MS Theory and Operation via

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

pubs.acs.org/jchemeduc

Teaching Undergraduates LC−MS/MS Theory and Operation via Multiple Reaction Monitoring (MRM) Method Development Thomas A. Betts and Julie A. Palkendo* Department of Physical Sciences, Kutztown University, Kutztown, Pennsylvania 19530, United States S Supporting Information *

ABSTRACT: This laboratory experiment employs an innovative approach to introduce undergraduates to liquid chromatography−tandem mass spectrometry (LC−MS/MS) by developing a method based on multiple reaction monitoring (MRM). During this qualitative investigation, students operated a triple quadrupole mass spectrometer in the MS scan and product ion scan modes to determine precursor and product ions, respectively, for a vanillin, ethyl vanillin, and coumarin standard mixture. After identifying precursor → product ion transitions, students conducted a 2 min liquid chromatography run in MRM mode. Students were astonished by the speed, selectivity, and sensitivity of the LC−MS/MS method compared to that of a liquid chromatograph with ultraviolet detection. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Chromatography, Mass Spectrometry, Consumer Chemistry

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eter has been briefly presented to students in the popular analytical textbook Quantitative Chemical Analysis in as early as the sixth edition.5 This small number of experiments tailored to the academic setting pales in comparison to its use in research, industrial, and clinical settings. A SciFinder search on the topics “multiple reaction monitoring mode” or “selected reaction monitoring mode” returned nearly 1,500 articles since 2012 with the highest number of publications occurring in 2014. As any novel technique becomes refined, routine, and highly automated, it is easy for operators and analysts to lose sight of the theoretical underpinnings of the technique. The unique aspect of this laboratory experiment is to provide an opportunity for undergraduates to build an LC−MS/MS MRM method. Interestingly, the stepwise process of building an MRM method inherently fosters learning aligned with various modes of MS/MS instrument operation as well as foundational knowledge involved in tandem mass spectrometry. Specifically, students are expected to identify precursor ions in MS scan mode and product ions in product ion scan mode. Students also gain a deeper understanding of the function of each mass analyzer and the sequence of events in their MRM method. To provide a benchmark for comparison, this experiment is paired with a set of experiments that employ high performance liquid chromatography with ultraviolet detection (HPLC-UV) for the analysis of the same analytes.

iquid chromatography−tandem mass spectrometry (LC− MS/MS) is commonly used in research and industrial settings for applications that span pharmaceutical, clinical, food and beverage, environmental, forensic, and toxicology fields; however, LC−MS/MS instrumentation is still not routinely found in academic curricula, especially at primarily undergraduate institutions. This is due undoubtedly to the large initial investment in the instrument itself, lack of laboratory technician(s) and/or faculty expertise, need for laboratory space and renovations, and costs of preventative maintenance and service contracts. As undergraduate institutions continue to invest in modern instrumentation, strategies must be developed to incorporate these techniques into the curriculum to foster both theoretical and practical understanding. While there are a number of LC−MS and electrospray ionization mass spectrometry (ESI-MS) experiments published in this Journal, very few examples introduce students specifically to LC−MS/MS with the use of the multiple reaction monitoring (MRM). Stock et al. was the first group to do so in 2007 with the analysis of perfluorinated surfactants in fish liver.1 Most recently, Parker et al. introduced quantitation with MRM for drugs of abuse on paper currency.2 Stock and March published a hands-on experiment for upper-level undergraduate and graduate students that focuses on operating mass spectrometers in various modes, including MRM, on a standard caffeine sample.3 Homem et al. have also introduced undergraduates to a direct injection LC−MS/MS experiment using an ion trap, in a mode similar to MRM, for the analysis of amoxicillin in river waters.4 MRM, also called selected reaction monitoring (SRM), using a triple quadrupole mass spectrom© XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: November 29, 2017 Revised: April 13, 2018

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DOI: 10.1021/acs.jchemed.7b00914 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

injections. Students performed serial dilutions by factors of 10 or 100 to estimate the limit of detection.

Through this comparison, students are able to discover for themselves the benefits of tandem mass spectrometry in terms of selectivity, speed, and limit of detection.

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Instrumental Parameters

The electrospray ionization source of the Shimadzu LCMS8040 triple quadrupole mass spectrometer was operated in positive ion mode using nitrogen nebulizing and drying gas flows of 2 and 15 L/min, respectively. The desolvation line and heat block temperatures were set to 250 and 400 °C, respectively. An injection volume of 2 μL and isocratic mobile phase composition of 90% methanol with 0.1% FA/10% water with 0.1% FA were used for all acquisitions. Data were acquired for 0.5 min with a flow rate of 0.20 mL/min when no column was installed during MS scan and product ion scans. For MRM mode, a Phenomenex Kinetex XB-C18 column (100 mm × 4.60 mm; 2.6 μm particle size) was installed; the column oven temperature was set at 30 °C, and the flow rate was increased to 0.60 mL/min. The acquisition time was extended to 2.0 min. The mass spectrometer parameters varied depending on the acquisition mode. In the MS scan, a mass range of m/z 50−200 was scanned over a 50 ms time period. For product ion scans, a mass range of m/z 30−140 was scanned, again over a 50 ms time period; collision cell potentials (which are directly related to collision energies) were set at −10, −15, and −27 V for vanillin, ethyl vanillin, and coumarin, respectively. The same collision cell potentials were used in the MRM mode with 100 ms dwell times for each precursor → product ion transition. All students selected precursor ions at m/z 153, 167, and 147 for vanillin, ethyl vanillin, and coumarin, respectively; however, product ion selection varied between student pairs.

PEDAGOGICAL APPROACH The experiment described herein is an extension of a laboratory exercise using HPLC-UV of vanilla flavoring, which was derived from articles published in6,7 and outside8,9 of this Journal and was performed by student pairs in an advanced analytical course using a round-robin style. Enrollments in this course typically range between 14 and 16 undergraduates, and students meet weekly for two laboratory periods each of 3 h duration. During the first lab period, students determined an appropriate UV detection wavelength and investigated the effect of mobile phase composition on chromatographic resolution. In the second lab period, students used their HPLC method to quantify vanillin, ethyl vanillin, and coumarin in vanilla extract samples. It should be noted that vanilla flavorings are interesting to students because a majority of them consume these products on a daily basis. In addition, vanilla bean prices skyrocketed by 890% between 2008 and 2016 due primarily to consumers demanding “all natural” products from food manufacturers in place of synthetic or artificial flavorings.10 Most recently, the MRM method development was incorporated into this set of HPLC vanilla experiments; a third lab period was devoted to this innovation. By using familiar samples and chromatography, students could focus on identifying precursor → product ion pairs and making comparisons between the UV and MS/MS detectors. Chromatography and gas chromatography−mass spectrometry (GC−MS) concepts were previously covered in the first semester analytical course, but this experiment was the students’ first encounter with LC−MS/MS. Depending on the timing of the lab rotation schedule, tandem mass spectrometry concepts may not have been introduced in lecture. Therefore, before embarking on MRM method development, students were expected to read an LC−MS/MS and MRM method development theory handout (see Supporting Information). The theory handout was specifically designed to build upon their recent HPLC-UV experience, provide an overview of LC−MS/MS operational modes, describe the various functions of the quadrupoles, and clearly outline the conceptual steps of MRM method development.

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HAZARDS There are no significant hazards in this experiment. Methanol is a flammable solvent and may be hazardous if inhaled, ingested, or adsorbed through the skin. Students should wear appropriate personal protective equipment (PPE).

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RESULTS AND DISCUSSION Students first obtained a mass spectrum of the 3-component mixture to identify the precursor ions of the vanillin, ethyl vanillin, and coumarin (see Figure 1A) using MS scan mode. The dominant [M + H]+ ions at m/z 147, 153, and 167 were easily detected as well as [M + 32]+ ions at m/z 179, 188, and 199 likely due to methanol adducts resulting from the 90% methanol mobile phase. While the MS scan may seem rudimentary, this was the first mass spectrum that our students ever observed using electrospray ionization. All of their previous mass spectral experiences were based upon electron ionization using GC−MS. Product ion scans for each precursor ion (see Figure 1B−D) were performed next to identify product ions. A great deal of student discussion surrounded how to select the “best” product ion. Some students decided to use ions with the greatest abundance while others considered patterns of dissociation and how different structures of protonated molecules resulted in product ions of both similar and different mass/charge ratios. Students also debated whether or not it would be acceptable to select the same mass/charge ratio for two different compounds (i.e., m/z 93 for both vanillin and ethyl vanillin). This allowed instructors to emphasize the selectivity of MRM mode. Figure 2 shows the resulting mass chromatograms from a student’s MRM method with all compounds eluting in well under 2 min. As students obtained this data, the instructor

EXPERIMENTAL OVERVIEW

Reagents

Optima LC−MS grade methanol, 0.1% formic acid (FA) in water, and concentrated formic acid ampules were purchased from Fisher Scientific. HPLC grade methanol was also purchased from Fisher Scientific, and deionized water (18 MΩ cm) was obtained from a Barnstead EASYpure UF water purification system. Vanillin and ethyl vanillin were purchased from Acros Organics, and coumarin was acquired from Aldrich. Sample Preparation

The instructor prepared separate stock solutions of 10 mg/mL vanillin, ethyl vanillin, and coumarin in HPLC grade methanol. Students prepared a mixture by micropipetting 20 μL of each stock solution into an autosampler vial and diluting with 1000 μL of 50% methanol/50% deionized water (v/v) solvent. (This standard mixture was also used for the HPLC-UV experiment in previous lab periods.) A 100-fold dilution of the mixture was prepared and used for all MRM method development B




reviewed with students and questioned students about the instrument’s sequence of events within the MRM method. Students easily recalled setting the first quadrupole (Q1) to allow only the precursor ion into the collision cell (Q2) and then setting the third quadrupole (Q3) to allow only their selected product ion to be detected for each analyte event as depicted at the top of Figure 2. Where students needed more guidance was in linking each precursor → product ion transition to their seemingly overlapped mass chromatograms. The instructor helped students make this important connection by pointing out the sequential nature of detection for each product ion. The vanillin event occurred in the first 100 ms, followed by the ethyl vanillin event in the second 100 ms, and, finally, the coumarin event in the last 100 ms. This loop was repeated roughly 400 times in the 2 min chromatographic run. By simply highlighting that product ion detection is sequential and not simultaneous, students recognized each analyte event as an independent segment enabling the generation of independent mass chromatograms for each analyte. The tools in the data analysis software provided students a powerful visualization of this concept by turning off and on the mass chromatogram for each precursor → product ion transition. When students were asked to compare the results of their MRM method to the HPLC-UV method that they recently completed for the same analytes, two differences were evident: selectivity and speed. Depending on the column used in the HPLC-UV experiments, students were able to baseline resolve all three components in about 5 min. In the MRM method, retention times were cut dramatically to under 2 min. In their reports, students related the increased speed of analysis to the fact that each analyte did not need chromatographic separation due to the selective, sequential nature of the MS/MS detector. In comparison, the UV detector required excellent but timeconsuming chromatography because it has no way of discriminating between analytes that elute simultaneously. Instructors emphasized the advantage of obtaining partially

Figure 1. MS scan of the vanillin, ethyl vanillin, and coumarin mixture (A) and product ion mass spectra of protonated vanillin (B), ethyl vanillin (C), and coumarin (D).

Figure 2. Student-generated mass chromatogram of vanillin, ethyl vanillin, and coumarin mixture with a diagram to show the sequence of events for detection of each precursor → product ion transition. Note that each black dot represents a collected data point from the MRM transition for vanillin, each pink dot represents a data point collected from the MRM transition for ethyl vanillin, and each blue dot represents a data point collected from the MRM transition for coumarin. Dots are not shown to the true time scale and only serve as a model to emphasize the event sequence. C




covering a wide range of mass spectrometry concepts. The MS scan mode introduces the function of a quadrupole as a mass filter and exposes students to a simple ESI mass spectrum. The product ion scan introduces collision-induced dissociation and the operation of multiple mass analyzers. Finally, the MRM method showcases speed, selectivity, and lower limits of detection, and with this pedagogical approach, allows direct comparisons to be made to HPLC with UV detection.

resolved components in LC−MS/MS screening methods where hundreds of pesticides, for example, are eluted very quickly with overlap, but again, the power of the MS/MS detector allows for selective and sensitive detection.11 It should be noted that the experimental procedure could be modified for a flow injection analysis without a column where each compound would elute simultaneously within 0.5 min; however, use of the column to partially resolve the components seemed to foster a better understanding of the capabilities of tandem mass spectrometry. In addition, it is always good standard practice to perform chromatographic separations with acceptable resolution. The two-stage requirement of precursor and product ion selection by Q1 and Q3 in the MRM method prevents many ions, which would otherwise contribute to background noise, from reaching the detector. This reduction in noise enhances the signal-to-noise ratio, resulting in enhanced sensitivity. In order for students to compare the sensitivity of their MRM method to that of HPLC-UV, they estimated the instrument detection limits. Students prepared serial, 10-fold dilutions of their approximate 2 μg/mL working solution and analyzed each solution until the signal was 3 times the magnitude of the noise. Most students reported 2 ng/mL as the approximate detection limit for this mixture, and a few groups were able to break into the 0.2 ng/mL range, which was highly dependent on (1) the selection of precursor → product ion transitions and (2) the quality of students’ micropipetting skills. Nonetheless, students were able to demonstrate the significant improvement in detection limits of their MRM method compared to HPLC-UV where instrument detection limits were in the 100 ng/mL range. There are numerous extensions of this experiment that students may pursue with more instructional time. Most obviously, students may be asked to more formally determine the limits of detection and quantitation. Students may select different product ion mass/charge ratios and optimize collision cell potentials manually or by utilizing a method optimization wizard within the data acquisition software. Additionally, LC conditions and ion source parameters may be adjusted. If decoupling this experiment from the HPLC analysis, students may be asked to apply the optimized MRM method to quantify vanillin, ethyl vanillin, and coumarin in vanilla extract products with or without an internal standard. Assessments of precision, linearity, and accuracy would also be appropriate add-ons for advanced analytical undergraduate students. Students’ conceptual understanding was evaluated through on-the-fly conversations during the experiment, and in summary reports that included comparisons of their MRM and HPLC-UV methods. Students discovered that MRM methods offer increased speed and selectivity due to the ability of MRM to independently detect overlapping chromatographic peaks. They also discovered limits of detection that were much lower for the MRM method. While operating the LC−MS/MS, students often asked about the price tag of this instrument compared to HPLC-UV, leading to the understanding that these benefits come at a cost. Perhaps the greatest evaluation of student understanding came later in the semester when students revisited this instrument to develop MRM methods for other analytes with minimal instruction.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00914. Student handouts on (1) LC−MS/MS and MRM theory, (2) MRM method development instructions using Shimadzu LCMS-8040, and (3) HPLC method development for vanilla flavoring; and instructor notes (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Julie A. Palkendo: 0000-0002-7321-2771 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the students in the spring 2017 semester of CHM340 for their participation and feedback. Additionally, we would especially like to acknowledge Dr. Jonathan Ho from Shimadzu Scientific Instruments, Inc., for his guidance and training on LabSolutions and MRM method development.

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REFERENCES

(1) Stock, N. L.; Martin, J. W.; Ye, 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 (2), 310−311. (2) Parker, P. D.; Beers, B.; Vergne, M. J. What is in Your Wallet? Quantitation of Drugs of Abuse on Paper Currency with a Rapid LCMS/MS Method. J. Chem. Educ. 2017, 94 (10), 1522−1526. (3) Stock, N. L.; March, R. E. Hands-On Electrospray IonizationMass Spectrometry for Upper-Level Undergraduate and Graduate Students. J. Chem. Educ. 2014, 91 (8), 1244−1247. (4) Homem, V.; Alves, A.; Santos, L. Development and Validation of a Fast Procedure to Analyze Amoxicillin in River Waters by DirectInjection LC-MS/MS. J. Chem. Educ. 2014, 91 (11), 1961−1965. (5) Harris, D. L. Mass Spectrometry. Quantitative Chemical Analysis, 6th ed.; W.H. Freeman: New York, 2003; Chapter 22. (6) Beckers, J. L. The Determination of Vanillin in a Vanilla Extract. J. Chem. Educ. 2005, 82 (4), 604−606. (7) Sparks, L.; Bleasdell, B. D. A Comparison of Separation Techniques: Analysis of Vanilla for Coumarin Contamination. J. Chem. Educ. 1986, 63 (7), 638−639. (8) DeJager, L. S.; Perfetti, G. A.; Diachenko, G. W. Determination of coumarin, vanillin, and ethyl vanillin in vanilla extract products: liquid chromatography mass spectrometry method development and validation studies. J. Chromatogr., A 2007, 1145, 83−88. (9) AOAC, Official Methods of Analysis, 18th ed.; AOAC International: Gaithersburg, MD, revised 2006.

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CONCLUSION Teaching the process of developing an MRM method on a triple quadrupole mass spectrometer is a great strategy for D




(10) Fraser, I. How Picky Consumers Have Made Vanilla Prices Soar. Newsweek March 28, 2017. http://www.newsweek.com/pickyconsumers-vanilla-prices-spike-1000-percent-575253 (accessed Apr 2018). (11) Wittrig, B.; Schreiber, A. Comprehensive Pesticide Residue Analysis by LC/MS/MS Using an Ultra Aqueous C18 Column. Restek Application Note 2011. http://www.restek.com/adv004 (accessed Apr 2018).

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