Introduction to Liquid Phase Mass Spectrometry Andrew Baca

Introduction to Liquid Phase Mass Spectrometry Andrew Baca Business Development Specialist Environmental and Food Markets Proprietary & Confidential The world leader in serving science 1 Proprietary & Confidential

Topics • Detectors • Liquid Phase • Example Applications 2 Proprietary & Confidential

LCMSMS Detectors • Triple Quadrupoles • Ion Traps • HRAM Benchtop Orbitraps ™ • Hybrid Orbitraps ™ 3 Proprietary & Confidential

Quadrupole vs. Ion Traps Single Quadrupole VS Filter 4 Bucket Proprietary & Confidential

Single Quadrupole Principles of Operation Full Scan (FS) Ion Source Detector the quadrupoles at a time. Single Ion Monitoring (SIM) Ion Source 5 Diagonal line represents the energies that are scanned to allow one ion at a specific mass to charge ratio to go through Detector Proprietary & Confidential Straight line represents the energy that is applied to allow only one ion at a specific mass to charge ratio to go through the quadrupoles.

Triple Quad MS – Principle of Operation Quantitation of target compounds in matrix samples Q 1 selects the precursor ion DDT ion: m/z 234. 94 6 Detector Fragment Ion Source Select Argon Collision Gas Proprietary & Confidential Q 3 selects the product ion fragments to m/z 164. 96

Ion Trap Principle of Operation • Trap all m/z ions simultaneously. • Mass-selectively eject them. Ion Injection 7 Ion Trapping Proprietary & Confidential Mass-selective Ejection

Orbitrap – Principle of Operation Kingdon Trap 1923 r z φ Hyper-logarithmic potential distribution: “ideal Kingdon trap” § Characteristic frequencies: • Frequency of rotation ωφ • Frequency of radial oscillations ωr • Frequency of axial oscillations ωz Makarov A. Anal. Chem. 2000, 72, 1156 -1162. 8 Proprietary & Confidential

Triple Stage Quadrupole Systems Target market: • Most sensitive system • Most demanding quantitative requirements. Performance TSQ Vantage 3, 000 timed SRMs per LC run EMR for peptide Quan > 4 orders linear dynamic range Sensitivity: ESI/APCI 500 fg injected on column s/n 500: 1 Target market: • Routine quantitation • highly selective reaction monitoring (H-SRM) for greater TSQ Quantum Ultra analytical selectivity • Sensitivity: ESI/APCI 500 fg injected on column s/n 150: 1 Target market: • Environmental and food safety • Entry level quantitation, best in Class, extremely good value • Customers with limited budgets TSQ Quantum Access Max • Mass range m/z 10 -3000 – Small molecules to intact proteins • Fast +/- switching for high-speed • Sensitivity: ESI/APCI 1 pg injected on column s/n 200: 1 Value 9 • • Proprietary & Confidential

HSRM Matrix background peak! Unit Resolution S/N 289 10 HSRM Chromatogram on the right (HSRM) shows improved S/N over left (unit resolution). By increasing the resolution on Q 1 to 0. 1 FWHM the TSQ Vantage can eliminate matrix background noise as well as interference peaks. HSRM provides enhanced selectivity for the ion of interest resulting in fewer false positives! Proprietary & Confidential S/N 3918 Matrix peak removed by HSRM

Ion Trap Systems Target market: • Peptide ID and Quan in one system • Metabolite ID and Quan in one system • Premium Ion Trap System Performance Velos Pro Mass range m/z 50 -2000 Worlds first quantitative Ion Trap Worlds fastest most sensitive Ion Trap CID, PQD, ETD, HCD for MSN Target market: • Peptide ID and Metabolite ID • Small molecule structural elucidation • Ultimate flexibility – upgrade to MALDI, ETD, Orbitrap LTQ XL • Mass range 50 -2000 m/z • CID, PQD, ETD for MSN • Upgrade to Orbitrap Target market: • Ion trap of choice for routine analysis of complex matrices & multi-component samples • Single Quad Replacement • Ease of use for non-MS operators LCQ Fleet • Mass range m/z 50 -2000 – Small molecules to intact proteins • CID, PQD for MSN Value 11 • • Proprietary & Confidential

Ion Trap: Data Dependent MSN • At each point in the chromatogram we obtain a unique MSN spectrum. MS 3 121. 25 100 177. 26 Relative Abundance 50 147. 29 40 30 57. 08 20 165. 17 93. 06 50 100 70 m/z 150 • In this example, we have MS 2, MS 3 and up to MS 4 (not shown) 80 70 60 286. 62 50 40 • The full collection of MSN spectra is referred to as a Spectral Tree. 30 20 110. 23 10 93. 26 132. 42 0 100 150 60 50 195. 38 200 m/z 250 300 • The Spectral Tree uniquely defines the structure of a molecule – just like a fingerprint. 40 30 303. 69 20 272. 58 10 0 200 485. 93 100 90 60 0 80 177. 31 100 70 10 90 MS 1 80 Relative Abundance MS 100 Relative Abundance 90 2 • The high intensity ion in MS 1 triggers data dependent acquisition to any desired level of MSN. 300 m/z 391. 81 400 500 7 8 Relative Abundance 90 80 70 60 50 40 30 20 10 0 300 0 1 400 500 m/z 2 600 3 700 4 5 6 9 10 11 12 13 14 15 16 17 Time (min) 12 Proprietary & Confidential 18 19 20 21 22 23 24 25 26 27 28 29

HRAM Benchtop Orbitrap ™ Systems Performance Target market: • Q-TOF competitor • Little Method Development • Environmental and Food Safety Q Exactive Mass Range m/z 50 - 6000 Mass Accuracy: <1 ppm MS/MS for confirmation and ID Mass resolution: 140, 000 Target market: • TOF competitor • Multiple component screening and quantitation • Ease of use for non-MS operators – no method development Exactive Plus • Mass Range m/z 50 - 6000 • Mass Accuracy: <1 ppm • Mass Resolution: 140, 000 (1. 5 scan/sec); 17, 500 (12 scans/sec) Value 13 • • Proprietary & Confidential

Resolution and Mass Accuracy § Resolution FWHM 10% § Mass accuracy 14 Proprietary & Confidential

510 Pesticides analysis in 12 Minutes 15 Proprietary & Confidential

Hybrid Orbitrap Systems Target market: • Complete Proteomics Solution • Metabolite ID • Worlds Best mass spectrometer Performance Orbitrap Elite Mass range m/z 50 -4000 Velos Pro Ion Trap CID, PQD, ETD, HCD for MSN HR/AM >240, 000 FWHM Target market: • Peptide ID and Metabolite ID • Small molecule structural elucidation • Ultimate flexibility – upgrade to MALDI, ETD, Orbitrap Velos Pro • • Mass range m/z 50 -4000 Velos Pro Ion Trap CID, PQD, ETD, HCD for MSN HR/AM >100 000 FWHM Target market: • Amazing analytical performance at this price point • Peptide ID and Small Molecule structural elucidation • MALDI option LTQ Orbitrap XL • • Mass range m/z 50 -4000 LTQ XL Ion Trap CID, PQD, ETD, HCD for MSN HR/AM >100 000 FWHM Price 16 • • Proprietary & Confidential

Example Application LC Triple Quad Vitamins Istds Vitamins Niacinamide Pyridoxine Pantothenic Acid Thiamine Folic Acid Biotin Riboflavin 17 Proprietary & Confidential

Example Application HRAM Benchtop Orbitrap ™ Systems Mass Accuracy 0. 595 ppm Detection from Blank Simulation 18 Proprietary & Confidential

After Melamine DEHP, what is the next one? ? Mela § Is a mine n ind § No ustri al ch emic rove al d for use in fo od t app DEH P § Pla stici zer § No t app rove d for use 19 Proprietary & Confidential in fo od