New Capabilities for Analysis of Biogenic Amines using Ion Biogenic - PowerPoint PPT Presentation

New Capabilities for Analysis of Biogenic Amines using Ion Biogenic Amines using Ion Chromatography Rosanne Slingsby and Maria Rey R&D, Sunnyvale, CA

Outline • Properties of biogenic amines • Alkyl, unsaturated/aromatic, ion-pair formers y p • Low-to-moderate water solubility • Cationic at low pH • Achieving retention and separation • Achieving retention and separation • Cation exchange columns with mixed selectivities • Options for detection p • Electrical conductivity • ESI-MS/MS • Capabilities of Capillary IC MS/MS • Capabilities of Capillary IC-MS/MS • High Ionic Matrix Challenges Proprietary & Confidential 2

Biogenic amines • Involved in • Cellular growth, regulation, repair and breakdown Cellular growth, regulation, repair and breakdown • Protein and nucleic acid synthesis • Implicated in • Allergic responses • Properties of biogenic amines • Chains of aminopropyl units with pkas above physiological pH • Alkyl, unsaturated/aromatic, ion-pair formers • Other amine structures with biological activity • Other amine structures with biological activity • Low-to-moderate water solubility • Cationic at low pH Proprietary & Confidential 3

Cation exchange selectivity • Weak cation exchange sites have high selectivity for H + • allows control of charge density and capacity with pH, g y p y p , • eliminates the need for a divalent eluent cation • simplifies use of eluent suppressor • Substrate particles are formulated with varying surface • Substrate particles are formulated with varying surface area, pore size and hydrophilicity • Cation exchange capacity can be increased to allow g p y • better peak resolution • wider sample pH range, • higher matrix ionic strength higher matrix ionic strength • more diverse analyte concentrations • Column cleanup is easy Proprietary & Confidential 4

Comparison of IonPac CS17 and IonPac CS19 CS17 CS19 Comment Efficiency < 50% Higher C Capacity it < 65% Hi h 65% Higher Selectivity more hydrophilic Selectivity phase differences for hydrophobic analytes Solvent 100% 100% Acetone, compatibility Acetonitrile, IPA Proprietary & Confidential 5

Comparison of two cation exchange phases First layer supermacroporous supermacroporous macroporous Hydro-COOH COOH CS17 CS17 CS19 CS19 More hydrophilic Higher capacity Proprietary & Confidential 6

Selectivity comparison – IonPac CS17 eluent and standard Column: IonPac CS19, 2x250 mm 1.80 Eluent: 6 mM MSA at 0.25 mL/min 4 3 Higher Temperature: 30 o C capacity Peaks: 1 Li + 1. Li 2 2 7 7 5 1 2. Na + µS + 3. NH 4 6 4. K + and Dimethylamine 5. Mg 2+ 0 0 0.0 6 T i th l 6. Triethylamine i 7. Ca 2+ 0.0 10.0 20.0 Minutes Column: IonPac CS17, 2x250 mm 3.00 3 00 Eluent: 6 mM MSA at 0.25 mL/min El t 6 M MSA t 0 25 L/ i 2 2 Temperature: 30 o C More hydrophilic Peaks: 3 4 1. Li + 2. Na + 1 1 8 8 µS µS 6 6 + 3. NH 4 7 5 4. K + 5. Dimethylamine 0.0 6. Triethylamine 7 Mg 2+ 7. Mg 2 0.0 10.0 20.0 8. Ca 2+ Minutes Proprietary & Confidential 7

Mono/divalent cation exchange selectivity of the IonPac CS19 3.0 Column: IonPac CS19, 2 x 250 mm Eluent: 8 mM MSA at 0.25 mL/min 4 4 Temperature: 30ºC Temperature: 30 C 5 6 Detection: Suppressed Conductivity Injection volume: 5 µL 2 3 Peaks: 1 1. Li + 0.25 mg/L 2. Na + 1.00 + µS 3. NH 4 1.25 4. K + 2.5 5 Mg 2+ 5. Mg 1 25 1.25 6. Ca 2+ 2.5 NOTE: Concentrations are approximate. 0.0 0.0 5.0 10.0 Minutes Minutes Proprietary & Confidential 8

Dual microbore/capillary ion chromatograph • ICS-5000 RFIC TM System • Capable of performing both conventional and capillary- • Capable of performing both conventional and capillary- scale IC separations • Includes a Dual-Pump module (DP), an Eluent Generator (EG), and a Detector/Chromatography module (DC) module (DC) • Modular design odu a des g Dual-channel capillary RFIC system, • • Dual-channel conventional RFIC system, • D Dual-channel hybrid RFIC system l h l h b id RFIC t Proprietary & Confidential 9

IC-CD-ESI-MS/MS System Pump Post-column Eluent solvent solvent Generator and ESI-MS/MS ground DC module (with IC-Cube unit, inc Conductivity inc. Conductivity Detector) Proprietary & Confidential 10

ICS-5000 IC Cube TM • Small housing that resides in the DC • Holds an EG degas cartridge, capillary column g g , p y cartridge, injection valve, capillary suppressor cartridge, optional Carbonate Removal Device IC C b ™ IC Cube™ • Unique plumbing reduces the number of operator-made fluidic connections by 50% Proprietary & Confidential 11

ESI-MS/MS detection of biogenic amines Name Formula Q1/Q3 pK a Putrescine C4H12N2 89.1/72.1 9.35 aliphatic aliphatic Cadaverine C5H14N2 103.1/86.1 10.05 aliphatic Histamine Histamine C5H9N3 C5H9N3 112/95 112/95 9 75 9.75 aromatic Agmatine C5H14N4 131/72.1 12.5, 9.07 decarboxylated y arginine Phenethylamine C8H11N 122/105 9.8 Spermidine Spermidine C7H19N3 C7H19N3 146 1/72 1 146.1/72.1 10 88 9 81 8 34 10.88, 9.81, 8.34 aliphatic Spermine C10H26N4 203.1/129.1 10.02, 9.21, 4.42 aliphatic 203.1/112 Proprietary & Confidential 12

Main fragments of spermine and spermidine NH2 NH2 NH NH NH H+.H2N NH2.H+ Spermine p H2N NH m/z 203 Spermidine m/z 146 NH2. H+ m/z 72 H+ NH H+.H2N NH m/z 112 m/z 129 Proprietary & Confidential 13

Effect of post-column solvent compositions on ESI cadaverine cadaverine 2.1e4 A. Ammonium Acetate histamine ACN putrescine agmatine g In tensity, cps Elution of inorganic cations 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min 4.6e4 histamine hi t i B. Propanoic Acid 2-propanol putrescine Intensit agmatine cadaverine spermidine spermidine ty, cps spermine 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min Proprietary & Confidential 14

Fast determination of biogenic amines using CS17 and CapIC-MS/MS Column: Prototype Capillary CS17, 0.4 x 250 mm Suppressor: CCES 0.90 Flow rate: 15 µL/min A Gradient: 10-55 mM MSA C CD_1, µS Postcolumn: 1-4 10% propanoic acid/25% IPA/65% water PC flow rate: 10 µL/min 5,6 7,8 11 12 10 Detection: A,Conductivity; B, ESI-MS/MS, API2000 9 9 13 3 Injection volume: 0.4 µL Injection volume: 0 4 µL 0.00 0.01.0 3.0 5.0 7.0 9.0 11.0 13.0 15.0 17.0 19.0 Peaks: 1-4. Li, Na, NH 4 , K 9 1.20e5 5,6. Mg, Ca B B 7. Putrescine, 1mg/L Intensity, cps 8. Cadaverine, 1 mg/L 7 13 9. Histamine, 3 mg/L 10. Agmatine, 1 mg/L 8 10 11 11. Spermidine, 2 mg/L 11. Spermidine, 2 mg/L s 12. Spermine, 4 mg/L 12 13. Phenethylamine, 5 mg/L 0.00 1.0 3.0 5.0 7.0 9.0 11.0 13.0 15.0 17.0 19.0 Time, min Proprietary & Confidential 15

Fast determination of biogenic amines using CS19 and CapIC-MS/MS Column: Prototype Capillary CS19, 0.4 x 250 mm 0.58 Suppressor: CCES Flow rate: 15 µL/min A Gradient: 10-55 mM MSA Gradient: 10-55 mM MSA 1 6 1-6 CD_1, µS Temp.: 30 o C 7 12 13 Postcolumn: 10% propanoic acid/25% IPA/65% water 10 8 PC flow rate: 10 µL/min 9 9 Detection: A,Conductivity; B, ESI-MS/MS, API2000 11 0.0 Injection volume: 0.4 µL 1.0 3.0 5.0 7.0 9.0 11.0 13.0 15.0 17.0 19.0 Peaks: 9 1-6. Li, Na, NH 4 , K, Mg, Ca 1 6. Li, Na, NH 4 , K, Mg, Ca 1.20e5 1 20 5 7 7 7. Putrescine, 1mg/L B 8. Cadaverine, 1 mg/L Intensity, cps 11 9. Histamine, 3 mg/L 8 10 10. Agmatine, 1 mg/L 12 12 s 11 Pheneth lamine 5 mg/L 11. Phenethylamine, 5 mg/L 13 12. Spermidine, 2 mg/L 13. Spermine, 4 mg/L 0.0 1.0 3.0 5.0 7.0 9.0 11.0 13.0 15.0 17.0 19.0 Ti Time Proprietary & Confidential 16

Detection of biogenic amines in 10% seawater using capillary IC-CD-ESI-MS/MS 2.0 Column: Prototype Capillary CS17, 0.4 x 250 mm A Suppressor: CCES Flow rate: 15 µL/min µ µS S Gradient: 10-55 mM MSA 1 Postcolumn: 2 3 4 10% propanoic acid/25% IPA/65% water 5 PC flow rate: 10 µL/min 6 Detection: A Conductivity Detection: A, Conductivity 0.0 B, ESI-MS/MS, API2000 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.011.012.013.014.0 Injection volume: 400 nL Sample: San Francisco Seawater diluted 1/10 3 5.4e4 1 1 B B Peaks (spiked): 2 Intensity 1. Putrescine, 1mg/L 4 2. Cadaverine, 1 mg/L 3 Histamine 3 mg/L 3. Histamine, 3 mg/L , cps 5 4. Agmatine, 1 mg/L 6 5. Spermidine, 2 mg/L 6. Spermine, 4 mg/L 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.011.012.013.014.0 Time, min Proprietary & Confidential 17

Signal suppression in ESI-MS/MS from very high ionic strength . Column: Prototype Capillary CS17, 0.4 x 250 mm 2400 5 Suppressor: CCES 1 Flow rate: 15 µL/min µ Gradient: 10-55 mM MSA 3 2 Postcolumn: 4 10% propanoic acid/25% IPA/65% water PC flow rate: 10 µL/min Detection: A, Conductivity Detection: A Conductivity Intensity, cps B, ESI-MS/MS, API2000 Injection volume: 400 nL Sample matrix: Seawater, undiluted Peaks (spiked): 1. Putrescine, 1mg/L 2. Cadaverine, 1 mg/L 3. Histamine, 3 mg/L 4. Agmatine, 1 mg/L 6 5 Spermidine 2 mg/L 5. Spermidine, 2 mg/L 6. Spermine, 4 mg/L 0 7.0 14.0 Time, min Proprietary & Confidential 18