Laboratory Experiment pubs.acs.org/jchemeduc
Hands-On Electrospray Ionization-Mass Spectrometry for UpperLevel Undergraduate and Graduate Students Naomi L. Stock*,† and Raymond E. March†,‡ †
Water Quality Centre, Trent University, 1600 West Bank Drive, Peterborough Ontario K9J 7B8 Canada Department of Chemistry, Trent University, 1600 West Bank Drive, Peterborough Ontario K9J 7B8 Canada
‡
S Supporting Information *
ABSTRACT: Electrospray ionization-mass spectrometry (ESI-MS) is a powerful technique for the detection, identification, and quantification of organic compounds. As mass spectrometers have become more user-friendly and affordable, many studentsoften with little experience in mass spectrometryfind themselves needing to incorporate mass spectrometry into their research. Herein, a hands-on laboratory experiment for upper-level undergraduate and graduate students to investigate ESI-MS is described. This experiment provides students with the opportunity to observe and use instrumentation discussed in class, to investigate various modes of operation, to compare triple-stage quadrupole (TSQ) with quadrupole linear ion trap (QLIT) instrumentation, and to decide upon the optimum approach for incorporation of mass spectrometry into their research. KEYWORDS: Upper-Division Undergraduate, Graduate Education/Research, Analytical Chemistry, Environmental Chemistry, Laboratory Instruction, Hands-On Learning, Mass Spectrometry
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chromatography; instead it focuses only on mass spectrometry. Second, as opposed to examining how a mass spectrometer functions for a particular analysis, this hands-on laboratory exercise provides graduate students with an overview of the range of capabilities of the mass spectrometer. The experiment is also appropriate for upper-level undergraduate students. This hands-on experiment, which has been incorporated into our graduate course in mass spectrometry for the past four years, was designed to provide students with an overview of the capabilities of electrospray ionization-mass spectrometry (ESIMS) and to compare the performance of a triple-stage quadrupole (TSQ) mass spectrometer with that of a linear quadrupole ion trap (QLIT) mass spectrometer. As described by March and Todd,6 the function of a trapping device is to enhance the duration of observation of ions; an ion is “trapped” when its residence time within a defined spatial region exceeds that had the motion of the ion not been impeded in some way. Students were encouraged to investigate the various scan modes in which these instruments can be operated. Perhaps most importantly, this hands-on experiment prompted graduate students to think about how they can best incorporate mass spectrometry into their research projects. This experiment has proven useful for all graduate students enrolled in the course, regardless of their analytical chemistry background.
n recent years, the mass spectrometry community has expanded as instruments have become more user-friendly and affordable, with opportunities for novice users to employ highly advanced, state-of-the-art instrumentation.1 Often these novice users are students who desire to incorporate mass spectrometry into their research projects. During this period, we have observed an increase in the number of students enrolling in our graduate course in mass spectrometry who do not have a strong background in analytical chemistry, for example, biochemistry students who wish to use mass spectrometry for protein analysis; biology students wanting to use mass spectrometry to identify chemical cues in predator− prey relationships; and toxicology students who would like to quantify potentially harmful chemicals. Recent articles have reported the successful inclusion of liquid chromatography-electrospray ionization-mass spectrometry (LC−ESI-MS) in both graduate and undergraduate courses. Bergen et al.2 created an experiment for students to measure aspartame and caffeine in carbonated beverages using LC−ESI-MS, while Stock et al.3 provided undergraduate students with the opportunity to employ LC−ESI-MS to measure perfluorinated surfactants in fish liver. Students of Fenk et al.4 used LC−ESI-MS for the identification and quantitative analysis of acetaminophen, acetylsalicyclic acid, and caffeine in commercial analgesic tablets. Finally, Rosado et al.5 prepared a one-week mini-course, including both lectures and hands-on activities, to train students in the practical aspects of modern mass spectrometry. The approach discussed in this paper is a little different. First, this laboratory experiment does not employ any liquid © XXXX American Chemical Society and Division of Chemical Education, Inc.
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Table 1. Different Scan Types of the Hybrid Triple Quadrupole/Linear Ion Trap Instrument Scan Types in Triple Stage Quadrupole (TSQ) Modea First Quadrupole Mass Scan (Q1MS) Third Quadrupole Mass Scan (Q3MS) Product Ion (MS2) Precursor Ion (PREC) Multiple Reaction Monitoring (MRM) Scan Types in Quadrupole Linear Ion Trap (QLIT) Modea Enhanced Mass Scan (EMS) Enhanced Resolution (ER) Enhanced Product Ion (EPI) MS/MS/MS (MS3)
a
Descriptiona A full scan using the first quadrupole (Q1). A full scan using the third quadrupole (Q3). A tandem mass spectrometry (MS/MS) scan where Q1 is fixed to transmit a precursor ion, product ions are generated in the second quadrupole (Q2) collision cell via collision with nitrogen, and a defined mass range is scanned in Q3. MS/MS scan where a defined mass range is scanned by Q1, product ions are generated in Q2, and Q3 is fixed to transmit a product ion. Ions of a given mass in Q1 must fragment or dissociate to give a product ion of specific mass in Q3 in order for a response to be detected.
Descriptiona
Ions are scanned in Q1 to the QLIT where they are collected. The ions are scanned from Q3 to produce a mass scan type spectrum. Ions are scanned in Q1 to the QLIT where they are collected. Ions in a 30 Da window around the precursor ion are then scanned from Q3. MS/MS scan where Q1 is fixed to transmit a precursor ion, product ions are generated in Q2 and collected in the QLIT, and Q3 scans a defined mass range. MS3 scan where Q1 is fixed to transmit a precursor ion, and product ions are generated in Q2 and collected in the QLIT. A specific product ion is then isolated in the QLIT, all other ions are removed, a voltage is applied to the trap, and the selected product ion collides with residual nitrogen in Q3, further fragmenting the ion and producing MS3 product ions.
Adapted From AB Sciex.10
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EXPERIMENTAL DETAILS This hands-on experiment was designed for a 2 h laboratory period. Students were divided into two groups, typically ranging from two to four students. Each student group was assigned a mass spectrometer. Both mass spectrometers used in this experiment were hybrid triple quadrupole/linear ion trap instruments, either the Qtrap or the 5500 Qtrap (both AB Sciex, Concord, ON, Canada). Hybrid triple quadrupole/linear ion trap instruments can be operated in either TSQ or QLIT mode. In TSQ mode, the ion beam is continuous, whereas in QLIT mode, the third quadrupole (Q3) functions as a quadrupole linear ion trap where the ion beamed is pulsed and ions can be trapped and stored before being scanned out. Within each mode, the instrument can perform several different scan types as outlined in Table 1. Using a syringe infusion pump (Harvard Apparatus, Holliston, MA) and solutions of caffeine (1,3,7-trimethylpurine-2,6-dione) in methanol, students obtained mass spectra operating the mass spectrometer in both TSQ and QLIT modes and using all nine scan types outlined in Table 1. Caffeine (Figure 1), purchased from Sigma-Aldrich (Oakville,
HAZARDS There are no significant safety issues associated with this laboratory exercise. Students should follow instruction when using the high voltage ion source on the mass spectrometer and wear protective clothing and eyewear in the lab. Detailed information on the hazards and safe handling procedures of methanol and caffeine are available on material safety data sheets (MSDS). All chemicals used in this experiment should be collected in labeled waste containers and disposed appropriately.
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RESULTS AND DISCUSSION Students first obtained mass scans using three scan types: Q1MS, Q3MS, and ER; results are shown in Figure 2. All three mass scans consist of a protonated molecule [M+H]+ at m/z 195 and are consistent with previously published data.7−9 Students observed very little difference between the Q1MS and Q3MS scans. However, the ER scan using the QLIT produced a mass scan with greater ion signal intensity and enhanced resolution. Students were also able to identify the [M+1+H]+ and [M+2+H]+ isotope peaks. Using the MS2 and EPI scan types, students obtained two product ion scans (Figure 3). The precursor ion at m/z 195 gives a major product ion at m/z 138 and minor product ions at m/z 110, 83, and 69; these product ions are consistent with previously published data.7−9 The EPI scan using the QLIT produced a product ion scan with increased ion signal intensity and resolution. In this scan, additional minor product ions, including those at m/z 163, 151, and 123 were observed. When considering possible fragmentation patterns, generally students suggested the following: the ion at m/z 138 corresponds to the loss of OCNCH3 (57 Da). The ions at m/z 110, 83, and 69 originate from the ion at m/z 138 and are the result of the losses of CO (28 Da), HCN or CNH (27 Da), and C2H2N (40 Da), respectively. This fragmentation is consistent with the literature8,9 and is shown in Figure 3A. To illustrate that the mass spectrometer can also operate in reverse, students obtained a precursor ion scan using the PREC scan type (Figure 4). In this scan, students selected one of the
Figure 1. Caffeine.
ON, Canada), was selected as the analyte of interest as is it inexpensive, readily available, and nontoxic at the concentrations needed for the experiment. In addition, both the fragmentation and electrospray ionization parameters are well documented.7−9 For product ion and MS/MS/MS (or MS3) scans, students were asked to consider fragmentation pathways and suggest elemental compositions for the product ions observed. B
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Figure 3. Two product ion scans of caffeine, m/z 195. The spectrum 3A was acquired using the MS2 scan type and the spectrum 3B was obtained using the EPI scan type (QLIT).
Figure 2. Mass scan of caffeine, m/z 195. Spectrum 1A was acquired using the first quadrupole (Q1), whereas spectrum 1B was acquired using the third quadrupole (Q3) and shows little difference. Spectrum 1C was obtained using the ER scan type (QLIT). Isotope peaks [M +1+H]+ and [M+2+H]+ were also identified. The small peak observed at m/z 193 is likely an impurity in the caffeine solution.
product ions identified above and confirmed that the instrumentation was able to obtain m/z 195 as the precursor ion. It was discussed that such a scan may be useful when trying to identify if a suite of compounds (for example, degradation products or metabolites) originate from a single source. As many graduate students use mass spectrometry as a quantitation tool, students explored multiple reaction monitoring (MRM) mode. With this technique, students created a method where the transition of either m/z 195 → 110 or m/z 195 → 138 was monitored. It was discussed that this scan type is extremely useful in increasing signal/noise ratios and, therefore, useful for the quantitation of specific analytes. For their final hands-on exercise, students obtained an MS3 scan using the QLIT. Students selected the precursor ion m/z 195 and one of the product ions observed in the product ion spectra (Figure 3). A typical MS3 spectra of precursor ion m/z 195 and product ion m/z 138 is shown in Figure 5. These results are particularly interesting as the major MS3 product ions, m/z 110, 83, and 69, are the same as those observed using MS2 and confirm the students’ suggestions that m/z 110, 83, and 69 originate from the ion at m/z 138. Ions at m/z 123, 97, 95, and 56 are also observed. These results confirm the usefulness of the MS3 scan to investigate fragmentation patterns.
Figure 4. Precursor ion scan of caffeine using product ion m/z 138.
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ADAPTIONS FOR OTHER INSTRUMENTATION If hybrid triple quadrupole/linear ion trap instruments are not available, the experiment could be adapted to other instruments including triple-stage quadrupole, three-dimensional quadrupole ion trap, time-of-flight, or orbitrap instruments. In each case, students should acquire as many scan types as possible so as to appreciate fully the capabilities of the instrumentation.
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CONCLUSIONS Graduate students, many with little to no experience with mass spectrometry, were able to participate in hands-on experiments using state-of-the-art ESI-MS instrumentation. They were able to experience first-hand the capabilities of two hybrid mass spectrometers and understand the usefulness of each scan type. Most importantly, these hands-on experiments provided C
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Spectrometry; March, R. E., Todd, J. F. J., Eds.; CRC Press: Boca Raton, FL, 2010; Vol. 4, Ch. 1, p 7. (7) Tuomi, T.; Johnsson, T.; Reijula, K. Analysis of Nicotine, 3Hydroxycotinine, Cotinine, and Caffeine in Urine of Passive Smokers by HPLC-Tandem Mass Spectrometry. Clin. Chem. 1999, 45, 2164− 2172. (8) Bioanco, G.; Abate, S.; Labella, C.; Cataldi, T. R. I. Identification and Fragmentation Pathways of Caffeine Metabolites in Urine Samples via Liquid Chromatography with Positive Electrospray Ionization Coupled to a Hybrid Quadrupole Linear Trap (LTQ) and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry. Rapid Commun. Mass Spectrom. 2009, 23, 1065− 1074. (9) Bier, D.; Hartmann, R.; Holschbach, M. Collision-Induced Dissociation Studies of Caffeine in Positive Electrospray Ionisation Mass Spectrometry using six Deuterated Isotopomers and one N1ethylated Homologue. Rapid Commun. Mass Spectrom. 2013, 27, 885− 895. (10) Getting Started Guide Analyst Software D1000064245 B; AB Sciex: Concord, Ontario, 2008.
Figure 5. MS3 scan of caffeine using precursor ion m/z 195 and product ion m/z 138.
students with the opportunity to understand how mass spectrometry may play a role in their research and give them the confidence to include the technique.
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ASSOCIATED CONTENT
S Supporting Information *
A student handout and detailed information on the experimental and mass spectrometer conditions. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank all students in ENLS 5088: Mass Spectrometry at Trent University from 2009−2012 who participated in this laboratory session. The authors also thank the Water Quality Centre at Trent University for use of their mass spectrometers.
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REFERENCES
(1) Duncan, M. W. Good Mass Spectrometry and its Place in Good Science. J. Mass Spectrom. 2012, 47, 795−809. (2) Bergen, H. R., III; Benson, L. M.; Naylor, S. Determination of Aspartame and Caffeine in Carbonated Beverages Utilizing Electrospray Ionization-Mass Spectrometry. J. Chem. Educ. 2000, 77, 1325− 1326. (3) Stock, N. L.; Martin, J. W.; Yun, Y.; Mabury, S. A. An Undergraduate Experiment for the Measurement of Perfluorinated Surfactants in Fish Liver by Liquid Chromatography-Tandem Mass Spectrometry. J. Chem. Educ. 2007, 84, 310−311. (4) Fenk, C. J.; Hickman, N. M.; Fincke, M. A.; Motry, D. H.; Lavine, B. Identification and Quantitative Analysis of Acetaminophen, Acetylsalicylic Acid, and Caffeine in Commercial Analgesic Tablets by LC-MS. J. Chem. Educ. 2010, 87, 838−841. (5) Rosado, D. A., Jr.; Masterson, T. S.; Masterson, D. S. Using the Mini-Session Course Format To Train Students in the Practical Aspects of Modern Mass Spectrometry. J. Chem. Educ. 2011, 88, 178− 183. (6) March, R. E.; Todd, J. F. J. An Appreciation and Historical Survey of Mass Spectrometry. In Practical Aspects of Trapped Ion Mass D
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