Mass Spectrometry for Forensic Analysis: An Interview with Glen Jackson

As part of “The Future of Forensic Analysis” content series, Spectroscopy sat down with Glen P. Jackson of West Virginia University to talk about the historical development of mass spectrometry in forensic analysis.

Mass spectrometry (MS) is an analytical technique used in analytical chemistry and spectroscopy. It measures the mass-to-charge (m/z) ratio of ions to identify and quantify molecules in a sample (1). This method involves ionizing chemical compounds to generate charged molecules or molecule fragments, then measuring their m/z ratios. The resulting data, typically presented as a spectrum, provides detailed information about the molecular weight and structure of the analytes, making it a powerful tool for chemical analysis.

MS has been used in forensic and criminal investigations to analyze many substances. In particular, MS analysis has been used to study biological samples such as urine, hair, and blood to identify drugs and other toxic samples (1). MS can help uncover evidence in cases of poisoning or drug abuse.

MS is also employed in the examination of environmental samples, such as soil and water, to detect pollutants and residues related to criminal activities. Furthermore, MS analysis contributes to uncovering biological evidence, including DNA and proteins, aiding in the identification of individuals and establishing connections between suspects, victims, and crime scenes (1).

Glen Jackson, a Ming Hsieh Distinguished Professor of Forensic and Investigative Science at West Virginia University, has been conducting extensive research in forensic analysis applications. Jackson earned his BS in Chemical and Analytical Science from the University of Wales Swansea, an MS degree in Analytical Chemistry from Ohio University, and a PhD in Analytical Chemistry from West Virginia University. After receiving his PhD, Jackson conducted his postdoc at Oak Ridge National Laboratory before starting his career as a member of the chemistry faculty at Ohio University. Following his tenure at Ohio University, he returned to West Virginia University, where he is currently exploring MS instrumentation development in forensic and biological applications.

As part of “The Future of Forensic Analysis” content series, we invited Jackson to talk about his laboratory group’s current research in forensic analysis. Our interview with Jackson covers a breadth of topics, including the historical developments of MS in forensic analysis, current research efforts in this field, and how the role of MS is currently evolving.

Dr. Glen P. Jackson of West Virginia University. | Photo Credit: © Glen Jackson.

Can you elaborate on the major historical developments in MS that have significantly impacted its application in providing evidence in the criminal justice system?

The three most important developments have been: 1) the electron ionization (EI) source, which was driven by Bleakney and others in 1937 (2); 2) the coupling of MS with gas chromatography (GC–MS), which was demonstrated by Gohlke in 1959 (3); and 3) the invention of the quadrupole mass analyzer by Paul and Steinwedel in 1953 (4). The hyphenated GC-EI-MS instrument, which was first commercialized by Finnigan in 1968, became the technology of choice for many areas of chemical analysis, and it is still by far the most trusted and commonly used instrumental method of analysis for seized drugs and ignitable liquid residues.

How has MS been utilized in casework involving trace metal impurities in hair and glass? Could you provide specific examples?

The oldest case that I know of involving MS analysis of trace metals in hair involved ion microprobe mass spectrometry (IMSS), now called secondary ion mass spectrometry (SIMS). The technique was first presented by R. Castaing and G. Slodzian in 1962 (5,6). In its first forensic application in 1977, Walter McCrone used IMSS in United States v. Brown to link a suspect’s hair with hair strands found at a Planned Parenthood clinic that had been bombed (7). The court struggled with the validity and application of IMSS for human hair because it had never been applied for this purpose before. Although IMSS was found to reliable as a scientific technique, its application to human hair did not meet admissibility standards because it had not gained the level of general acceptance in the scientific community for this purpose (7).

The first casework applications to glass that I know of occurred after Houck and others successfully coupled MS to an inductively coupled plasma (ICP-MS) instrument in 1980 (8). Following commercialization of ICP-MS instruments a few years later, the forensic community demonstrated that the intra- and inter-variability of different elements in glass were sufficient to distinguish glasses from different sources because of their impurity differences. Today, both ICP-MS and laser-ablation (LA)-ICP-MS are widely used to compare glass samples in forensic contexts (9–11).

What applications of mass spectrometry are most common and successful for criminal investigations?

By far, the most common application of MS is the use of GC-EI-MS to confirm the identity of seized drugs. A recent white box study involving 71 laboratories showed that they all used GC–MS to help with the identification of a controlled substance (12). GC–MS is also the method of choice for ignitable liquid residues to support suspected arson cases (13). Finally, although GC–MS was also the method of choice to detect drugs and drug metabolites in human body fluids for several decades, liquid chromatography–MS (LC–MS) now tends to dominate the market in toxicology laboratories.

What are some notable cases where MS has been used to identify drugs, explosives, polymers, and ignitable liquids?

Gosh! There are so many. I recently wrote a review article on this topic in which I attempted to include all the notable early cases (14). One of the earliest examples for seized drugs was by Martin and Alexander at the U.S. Food and Drug Administration (FDA) in 1968. They published a report that explained how they used cracking patterns and high-resolution mass spectrometry (HRMS) to identify the hallucinogen dimethyltryptamine (DMT) in a casework sample (15). They noted that the use of mass spectrometry changed the project from a major research project, which would have taken weeks or months using conventional techniques, to “an exercise problem in spectroscopic identification” (15).

For polymers, the earliest application of MS to casework I could find was the use of pyrolysis-GC–MS (Pyr-GC–MS) by Zoro and Hadley in 1976. They described a case where they linked an antioxidant in the trace fragments of a polymer in blades of a hacksaw to those of a stolen cable that was coated with a polymer (16).

How do the scientific foundations of MS compare to other forensic techniques like pattern-based methods and physical matching?

The theory and practice of MS is based on physics and the fundamental properties of matter. In an EI source, the branching patterns of a molecule can be tied to statistical mechanics through the Rice–Ramsperger–Kassel–Marcus (RRKM) theory. In these ways, our measurements have scientifically rigorous foundations. However, the result of our measurements is usually a mass spectrum of a substance, which can be thought of as a pattern. Although the spectral patterns can be linked to chemical structures through spectral interpretation, our comparisons of one spectrum to another—such as in a database search—are hardly distinguishable or superior to pattern-based disciplines. Sure, we have a plethora of algorithms for comparing and interpreting spectra, but even the most advanced algorithms struggle to provide meaningful statistics, probabilities, or likelihood ratios for specific compound identifications. Quite simply, the spectra of some drugs are more unique than others. For these reasons, we should not rest on our laurels, and we should continue to improve the evidential power of our MS results.

Can you discuss any landmark legal precedents where mass spectrometric evidence played a critical role in the outcome of the court’s decision-making process?

Again, I would refer you to my recent review that specifically covers influential legal precedents in a wide variety of forensic MS applications (14). If I had to pick two favorites, one would be GC–MS identification of cocaine. For most of the 1970s, GC–MS was basically useless for the identification of l-cocaine—the controlled isomer—because it could not distinguish l-cocaine from the uncontrolled d-cocaine. For most of the 1970s, analysts often struggled on cross-examination about how many isomers of cocaine exist and which ones they had identified—there are four chiral centers in cocaine, so eight theoretical isomers in total, but only two are commonly considered (17). In 1981, a Federal Bureau of Investigation (FBI) analyst testified that because plants make exclusively l-cocaine and humans make racemic mixtures of d- and l-cocaine via laboratory-based synthetic methods, d-cocaine had never been observed independently from l-cocaine (18). Therefore, if any cocaine is identified, at least 50% will be l-cocaine. Since then, analysts have never really had to identify the isomeric form of cocaine, so GC–MS regained its use.

One case in which MS proved essential was in 1991, when a mother was found guilty of poisoning her 5-month-old child with ethylene glycol. Ethylene glycol had been identified based on GC retention time data using a flame ionization detector (GC-FID) (19). She gave birth to a second child in prison, and after that child also became ill, doctors were able to diagnose a genetic disorder in the newborn called methylmalonic acidemia. Scientists then re-analyzed the serum from the first child using the same GC-FID method, and of course, they still found ethylene glycol. Methylmalonic acidemia does not cause a buildup in ethylene glycol, so there was no evidence to overturn the conviction for poisoning her first child. Finally, a toxicologist named Jim Shoemaker whose laboratory had worked on the original case developed a more selective GC–MS approach that proved that the toxin was in fact propionic acid. Propionic acid has the same GC retention time as ethylene glycol, but it has a different fragmentation pattern. Unlike ethylene glycol, propionic acid could be linked to the genetic disorder, so, thankfully, the mother was ultimately exonerated (19,20).

Despite its reliability, MS evidence has been challenged in court. What are some reasons for these challenges, and how have courts typically responded?

In 1981, mass spectrometric evidence was discussed at length in a case against 2,116 boxes of boned beef (21). The U.S. District Court of Kansas was appalled at the GC–MS expert witnesses in the case. The case concerned the alleged adulteration of beef with the hormone diethylstilbestrol. Apparently, the court witnessed mass spectrometrists from each side arguing for opposite conclusions about the exact same data. The Court bemoaned the GC–MS experts and reported that “they are disregarded as being of any scientific assistance to the Court. Simply stated, a review of these exhibits suggests that the experts can read into them about what they want to read, the Court perceiving nothing and is totally helpless” (21). The example above from the McCrone institute in 1977 regarding the analysis of trace elements in human hair showed that although MS techniques can be perfectly valid for the scientific community, their application to a particular case needs to be fit for purpose to be accepted by the court. This criterion is an important part of Daubert standards and the Federal Rules of Evidence that help judges assess the validity of forensic evidence.

What future developments or improvements do you foresee in the application of MS within the criminal justice system?

I’m quite sure that vendors will continue to deliver instruments with lower limits of detection, greater signal-to-noise (S/N) ratios, and shorter analysis times than we have today. These instruments will have trivial effects on most applications, since in most applications, we already have the required sensitivity to identify substances at biologically relevant levels. However, portable MS instruments have long been proposed to speed up the analysis of seized drugs and enable on-site detection; in so doing, they could save a fortune for the criminal justice system. One might wonder why they have struggled to fulfill their potential. I suspect that one problem could be that cost saving is most beneficial for the criminal justice system and not directly for the crime laboratory. For example, on-site testing could help reach plea deals with suspected drug dealers/possessors in a matter of days, thereby avoiding the average wait of approximately 100 days in jail at an average cost of ~$150 per day. That is a total of ~$15,000 per case in jail costs alone, not including legal fees and laboratory expenses. If a portable GC–MS instrument could be used to conduct on-site confirmatory testing, thusly circumventing jail time, legal proceedings, and legal fees, the criminal justice system could save a fortune. The eventual savings would more than compensate for the upfront and maintenance costs.

I believe that we have the most work to do with data analysis. We have barely scratched the surface in terms of maximizing the evidential value of mass spectrometric evidence. We need to help the forensic community by providing more meaningful probabilities or error rates with our mass spectral identifications.

Can you talk about some of the current projects your laboratory group is working on?

The forensic applications in our laboratory have two main themes: spectral interpretation and spectral identification. For spectral interpretation, we help analysts understand the mechanisms of fragmentation that lead to the observed product ion spectra of certain seized drugs. We typically use a variety of age-old techniques, including isotope labeling, MSn, and HRMS, to help identify the observed fragmentation pathways of various synthetic drugs, like cannabinoids, cathinones, and fentanyl analogs. We’ve identified novel structures and pathways for cathinone fragmentation (22), and we’ve identified some unprecedented skeletal re-arrangements for fentanyl analogs (23).

Regarding spectral identification, we’re developing a new algorithm that can help overcome the problems caused by spectral variance when substances are analyzed on different days or on different instruments (24,25). The algorithm will allow analysts to identify substances in the absence of a standard analyzed contemporaneously with the casework sample. It’s still a work in progress, but we’ve made some promising developments.

Can you explain the importance of your research within the broader field of spectroscopy or in a specific industry or application?

Our new algorithm for spectral identification has the most potential outside of forensic science. We are currently demonstrating that the algorithm is applicable to protonated precursors from electrospray ionization (ESI) and direct analysis in real time (DART) ion sources in addition to EI sources. Therefore, the algorithm should be applicable to identifying peptides, lipids, metabolites, and almost any substances for which replicate spectra can be (or have been) acquired.

How do you stay updated with advancements in mass spectrometry and spectroscopy techniques and technologies?

I read the table of contents for most MS journals when they arrive in my inbox. I find most articles that way. My students occasionally share new and interesting articles with me, too. Sometimes, I will come across other articles when I perform targeted literature searches during a literature review for a manuscript or grant proposal. I also keep up to date with developments by paying attention to the work of my colleagues at conferences, meetings, and college visits.

Can you discuss a recent innovation or development that you find particularly impactful or exciting in mass spectrometry and forensic analysis?

I think ion mobility spectrometry has some untapped potential in forensic science, especially if differences in drift times enable the resolution of isomers that are difficult to resolve by GC–MS or LC–MS. For example, ortho-, meta-, and para-isomers of fentanyl analogs are often difficult to resolve with tandem mass spectrometry, so it would be helpful—and apparently no slower—if IMS-MS could resolve such isomers as they eluted from a chromatographic column. A couple of groups have recently shown that the site of protonation within a fentanyl analog—such as on the amide or amine group—can significantly impact the drift time of the protomer (26–28). Therefore, in contrast to most drugs, many fentanyl analogs show a bimodal distribution of arrival times in IMS-MS. However, the work is strictly academic now and has yet to be generalized or made practicable. So, although IMS-MS is popular in areas like structural biology, there are very few researchers exploring and maximizing opportunities in forensic science.

References

(1) Edwards Vacuum. Mass Spectrometry in Forensic Science – A Brief History. EdwardsVacuum.com 2020. https://www.edwardsvacuum.com/en-us/vacuum-pumps/knowledge/applications/mass-spectrometry-in-forensic-science (accessed 2024-08-02)

(2) Bleakney, W.; Condon, E. U.; Smith, L. G. Ionization and Dissociation of Molecules by Electron Impact. J. Phys. Chem. 1937, 41 (2), 197–208.

(3) Gohlke, R. S. Time-of-Flight Mass Spectrometry and Gas-Liquid Partition Chromatography. Anal. Chem. 1959, 31 (4), 535–541.

(4) Paul, W.; Steinwedel, H. Ein Neues Massenspektrometer Ohne Magnetfeld. Zeitschrift für Naturforschung 1953, 8a, 448.

(5) Castaing, R.; Slodzian, G. Microanalyse Par Émission Ionique Secondaire (Microanalysis by Secondary Ion Emission). J. Microscopie 1962, 1, 395–410.

(6) Andersen, C. A.; Hinthorne, J. R. Ion Microprobe Mass Analyzer. Science 1972, 175 (4024), 853–860.

(7) United States of America, Plaintiff-Appellee, v. Hayward Leslie Brown, Defendant-Appellant. United States court of Appeals for the Sixth Circuit, 557 F.2d 541; 1977 U.S. App. LEXIS 12945: 1977.

(8) Houk, R. S.; Fassel, V. A.; Flesch, G. D.; Svec, H. J.; Gray, A. L.; Taylor, C. E., Inductively Coupled Argon Plasma as an Ion Source for Mass Spectrometric Determination of Trace Elements. Anal. Chem. 1980, 52 (14), 2283.

(9) Duckworth, D. C.; Bayne, C. K.; Morton, S. J.; Almirall, J. Analysis of Variance in Forensic Glass Analysis by ICP-MS: Variance Within the Method. J. Anal. Atom. Spectrom. 2000, 15 (7), 821-828. DOI: 10.1039/A908813J

(10) Parouchais, T.; Warner, I. M.; Palmer, L. T.; Kobus, H. The Analysis of Small Glass Fragments Using Inductively Coupled Plasma Mass Spectrometry. J. Forens. Sci. 1996, 41 (3), 351–360.

(11) Zurhaar, A.; Mullings, L. Characterisation of Forensic Glass Samples Using Inductively Coupled Plasma Mass Spectrometry. J. Anal. Atom. Spectrom. 1990, 5 (7), 611–617.

(12) Triplett, J. S.; Salyards, J.; Rodriguez-Cruz, S. E.; Morris, J. A.; Creel, D.; Zemmels, J.; Grabenauer, M. Evidence-Based Evaluation of the Analytical Schemes in ASTM E2329-17 Standard Practice for Identification of Seized Drugs for Methamphetamine Samples. Forensic Chem. 2024, 38, 100560.

(13) ASTM-E1618-19: Standard Test Method for Ignitable Liquid Residues in Extracts from Fire Debris Samples by Gas Chromatography-Mass Spectrometry. 2019.

(14) Jackson, G. P.; Barkett, M. A. Forensic Mass Spectrometry: Scientific and Legal Precedents. J. Am. Soc. Mass Spectrom. 2023, 34 (7), 1210–1224.

(15) Martin, R. J.; Alexander, T. G. Analytical Procedures Used in FDA Laboratories for the Analysis of Hallucinogenic Drugs. J. AOAC Int. 1968, 51, 159–163.

(16) Zoro, J. A.; Hadley, K. Organic Mass Spectrometry in Forensic Science. J. Forens. Sci. Soc. 1976, 16 (2), 103–114.

(17) United States of America, Plaintiff-Appellee, v. Martin Ross, Defendant-Appellant. United States Court of Appeals for the Second circuit, 719 F.2d 615; 1983 U.S. App. LEXIS 16076: 1983.

(18) United States of America, Plaintiff-Appellee, v. Gary Dale Posey, Defendant-Appellant. United States Court of Appeals, Tenth Circuit, 647 F.2d 1048; 1981 U.S. App. LEXIS 13733; 8 Fed. R. Evid. Serv. (Callaghan) 228: 1981.

(19) McClellan, B., Refusal to Accept Odd Coincidence Saved Stallings. St. Louis Post Dispatch 25 Sept, 1991.

(20) Shoemaker, J. D.; Lynch, R. E.; Hoffmann, J. W.; Sly, W. S. Misidentification of Propionic Acid as Ethylene Glycol in a Patient with Methylmalonic Acidemia. J. Pediatr. 1992, 120 (3), 417–421.

(21) United States of America, Plaintiff, v. 2,116 Boxes of Boned Beef Weighting Approximately 154,121 Pounds, and 541 Boxes of Offal Weighing Approximately 17,732 Pounds, Defendant. United States District Court for the District of Kansas, 516 F. Supp. 321; 1981 U.S. Dist. LEXIS 18559: 1981.

(22) Davidson, J. T.; Piacentino, E. L.; Sasiene, Z. J.; Abiedalla, Y.; DeRuiter, J.; Clark, C. R.; Berden, G.; Oomens, J.; Ryzhov, V.; Jackson, G. P. Identification of Novel Fragmentation Pathways and Fragment Ion Structures in the Tandem Mass Spectra of Protonated Synthetic Cathinones. Forensic Chem. 2020, 19, 100245.

(23) Davidson, J. T.; Sasiene, Z. J.; Jackson, G. P. The Characterization of Isobaric Product Ions of Fentanyl Using Multi-Stage Mass Spectrometry, High-Resolution Mass Spectrometry and Isotopic Labeling. Drug Test. Anal. 2020, 12, 496–503.

(24) Jackson, G. P.; Mehnert, S. A.; Davidson, J. T.; Lowe, B. D.; Ruiz, E. A.; King, J. R. Expert Algorithm for Substance Identification Using Mass Spectrometry: Statistical Foundations in Unimolecular Reaction Rate Theory. J. Am. Soc. Mass Spectrom. 2023, 34, 1248–1262.

(25) Mehnert, S. A.; Davidson, J. T.; Adeoye, A.; Lowe, B. D.; Ruiz, E. A.; King, J. R.; Jackson, G. P. Expert Algorithm for Substance Identification Using Mass Spectrometry: Application to the Identification of Cocaine on Different Instruments Using Binary Classification Models. J. Am. Soc. Mass Spectrom. 2023, 34, 1235–1247.

(26) Johnson, C. R.; Sabatini, H. M.; Aderorho, R.; Chouinard, C. D. Dependency of Fentanyl Analogue Protomer Ratios on Solvent Conditions as Measured by Ion Mobility-Mass Spectrometry. J. Mass Spectrom. 2024, 59 (8), e5070.

(27) Hollerbach, A. L.; Ibrahim, Y. M.; Lin, V. S.; Schultz, K. J.; Huntley, A. P.; Armentrout, P. B.; Metz, T. O.; Ewing, R. G. Identification of Unique Fragmentation Patterns of Fentanyl Analog Protomers Using Structures for Lossless Ion Manipulations Ion Mobility-Orbitrap Mass Spectrometry. J. Am. Soc. Mass Spectr. 2024, 35 (4), 793–803.

(28) Aderorho, R.; Chouinard, C. D. Determining Protonation Site in Fentanyl Protomers Using Ion Mobility-Aligned MS/MS Fragmentation. Int. J. Mass. Spectrom. 2024, 496, 117185.