LCGC International spoke to Heather Barkholtz and Maia Bates of the University of Wisconsin-Madison about their recent evaluation of a broad screening technique for high-priority drugs of abuse in whole blood.
A recent study conducted at the University of Wisconsin-Madison evaluated an untargeted workflow for broad toxicological screening of the most recent Tier I drugs of abuse and metabolites using liquid chromatography-high-resolution mass spectrometry (LC–HRMS), a popular technique for untargeted screening and presumptive identification of drugs of abuse because of its superior sensitivity and detection capabilities in complex matrices. The method proposed includes the untargeted extraction of analytes at biologically relevant concentrations from whole blood, and, to more deeply examine the strengths and weaknesses of the method, extraction recovery, ion suppression or enhancement, and precision were also assessed. LCGC International spoke to Heather Barkholtz and Maia Bates, two of the authors of the article resulting from this work.
In your paper (1), you state that the ever-changing landscape of illicit substances (street drugs) makes analysis complicated for forensic and clinical laboratories expected to provide a rapid screening of samples for a wide range of analytes. Can you briefly state some examples?
Heather Barkholtz: The unregulated drug market is experiencing a rapid proliferation of novel psychoactive substances (NPS). These are synthetic compounds designed to mimic the effects of traditional drugs like opioids, stimulants, or hallucinogens but often have slightly altered chemical structures to evade legal restrictions. A prominent example of novel psychoactive substances (NPS) is the rise of nitazenes, a new class of synthetic opioids. These compounds, designed to mimic traditional opioids, have slightly altered chemical structures that allow them to bypass legal restrictions. Nitazenes emerged in the illicit drug market after the U.S. and China implemented core-structure scheduling for fentanyl-related compounds. Because nitazenes lack fentanyl’s core structure, they initially evaded regulation. Their extreme potency, often surpassing fentanyl, poses a significant public health threat.
What, in your opinion, were the shortcomings of previous attempts to overcome these complications?
HB: Traditional methods often focus on a predefined list of analytes. With the constant emergence of new drugs, these targeted methods may miss substances not included in their panels. You will never find something if you don’t look for it.
Why do you believe that liquid chromatography-high-resolution mass spectrometry (LC–HRMS) has become a popular technique for untargeted screening and presumptive identification of drugs of abuse?
HB: I think LC–HRMS represents a paradigm shift in qualitative screening through the emergence of data-independent acquisition (DIA). This technique surveys the entire chemical environment and enables the detection of unknown substances without prior selection. When combined with high sensitivity and specificity and exact mass measure, toxicologists have new powerful tools to aid in elucidating NPS.
Briefly state your overall findings in your research.
HB: Our research demonstrated that an untargeted extraction and data acquisition method, using supported liquid extraction (SLE) and LC–HRMS is effective for broad screening of drugs of abuse and their metabolites. We were able to include multiple diverse analytes with acceptable selectivity and sensitivity for qualitative applications. That said, we observed variability in ionization and extraction recovery measures–which highlights the challenges in developing a universal method.
Do your findings correlate with what you had hypothesized or any preconceptions you might have had prior to your work?
HB: This method effectively screened a wide range of substances, supporting our hypothesis that a single extraction and data acquisition method could be used. We applied our method to a suite of 166 authentic specimens, which demonstrated its practical utility in forensic and clinical toxicology settings.
Was there anything particularly unexpected that stands out from your perspective?
HB: Given all the hype in the literature around LC–HRMS, we were surprised to see the variability in ionization and analyte recovery. Qualitative screening assays rarely include this level of method performance measures when published in the literature. That said, the point of our work was to scope both the strengths and weaknesses of these wide-scope screening approaches.
What were the major challenges you encountered in your work?
Maia Bates: A major challenge throughout this work was trying to maximize extraction efficiency while minimizing sample preparation and time. Due to the diversity of physiochemical properties within and across drug classes, developing an extraction method that could effectively extract every potential analyte is difficult. For our particular supported liquid extraction (SLE) method, cannabinoids were difficult to extract simultaneously alongside all the other analytes at the low concentrations considered.
What best practices can you recommend in this type of analysis for both instrument parameters and data analysis?
MB: We used a Waters Xevo G2-XS QToF mass spectrometer with an electrospray ionization source in positive and negative ionization mode, and data-independent acquisition with three MS functions (MSE) for our analysis. These parameters give us a comprehensive view of a sample with as much information as possible.
Can you please summarize the feedback that you have received from others regarding this work?
MB: The feedback has largely been positive and encouraging! Other forensic and clinical toxicology labs have been very interested in our findings as they are familiar with the limitations of modern drug screens. The adaptability and rapidness of our untargeted extraction and data acquisition method is promising for the high throughput demands of those labs.
The impetus for your research was the belief that there is still a lack of one extraction and data acquisition method that works well for all analytes and can meet the high throughput demands of a toxicology laboratory. Do you believe that you’ve overcome that challenge with your work?
MB: While this work has great potential to address the previously stated challenges, there is still much work to be done, particularly in the broad extraction portion of this work.
The proposed method includes the untargeted extraction of analytes at biologically relevant concentrations from whole blood. Do you believe that the method can be adapted to perhaps other bodily fluids, or perhaps skin, muscle, or bone?
MB: The proposed method could be tailored to extract analytes from alternative matrices, for instance, we have successfully used this method with urine and vitreous humor for presumptive drug screening. However, any solid samples such as tissue or bone would require more sample preparation than is outlined.
What are your next steps in this research?
MB: Our next step is to turn our focus to the more obscure and novel Tier II substances and evaluate the performance of our proposed method. A new class of synthetic opioids, benzimidazoles or “nitazenes,” have emerged and very little is known about them aside from their dangerous potency. We are also exploring using molecular networking as a powerful computational strategy that may help with visualization and interpretation of the complex data arising from HRMS analysis.
References
1. Bates, M. N.; Helm, A. E.; Barkholtz, H. M. Screening for Forensically Relevant Drugs Using Data-Independent High-Resolution Mass Spectrometry. Chem. Res. Toxicol. 2024, 37 (4), 571–579. DOI: 10.1021/acs.chemrestox.3c00379
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