Introduction to Mass SpectrometryNavnath Jaybhaye Manager- Product development & Application BioAnalytical Technologies (I) Pvt. Ltd. 1 Agenda • • • • • • Introduction Overview of MS Ionization Techniques Mass Analyzers Quadrupole Operations Applications Of LCMS 2 Analytical Assays used in Pharmaceutical Industry Labs for New Chemical Entities Method HPLC (UV &Fluorescence) 1990 75% 12% 3% 10% 1998 50-60% 3% 40-50% 10% 2000 20% 2% 60-75% 10% 2006 2% 0 98% 0 3 GC/MS LC/MS/MS Immunoassay (ELISA/FPIA etc.) Mass Spectrometers • Separate and measures ions based on their mass-tocharge (m/z) ratio. • Operate under high vacuum (keeps ions from bumping into gas molecules) • Key specifications are resolution, mass measurement accuracy, and sensitivity. • Several kinds exist: for analysis, quadrupole, time-offlight (TOF) and ion traps are most used. 4 MS vs. MS/MS GC HPLC CE Inlet Ionize Mass Analyze Detect Separation MS Mass Analyze MS1 Identification Inlet Ionize Fragment Collision Cell Mass Analyze MS2 Detect MS/MS 5 Mass Spectrometry +CH 3 +COH CH3COCH3 CH3+COCH3 CH3C+OCH3 +COCH 3 Sample Inlet Ionization Fragmentation & Adsorption (Dissociation) of Excess Energy Mass Analysis Detection 6 Components of Tandem Mass Spectrometer Ionization Source Mass Spectrometer ESI APPI APCI MALDI Quatrupole Magnetic Sector Collision Cell Argon Xenon Mass Detector Spectrometer Quatrupole Magnetic Sector Time-of-flight MS1 Collision cell MS2 7 Sample introduction • Ion Souce – Transforms sample molecules to ions – Soft ionization • Places positive or negative charge on the analyte without significantly fragmenting the analyte • M+1 ion (or M-1 ion) • No need to volatilize • Down to fmol detection limits – Atmospheric Pressure Ionization (API) • • • • Electrospray MALDI APCI APPI 8 The Macabre History of Electrospray The Abbé Nollet experimented with electrified liquids in the 18th century ! He observed that when a person was connected to a high-voltage generator he/she would not bleed normally after cutting ...blood “sprayed” from the wound ! F. Lemière, LC•GC Europe “LC-MS Supplement”, December 2001, p29-35 9 How Sample is Introduced Sample Inlet Ion Spray Inlet Ion Spray Heater Ion Spray Probe Heater The sample is held on the surface of a capillary tube, a fine wire, or a small cup. 10 The Electrospray Phenomenon J. Zelene, Phys. Rev., 10, 1-6 (1917) 11 Ionization Source 12 Ionization Source Spraying Needle Sample Cone Orifice = 400µm Vacuum Isolation Valve 13 Ionization Methods. • Gas Phase Ionization • Electron Impact (EI) • Chemical Ionization (CI) Spray Ionization / Atmospheric Pressure Ionization (API) • Electrospray (ESI) / API • Atmospheric Pressure Chemical Ionization (APCI) • Atmospheric Pressure photo Ionization (APPI) • • Desorption Ionization 14 II. Spray Ionization/API The compound of interest in a volatile buffer mobile phase, is passed through heated, narrow bore tubing directly into the ion source of a mass spectrometer. The solution is vaporized in the tubing, and analyte ions desorb into the gas phase and pass into the mass analyzer. • Electrospray (ESI) / API • Atmospheric Pressure Chemical Ionization (APCI) • Atmospheric Pressure photo Ionization (APPI) 15 Electrospray Ionization(ESI) 16 Ion Sources make ions from sample molecules Electrospray ionization: Pressure = 1 atm Inner tube diam. = 100 um Sample Inlet Nozzle (Lower Voltage) Partial vacuum N2 + + + + ++ ++ + + ++ ++ + + ++ + ++ + ++ + ++ + ++ + ++ + ++ MH+ + + + + + + + + + + Sample in solution N2 gas MH2+ MH3+ High voltage applied to metal sheath (~4 kV) Charged droplets 17 TURBOIONSPRAY (TIS) 18 API 4000 Turbo Ion Spray TM 19 ESI Spectrum of Trypsinogen (MW 23983) 1599.8 M + 15 H+ M + 14 H+ M + 16 H+ 1499.9 1714.1 M + 13 H+ 1411.9 1845.9 1999.6 2181.6 m/z Mass-to-charge ratio 20 APCI 21 APPI 22 MALDI: Matrix Assisted Laser Desorption Ionization Sample plate h Laser MH+ 1. Sample is mixed with matrix (X) and dried on plate. 2. Laser flash ionizes matrix molecules. 3. Sample molecules (M) are ionized by proton transfer: XH+ + M MH+ + X. +/- 20 kV Grid (0 V) 23 The mass spectrum shows the results MALDI TOF spectrum of IgG Relative Abundance 40000 MH+ 30000 (M+2H)2+ 20000 10000 (M+3H)3+ 0 50000 100000 150000 200000 Mass (m/z) 24 Differential vacuum in MS OR IQ1 DP-FP RNG Collisional Focussing QO Q1 Analyzer IQ2 Collision Cell (N2) RO2 IQ3 Q3 Analyzer DET ST RO1 RO3 DF 8 x 10-3 torr 1 torr 1x10-5 torr 1-5 x 10 torr -4 1-2 x 10-5 torr Turbo Roughing pump 25 Significance of vacuum • A vacuum is necessary to permit ions to reach the detector. • It reduces or eliminates the chances of ion collisions with mass analyzer. • The major reason for maintaining high vacuum is to increase the mean-free path of ions. • Increases sensitivity and resolution of Mass Spectrum. 26 Types Of Analyzers • Quadrupole • Ion Trap • Time-of-flight 27 Analytical Quadrupole 28 Schematic Diagram of Triple quad Instrument 29 Quadrupole Theory Pre-filter Quadrupole Mass Filter Stable Trajectory Unstable Trajectories Only ions with the correct m/z values have stable trajectories within an RF/DC Quadrupole field. Ions with unstable trajectories collide with the rods, or the walls of the vacuum chamber, and are neutralised. 30 1. Quadrupole 31 Quadrupole: Pros & Cons • Advantages – Relatively small and low-cost systems – Low-energy collision-induced dissociation (CID) MS/MS – Triple quadrupole and hybrid mass spectrometers. Limitations Limited resolution Peak height vs. mass response must be 'tuned'. Not well suited for pulsed ionization methods Applications – Majority of benchtop GC/MS and LC/MS systems Triple quadrupole MS/MS systems quadrupole hybrid MS/MS systems 32 • • Tandem Quadrupole MS1 Collision cell MS2 33 Components of Tandem Mass Spectrometer Ionization Source Mass Spectrometer ESI APPI APCI MALDI Quatrupole Magnetic Sector Collision Cell Argon Xenon Mass Detector Spectrometer Quatrupole Magnetic Sector Time-of-flight MS1 Collision cell MS2 34 Operation Modes • Product Ion Scanning – Analyzes all products of a single precursor • Precursor Ion Scanning – Analyzes all precursors of a single charged product • Neutral Loss Scanning – Analyzes all precursors of a single uncharged product • Multiple Reaction Monitoring – Analyzes for specific precursors producing specific products. 35 Full Scan Acquisition Mode MS1 Collision cell MS2 MS1 Collision Cell MS2 Scanning Rf only, pass all masses SCANNING MODE: The first quadrupole mass analyzer is Scanning over a mass range. The collision cell and the second quadrupole mass analyzer allow all ions to pass to the detector. 36 Full Scan Acquisition Mode Mass Spectrum: Progesterone [M+H]+ 100 315.1 CH3 O CH3 CH3 O % 316.1 0 200 220 240 260 280 300 320 340 360 380 m/z 400 37 MS1 Collision cell MS2 Product ion scanning Argon gas Products Precursor Static (m/z 315.1) Scanning The first quadrupole mass analyzer is fixed at the mass-to-charge ratio (m/z) of the precursor ion to be interrogated while the second quadrupole is Scanning over a user-defined mass range. 38 Product Ion Spectrum: Progesterone Product ion scanning O 100 CH3 CH3 CH3 315.1 % 0 300 97.0 O Precursor ion 305 CH2 316.1 Mass Spectrum from MS1 m/z 330 310 O CH3 315 320 325 100 % 0 109.0 O CH2 H3C CH3 Product ions 100 125 150 175 Product ion spectrum from MS2 m/z 200 225 250 275 300 325 39 Precursor ion scanning Precursor Ion Scan MS1 Collision cell MS2 Argon gas Product Precursors Scanning Static The first quadrupole mass analyzer is Scanning a mass range while the second quadrupole is fixed, or Static, at the mass-to-charge ratio (m/z) of a product ion known to be common to the analytes in a mixture. 40 Acylcarnitines R=0 to 18 carbon alkyl chain. Derivatization and Fragmentation H CH COOH Precursor ion scanning RCOO (CH3)3N CH2 CH Butylation RCOO (CH3)3N CH2 CH H CH - RCOOH -(CH3)3N -C4H8 COOC4H8 CID All compounds of this type fragment to produce the 85 ion. [ CH2 CH (m/z 85) CH COOH ]+ 41 Normal Acylcarnitine Profile Precursor ion scanning 100 d3-free carnitine d3-C16 carnitine C2 carnitine % d3-C3 carnitine C16 carnitine d3-C8 carnitine 0 225 250 275 300 325 350 375 400 425 450 475 500 42 m/z MS1 Collision cell MS2 Neutral loss scanning Argon gas Products Precursors Scanning (M) Scanning (M-102) In a neutral loss scan the two quadrupole mass filters are Scanning synchronously at a user-defined offset. The neutral loss is known to be common to the analytes in a mixture. 43 Neutral and Acidic Amino Acids Derivatization and Fragmentation O R OH NH2 (Generic) O R NH2 O CH3 HO + CH3 HCl Neutral or Acidic AA O R NH3 + Butanol Amino acid butyl ester O Fragmentation R O CH3 NH2 + + HO CH3 Neutral or Acidic AA Fragment Butyl formate Neutral loss of 102Da 44 Normal Amino Acid Profile Neutral loss scanning Pro 100 Deuterated internal standards for quantification d3-Leu d5-Phe % d6-Tyr d4-Ala Ser Gly d8-Val d3-Met Glu 0 140 16 0 180 200 220 240 2 60 m /z 280 45 Multiple Reaction Monitoring MS1 Collision cell MS2 Argon gas Product(s) Precursor(s) Static (m/z 315.1) Static (m/z 109.0) Both the first and second quadrupole mass analyzers are held Static at the mass-to-charge ratios (m/z) of the precursor ion and the most 46 intense product ion, respectively. Collision induced dissociation Argon gas O CH3 CH3 H3C CH2 CH2 O CH3 CH3 O O CH3 Precursor ion Product ions • In the collision cell, the TRANSLATIONAL ENERGY of the ions is converted to INTERNAL ENERGY. • Collision conditions (FRAGMENTATION) is controlled by altering: – The collision energy (speed of the ions as they enter the cell) – Number of collisions undertaken (collision gas pressure) 47 2. Quadrupole Ion Trap • • • • In an Ion trap the ions are trapped in a radio-frequency quadrupole field. The ions are then ejected detected as the radio frequency field is scanned. Ions are dynamically stored in a three-dimensional quadrupole ion storage device. The RF and DC potentials can be scanned to eject successive mass-to-charge ratios from the trap into the detector. 48 QTRAP Linear ion trap Ion accumulation N2 CAD Gas Exit lens Q0 Q1 Q2 Q3 ion selection Fragmentation linear ion trap 3x10-5 Torr 49 3-D Ion Trap Schematics Heated quartz capillary MS Scan Product Ion Scan MSn Ion Trap ID ~ 10 mm 50 Q-Ion Trap- Pros & Cons • Benefits High sensitivity Multi-stage MS • Limitations Poor quantitation. Subject to space charge effects and ion molecule reactions. Collision energy not well-defined in CID MS/MS. • Applications Compact mass analyzer 51 3. Time-of-Flight (TOF) • Time of flight mass spectrometer measures the massdependent time • • It takes ions of different masses to move from the ion source to the detector. Ions are either formed by a pulsed ionization method (usually MALDI), or various kinds of rapid electric field switching are used as a 'gate' to release the ions from the ion source in a very short time. 52 TOF - Pros & Cons • Benefits Fastest MS analyzer Well suited for pulsed ionization methods High ion transmission Highest practical mass range of all MS analyzers Limitations Requires pulsed ionization method or ion beam switching Fast digitizers used in TOF can have limited dynamic range Limited precursor-ion selectivity for most MS/MS experiments Applications MALDI systems Very fast GC/MS systems • • 53 DETECTORS Mass analysis is the separation of bunches or streams of ions according to their individual mass-to-charge (m/z) ratio The mass analyzer sorts the ions according to m/z and the detector records the abundance of each m/z. 54 Detectors are ‘eyes’ of the Instrument Once the ion passes through the mass analyzer it is then detected by the ion detector, The final element of the mass spectrometer. 55 METHODS OF ION DETECTION The detector generates a signal from incident ions by two ways: 1. Inducing current generated by a moving charge 2. Generating secondary electrons, which are further amplified. 56 Current generated by a moving charge. •Flow of electrons in the wire is detected as an electric current which can be amplified and recorded. •The more ions arriving, the greater the current. •Variation in the magnetic field, changes the flow of ion stream to the detector which produce a current proportional to the number of ions arriving. •Timing mechanisms, which integrate those signals with the scanning voltages, allow the instrument to report which m/z strikes the detector 57 Current generated by secondary electrons • Detector operates by producing a signal current from incident ions by generating secondary electrons, which are further amplified • Key part of such type of detectors is a dynode. • Dynode is electron-multiplying electrode. Incoming Ion Secondary Electrons 58 Contd … Secondary electrons Electrode surface(dynode) Dynodes For Amplification Process of Secondary electron emission. • Electrons accelerated, and strike the surface of electrode (dynode) • Energy deposited by the incident electrons result in re- emission 59 CHARECTERISTICS • Certain of these characteristics are common, like : • high sensitivity • linear, quantitative response • Some detectors are designed for specific functions or applications. 60 Electron Multipliers contd… • Electron multiplier is made up of a series of dynodes maintained at ever increasing potentials. • Typical amplification or current gain of an electron multiplier is one million. 61 Electron Multipliers contd… • Two major modifications: • Multiple-dynode type • Continuous-dynode type 62 1. Multiple-dynode type EM Incident ions Cathode Anode • Incoming ion reaches the first dynode • Ejects several other electrons by secondary emission. • This process repeated at each succeeding dynode having a higher potential than the preceding dynode. • When it arrives at the anode, the electron flow is significantly amplified 63 2. Continuous Electron Multiplier resistive-material inner coating • Contains a glass pipe with a coating on its inner surface • The electron flow moves along the pipe, reflecting from the inner wall and progressively gaining electrons • The electrical field accelerating the flow is formed by the high voltage applied across the two ends 64 Features of Electron Multipliers • Advantages: • Very high current gain • Sensitive • Fast • Disadvantage: Short lifetime • Requires good vacuum to operate • • Electron multipliers are widely used in Quadrupole and Ion trap Instruments. 65 Channel Electron Multiplier • A modified continuous dynode electron multiplier • Comprised of "the channel," a hollow, cornucopia-shaped tube made of semi conductive glass. 66 Channel Electron Multiplier contd.. IONS • The primary incoming ions passes through the inlet and strikes the surface of the CEM • The collision energy eject an electron from CEM wall • Ejected electrons accelerated into interior of CEM • Trigger secondary emission and the process continues to produce an output electron 67 RECORDER • Analogue signal is produced by the detector. • Analogue Digital Converter sends the output to the computer. Detector ADC Recorder 68 IDENTIFICATION CHARACTERIZATION Single MS Specificity Precursor Ion Neutral Loss Enh.Multi Charged High Sensitivity Full Scan MSMS MS3 Capabilities Plug & Play Sources QUANTITATION SIM or MRM •TurboIonspray •Heated Nebulizer (APCI) •Nanospray Integrated Syringe pump Built-in divert valve AUTOMATION Info. Dep. Acquisition MetID 69 Where are Mass Spectrometer Used •Biotechnology: the analysis of Proteins, Peptides, Oligonucleotides •Pharmaceutical: Drug Discovery, Combinatorial Chemistry, Pharmacokinetics, Drug Metabolism •Environmental: PAH’s, PCB’s, water quality. Food contamination •Clinical: neonatal Screening, Hemoglobin analysis, drug testing •Geological: Oil composition 70 How can Mass Spectrometry help Biochemists •Accurate molecular weight measurements: sample confirmation, to determine the purity of a sample, to verify amino acid substitutions, to detect post-transnational modifications, to calculate the number of disulfide bridges •Reaction monitoring:to monitor enzyme reactions, chemical modification, protein digestion •Amino acid sequencing:sequence confirmation, de novo characterization of peptides, identification of proteins by database searching with a sequence “tag” from a proteolytic fragment •Oligonucleotide sequencing:the characterization or quality control of Oligonucleotides •Protein structure:protein folding monitored by H/D exchange, proteinligand complex formation under physiological conditions, macromolecular structure determination 71 Application Example 1 LC-MS/MS Selectivity and Sensitivity comparisons Application Area: Environmental 72 LC/MS or LC/MS/MS? Selectivity & Sensitivity • Quantitation of 3 pesticides in a surface water extract – Simazine; Atrazine and Metabenzthiazuron • Chromatographic and Mass Spec conditions: – Ionization technique : APCI 73 Product Ion Spectra MSMS Product Ion Scan= N2 NO2 NO2 NO2 NO2 Q1 fixed, = CAD Collision Q3 scanning MH+ Simazine (202-132) Metabenzthiazuron (222 MH+ 165) MH+ Atrazine (216-174) 74 Simazine in surface water extract (50 pg injected on column) API 150EXTM LC/MS System (SIM MODE; m/z 202) API 2000TM LC/MS/MS System (MRM MODE; m/z 202-174) 75 Application Example 2 LC-MS/MS Impurity profiling 76 Impurity/Degradation Product Profiling Experimental Conditions: – 4 Commercially available OTC Melatonin Tablet preparations – LC Column: 4.6 x 50 mm Chromolith SpeedRod RP-18 – Targeted analysis using MRM as survey for known impurities and EMS for profiling in IDA followed by EPI – Confirmation of impurity/degradation product by MS/MS using EPI 77 Impurity/Degradation Product Profiling Multiple MRM’s (1) EMS (2) Enhanced Resolution • IDA Criteria Level 1 For known impurities, targeted MRM analysis can be used. At the same time an EMS survey scan can search for unknown impurities… …for unknown impurities, IDA triggered MS/MS and MS3 information can be collected to aid in identification. Dependent Scan (s) Dependent Scan (s) Dependent Scan (s) Dependent Scan (s) Add to Exclusion List • IDA Criteria Level 2 Second Dependent Second Dependent Second Dependent Scan (s) Scan (s) Scan (s) 78 Impurity/Degradation Product Profiling Targeted MRM Known Impurities H 3C O OH H N oxidation Impurities O 249 m/z N H HO H N OH di-oxidation Impurity O 265 m/z N H H 3C O EMS Unknown Impurities 79 Impurity/Degradation Product Profiling + E P I (4 7 7 .0 0 ) C h a rg e (+ 2 ) C E (3 0 ): E xp 3 , 5 .5 6 8 m in fro m S a m p le 1 (S tre s s e d 1 0 n g /u L ) o f S tre s s ... 100% 95% 90% 85% 80% 75% 70% 65% 60% 55% 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 60 80 100 120 140 160 180 200 2 2 8 .0 1 6 0 .0 2 3 3 .0 2 6 7 .1 2 7 3 .0 220 240 260 280 300 m /z , a m u 3 2 9 .0 320 340 360 380 400 4 0 5 .2 4 1 8 .3 420 440 460 480 500 1 8 6 .0 2 0 2 .9 2 4 5 .0 M a x. 1 .6 e 5 c p s . EPI Melatonin-Formaldehyde Impurity 477 m/z H 3CO N N O H 3CO N H 4 7 7 .2 H N O 245 80 Impurity/Degradation Product Profiling X IC o f + E M S : E x p 1 , 2 4 9 .0 a m u f r o m S a m p le 9 ( M e la to n in T a b le t (1 0 0 n g /u L ) I D A E M S _ E R _ E P I w i. .. 8 .3 e 6 8 .0 e 6 7 .5 e 6 7 .0 e 6 6 .5 e 6 6 .0 e 6 5 .5 e 6 5 .0 e 6 4 .5 e 6 4 .0 e 6 3 .5 e 6 3 .0 e 6 2 .5 e 6 2 .0 e 6 6 .9 1 1 .5 e 6 1 .0 e 6 5 .0 e 5 0 .0 1 2 3 4 T im e , m in 5 6 7 8 9 3 .8 1 7 .2 1 …software can help with data analysis through sample and control comparison for degradation products… 2 .8 6 M a x . 8 .3 e 6 c p s . XIC’s of peaks present in Sample, but not Control. MS/MS information collected through IDA 81 Specificity of Detection for LC • UV – chromophore – all compounds with a chromophore responding at the selected wavelength will interfere • MS – molecular mass – interference from isobaric compounds – chemical noise • MS/MS – molecular mass and structural information – interference from structural isomers only 82 HPLC-UV Analysis of Sirolimus in Whole Blood 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Wash all glassware in methanol x2 and tert-butyl methyl ether (TBME) x2. Place 50L of internal standard (in methanol) into each screw-cap glass tube. Add 200L Sirolimus calibrator (5x concentrated in methanol) or 200L methanol for patient samples. Add 1.0mL blank whole blood to calibrators or 1.0mL patient whole blood. Add 2.0mL 0.1M ammonium carbonate buffer. Mix thoroughly. Add 7.0mL TBME and extract for 15min. Transfer upper layer to clean tube and re-extract lower layer with 7.0mL TBME. Combine TBME extracts and evaporate to dryness. Redissolve residue in 5.0mL ethanol and evaporate to dryness. Redissolve residue in 1.0mL ethanol, transfer to Eppendorf tube and evaporate to dryness. Redissolve residue in 100L 85% methanol. Inject 80L (equivalent to 800L whole blood) and analyse using two 4.6mm x 250mm C18 columns connected in series (30min run time). 83 Sirolimus: HPLC - UV Example 84 Immunosuppressant Sample Preparation LC-MS/MS Analysis Whole Blood (10L - 40µL) Add ZnSO4 Soln. Add 2 volumes MeCN with IS, Seal & Vortex Mix Centrifuge, Inject 5 - 20L 85 Full Scan Acquisition Mode Sirolimus: MS Spectrum [M+NH4]+ 100 821.5 822.5 [M+Na]+ % [M+Li]+ [M+H]+ 810.5 826.5 827.5 [M+K]+ 0 790 795 800 805 810 815 820 825 830 835 840 845 m/z 850 86 Sirolimus: Single ion monitoring (MS) LC-MS (SIM) vs LC-UV 100 30µg / L HPLC-MS SIR m/z 821 % 0 1.5 min HPLC-UV 87 Full Scan Acquisition Mode Sirolimus: MS Spectrum [M+NH4]+ 100 821.5 822.5 [M+Na]+ % [M+Li]+ [M+H]+ 810.5 826.5 827.5 [M+K]+ 0 790 795 800 805 810 815 820 825 830 835 840 845 m/z 850 88 Product ion scanning MS1 Collision Cell Ar (2.5 – 3.0e-3mBar) MS2 Products Precursor Static (m/z 821.5) Scanning The first quadrupole mass analyzer is fixed, or Static, at the mass-to-charge ratio (m/z) of the precursor ion to be interrogated while the second quadrupole is Scanning over a user-defined mass range. 89 NH4+ Product ion scanning 100 821.5 Mass spectrum from MS1 822.5 % 826.5 827.5 810.5 0 790 795 800 805 810 815 820 825 830 835 840 845 m/z 850 100 768 Product ion spectrum from MS2 % 576 548 558 718 750 786 821 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 m/z 900 90 Multiple Reaction Monitoring MS1 Collision Cell Ar (2.5 – 3.0e-3mBar) MS2 Precursor(s) Product(s) Static (m/z 821.5) Static (m/z 768.5) MS/MS : Compound-Specific Monitoring 91 Multiple Reaction Monitoring Sirolimus LC-MS(SIM) vs LC-MS/MS (MRM) 100 100 3µg / L % % 30µg / L SIR m/z 821 0 100 0 100 MRM m/z 821>768 % % 0 0.50 1.00 1.50 Time 0 0.50 1.00 1.50 Time 92 Questions please?... 93