Phillip M. Mach

Primary human hepatocytes (PHHs) are widely used as in vitro models for liver function and drug metabolism studies, yet their metabolic stability post-thawing remains an open question. To better characterize early metabolic changes, we conducted a time-course experiment using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze metabolic shifts in PHHs cultured in suspension. Unexposed and exposed (acetaminophen-treated) samples were evaluated, and TITAN analysis was applied to determine the time point of maximal metabolic change at both individual metabolite and global metabolic profile levels. Our results indicate that the majority of metabolic shifts occur within the first five hours post-thawing. In the early culture time points, substantial metabolic overlap was observed between unexposed and exposed cells, suggesting a conserved biological response likely related to cellular recovery. However, at later time points, metabolite profiles diverged, with acetaminophen treatment-specific metabolic changes emerging, potentially reflecting differences in homeostatic restoration versus hepatotoxic responses. Our study highlights the importance of considering early post-thawing metabolic dynamics in experimental design and offers insights for optimizing hepatocyte culture protocols to better replicate in vivo physiological conditions.

Illicit drug use causes over half a million deaths worldwide every year. Drugs of abuse are commonly smuggled through customs and border checkpoints and, increasingly, through parcel delivery services. Improved methods for detection of trace drug residues from surfaces are needed. Such methods should be robust, fieldable, sensitive, and capable of detecting a wide range of drugs. In this work, commercially produced paper with a pressure-sensitive adhesive coating was utilized for the collection and analysis of trace drug residues by paper spray mass spectrometry (MS). This modified substrate was used to combine sample collection of drug residues from surfaces with rapid detection using a single paper spray ticket. The all-in-one ticket was used to probe different surfaces commonly encountered in forensic work including clothing, cardboard, glass, concrete, asphalt, and aluminum. A total of 10 drugs (acetyl fentanyl, fentanyl, clonazolam, cocaine, heroin, ketamine, methamphetamine, methylone, U-47700, and XLR-11) were evaluated and found to be detectable in the picogram range using a benchtop mass spectrometer and in the low nanogram range using a portable ion trap MS. The novel approach demonstrates a simple yet effective sampling strategy, allowing for rapid identification from difficult surfaces via paper spray mass spectrometry.

The inhibition of acetylcholinesterase is regarded as the primary toxic mechanism of action for chemical warfare agents. Recently, there have been numerous reports suggesting that metabolic processes could significantly contribute to toxicity. As such, we applied a multi-omics pipeline to generate a detailed cascade of molecular events temporally occurring in guinea pigs exposed to VX. Proteomic and metabolomic profiling resulted in the identification of several enzymes and metabolic precursors involved in glycolysis and the TCA cycle. All lines of experimental evidence indicated that there was a blockade of the TCA cycle at isocitrate dehydrogenase 2, which converts isocitrate to α-ketoglutarate. Using a primary beating cardiomyocyte cell model, we were able to determine that the supplementation of α-ketoglutarate subsequently rescued cells from the acute effects of VX poisoning. This study highlights the broad impacts that VX has and how understanding these mechanisms could result in new therapeutics such as α-ketoglutarate.

Microneedles have been demonstrated to be a minimally invasive technique for sampling dermal interstitial fluid (ISF). Shotgun quantitative proteomics has already identified hundreds of proteins in ISF and quantitatively compared the proteome to matching serum and plasma. Interstitial fluid was determined to be a viable minimally invasive alternative to blood-derived fluids. In this communication, we re-examined the proteomic data from previous work to determine the diversity of immunoglobulins present compared to serum and plasma. Similar to our previous findings regarding the proteomic content across fluid types, ISF had a similar composition of IgG, IgA, IgD, and IgE antibodies as plasma or serum. However, IgM was expressed in lower quantities, which could limit utility of this fluid type for certain diagnostic applications. The work offers a proof of principle result of the approach previously developed towards the potential of ISF to be used in diagnostic purposes. Data are available via ProteomeXchange with identifier PXD012658.

Rationale: The analysis of chemical warfare agents (CWAs) from ambient atmosphere presents an analytical challenge due to their ease of degradation and volatility. Herein is described a method for derivatizing CWAs directly onto a paper spray substrate prior to analysis. This derivatization allows for much longer times of analysis without sample degradation and with little to no sample preparation. Methods: Derivatization was performed using 2‐[(dimethylamino)methyl] phenol both in‐vial and directly on paper spray cartridges. Solution studies were carried out over time and samples were analyzed via liquid chromatography/tandem mass spectrometry (LC/MS/MS) operated in positive ion mode. Paper spray substrates impregnated with the derivatizing agent prior to CWA vapor capture were also analyzed over time using a mass spectrometer operated in positive ion mode. Results: Use of 2‐[(dimethylamino)methyl] phenol as a paper spray substrate dopant enables derivatization of G‐series compounds into lower volatility complexes. The reaction occurs in solution and in the vapor phase. This new technique effectively traps and captures G‐series agents for analysis while extending the time for which the compound remains absorbed. The complex is highly suitable for direct analysis via paper spray mass spectrometry. Conclusions: Derivatization of paper spray substrates was shown to greatly increase the time for analysis of CWAs. This technique, combined with the vapor phase capture stage outlined previously, allows for rapid, quantitative CWA detection by paper spray ionization with little or no sample preparation.