Drug abuse and involuntary drug exposure have far-reaching consequences for individuals, families, and communities. Investigators often need forensic toxicologists to provide evidence of an individual's history of drug use or exposure, even if it dates further back than commonly used tests can determine. Such testing can detect drug-facilitated sexual assault, compliance with probation and parole, or compliance with addiction rehabilitation programs.
When a human body breaks down a drug, it forms drug metabolites that can be analyzed in blood or urine, among other samples. Forensic toxicologists assess drug use and drug exposure by looking for the presence of these metabolites and measuring them. However, the body tends to clear drug metabolites within one week, and often sooner. Therefore, detecting drug use after the fact (sometimes known as retrospective drug monitoring) presents a challenge.
Improving Retrospective Drug Monitoring
To increase the window of time that a drug exposure can be detected, researchers from Florida International University have developed an innovative, sensitive, and specific method to detect drug exposure which is fundamentally different than traditional toxicological drug testing.
The research team, led by Dr. Anthony DeCaprio, developed and validated the procedure (or assay) to detect and measure drug exposure based on modifications to blood proteins caused by the body’s reaction to a drug. The assay is based on the premise that drugs modify blood proteins. Specifically, drugs can add small chemical groups to proteins, which alter the proteins’ structures and functions. Researchers can identify these changes using mass spectrometry. Although widely used in studies of environmental and occupational chemical exposure, scientists have not used this technique to detect drug use before now.
Researchers from Dr. DeCaprio’s group focused on a specific type of protein modification known as protein adduction. Protein adducts form when proteins are exposed to certain drugs, the protein and the reactive metabolites of the drug bond together.
This helps detect drug use that occurred further in the past because these bonds typically persist for the life of the protein; therefore, researchers can detect drugs for much longer than metabolites that are measured in blood or urine. (See “Stable vs. Reactive Metabolites.”)
Dr. DeCaprio and colleagues hypothesized that they could conduct retrospective drug monitoring by measuring changes in blood proteins. In 2015, the research team showed that they could modify a specific protein called glutathione by using the reactive metabolites of 16 select drugs.
Based on that work, the team created a stronger method for retrospective drug monitoring in 2017. They added two more proteins — hemoglobin and serum albumin — to their assay and looked at both reactive and stable drug metabolites. Specifically, they wanted to examine the thiol group , a chemical structure attached to every hemoglobin protein, to see if they could use it as a target for assessing retrospective drug exposure. They chose hemoglobin and serum albumin because both proteins persist for about four months (the life of a red blood cell) — far longer than the one-week capabilities of previous testing methods.
The researchers examined the following commonly abused drugs: methamphetamine, MDMA, Δ9-THC, oxycodone, cocaine, and diazepam. They also used acetaminophen and clozapine, which are well-characterized drugs that form adducts, as positive controls to ensure proper adduct formation occurred.
The researchers successfully:
- Developed and adapted assays to generate and identify stable and reactive metabolites of eight drugs.
- Confirmed that hemoglobin was modified by acetaminophen, clozapine, oxycodone, cocaine, ∆9-THC, and diazepam (and was not modified by MDMA or methamphetamine).
- Identified the adduct structure created in the modified proteins.
- Developed a novel procedure to enrich drug-modified hemoglobin and remove unmodified proteins, an approach that greatly increases sensitivity for detecting drug modified proteins.
Due to delays from the COVID-19 pandemic, the researchers were unable to complete the final phase of their proposed research, which included developing a screening method for routine detection of drugs adducted to hemoglobin and serum albumin and testing the method with authentic samples. However, they are making progress toward this final goal under NIJ award 2020-R2-CX-0023 and other sources.
Advantages: Sensitive, Specific, and Wide-Ranging
The novel blood protein modification assay developed by the researchers has some advantages over currently available methods for detecting drug exposure. First, the assay is highly sensitive and specific, meaning that it can detect drug exposure even in cases where the drug is no longer detectable in blood or urine samples. Second, the assay is based on modifications to blood proteins rather than the drug itself or its metabolites. This means that it can detect exposure to a wider range of drugs and can potentially identify obscured or disguised drug exposure.
“Basically, this is a potential answer to the problem of delays in reporting potential drug-facilitated crimes and drug-facilitated sexual assaults. As we know, sexual assault victims often do not report right away, if at all. If there is a question of a drug having been administered surreptitiously, that delay usually means that the window for traditional blood drug testing has passed.”
—Frances Scott, Ph.D., physical scientist at the National Institute of Justice
The assay has important implications for forensic investigations, particularly in cases where drug use is relevant but difficult to detect. For example, investigators could use the assay to retrospectively detect drug use in cases involving impaired driving, workplace accidents, or criminal investigations. Substance abuse treatment programs could also use the assay to help monitor individuals.
Researchers anticipate that this novel approach will significantly benefit criminal justice research by providing an additional tool to detect and quantify important agents of forensic interest over longer periods of time.
Sidebar: Stable vs. Reactive Metabolites
Metabolite: A metabolite is a substance made when the body breaks down a drug.
Reactive Metabolites: Reactive metabolites are unstable in the body and may react further, such as binding covalently (with a strong bond) to molecules such as proteins or DNA. In this study, researchers looked for reactive drug metabolites that bind to the hemoglobin and serum albumin proteins (a process called adduction), thereby altering the structure of the protein. Mass spectrometry can identify this altered protein. The researchers used acetaminophen and clozapine as positive controls in their experiment because their ability to adduct is well characterized.
Stable Metabolites: Stable metabolites, by contrast, are stable in the body and will not react further. They will be excreted unchanged and can be detected directly if present in sufficient quantity.
About This Article
The work described in this article was supported by NIJ grant number 2017-MU-BX-0002, awarded to The Florida International University board of Trustees. This article is based on the grantee report “Novel Blood Protein Modification Assay for Retrospective Detection of Drug Exposure” (pdf, 63 pages), by Anthony P. DeCaprio, Ph.D.
[note 1] Drug use can also be measured from hair, but this method is not without its own set of problems, including differences in hair growth rates, differences in drug binding based on melanin content of hair (and related potential racial/ethnicity equity concerns), and the need to then wait four to eight weeks to get an appropriate sample.
[note 2] Thiol groups, also known as sulfhydryl groups, consist of a sulfur atom bonded to a hydrogen.
[note 3] Researchers used two types of mass spectrometry: 1) Liquid Chromatographic-Quadrupole Time-of-Flight-Mass Spectrometry (LC-QTOF-MS) and 2) Liquid Chromatographic-Triple Quadrupole-Tandem Mass Spectrometry (LC-QqQ-MS).