Trends in 2006 Introduction to LC/MS/MS By Crystal Holt, LC/MS Product Specialist, Varian Inc. Toxicology laboratories Increased use of LC/MS Excellent LD Cheaper (still expensive) Much more robust Solves many limitations of GC/MS Simple sample preparation Pharma laboratories Increased (although still limited) use of GC/MS Less FDA inspection issues (helium vs. LC mobile phases) Significantly lower cost than LC/MS 2 Factors to Consider Polarity, MW and Volatility Experience and preference of users Existing methodologies Budget constraints GC/MS $50K - $200K LC/MS $150K - $400K what can you afford? Sample prep, labor, and material costs Analytical requirements Molecular weight, volatility and polarity of analytes Sensitivity and linearity Matrix Sample preparation requirements Analytical requirements by regulatory agencies verlap: GC/MS or LC/MS 3 4.D. Sparkman and F.E. Klink, "Chromatography/Mass Spectrometry: Principles and Practice", American Chemical Society Continuing Education Courses, 2001.
LC/MS Triple Quad Components LC/MS/MS Analyzer Q1 Mass Analyzer Q2 Collision Ce Ionization Source Solvent Delivery System AutoSampler MS Detector Vacuum System Q3 Mass Analyzer 5 6 LC-API-MS Revolution Prof. John Fenn, Nobel Laureate Early Interface Technologies Moving belt Flow-FAB Thermospray Particle Beam None of these interfaces achieved the sensitivity and robustness required for wide application of LC/MS Atmospheric Pressure Ionization Electrospray (ES, ESI, APESI) Chemical Ionization (APCI) Photoionization (APPI) Modern API sources have helped LC/MS transition from a research tool to a routine analytical instrument John Fenn and co-workers first published the successful ionization of large synthetic polymers (PEGs), and subsequently proteins, using the electrospray ionization (ESI) technique in the late 1980's while working at Yale University. Fenn s work built on that of Malcolm Dole from Northwestern University in the late 1960's. Fenn added a counter-current dry gas to assist with the drying of the electrosprayed droplets that greatly assisted in the formation and detection of ions. 7 8
Steps in Electrospray Process Simple Electrospray Description Production of charged droplets from a capillary tip Under influence of strong electric field Reduction of droplet size Rapid solvent evaporation Repeated coulombic explosions (fission) Transfer of ions from surface of small droplets to gas phase No heat of evaporation 9 10 ESI: Evaporative Cooling Declustering with Drying Gas Hot Drying Gas solvent solvent solvent solvent Drying gas serves two functions N 2 Gas Heater 1. Droplet size reduction (desolvation) N 2 drying gas Analyte inside the droplet never experiences the drying gas temperature No thermal degradation until release into gas phase ions Vacuum Restriction Droplets Very Large Droplets Large Droplets 2. Dissociating ion/solvent clusters before the sampling orifice 11 12
Electrospray Droplets ESI Ionization of the Analyte Distribution of droplet sizes Number Higher Probability f Thermal Degradation Useable Droplets Desolvated and Declustered Blown Away By Drying Gas Direct Away (ff-axis needle) Analyte forms an ion in the mobile phase Positive Ion ESI Cation attachment: H M (MH) also Na, K, NH 4 May produce a mixture of cations Negative Ion ESI Proton loss: M (M-H) - H Anion attachment: A - M (MA) - May produce a mixture 1 micron Droplet Diameter 300 micron 13 14 ESI: Ion Suppression Mechanisms Ion suppression = reduction of analyte signal Ion Pairing Avoid reagents that strongly pair with the ion of interest and effectively neutralize the ion in solution (reduced activity for ion evaporation) Competitive Ion Evaporation Large concentrations of similarly charge ions from the buffer, matrix, etc. that compete for ion evaporation from the micro-droplet Ion Suppression of Signal Above 6 mm NH 4 AC, signal decreases 15 16
Ion Suppression of Signal ESI Advantages MW Information Higher MW possible due to multiple charges Much lower signal at higher buffer concentration High sensitivity (versus particle beam, thermospray) Good for volatile and non-volatile analytes Good for polar and ionic (very polar) analytes Pneumatic nebulization and thermally assisted desolvation, flow rate up to 1000 µl/min 17 18 ESI Disadvantages Minimal qualitative information (MH) only is typical Skimmer CID or MS/MS for additional fragments Best mobile phase for chromatography may not be best for ESI Poor performance with high aqueous mobile phases Problems with involatile buffers Introduction to APCI Assumes total vaporization Mobile phase, analyte and matrix APCI hot evaporation of total droplet ESI cold ion evaporation from microdroplet Requires appropriate gas phase chemistry Mobile phase selection can affect response pens new options for chromatography Susceptible to ion suppression Sensitivity inhibited by high salt concentrations Sample matrix ions may compete with analyte ions 19 Flow dependent precision 20
What Applications Need APCI? APCI Vaporization APCI works well for small molecules that are moderately polar to non-polar APCI works well for samples that contain heteroatoms Avoid samples that are typically charged in solution Avoid samples that are very thermally unstable or photosensitive APCI is a gas phase reaction Analyte must be available as a stable, gas phase molecule Matrix components may or may not undergo non-destructive evaporation Evaporation driven by a very hot auxiliary gas gas liquid gas Quartz Tube Vortex flow of hot gas 21 22 APCI Ionization Process Declustering with Drying Gas Corona discharge ionizes atmospheric gases e - N 2 N 2 2 e - N 2 N 2 N 4 Vaporizer Assembly Charge transferred to water N 4 H 2 2N 2 H 2 H 2 H 2 H 3 (H 2 cluster) H - Charge transferred to organic solvent H 3 CH 3 H CH 3 H 2 H2 Corona needle N 2 drying gas ion/solvent Gas Heater ions N 2 Charge transferred to analyte (M) CH 3 H 2 M MH CH 3 H For APCI, the drying gas flow is lower than ESI, but it is still critical to the dissociation of ion/solvent clusters before the sampling orifice Capillary Restriction 23 24
APCI Advantages APCI Disadvantages MW information (classical CI) Easy to use, rugged May have higher sensitivity than ESI Ion suppression much less of a problem Good for moderately volatile analytes Accommodates high flow rates (> 1 ml/min) Works well with water Good results in positive- and negative-ion modes Adduct formation Excellent precision for quantitation No information from controlled fragmentation Skimmer CID (scid) or MS/MS for additional fragments Problems with involatile buffers Single-charge ions limits mass range Low MW (<150 Da) analytes face problems with chemical noise (mobile phase ions) Thermal degradation of analytes can result 25 26 return to APCI menu ESI vs. APCI MS/MS what s that? ESI APCI Drying gas 7 l/min @ 350 C 2 l/min @ 150 C Nebulizing gas 60psi 60psi Auxiliary gas N/A 2 l/min @ 550 C Inner needle adjustable fixed Needle assy up/down adjustable fixed Needle assy translation adjustable adjustable Needle setting 5000V 5μA ptimum liquid flow rate 200-400μl/min 1 ml/min MS/MS is when you have more than one step of mass filtration/separation and an additional step of fragmentation. MS/MS is used to give an additional level of confirmation to an analysis or when very specific results are required. MS/MS can also help determine the molecular structure of a molecule. 27 28
MS/MS in a Triple Quad Triple Quad Step 1 Ionization GCMS or LCMS Step 2 Isolation Select mass to analyze using Q1. Removes all other ions that we aren t interested in. 29 30 Triple Quad Triple Quad Step 3 Fragmentation Excite ions in Q2 and smash them into Ar Collision Induced Dissociation (CID) The original ion is smashed into smaller pieces. Step 4 Detection Use the third quadrupole to select fragment ions. Pass those product ions to the detector for quantitation. 31 32
Limitation of API Interfaces Why MS/MS for LC/MS? Atmospheric Pressure Ionization (API) dominate LC/MS Electrospray (ES, ESI, APESI) Chemical Ionization (APCI) Photoionization (APPI) All API mechanisms are soft Minimal fragment ions formed during ionization Typically only molecular ion information Pseudo- or quasi-molecular ion(s) Minimal qualitative information for confirmation Adduct ions (Na, K, etc) are not useful for confirmation 34 EI Full Scan MS of Reserpine ESI () Full Scan MS of Reserpine EI-GC/MS produces a wide range of ions Information Rich (MH) 100 195 HN N API-LC/MS creates very few ions Limited information 212 50 141 395 251 77 109 359 381 608 226 265 301 413 448 0 40 90 140 190 240 290 340 390 440 490 540 590 (mainlib) Reserpine Na adduct Electron Ionization spectrum from NIST; probe sample introduction 35 36
ESI () MS/MS of Reserpine After MS/MS dissociation More ions = more information CID Before the Skimmer Literature has give it many names In-source CID Skimmer CID s-cid Cone CID Pseudo-MS/MS What is it? How does it occur? 37 Product Ion Scan Spectrum 38 What Is Skimmer-CID? Limitations of Skimmer CID Q1 Ions Ion Guide 3.5 mtorr 0.8 Torr Region for skimmer CID Pressure in the first stage of vacuum is still relatively high. The distance between the capillary and skimmer is short, but appropriate acceleration ions in this region can induce CID. Dissociation occurs BEFRE the analyzer Skimmer CID is less selective than true MS/MS All eluting ions may be dissociated Analytes and matrix components Full scan spectra shows a mix of product ions from the analyte and the matrix SIM can focus on the desired product ions SIM can ignore matrix ions with different m/z SIM does NT eliminate interference if common product ions are derived from coeluting precursors Voltage setting may give limited selectivity 39 40
Ion Intensity Ion Intensity Ion Intensity s-cid: Product Ion Interference Skimmer CID: No Noise Reduction Consider an analyte ion A and an interfering isobar from compound B. With s-cid, A and B may dissociate to yield different product ions... but C A C A B B A C s-cid product ions are measured against chemical noise of the background B B E E E D D A A D B A C C C 41 m/z another interfering ion can originate from any m/z from any coeluting compound such as C 42 m/z Signal for A often less than A, and noise at lower m/z A often greater than at A; therefore, S/N for A less than A What Is True MS/MS? MS/MS Ion Isolation Three step process Mass analysis isolation of precursor m/z CID accelerate of the precursor into a collision gas Key difference: CID occurs AFTER mass analysis Mass analysis separation of the product ions Typically SIM process for transmission quads Full scan for ion traps and TFs Ion isolation BEFRE CID, eliminates ions other than the precursor ion B A Ion isolation does not eliminate common precursor ions Low m/z ions eliminated m/z High m/z ions eliminated 43 44
Ion Intensity Noise Advantage for MS/MS MS/MS: Ultra-Low Noise Detection In full scan or SIM, chemical noise can appear across the m/z range Isolation step eliminates all chemical noise ther than noise at the precursor ion Product ions of lower m/z can be measured against zero background If chemical noise decreases faster than signal, detection limits improve MS/MS product ions are measured in a noise free region A B B A 45 46 m/z Signal for A often less than A, but noise free region yields larger S/N at A than at A ESI () MS/MS of Reserpine Product Ions - Lowest probability for interference Precursor Ion - Highest probability for interference Why MS/MS for LC? More qualitative information Increased Signal/Noise Enhanced selectivity of product ion High quality analytical results 47 48
Advantages of MS/MS MS/MS offers increased selectivity for dirty samples. It helps the users look for small amounts of sample that would otherwise be hidden by all the background noise. Same Molecule, MS Vs. MS/MS Questions?? MS/MS capabilities cost more due to additional hardware and software requirements in a transmission quadrupole, but often times the performance is worth the price increase. 49 50 51