS Urea P V 40 60 80 100 120 140 160 180 200 m/z Courtesy of Robert “Chip” Cody, JEOL USA

  45. Promazine in UrineChlorpromazine internal standard

  46. 900.00 800.00 2 R = 0.9991 700.00 600.00 500.00 Series1 [GHB]/[IS] Linear (Series1) 400.00 300.00 200.00 100.00 0.00 0 100 200 300 400 500 600 700 800 900 ppm Gamma Hydroxybutyrate (GHB) in Urine (Deuterated I.S.)

  47. [M+H]+ M+. 140 160 180 200 220 240 260 280 300 320 340 360 380 m / z Solvent for non-polar compounds He DART Flow injection of 75 ppm triphenylene in Hexane/MeCl2 No triphenylenesignal is observed if mobile phase is acetonitrile Normal-phase chromatography should work

  48. Conclusions • I POD Touch controlled Direct Analysis in Real Time provides a methods driven platform for solving problems

  49. Questions?

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