1	Mass Spectrometry Concepts Tina Wu Chang DEA Western Laboratory San Francisco, CA Presented at SAFS 2007 Atlanta, GA September 10, 2007
2	OVERVIEW Overview of the components of a GC-MS system Thorough description of mass spectrometer components Focus is on quadrupole system in EI mode Mass spectrometer calibration Reference comparison tips
3	GC-MS System Components
4	Gas Chromatograph This is where you can achieve separation of sample components Separation depends on various factors Injection technique Column type (coating, length, dimensions) Oven temperature program Carrier gas type and flow rate
5	Quadrupole Mass Spectrometer
6	Electron Ionization (EI)
7	Fragmentation Molecular ion creation Unimolecular decomposition
8	Ion Source
9	Ion Source In Action
10	Quadrupole Mass Filter
11	Change In Potentials During Scan
12	Ion Trajectories In A Quadrupole Small ion greatly affected by alternating field Large ion drifts in constant part of field Ion of right size drifts in constant field, but is also pulled back by alternating field
13	Quadrupole As A Mass Filter Positive rods form high-pass mass filter Negative rods form low-pass mass filter When both sets work together, a narrow-band filter is the result
14	Ion Stability Diagram
15	Detector (HED/EM)
16	Mass Spectrometer Calibration Mass spectrometer will produce no meaningful information if analyzer is not set up correctly Actual relationship between voltage and m/z is nonlinear Need to adjust for variations in the source, analyzer, and EM surfaces that occur during routine use Achieved by TUNING
17	Tuning Calibration compound introduced into instrument Variables are adjusted until sensitivity and resolution are within acceptable limits Four basic processes Adjust ion ratios Adjust mass resolution Adjust overall sensitivity Mass axis calibration
18	Compound Used For Tuning Perfluorotributylamine (PFTBA) Chemically stable Volatile Simple spectrum
19	#1: Adjust Ion Ratios Ramping source element voltages Ion focus lens Entrance lens Repeller
20	#2: Adjust Mass Resolution By setting AMU gain and offset for proper mass peakwidths
21	#3: Adjust Overall Sensitivity By setting EM voltage
22	#4: Mass Axis Calibration Ensures correct mass assignment by adjusting mass axis gain and offset
23	Tune Sequence Of Events Find mass peaks (69, 219, 502) Coarse adjust EM voltage/peak widths Adjust ion source elements to optimize 502 Fine adjust EM voltage/peak widths Mass axis calibration Save tune file
24	Why Is Tuning Important? Tune reports serve as a partial record of the history of your system Troubleshooting/diagnostic tool Can indicate leaks Chemical contamination Dirty source Old EM horn Incorrect/malfunctioning heated zones
25	Different Tune Programs Autotune Maximizes sensitivity across entire scan range Standard Spectra Tune Has set targets to ensure a standard response across entire scan range GOOD CHOICE IF SEARCHING COMMERCIAL LIBRARIES Target Tune Used with EPA methods Low Mass Autotune Uses 69, 131, 219 to tune (for low MW applications) Manual Tune User-controlled to meet defined criteria
26	PFTBA—Atune vs. Stune
27	Heroin—Atune vs. Stune
28	Spectral Library Searching Beware and use judgment when searching Spectral tilting Library selection Understand how library searches work Contamination? Run blanks Subtract background Beware of isomers Other problems
29	Spectral Tilting
30	Library Selection 2 main spectra collections NIST/EPA/NIH mass spectral library Wiley Registry of Mass Spectral Data Smaller specialized libraries exist Create an in-house library Important to have data from standards run under similar conditions Use authenticated, traceable standards
31	Library Searches Quick and easy way to “identify” an unknown mass spectrum Presearch of library occurs first Most computer libraries contain only condensed spectra Two different search algorithms Forward-search Reverse-search (Probability Based Matching)
32	Forward Search Unknown spectrum is condensed Common weighting factor is square root of the mass The weighted condensed unknown spectrum is compared with the subset of library spectra selected during presearch Possible errors Unknown could have high mass contamination Assumes all spectra are of the highest quality
33	Reverse Search (PBM) Each spectrum in the library subset is compared with the unknown spectrum “Is the library spectrum present in the unknown spectrum?” The full unknown spectrum is used here Unknowns can frequently have contaminated or co-eluting peaks present Uniqueness + Abundance factors
34	Common Contaminant Ions
35	Column Bleed Example
36	Stereoisomers
37	Positional Isomers
38	Another Isomer Example
39	More 58 Base Peaks
40	Other Problems With Library Searches Correct match for unknown may not be in library at all If spectrum is not clean, search results may reflect presence of extraneous ions rather than that of unknown Errors DO occur with respect to data included in mass spectral databases
41	YOU CAN NEVER JUST RELY ON YOUR LIBRARY SEARCH RESULTS…YOU MUST AT LEAST VISUALLY INSPECT BOTH THE UNKNOWN AND THE LIBRARY SPECTRUM!!!
42	What To Do If You Have A Complete Unknown Library search can possibly give structural clues Hit the books! Instrumental Data for Drug Analysis Eight Peak Index of Mass Spectra Interpretation Of Mass Spectra by McLafferty Analyze sample with other techniques to get clues on identity Always verify results with standard
43	Any Questions? How a quadrupole mass spectrometer works in EI mode Optimization of mass spectrometer performance through tuning Spectral library searching of data Contact info tina.wu.chang@usdoj.gov 415-744-7051 ext. 299
44	References Watson, J. T. Introduction to Mass Spectrometry; Lippincott-Raven: Philadelphia, 1997; p 75. Watson, J. T. Introduction to Mass Spectrometry; Lippincott-Raven: Philadelphia, 1997; p 2. Intro to HP 5973 GC-MSD Troubleshooting and Preventative Maintenance, Course # H5947A Student Manual; Hewlett-Packard Company: Atlanta, 1997; p 112. Watson, J. T. Introduction to Mass Spectrometry; Lippincott-Raven: Philadelphia, 1997; p 140. http://www.chem.vt.edu/chem-ed/ms/quadrupo.html Skoog, D. A.; Leary, J. J. Principles of Instrumental Analysis; Saunders College Publishing: Fort Worth, 1992, 4^th ed.; p 432. http://www.jic.bbsrc.ac.uk/SERVICES/metabolomics/lcms/single1.htm Skoog, D. A.; Leary, J. J. Principles of Instrumental Analysis; Saunders College Publishing: Fort Worth, 1992, 4^th ed.; p 431. http://www.jic.bbsrc.ac.uk/SERVICES/metabolomics/lcms/single2.htm HP GC-MSD ChemStation and Instrument Operation, Course # H4043A Student Manual; Hewlett-Packard Company: Atlanta, 1997; Vol. 1, p 27. Intro to HP 5973 GC-MSD Troubleshooting and Preventative Maintenance, Course # H5947A Student Manual; Hewlett-Packard Company: Atlanta, 1997; p 24. Intro to HP 5973 GC-MSD Troubleshooting and Preventative Maintenance, Course # H5947A Student Manual; Hewlett-Packard Company: Atlanta, 1997; p 125. Smith, R. M.; Busch, K. L. Understanding Mass Spectra--A Basic Approach; John Wiley & Sons, Inc.: New York,1999, p 24. HP GC-MSD ChemStation and Instrument Operation, Course # H4043A Student Manual; Hewlett-Packard Company: Atlanta, 1997; Vol. 1, p 48. Watson, J. T. Introduction to Mass Spectrometry; Lippincott-Raven: Philadelphia, 1997; p 392. HP 5973 Mass Selective Detector Hardware Manual; Hewlett-Packard Company, 1996; p 89. Smith, R. M.; Busch, K. L. Understanding Mass Spectra--A Basic Approach; John Wiley & Sons, Inc.: New York,1999, p 34. Noggle, F. T.; Clark, C. R.; Bouhadir, K. H.; DeRuiter, J. J. Chromatogr. Sci. 1991, 29, 31-36. Smith, R. M. Understanding Mass Spectra--A Basic Approach; John Wiley & Sons, Inc.: Hoboken, NJ, 2004, 2^nd ed.; p 246.