Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX-XXX
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Introducing Graduate Students to High-Resolution Mass Spectrometry (HRMS) Using a Hands-On Approach Naomi L. Stock* Water Quality Centre, Trent University, Peterborough, Ontario K9L 0G2, Canada S Supporting Information *
ABSTRACT: High-resolution mass spectrometry (HRMS) features both high resolution and high mass accuracy and is a powerful tool for the analysis and quantitation of compounds, determination of elemental compositions, and identification of unknowns. A hands-on laboratory experiment for upper-level undergraduate and graduate students to investigate HRMS is described. This experiment was developed in response to students’ requests and provides a hands-on overview of the range of capabilities of an HRMS instrument. Students were encouraged to explore and investigate all the various modes in which the instrument can be operated. This experiment prompted students, even those with little experience in mass spectrometry, to think about how HRMS could be best incorporated into their research projects. KEYWORDS: Upper-Division Undergraduate, Graduate Education/Research, Laboratory Instruction, Hands-On Learning/Manipulatives, Mass Spectrometry, Isotopes, Analytical Chemistry
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INTRODUCTION High-resolution mass spectrometry (HRMS) uses mass spectrometers capable of high resolution, as well as high mass accuracy measurements. These instruments can be used to distinguish between compounds with the same nominal mass, determine elemental compositions, and identify unknowns. Historically, HRMS was limited to double-focusing magnetic sector or Fourier transform ion−cyclotron resonance (FTICR) instruments1 but, recently, has become more accessible and affordable for many laboratories,2 with benchtop time-of-flight (TOF) and Orbitrap instruments. Many of these instruments are now quite user-friendly, presenting opportunities for even novice users to employ highly advanced, state-of-the-art instrumentation.2 Correspondingly, over the past few years, there has been a sharp increase in the number of fourth-year undergraduate and graduate students, often with little experience in mass spectrometry, who request to use the HRMS instruments in the Water Quality Centre at Trent University, for their research projects. Inclusion of hands-on HRMS at the upper-undergraduate or graduate level is relatively new; two articles have recently reported the successful addition of the technique in laboratory courses. Walsh et al.3 designed an experiment where students isolated parthenolide from the plant feverfew (Tanacetum parthenium), and then used various spectroscopy techniques, including HRMS, to elucidate the structure of parthenolide. Students were provided with an HRMS spectrum from which they could obtain the accurate mass of parthenolide and its corresponding molecular formula. Alty and LaRiviere4 created an experiment in an upper-undergraduate biochemistry laboratory course, where students used HRMS to determine © XXXX American Chemical Society and Division of Chemical Education, Inc.
the accurate mass of peptides digested from egg whites. These peptide masses could then be matched to peptide sequences and individual proteins using instrumental software and online databases. The hands-on experiment described here was developed in response to students’ requests to use HRMS, and was incorporated into a graduate course in mass spectrometry for the past two years. The experiment was developed on the basis of the unique experimental design recently reported by Stock and March,5 in a hands-on laboratory experiment where students investigated and compared the performance of a triple quadrupole mass spectrometer and a linear quadrupole ion trap mass spectrometer. In both experiments, there is no chromatography, only mass spectrometry. In addition, rather than examining how a mass spectrometer functions for a specific analysis, the experiments provide students with an overview of the range of capabilities of a specific instrument. In the hands-on experiment described here, students were encouraged to explore HRMS and to investigate the various modes in which an HR−MS instrument can be operated. The experiment prompted students, even those with little experience in mass spectrometry, to think about how HRMS could be best incorporated into their research projects. Received: July 28, 2017 Revised: September 29, 2017
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DOI: 10.1021/acs.jchemed.7b00569 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Figure 1. Atenolol, C14H22N2O3 (left), and metoprolol, C15H25NO3 (right).
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EXPERIMENTAL DETAILS Atenolol and metoprolol (Figure 1) were purchased from Sigma-Aldrich (Oakville, ON, Canada), and selected for this experiment as the masses of the two analytes differ by only 1 amu; metoprolol and the M + 1 isotope of atenolol have the same nominal mass. Additionally, the fragmentation of these compounds is well-understood,6,7 and these compounds have previously been used in hands-on experiments for undergraduate students.8 Prior to the hands-on laboratory session, students completed a prelaboratory assignment that had them calculate the nominal and accurate monoisotopic masses for the protonated molecules [M + H]+ and deprotonated molecules [M − H]− for both atenolol and metoprolol and their M + 1 isotopes, [M + 1 + H]+ and [M + 1 − H]−. Only the contributions from 13C were considered for the M + 1 isotopes, 13C12C13N2O3 and 13 12 C C14NO3 for atenolol and metoprolol, respectively. These calculated masses are found in Table 1. Students were also
HAZARDS There are no significant safety issues associated with this laboratory activity. Students should follow instructions when using the high-voltage electrospray ionization 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 (CAS 67-56-1), atenolol (CAS 2912268-7), and metoprolol (CAS 37350-58-6) are available on safety data sheets (SDS). All chemicals used in this experiment should be collected in labeled waste containers and disposed appropriately.
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RESULTS AND DISCUSSION Students obtained mass scans of the atenolol and metoprolol solution in both positive and negative ion modes using an instrument resolution of 17,500 (Figure 2). In positive ion mode (Figure 2A), the mass scan consists of three major peaks: the protonated molecule [M + H]+ of atenolol at m/z 267.1698, a peak corresponding to the M + 1 isotope [M + 1 + H]+ of atenolol and the protonated molecule [M + H]+ of metoprolol at m/z 268.1910, and the M + 1 isotope of metoprolol [M + 1 + H]+ at m/z 269.1940. The mass scan obtained in negative ion mode (Figure 2B) consists of the deprotonated molecule [M − H]− of atenolol at m/z 265.1482 and the M + 1 isotope [M + 1 − H]− at m/z 266.1512. Metoprolol was not observed in negative ion mode. The ion signal intensity of the base peak in the spectrum obtained using positive ion mode (Figure 2A) is ∼1.7 × 108, and much greater than the ion signal intensity of the base peak in the spectrum obtained using negative ion mode (Figure 2B), which is ∼9.7 × 106. Most students hypothesized that positive ion mode would be more sensitive for the analysis of these compounds due to multiple sites on the molecules where protonation could occur. To investigate the importance of HRMS, students obtained a second mass scan in positive ion mode using an instrument resolution of 140,000 and compared this to the initial mass scan obtained in positive ion mode using an instrument resolution of 17,500 (Figure 3). Using an instrument resolution of 17,500 (Figure 3A) was not sufficient to separate the peaks of the M + 1 isotope of atenolol and the protonated molecule [M + H]+ of metoprolol; only one peak is observed at m/z 268.1910. Using a resolution of 140,000 (Figure 3B), the two peaks could be resolved; the M + 1 isotope [M + 1 + H]+ of atenolol is observed at m/z 268.1704, and the protonated molecule [M + H]+ of metoprolol is observed at m/z 268.1902. On the basis of the prelaboratory assignment, most students were expecting to observe four peaks in all their mass scans and were surprised to observe only three peaks in the scan obtained using an instrument resolution of 17,500. Obtaining mass scans at two different resolutions was very effective at illustrating to the students the importance of resolution and the role HRMS can play in resolving overlapping peaks. As mass accuracy is an important component of HRMS, students were asked to calculate the mass accuracy for either atenolol or metoprolol using the equation
Table 1. Nominal and Monoisotopic Masses for the Protonated and Deprotonated Molecules of Atenolol, Metoprolol, and Their M + 1 Isotopes Ion
a
[M [M [M [M
+ H]+ + 1 + H]+a − H]− + 1 − H]−a
[M [M [M [M
+ H]+ + 1 + H]+a − H]− + 1 − H]−a
Atenolol, m/z Nominal Masses 267 268 265 266 Monoisotopic Masses 267.1703 268.1737 265.1558 266.1591
Only the contributions from isotopes.
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Metoprolol, m/z 268 269 266 267 268.1907 269.1941 266.1762 267.1795
13
C were considered for the M + 1
asked to hypothesize whether electrospray ionization in positive or negative ion mode would be most appropriate for the analysis of these analytes, and to examine the literature or an online mass spectral database to investigate the fragmentation of atenolol and metoprolol. The hands-on experiment was designed for a 2 h laboratory period, for a group of two, three, or four students. Students worked on a Thermo QExactive Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with both a quadrupole and a higher-energy collisional dissociation (HCD) cell. Using a syringe infusion pump (Harvard Apparatus, Holliston, MA), and a mixed solution of atenolol and metoprolol, 500 ppb each in methanol (HPLC grade; VWR, Mississauga ON, Canada) was used for all experiments. Students were required to answer all questions on the student handout and submit it at the completion of the laboratory session. B
DOI: 10.1021/acs.jchemed.7b00569 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Figure 2. Mass scans of a 500 ppb solution of atenolol and metoprolol in methanol. Spectrum A was acquired in positive ion mode and consists of the protonated molecule [M + H]+ of atenolol, a peak corresponding to the M + 1 isotope [M + 1 + H]+ of atenolol and the protonated molecule [M + H]+ of metoprolol, and the M + 1 isotope of metoprolol [M + 1 + H]+. Spectrum B was acquired in negative ion mode and consists of the deprotonated molecule [M − H]− and the M + 1 isotope [M + 1 − H]− of atenolol; metoprolol was not observed. The small peaks observed in spectrum B at m/z 267.1433 and 267.2328 are impurities of unknown origin.
Figure 3. Mass scans of a 500 ppb solution of atenolol and metoprolol in methanol in positive ion mode. Spectrum A was acquired using an instrument resolution of 17,500. Spectrum B was acquired using an instrument resolution of 140,000. Using an instrument resolution of 140,000 the peaks corresponding to the M + 1 isotope [M + 1 + H]+ of atenolol and the protonated molecule [M + H]+ of metoprolol are resolved.
additional product ions at nominal m/z 121, 159, 191, and 226 (Figure 4B). Product ions observed by the students are consistent with previously published data6,7 and in agreement with an online mass spectral database.9 It should be noted that the intensity of individual product ions is dependent on the collision energy selected by students. A thorough investigation of the fragmentation patterns of atenolol and metoprolol was beyond the expectations of this laboratory session; however, it was discussed that fragmentation is crucial when determining the structure of a compound, even if the molecular formula of the compound is known. For those who may be interested, the probable identity and possible structures of observed products ions is included in the Supporting Information. For their second fragmentation scan, students obtained an all ion fragmentation (AIF) scan (Figure 5). This mode is similar to a product ion scan; however, all precursor ions are fragmented without preselection in the quadrupole. The mass spectrum obtained using AIF scan mode consists of both precursor ions, at nominal masses m/z 267 and 268, in addition to the product ions observed in the MS/MS scans (Figure 4). Most students described the AIF mass spectrum as a “combination of the product ion scans obtained for atenolol and metoprolol” and noted that more collision energy was
(observed mass − theoretical mass) × 106 theoretical mass
For the mass scan obtained using an instrument resolution of 140,000 (Figure 3B), and using the theoretical masses in Table 1, the mass accuracies of atenolol and metoprolol are −1.12 and −1.86 ppm, respectively. All students obtained mass accuracy
