What Is in Your Wallet? Quantitation of Drugs of Abuse on Paper

Aug 15, 2017 - Laboratory experiments were developed to introduce students to the quantitation of drugs of abuse by high performance liquid chromatogr...
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Laboratory Experiment pubs.acs.org/jchemeduc

What Is in Your Wallet? Quantitation of Drugs of Abuse on Paper Currency with a Rapid LC−MS/MS Method Patrick D. Parker,† Brandon Beers,‡ and Matthew J. Vergne*,‡ †

Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27607, United States Department of Pharmaceutical Sciences and Department of Chemistry and Biochemistry, Lipscomb University, Nashville, Tennessee 37204, United States

‡

S Supporting Information *

ABSTRACT: Laboratory experiments were developed to introduce students to the quantitation of drugs of abuse by high performance liquid chromatography-tandem mass spectrometry (LC−MS/MS). Undergraduate students were introduced to internal standard quantitation and the LC−MS/MS method optimization for cocaine. Cocaine extracted from paper currency was analyzed with a simple and rapid LC−MS/MS method. Students in advanced laboratories determined the amount of amphetamine, methamphetamine, and oxycodone on currency in addition to cocaine. The LC−MS/MS method has a short run time (2.5 min) to allow for a high throughput of student samples. KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Drugs/Pharmaceuticals, Chromatography, Mass Spectrometry

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INTRODUCTION Drug abuse is an ongoing societal problem around the world. Due to the highly addictive nature of illicit drugs, they are closely monitored throughout the United States and much of the developing world.1 Forensic analysis of drugs as a lab experiment appeals to a wide audience of students in undergraduate analytical, instrumental, and environmental chemistry or forensic science.2 The analysis of cocaine and other drugs on domestic international paper currency offers an opportunity for physical science and social science to interface in the classroom.3 High performance liquid chromatography-tandem mass spectrometry (LC−MS/MS) is a technique that has yet to be incorporated in the undergraduate colloquia of most colleges.4−6 However, LC−MS/MS is a technique that is finding rapid acceptance in clinical and forensic laboratories for testing drugs of abuse.7 LC−MS/MS techniques are rapidly replacing GCMS techniques due to the higher throughput available with LC−MS/MS.8 Therefore, it is essential that undergraduate students learn about this powerful technique for quantifying drugs in complex matrices. The main objectives of this experiment are giving students the opportunity to learn about fundamentals of mass spectrometry, to provide a basic understanding of quantitation with selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) using LC−MS/MS, and to perform a quantitation experiment with a stable isotope labeled internal © XXXX American Chemical Society and Division of Chemical Education, Inc.

standard (cocaine-d3). It should be noted that SRM and MRM refer to essentially the same technique of analyzing product ions as a result of fragmentation of precursor ions; however, MRM terminology is used by some MS instrument vendors (e.g., Shimadzu and Bruker). In addition to the objectives, some Instrumental Methods of Analysis classes used SRM analysis to quantitate multiple drugs of abuse in this lab experiment as a demonstration of the selectivity available with LC−MS/MS analysis.

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TEACHING CONSIDERATIONS The work described in this article was planned for undergraduate students enrolled in two courses: Introduction to Forensic Science and Instrumental Methods of Analysis. Each class emphasized different key chromatographic concepts to give students a better understanding of LC−MS/MS from the perspective of their respective course’s field. As part of each lab session, the instructor presented a brief seminar on chromatography and mass spectrometry, and the students were given instructions on the liquid extraction procedure. Students were also provided with a range of both U.S. and foreign currencies for analysis; students could also bring their own currency. Received: March 15, 2017 Revised: July 5, 2017

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DOI: 10.1021/acs.jchemed.7b00196 J. Chem. Educ. XXXX, XXX, XXX−XXX

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(LC−MS/MS). The HPLC instruments consisted of Shimadzu LC-20AD pumps and either a SIL20AC or SIL10A autosampler. For all experiments, separations were carried out using a Phenomenex (Torrance, CA) Kinetex reversed-phase C18-XB column (2.6 μm particle size, 5.00 mm × 2.1 mm i.d.) column. The mobile phase had a flow rate of 0.5 mL/min and consisted of A, 0.1% formic acid in water, and B, 0.1% formic acid in acetonitrile. The initial conditions were 10% B and 90% A. At injection, B was increased from 10% B to 40% B at 0.9 min; at 0.95 min, B was increased to 90% and held at 90% until 1.50 min; and at 1.51 min, B was decreased to 10%. The injection volume was 5 μL. The HPLC column eluent was coupled to a Shimadzu LCMS 8040 triple stage quadrupole (3Q) mass spectrometer or a Waters Micromass Quattro Micro 3Q mass spectrometer. Selected reaction monitoring (SRM) was used on the 3Q instruments. Chromatograms were acquired with Lab Solutions software (Shimadzu) or MassLynx (Waters). Peak areas were determined using the software.

Biannually since 2014, students in Introduction to Forensic Science analyzed the concentration of cocaine on both domestic and foreign currency in a 2 h lab session. The topics of presumptive and confirmatory drug tests are covered in the corresponding lecture course as a background for this experiment. Students worked in groups of two, and the lab was split into two sections of about 20 students each. Each pair of students set up an LC−MS/MS batch with an experimental procedure to run their samples. The LC−MS/MS run time for each sample was 2.5 min including column equilibration. During each run, students noted the retention times of each peak and the corresponding peak areas. The determination of drug concentration was performed using an external standard calibration curve (obtained prior to the lab session). This method of determination was a key laboratory concept. Students were also instructed to consider the origin of each note and to predict the relative amounts of cocaine on currency from various nations. Annually since 2014, students in Instrumental Methods of Analysis worked on the determination of cocaine concentration on currency by LC−MS/MS. There were about 15 students per session, and the lab was done as a two week experiment (one 3 h lab session per week). The first week involved the development of an LC−MS/MS and SRM method for cocaine quantitation. During the second week of lab, students prepared cocaine calibrants and samples with cocaine-d3 as an internal standard. In addition to the ability for students to learn about internal standard methods, the practical purpose of adding an internal standard in these LC−MS/MS experiments is to improve the accuracy and precision of the method by correcting run-to-run changes in MS response due to ionsuppression or enhancement.

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Student Procedure: Introduction to Forensic Science

In order to extract cocaine from currency, each student pair folded a bank note in accordion style and placed it in a vial with 10.00 mL of methanol. The vial was shaken by hand for 5 min and mixed with a vortex shaker for 2 min. The currency was removed, and the methanol was filtered with a 10 mL syringe connected to a disposable 0.45 μm filter; about 1 mL of the filtrate was dispensed directly into an HPLC sample vial. The samples were loaded into the LC−MS/MS autosampler, and the samples were run in a queue. The students used the area of the cocaine peak in the SRM chromatogram to calculate the concentration of cocaine on their currency based on comparison with the instructor-provided calibration curve data.

EXPERIMENTAL OVERVIEW

Student Procedure: Instrumental Methods of Analysis

The objectives of this lab are 2-fold for students: first, to optimize the mass spectrometer parameters for the quantitation of cocaine in the first lab session; second, to quantify cocaine on various paper currency notes in the second lab session. To this end, students determined the m/z for the cocaine precursor ion (304 m/z) either by flow injection analysis or syringe infusion of a 10 μg/mL solution of cocaine in 1:1:0.01 acetonitrile/water/formic acid. Students were given a default instrument parameters method with basic source parameters. From this method, students worked in groups of three to optimize mass spectrometer source parameters and collision energy (CE). Students observed product ion spectra at different CE voltages (5−50 eV, 5 eV steps) and determined the best product ion for cocaine. From the optimized parameters, students constructed a mass spectrometer instrument parameters method to use with the liquid chromatography method. Then, using the HPLC with the column installed, students ran a test injection of the 10 μg/mL cocaine standard to observe the peak shape and retention time. In order to extract cocaine from currency, each student folded a bank note in an accordion style and placed it in a vial with 10.00 mL of methanol. The vial was shaken by hand for 5 min and mixed with a vortex shaker for 2 min. The currency was removed, and 1 mL of the methanol from the extraction was dispensed into a 5 mL beaker; a 1000 μL aliquot of the internal standard, cocaine-d3, was added to the methanol, and then a 2 mL syringe was used to filter the sample with a disposable 0.45 μm filter directly into a 2 mL HPLC sample vial.

Reagents and Materials

For this experiment, 1 mg/mL standard solutions of methamphetamine, cocaine, cocaine-d3, and amphetamine were purchased from Cerilliant. LC−MS grade acetonitrile, water, methanol, and formic acid were used. Standards Preparation: Introduction to Forensic Science Class

The instructor prepared a 10 μg/mL stock solution of cocaine in methanol. Students prepared a series of seven calibration standards in methanol between 10.0 μg/mL and 156 ng/mL by 1/2 serial dilutions. The samples are then filtered with 45 μm nylon syringe filters. The solutions were stored at 4 °C. The instructor analyzed the standards before the lab session, and prepared a calibration curve based on the peak areas from the LC−MS/MS chromatograms. Standards Preparation: Instrumental Methods of Analysis Class

Students utilized an internal standard, cocaine-d3, prepared in a 1 μg/mL solution. Students prepared the eight calibration standards by 1/2 serial dilutions from a 10 μg/mL stock solution. Each calibration standard consisted of 500 μL of standard solution and 500 μL of cocaine-d3 internal standard. The samples are then filtered with 45 μm nylon syringe filters. The solutions were stored at 4 °C. Instrumentation

The analysis was performed at Lipscomb University using an HPLC coupled to a triple stage quadrupole mass spectrometer B

DOI: 10.1021/acs.jchemed.7b00196 J. Chem. Educ. XXXX, XXX, XXX−XXX

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During the second laboratory period, students analyzed the calibration standards and samples. As a group, students picked the best mass spectrometer instrument parameters to use as a class. The class-optimized mass spectrometer source parameters were used in conjunction with an HPLC method to analyze the samples from a queue.

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HAZARDS There are no significant hazards in this experiment. Methanol and acetonitrile are flammable, and they are hazardous if ingested, inhaled, or absorbed through skin contact. Good laboratory practices should be followed. Students should wear gloves, aprons, and protective eyewear. Solvents and standards should be handled in fume hoods.

Figure 2. Student-generated internal standard calibration curve of cocaine by LC−MS/MS. The peak area ratios of cocaine/cocaine-d3 are plotted on the y-axis. The internal standard, cocaine-d3, is the same concentration (1 μg/mL) in all standards and samples; therefore, the actual cocaine concentrations for the standards can be plotted on the x-axis instead of the concentration ratios (cocaine/cocaine-d3).

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RESULTS AND DISCUSSION For Instrumental Analysis Lab, the lab consisted of two 3 h lab periods. For the first lab period, students attended a presentation detailing electrospray ionization (ESI) theory and multiple reaction monitoring theory. Details on optimization of mass spectrometer parameters such as collision induced dissociation were covered as an introduction to the lab procedure. In the first laboratory period, students worked in groups of three to optimize the ESI source parameters to produce the most intense precursor ion signal for cocaine; the precursor ion was [M + H]+ at m/z 304. Students then selected the appropriate product ion by using collision induced dissociation (CID) to fragment the precursor ion at several different collision energies. There are two main product ions at m/z 182 (major product) and m/z 122 (Figure 1). The major

standards containing cocaine, methamphetamine, amphetamine, and oxycodone without internal standards. Students extracted drugs from money, but did not add internal standards to the samples. The same HPLC method was used as described in the experimental method; detailed MS parameters are in the Supporting Information. SRM transitions are shown in Table 1. Table 1. SRM Parameters Developed for the Shimadzu LCMS 8040 LC−MS/MS Compound

Precursor Ion (m/z)

Product Ion (m/z)

Collision Energy/V

Amphetamine Methamphetamine Oxycodone Cocaine Cocaine-d3

136.90 150.30 316.40 304.40 307.40

91.20 91.10 298.15 182.10 185.10

22 22 20 21 21

Figure 3 shows SRM chromatograms of a calibration standard containing four drugs and an extraction from a U.S. $10 bill. The students prepared external standard calibration curves for each drug for quantitation. From the quantitative results in Introduction to Forensic Science and Instrumental Methods of Analysis, the students drew conclusions about drug abuse in different nations based on the amount of drugs on bank notes. We analyzed currency from a number of countries including the U.S., Zimbabwe, Guatemala, Honduras, and China. On all Chinese Yuan bank notes analyzed, the amount of drugs were below the limit of detection (10 ng/note for cocaine). U.S. currency has the highest amount of cocaine on average. Students have detected methamphetamine and oxycodone on some U.S. bank notes, though in smaller amounts than cocaine. Students wrote lab reports describing method optimization, quantitation, and discussion of drug amounts on various currencies. One limitation to the lab is that some colleges may have an LC/ MS system that is incapable of SRM (for example, a single quadrupole mass analyzer); however, the experiment has been validated in our lab using selected ion monitoring (SIM) of the [M + H]+ ions of the drugs with a single quadrupole LC/MS instrument. Another limitation in the procedure presented here is that the students did not determine recovery, which could be done by multiple extractions from the same bank note. In addition the percent recovery for the extraction procedure

Figure 1. LC−MS/MS fragmentation of the cocaine molecule.

product ion can be assigned to the neutral loss of benzoic acid [M + H − 122] while the other product ion represents benzoic acid. Each student group spent about 30 min optimizing MS source parameters; students recorded the highest intensity of the product ion and saved the MS parameter file. At the beginning of the second laboratory period, students selected the best MS parameter file from those created by the student groups by evaluating the final product ion signal intensity. Students prepared calibration standards as a class and prepared samples individually. Students worked as a class to load the autosampler and create a batch queue for the HPLC autosampler. Cycle time can be as short as 2.5 min per run; a set of seven calibrants and samples can be analyzed in 50 min. Students evaluated and determined the peak area ratio of (calibrant peak area)/(internal standard peak area). Good linearity was observed from 0.156 to 10 μg/mL. A studentgenerated calibration curve is represented in Figure 2. The amounts of cocaine on different bank notes were determined by the students by multiplying the concentration determined from the LC−MS/MS analysis by 10 mL since the cocaine was extracted with a 10 mL aliquot of methanol. In certain advanced lab classes and as a supplement to instrumental methods lab, students prepared calibration C

DOI: 10.1021/acs.jchemed.7b00196 J. Chem. Educ. XXXX, XXX, XXX−XXX

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Figure 3. (A) SRM chromatograms of amphetamine, methamphetamine, oxycodone, and cocaine standards in a solution with a concentration of 10 μg/mL each. (B) SRM chromatograms obtained from a student-prepared sample from a U.S. $10 note. LC−MS/MS SRM parameters are in Table 1.

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could be determined by spiking an aliquot of an internal standard solution to a bank note in the procedure step before solvent extraction.1

Corresponding Author

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*E-mail: [email protected].

CONCLUSION This experiment provides students with hands-on experience in analytical method development for the analysis of drugs of abuse with LC−MS/MS instrumentation. The ability of students to optimize the LC−MS/MS instrument parameters and create their own SRM methods increased their understanding of the fundamentals of mass spectrometry and LC− MS/MS instrumentation. A short HPLC run time of 2.5 min allows a large number of samples to be run in a short period of time giving students the opportunity to experiment with optimizing the analysis during the lab periods. Students also gained knowledge about quantitation with a deuterated internal standard method which is universally used in clinical mass spectrometry laboratories. The resulting lab reports produced by students demonstrated that the learning objectives were broadly met, and students were excited to use an instrumental technique to measure drugs on money, a common item in their wallets.

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AUTHOR INFORMATION

ORCID

Matthew J. Vergne: 0000-0002-0841-0018 Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Wimmer, K.; Schneider, S. Screening for illicit drugs on Euro banknotes by LC-MS/MS. Forensic Sci. Int. 2011, 206 (1−3), 172−7. (2) Tarr, M. A. Solving a Mock Arsenic-Poisoning Case Using Atomic Spectroscopy. J. Chem. Educ. 2001, 78 (1), 61. (3) Heimbuck, C. A.; Bower, N. W. Teaching Experimental Design Using a GC-MS Analysis of Cocaine on Money: A Cross-Disciplinary Laboratory. J. Chem. Educ. 2002, 79 (10), 1254. (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) 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 (8), 838−841. (6) 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. (7) Nordgren, H. K.; Holmgren, P.; Liljeberg, P.; Eriksson, N.; Beck, O. Application of direct urine LC-MS-MS analysis for screening of novel substances in drug abusers. J. Anal. Toxicol. 2005, 29 (4), 234−9.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00196. Student and instructor instructions including instructions for Waters and Shimadzu LC−MS/MS instrumentation (PDF, DOCX) D

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(8) Grebe, S. K. G.; Singh, R. J. LC-MS/MS in the Clinical Laboratory − Where to From Here? Clinical Biochemist Reviews 2011, 32 (1), 5−31.

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DOI: 10.1021/acs.jchemed.7b00196 J. Chem. Educ. XXXX, XXX, XXX−XXX