Reinforcing Mass Spectrometry Concepts through an Undergraduate

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

pubs.acs.org/jchemeduc

Reinforcing Mass Spectrometry Concepts through an Undergraduate Laboratory Exercise Utilizing a Direct Analysis in Real Time Enabled Mass Spectrometer Rachel C. Beck† and Mitzy A. Erdmann*,‡ †

Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States

‡

S Supporting Information *

ABSTRACT: Identification and quantification of an unknown illicit drug (cocaine) from both simulated drug paraphernalia and simulated urine can be achieved via direct analysis in real time (DART) triple quadrupole linear ion trap (QTRAP) mass spectrometry (MS). We describe a novel, hands-on laboratory activity which utilizes the recent DART technology interfaced with a tandem mass spectrometer for the purpose of integrating recent technology into a quantitative analysis course, reinforcing chemical concepts, improving critical thinking and written communication skills, and providing a context for real-life analyses. The hands-on laboratory activity requires students to use good laboratory practice, perform common laboratory calculations, and think critically to perform both qualitative and quantitative analyses and answer real-life forensic questions. Assessments include prelaboratory activity questions, observations during the activity, postlaboratory activity written report, and anonymous summative activity. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-on Learning/Manipulatives, Applications of Chemistry, Calibration, Forensic Chemistry, Instrumental Methods, Mass Spectrometry, Quantitative Analysis

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INTRODUCTION While linking theory with applications is crucial for their professional success,1 it has been reported that many students, both undergraduate and graduate students, struggle to grasp the theory of mass spectrometry and how it can be applied to realworld settings.2,3 One way to strengthen this link is to offer authentic learning experiences with analytical instrumentation. Both students and instructors have long agreed that this approach is the most effective way to learn.4 To this end, a hands-on laboratory activity was developed that allows students to analyze samples on a direct analysis in real time (DART) enabled triple quadrupole linear ion trap (QTRAP) mass spectrometer (MS). The DART enabled QTRAP MS was selected for this laboratory exercise due to it being a newer technology, its availability, its documented and growing application in forensic casework5,6 and clinical research,7 and its ability to produce rapid results. The use of existing instrumentation that required little sample preparation kept © XXXX American Chemical Society and Division of Chemical Education, Inc.

the costs of the activity low. Additionally, course layout restricted the allotted time for this activity to one laboratory meeting (3 h). By coupling a real-life context (forensic application, drug identification) with the rapid analysis and data generation of the DART QTRAP MS (a recent technology), students were able to work through two experiments within the time confines. Increasing the instrumental hands-on exposure time and working with different data collection modes provided students with experience and data that both reinforced instrument functionality and its application. Student activities involving the DART technology exist, but are limited8,9 due to it being a relatively newer technology. This makes this experiment an exciting addition to the literature and Received: June 21, 2017 Revised: February 2, 2018

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DOI: 10.1021/acs.jchemed.7b00437 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Figure 1. Schematic of DART QTRAP MS. The figure walks students through key components of the DART QTRAP MS instrument.

benzoylecognine (MW 289.33 g/mol; CAS 519-09-5), cocaethylene (MW 317.38 g/mol; CAS 529-38-4), ecognine methyl ester (MW 199.25 g/mol; CAS 7143-09-1), and cocaethylened3 (MW 320.40 g/mol; CAS 136765-30-5) and were obtained from Cerilliant Corporation (Round Rock, TX). The instructor prepared three separate working stocks (calibration stock, control stock, and internal standard stock) at 10 μg/mL from the original 1 mg/mL reference materials. The control stock contained all analytes except the cocaethylene-d3. The calibration stock only contained cocaine, and the internal standard stock only contained cocaethylene-d3. These working stock solutions were prepared in reagent-grade methanol (CAS 67-56-1), secured from Fisher Scientific (Pittsburgh, PA), and were stored at 0 °C prior to the activity. Additional standard laboratory materials needed for this exercise include 2 mL autosampler vials and autosampler caps (substituted for 2 mL microcentrifuge tubes), transfer pipettes, rubber bulbs, pipettes (adjustable 10 μL, 100 μL, and 1.0 mL pipettes), disposable pipette tips, glass microscope coverslips, and disposable test tubes (13 × 100 mm2). The uses for these materials and the procedure used are included in the Supporting Information (SI). To prepare the simulated urine and drug paraphernalia the following items were necessary: yellow and orange food coloring, disposable syringe and needle, absorption cylindrical cell, aluminum foil, table salt, transfer pipettes, Parafilm, forceps, and cigarette lighter. All items were prepared by the instructor. A volume of 2 mL of simulated urine was prepared for each student group utilizing tap water and a combination of yellow and orange food coloring. (Note: the amount of food coloring varied to give different shades to the three specimens; actual amounts were not recorded.) The three unknown simulated urine specimens (A, B, and C) were prepared at concentrations of 3,000, 750, and 1,500 ng/mL, respectively, using the 10 μg/mL working control stock described previously (which contained cocaine and additional analytes). Additionally, a 50 mL batch of blank, simulated urine (absent of analytes and internal standard) was prepared for preparation of the student group calibration curves. Three types of simulated drug paraphernalia (homemade pipe, syringe, and foil packet) were prepared and are shown in Figure 2. The homemade pipe was constructed from a glass transfer pipette, Parafilm, and absorption cylindrical cell. The bottom of the absorption cell was exposed to an open flame to give a desired usage

highly relevant for the students that will soon be embarking on professional careers.3,10 Similar to an activity described by Stock and March,11 this exercise does not use chromatography, but rather, focuses on the different data collection modes of quadrupole mass spectrometers. Unlike Stock and March’s activity,11 this activity implements the DART ionization (a more recent technology) as part of data generation and asks students to solve real-world, forensically relevant questions. In addition to exposing students to research-grade instrumentation, this activity was designed to require students to think critically, further develop written communication skills, reinforce both chemical concepts and good laboratory practice, and provide context for how these concepts can be applied through assessment preactivity, during activity, and postactivity. To provide an authentic learning experience for students, they were instructed to design an experimental procedure to analyze samples that would be common in a forensic laboratory. This real-life context allowed students to observe the necessity for quality assurance and to experience what that looks like in a professional laboratory setting. Students were able to successfully complete the activity in one laboratory period and offered positive feedback about their learning gains.

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LEARNING GOALS In addition to reinforcing common laboratory practices, learning goals for the activity focused on improving student understanding of mass spectrometry. Progress toward these goals was measured using pre- and postactivity surveys and a postactivity written laboratory report. In addition to introducing students to the forensic laboratory, this activity strengthens their ability to 1. Recognize how mass spectrometry is utilized in a forensic laboratory setting. 2. Demonstrate the concept of mass spectrometry and its theory. 3. Prepare, identify, and quantify drugs in various sample types. 4. Apply critical thinking to real-world scenarios.

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MATERIALS

Chemicals

Certified drug standard reference materials analyzed in this activity included cocaine (MW 303.35 g/mol; CAS 50-36-2), B




Figure 2. Simulated drug paraphernalia for the DART QTRAP MS laboratory exercise included a homemade pipe, a syringe, and a piece of foil coated with a white residue and possessing burn markings.

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appearance. (Note the exposure to the open flame was performed in the hood using a hand-held cigarette lighter and a pair of forceps.) The transfer pipette and transfer pipette tip were secured inside the top of the absorption cylindrical cell using Parafilm. The syringe and needle were disposable. The needle was kept covered until the laboratory and was disposed of in the sharps container. For the third piece of simulated drug paraphernalia, aluminum foil was cut into small squares (∼2 in. × 2 in.) and was exposed to an open flame. (The foil was held with forceps.) Once usage appearance was established, table salt was placed on the foil, and the foil was folded. The foil was then unfolded for dosing. With all three types of paraphernalia constructed, each item was exposed to 10 μL of the 10 μg/mL cocaine calibration solution and allowed to dry inside the hood.

IMPLEMENTING THE ACTIVITY

Participant Profile

The activity was completed by third and fourth year undergraduate chemistry majors and first year chemistry and forensic science graduate students enrolled in a full-term quantitative analysis laboratory course that met for 3 h once a week. The laboratory course has a corresponding lecture, where students learn about many types of instrumentation including mass spectrometry. DART QTRAP MS technology is not specifically taught in this course, however, and students’ first interaction with this instrumentation was provided in the preactivity materials. The activity was completed in one period. All students were able to sufficiently prepare samples and collect data in this time frame.

Instrumentation

Preactivity Preparation and Instruction

A DART QTRAP MS was utilized (shown in Figure 1). The IonSense open air DART SVP (Saugus, MA) ionization source produced a protonated molecule, [M + H]+, in positive polarity.4 The AB Sciex 4000 QTRAP MS (Framingham, MA) utilized three quadrupoles in tandem and was capable of screening and confirming analytes through direct monitoring of protonated molecules and their dissociation12 for m/z up to 2800 Da. In this activity, the DART source was angled at 45° to minimize background noise. 13 Instrument configuration (setup) was covered in the preactivity instruction and again during the activity prior to sample analysis. This process consisted of switching the ionization source from the electrospray source to the DART interface and source (assembled, one unit). Student groups performed the actual configuration under instructor supervision at the beginning and ending of the activity. Sample analyses were performed by pipetting 2 μL of liquid sample onto glass microscope coverslips and inserting them into the DART gas stream. The specific parameters used in the methods for both experiments and the instrument configuration directions are included in the Supporting Information.

The week prior to the scheduled laboratory activity, students were delivered the learning goals, background material, experimental protocol, and a series of seven prelaboratory questions to answer based on the techniques to be employed. Students were given 1 week to prepare for this activity, which is standard for this course. This amount of time was sufficient to allow for adequate completion of prelaboratory notes, but could be tailored to any individual course as needed. The experimental protocol included details about the use of the instrument, what data to collect, and what supplies would be necessary while still allowing students to design the sample preparation protocol themselves. Class members were instructed to answer the seven prelaboratory questions designed to familiarize the students with the nomenclature and theory of the instrument before coming to the next class to complete the activity (Learning Goal 2). The seven preactivity questions are listed below. All preactivity materials are available in the Supporting Information. 1. Define mass spectrometry and what type of information does it provide (2−3 sentences). 2. Define selected ion monitoring (SIM) and scan mode. C




analyte identification based on this one analysis. Therefore, the students were then expected to analyze the sample again using the EPI method that corresponded to the appropriate protonated molecule (i.e., method “EPI304”). The EPI method provided the fragmentation pattern for the protonated molecule, m/z 304, which was then identified through library matching to a user created library. The user created library was generated by the instructor from spectra collected from certified reference materials for 17 common drugs of abuse. All materials mentioned are available in Supporting Information. In experiment 2, student groups prepared a calibration curve (minimum of 5 calibrator levels), 4 controls (3 positive and 1 negative), a matrix blank (urine without internal standard or analyte), and an unknown (A, B, or C). Quantification was achieved through internal standard correction using cocaethylene-d3 at a concentration of 400 ng/mL. Minimum acceptance criteria for an acceptable calibration curve required 5 calibrator levels within 20% of target concentration, 2 positive controls within 20% of target concentration, a negative control (internal standard only), a matrix blank (simulated urine without internal standard or analyte), and a coefficient of determination of 0.99. For all samples, students were asked to determine the correct volume of appropriate stock (concentrations given previously) to add to 0.5 mL of simulated urine using the dilution formula. (Concentration and volume are included in a table in the Supporting Information.) The internal standard was added to all samples except the matrix blank. The calibrator concentrations ranged from 300 to 4,000 ng/mL. The 3 positive controls were prepared at 400, 1,000, and 2,000 ng/mL. The matrix blank was included to ensure the simulated urine for the calibration curve was not contaminated with the target analyte. The calibration samples, control samples, unknown sample, and matrix blank were then analyzed by the DART QTRAP MS by both multiple reaction monitoring (MRM) and Q1 scanning methods (see Supporting Information for actual method parameters). Desired method names were “MRM304” and “304 scan”. The MRM method fragmented both m/z 304 (cocaine) and m/z 321 (cocaethylene-d3). The MRM transitions monitored for cocaine were m/z 304 → 182 and 304 → 150; MRM transitions monitored for cocaethylene-d3 were m/z 321 → 199. Quantification was achieved for cocaine in the simulated urine sample through the prepared calibrators: plotting the inverse of the calibrator concentrations (1/x weighting) versus the analyte/internal standard peak area ratio (peak area of m/z 304 → 182 divided by peak area of m/z 321 →199). The equation of the formed calibration curve was then used to solve for the concentration of the unknown. The Q1 scan was completed for the unknown, simulated urine sample and one of the controls in order to identify other potential analytes (cocaethylene, benzoylecgonine, and ecognine methyl ester) that could corroborate the cause of death conclusion (cocaine induced excited delirium). This analysis used the Q1 scan method, “304 scan”. “304 scan” differed from the Q1 scan method (Q1scan) used in experiment 1 by monitoring mass range intervals, focusing on the additional analytes (cocaine m/z 304, benzoylecgonine m/z 290, ecgonine methyl ester m/z 200, and cocaethylene m/z 318). The mass range intervals monitored were (m/z 190−210, m/z 289−290, m/z 300−330). The purpose for monitoring these mass range intervals rather than a continuous range was to aid student interpretations by enhancing the signal for the additional analytes while minimizing the potential for

3. Review the AB Sciex QTRAP MS and provide the number of quadrupoles it has. 4. What does DART stand for and how does it work in positive polarity? (Ionsense is manufacturer; 2 point bonus: provide an article on the theory of DART.) 5. Provide the definition for an internal standard and why it is important. 6. What is a calibration curve and what information does it provide? 7. Define the term extraction and provide 2 examples. The day of the laboratory activity, students submitted their preactivity questions and were given a 10 min, instructorprepared overview of DART QTRAP MS technology. The instructor was an expert on the instrument, and was able to comfortably walk students through its technical requirements. (An instrument expert is not necessary for completion of the activity, as other personnel in the department who are not experts have also been trained.) Following the preactivity lecture, students were divided into 3 groups of 2−3 students per group (total of 8 students). Each student group was provided with an instrument guide, a case scenario, simulated drug paraphernalia (Figure 2), and an unknown, simulated urine sample. These materials were used to complete two separate but related experiments: (1) analyte identification and (2) analyte quantification (Learning Goal 2). The case scenario provided to each student group was a narrative regarding the examination of a decedent by a medical examiner (ME). The scenario supplied clues (erratic behavior, white residue in nasal passage, injection sites on arms, and extremely high body temperature, nude) that when considered with the collected data from both samples (the simulated drug paraphernalia and simulated urine sample) could be used in the postlaboratory assessment (written laboratory report) to corroborate the cause of death (cocaine induced excited delirium). Learning Goal 4 was achieved through a correct cause of death determination. Activity Instruction

Following the laboratory activity both Learning Goals 1 and 3 were expected to have been met. For experiment 1, each group was asked to identify the analyte in one of three simulated drug paraphernalia (a homemade pipe, a syringe, and a piece of foil, shown in Figure 2) that contained drug residue. Student groups collected the residue using 0.2−0.5 mL of methanol. For example, the student group with the pipe pipetted the methanol into the pipe, swirled the methanol around inside the cylindrical absorption cell, and collected the solvent−analyte mixture (sample). These sample solutions were analyzed by both quadrupole 1 scanning (Q1) and enhanced product ion (EPI) methods on the DART QTRAP MS (see Supporting Information). Multiple “dummy” methods were created for both experiments 1 and 2 to prevent students from using deductive reasoning to work through the experiment. These “dummy” methods were blank methods, containing no parameters. By saving blank methods with a similar method naming scheme, students were prevented from using the list of available methods to determine the analyte identification. Students were expected to identify the base peak ion, which is the protonated molecule, in the spectrum (m/z 304) using the Q1 scanning method. Using the protonated molecule bank supplied at the beginning of the activity, student groups could postulate the identity of the analyte; however, the lack of chromatography and limitations of unit resolution associated with a quadrupole mass spectrometer prohibited definitive D




Figure 3. Student data from the syringe and needle (simulated drug paraphernalia) collected using Q1 scan (A) and EPI304 (B) methods on the DART QTRAP MS.

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confounding background ions from the open air source. This information was to be included in the postactivity assessment (written laboratory report).

RESULTS AND SURVEY OUTCOMES

Results

Both experiments 1 and 2 were completed within the 3 h laboratory period. All student groups successfully identified cocaine as the analyte in the simulated drug paraphernalia from experiment 1 and were able to achieve a library match quality of 80% or better to the user created library of certified reference materials. In Figure 3, student generated data is displayed for both “Q1 scan” and “EPI304” methods. Cocaine has a molecular weight of 303.4 g/mol; the base peak identified in Figure 3A is m/z 304.3. The mass difference was due to the addition of a proton during the ionization process. Collection of this data reinforced the theory of the DART source and how the QTRAP MS functions when both Q1 and Q3 are in scan mode, which was covered at the beginning of the activity. The fragmentation spectrum displayed in Figure 3B is the result of collisionally activated dissociation (CAD) which occurs in quadrupole 2, the collision cell, in the QTRAP MS. Again, students were directed to view their data as the result of instrument function (Q1 SIM, Q2 CAD gas added/ fragmentation, and Q3 scan), which was covered at the beginning of the activity. Both spectra (Figure 3A,B) reinforce Learning Goal 2. For experiment 2, students were expected to quantify the cocaine in a simulated urine sample, “unknown”, and scan the sample for the presence of any other potential metabolites. For quantification of cocaine, 2 out of the 3 student groups achieved calibration curves that met the minimum acceptance criteria. Minimum acceptance criteria for the calibration curve required 5 calibrator levels within 20% of target concentration, 2 positive controls within 20% of target concentration, a negative control (internal standard only), a matrix blank (simulated urine without internal standard or analyte), and a coefficient of determination of 0.99. A calibration table and

Postactivity Assessment

Upon completion of the laboratory activity, students were provided with copies of their group’s data. It was requested that the students explain any aberrant values and hypothesize possible explanations. The students were given 2 weeks to compose a written report summarizing the laboratory exercise (including instrument theory) and answer the following questions. 1. Discuss the 3 types of drug paraphernalia that were encountered in this laboratory and how they related. 2. Provide the identity of the unknown. 3. Explain the prominent ions in the fragmented spectrum from experiment 1. (HINT: Look at the structure and provide the structure or moiety loss that corresponds to each major fragment.) 4. Provide the concentration of the unknown in the simulated urine. 5. Were any additional compounds detected? If so, what? 6. What is the relationship of the unknown to the additional compounds? 7. Why did the decedent become unresponsive and expire? (HINT: Use the ME narrative.) The laboratory reports were used to assess student learning and whether the students met all 4 Learning Goals. In addition to the written report, 7 out of the 8 students who participated in this activity completed an anonymous postactivity survey (see Figure 6 for survey results and Supporting Information for survey contents). Course reflections and improvements were based upon instructor observations during the activity and upon the anonymous, summative survey. E




Figure 4. Student group calibration table and curve from experiment 2, quantification of unknown A. Actual concentration of unknown A was 3,000 ng/mL; student group calculated concentration of 3,560 ng/mL. Accuracy was determined to be 119% (calculated concentration divided by actual concentration and multiplied by 100).

curve generated by one student group is displayed in Figure 4. The calibrator level information is listed above the graphical curve and included sample type designation, analyte and internal standard peak areas, targeted analyte concentration, calculated concentrations, and accuracy. Accuracy was calculated by dividing the calculated concentration for the sample by the actual concentration and multiplying by 100. The acceptable 20% range is 80−120%. From the table it was observed that all but one control fall inside the desired 20% minimum acceptance criteria. Likewise, an acceptable coefficient of determination was also achieved. The two groups with successful calibration curves were able to quantify the cocaine in their unknown simulated urine achieving accuracies of 119% for unknown A (target concentration 3,000 ng/mL) and 73% for unknown C (1,500 ng/mL). While achieving calculated concentrations for the unknowns within 20% of the target concentration was a goal of this laboratory exercise, the

emphasis was the use and understanding of the instrumentation. Therefore, the two student groups that did not achieve acceptable values for the unknown were not penalized, but rather were asked to explain what errors occurred during the activity in the written assessment. Additionally, these student groups were provided with instructor data for calculation and discussion of the unknown. For the second part of experiment 2, students were expected to scan the “unknown” and a control to identify other possible analytes. Student group data for the “304 scan” of the positive control 1,000 ng/mL is displayed in Figure 5. From Figure 5, ions for ecgonine methyl ester (m/z 200.2), benzoylecgonine (m/z 290.2), cocaine (m/z 304.3), cocaethylene (m/z 318.1), and cocaethylene-d3 (m/z 321.1) are observed. The additional analytes (ecgonine methyl ester, benzoylecgonine, and cocaethylene) are metabolites of cocaine (benzoylecgonine and F




Figure 5. Student group data generated from “304 scan” method. Ions for additional analytes were m/z 200.2 (ecgonine methyl ester), m/z 290.2 (benzoylecgonine), and m/z 318.1 (cocaethylene). Ions m/z 304.3 and m/z 321.1 corresponded to cocaine and cocaethylene-d3, respectively.

Figure 6. Students’ perceived growth in comfort level pre- to postactivity.

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ecgonine methyl ester) and artifacts of concurrent cocaine and ethanol use (cocaethylene). These analytes were pipetted as part of the control preparation (included in control working stock solution, COC CTLA) with the analyte concentrations matching that of cocaine; the internal standard was maintained at 400 ng/mL. With concentrations being equal, the observed response differences were determined to be a product of differing ionization rates due to analyte chemical composition (i.e., proton affinities). Matching concentrations of cocaine and its metabolites in the simulated urine specimen is unrealistic due to biological metabolism; however, since these were qualitative analyses, the concentrations were kept equivalent for ease of the experiment. Successful identification of unknowns required that students meet Learning Goal 3 and work toward meeting (or meet) Learning Goal 4. The written assessment (laboratory reports) was due 2 weeks after completion of the laboratory activity. Students were expected to review the instrument theory, explain the activity procedure, show and discuss their group data, and answer the postactivity questions listed previously. The grading rubric used to assess the laboratory reports is available in Supporting Information. Detailed discussion of the written assessment is not included, but the grading rubric used to assess the laboratory reports is available in Supporting Information.

This addition will hopefully alleviate the stress of performing calculations during the limited time frame of the laboratory period. Despite these difficulties, student responses were overwhelmingly positive. One student wrote “I love when we can use instruments that are unfamiliar to us and be able to understand it [them] by applying a real-world situation.”

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HAZARDS Safety issues associated with this laboratory exercise included the following: use of standard laboratory supplies; high temperatures associated with instrumentation; exposure to methanol, cocaine, and its metabolites; and syringe needles (simulated drug paraphernalia). The exercise required the use of standard laboratory supplies (plastic pipette tips, glass autosampler vials, test tubes, etc.), requiring no special instruction. The DART QTRAP MS utilized high temperatures during analyses. Students were provided safety instruction prior to use and were supervised during use of the instrument. Exposure to the cocaine and metabolites was minimized by the low concentrations of the stock solutions and students wearing safety goggles and nitrile gloves. Methanol (CAS 67-56-1) poses a hazard if ingested, is an irritant to the eyes, and is flammable; care was taken when working with this solvent. The syringe needle was capped when provided to the student group and was disposed of in a sharps container after the residue had been collected. All students were required to don the appropriate (standard) personal protective equipment or PPE during the course of this laboratory exercise. The laboratory environment was equipped with a sharps container, hood, and proper waste disposal bins to which students were directed.

Survey Outcomes

The postactivity survey contained items aimed at determining students’ self-efficacy regarding mass spectrometry pre- and postactivity. See Supporting Information for summative activity. To maximize participation and minimize any instructor bias, students were instructed to keep the surveys anonymous and informed that handwritten student responses were to be recorded by a third party. Of the 8 students that completed the activity, 7 (87.5%) completed the survey (N = 7). Due to the limitations of a small sample size, statistical measures beyond percentages were not calculated as they are not meaningful. Student responses for changes in confidence between pre- to postactivity were compared and are presented in Figure 6. The student responses from the free response items highlighted three important observations. First, 85.7% (N = 6) of students expressed knowledge gains in DART theory and increased confidence when working with analytical equipment and analyzing data, while 100% of students felt more comfortable with mass spectrometry theory and how it is applied in forensic laboratories. Second, 71.4% (N = 5) of the students perceived an increase in critical thinking associated with sample preparation procedures, mass spectrometry, and forensic applications. Background information regarding the theory behind these topics was included in the prelaboratory materials. This perceived increase in critical thinking was expected due to learning objectives 1, 2, and 4. Even so, it is encouraging that students agreed the learning objectives were successfully met. Third, the majority of students reported comfort level gains on all survey items after completing this laboratory exercise, with the exception of preparing solutions. Students responded with “no change” for both “preparing a series of dilutions” and for “preparing samples for DART analysis”. Both of these survey items referred to a challenging part of the activity where mathematical calculations were required. This led the author to include instruction concerning the calculations for preparing the calibration curve in the prelaboratory activity, which is discussed in the Course Reflections and Improvement section.

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SUMMARY AND DISCUSSION This activity offered an authentic learning experience and access to newer instrumentation. In addition to its novelty in undergraduate laboratories, the DART QTRAP MS was also chosen for its availability and growing applications. Since access to recent technologies may be limited, this laboratory activity can be translated to other instrumentation including the following: an ESI source equipped tandem MS, ion trap, Orbitrap, quadrupole time-of-flight, etc. As this instrumentation was not used by the authors, additional details about these methods are not included. All teaching materials used by the authors have been provided in the Supporting Information to help others adapt for their institution. On the basis of the Learning Objectives, this activity was considered successful. Each student group was able to prepare solutions, configure the instrument, analyze their own samples, and collect and perform data analyses. In experiment 1, all student groups positively identified cocaine utilizing both Q1 scan and EPI methods. All student groups completed the first experiment of the activity with ease within the first 30−45 min of the laboratory period (∼15 min to collect the residue and ∼10 min per group at the instrument). The flow of the activity required student groups to complete experiment 1 before moving to experiment 2, but to keep the activity within the confines of 1 laboratory period, student groups were not required to stay in pace with other groups. Therefore, students were allowed to start on experiment 2 as soon as experiment 1 was completed. In experiment 2, students struggled with preparing the calibration and controls solutions needed, specifically using the pipette (presence of air bubbles) and performing calculations on the spot (incorrect volumes H




priori knowledge and also prevents cheating between the groups.

calculated). The preparation of samples for experiment 2 took the students over 1 h. The analyses of the samples took ∼30 min per group. The full 3 h period was necessary for activity completion, which was observed to add stress to the students. As a result of struggles and stress, only 2 student groups (66%) achieved a suitable calibration curve and only 1 student group (33%) was able to perform an acceptable quantification on the cocaine in the unknown. While this is not ideal, the success of the activity was determined based on the Learning Objectives, not on the student groups’ ability to calculate the correct concentration; therefore, students were not penalized for their data. These student groups were provided with instructor collected data. The students were required to interpret the instructor data as well as discuss the issues their group experienced during the activity. Despite the challenges associated with the activity, answers to the postactivity assessment (written reports and survey) were positive indicating that the activity increased their understanding of both mass spectrometry theory and the importance and application of instrumentation in nonresearch settings, such as a forensic laboratory. Furthermore, written laboratory reports revealed that students could identify small molecules using the DART QTRAP MS technique with 100% accuracy, and student survey responses indicated that students appreciated being offered the opportunity to complete a laboratory exercise that related to a real problem. Students felt that they were learning skills that they could take with them beyond the classroom. While not offered every semester, this activity is included as part of the revolving curriculum of the quantitative analysis course.

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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.7b00437. DART QTRAP MS lab protocol, standard curve preparation table, DART QTRAP MS bank of protonated molecules for common analytes, medical examiner (ME) case scenario, instructor DART QTRAP MS method parameters, DART QTRAP MS instrument configuration, cocaine fragmentation for prominent ions, and assessment items (DART QTRAP MS lab anonymous summative activity and DART QTRAP MS lab report grading rubric) (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Mitzy A. Erdmann: 0000-0001-9090-8730 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the professor of the instrumental analysis course, Dr. Sergey Vyazovkin, his teaching assistant, Dr. Rachel Prado, and Mr. James Stallworth, for supporting the development and implementation of this laboratory exercise. Additionally, we would like to thank the students for their participation in both the activity and feedback via the survey. The review and use of the student surveys is in compliance with the University of Alabama at Birmingham’s IRB protocols (protocol number E170504004).

Course Reflection and Improvement

Observed obstacles associated with this laboratory activity were issues with calculations and pipetting and undue stress because of the 3 h time limit. Several ideas were noted on how to improve the efficiency of the laboratory, thereby reducing stress on the students. These ideas included the following: creating the calibration table during the prelaboratory activity, reviewing proper pipetting technique, and allowing the student groups to divide duties between experiments 1 and 2 concurrently. Due to the issues with pipetting, students are now required to create their own dilution tables for preparing calibration curves in the prelaboratory preparation to decrease their time in laboratory. The instructor notes have also been amended to include a detailed demonstration of proper pipetting technique and to explain preparation of calibration curves where necessary for successful completion of the exercise. A completed calibration table is available in Supporting Information. These measures should decrease the stress of performing math on the spot during the laboratory period. To further increase the efficiency of the laboratory, student group sizes are increased to 4−6, which allows the group to divide between experiments allowing half the group to perform experiment 1 and half the group to perform experiment 2. By working concurrently on both experiments, the activity would be completed in a more efficient manner, and both halves of the group would still get the hands-on exposure of running the instrument. The group is expected to discuss and present the data as a whole. This change also accommodates larger class sizes. Other course improvements include changing the analyte of interest between student groups. The objective for switching analytes is to ensure students approach the laboratory without a

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REFERENCES

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Reinforcing Mass Spectrometry Concepts through an Undergraduate

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