Male lake char release taurocholic acid as part of a mating pheromone.

The evolutionary origins of sexual preferences for chemical signals remain poorly understood, due, in part, to scant information on the molecules involved. In the current study, we identified a male pheromone in lake char (Salvelinus namaycush) to evaluate the hypothesis that it exploits a non-sexual preference for juvenile odour. In anadromous char species, the odour of stream-resident juveniles guides migratory adults into spawning streams. Lake char are also attracted to juvenile odour but have lost the anadromous phenotype and spawn on nearshore reefs, where juvenile odour does not persist long enough to act as a cue for spawning site selection by adults. Previous behavioural data raised the possibility that males release a pheromone that includes components of juvenile odour. Using metabolomics, we found that the most abundant molecule released by males was also released by juveniles but not females. Tandem mass spectrometry and nuclear magnetic resonance were used to identify the molecule as taurocholic acid (TCA), which was previously implicated as a component of juvenile odour. Additional chemical analyses revealed that males release TCA at high rates via their urine during the spawning season. Finally, picomolar concentrations of TCA attracted pre-spawning and spawning females but not males. Taken together, our results indicate that male lake char release TCA as a mating pheromone and support the hypothesis that the pheromone is a partial match of juvenile odour.

In-line formation and identification of toxic reductive metabolites of aristolochic acid using electrochemistry mass spectrometry coupling.

Small-molecule metabolism has been extensively studied in the past decades, notably driven by the development of new pharmaceutical ingredients. The understanding of metabolism is critical to the anticipation of reactive metabolite formation in vivo that is often associated with toxicity. Electrochemistry has been proposed to simulate the oxidoreductive metabolism reaction catalyzed by cytochrome P450, a family of microsomal enzymes strongly involved in xenobiotic metabolism. The implementation of an electrochemical cell online with mass spectrometry allows for the fast formation and identification of the reaction end products. This study discusses the ability of the synthetic electrochemical approach to simulate a complex lactamization reaction that involves the formation of reactive metabolites. Aristolochic acid I was used as a model molecule to evaluate the ability of electrochemical simulation to generate nitroso, hydroxylamine, N-hydroxylactam, lactam, and nitrenium ion metabolites.

Bile acid production is life-stage and sex-dependent and affected by primer pheromones in the sea lamprey.

Pheromonal bile salts are important for sea lampreys (Petromyzon marinus Linnaeus) to complete their life cycle. The synthesis and release of a releaser/primer pheromone 3-keto petromyzonol sulfate (3kPZS) by spermiating males have been well characterized. 3kPZS evokes sexual behaviors in ovulatory females, induces immediate 3kPZS release in spermiating males, and elicits neuroendocrine responses in prespawning adults. Another primer pheromone released by spermiating males, 3-keto allocholic acid (3kACA), antagonizes the neuroendocrine effects of 3kPZS in prespermiating males. However, the effects of 3kACA and 3kPZS on pheromone production in prespawning adults is unclear. To understand the foundation of pheromone production, we examined sea lamprey bile salt levels at different life stages. To investigate the priming effects of 3kACA and 3kPZS, we exposed prespawning adults with vehicle or synthetic 3kACA or 3kPZS. We hypothesized that endogenous bile salt levels were life-stage and sex-dependent, and differentially affected by 3kACA and 3kPZS in prespawning adults. Using ultra-performance liquid chromatography tandem mass spectrometry, we found that sea lampreys contained distinct mixtures of bile salts in the liver and plasma at different life stages. Males usually contained higher amounts of bile salts than females. Petromyzonamine disulfate was the most abundant C27 bile salt and petromyzonol sulfate was the most abundant C24 bile salt. Waterborne 3kACA and 3kPZS exerted differential effects on bile salt production in the liver and gill, their circulation and clearance in the plasma, and their release into water. We conclude that bile salt levels are life-stage and sex-dependent and differentially affected by primer pheromones.

Evidence that male sea lamprey increase pheromone release after perceiving a competitor.

Sexual signals evolve via selective pressures arising from male-male competition and female choice, including those originating from unintended receivers that detect the signal. For example, males can acquire information from other males signaling to females and alter their own signal. Relative to visual and acoustic signals, less is known about how such communication networks influence chemical signaling among animals. In sea lamprey (Petromyzon marinus), the chemical communication system is essential for reproduction, offering a useful system to study a pheromone communication network that includes signalers and both intended and unintended receivers. Male sea lamprey aggregate on spawning grounds, where individuals build nests and signal to females using sex pheromones. We examined how exposure to a major component of the male pheromone, 3keto-petromyzonol sulfate (3kPZS), influenced male pheromone signaling, and whether females had a preference for males that altered their signal. Exposure to 3kPZS, at a concentration of 5×10-10 mol l-1, simulated the presence of other male(s) and led to increased 3kPZS release rates within 10 min, followed by a return to baseline levels within 30 min. Exposure also led to increases in hepatic synthesis and circulatory transport of pheromone components. In behavioral assays, females preferred the odor of males that had been exposed to 3kPZS; therefore, males likely benefit from upregulating 3kPZS release after detecting competition for mates. Here, we define how a specific pheromone component influences chemical signaling during intrasexual competition, and show a rare example of how changes in chemical signaling strategies resulting from male competition may influence mate choice.

Sex-dependent pheromonal effects on steroid hormone levels in sea lampreys (Petromyzon marinus).

Sea lampreys (Petromyzon marinus) are basal vertebrates that exhibit reproductive control via a hypothalamic-pituitary-gonadal axis. The function and evolution of the hypothalamic and pituitary peptide hormones are well studied in this species, whereas the functions of classical sex steroid hormones have not been well established due to their low or non-detectable plasma levels. Sea lamprey pheromone 3-keto petromyzonol sulfate (3kPZS) has been shown to increase while 3-keto allocholic acid (3kACA) decreases plasma 15α-hydroxyprogesterone (15αP) levels in prespermiating males (PSM) but not in preovulatory females (POF). However, spermiating male washings that contain both 3kPZS and 3kACA facilitate spawning in both sexes. Therefore, we wondered if the effects of pheromones on POF were elicited by classical steroid hormones such as progesterone, androstenedione, testosterone and estradiol. We hypothesized that waterborne 3kACA and 3kPZS differentially alter steroid hormone levels in prespawning sea lampreys. We determined the sex differences and pheromonal effects on steroid hormone levels in prespawning sea lampreys using sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods. Some PSM became spermiating (SM) at the time of sample collection, and those data were treated as a separate group. We found that males contained more androstenedione and progesterone in the gonad and more estradiol and testosterone in the plasma than POF, whereas POF contained more gonadal testosterone than males (no detectable gonadal testosterone). In POF, 3kPZS decreased gonadal androstenedione but increased gonadal progesterone and plasma estradiol, whereas 3kACA only increased gonadal progesterone levels. Exposure to 3kPZS for 4 h increased plasma 15αP in POF and SM, and gonadal 15αP in POF. Interestingly, 3kACA or 3kPZS depleted gonadal or plasma 15αP in PSM at various time points. On the other hand, both pheromones had no significant effect on androstenedione, progesterone or estradiol levels in males. Plasma testosterone levels did not change after pheromone exposure in both sexes. We conclude that sea lamprey pheromones 3kACA and 3kPZS induced differential steroidal responses in POF, PSM and SM.

Waterborne pheromones modulate gonadotropin-inhibitory hormone levels in sea lamprey (Petromyzon marinus).

The relationships between pheromone stimuli and neuropeptides are not well established in vertebrates due to the limited number of unequivocally identified pheromone molecules. The sea lamprey (Petromyzon marinus) is an advantageous vertebrate model to study the effects of pheromone exposure on neuropeptides since many pheromone molecules and neuropeptides have been identified in this species. Sexually mature male sea lamprey release pheromones 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3 keto-petromyzonol sulfate, 3kPZS) and 7α, 12α-dihydroxy-5α-cholan-3-one-24-oic acid (3-keto allocholic acid, 3kACA) that differentially regulate gonadotropin-releasing hormone (lGnRH) and steroid levels in sexually immature sea lamprey. However, the effects of these pheromones on gonadotropin-inhibitory hormones (GnIHs), hypothalamic neuropeptides that regulate lGnRH release, are still elusive. In this report, we sought to examine the effects of waterborne pheromones on lamprey GnIH-related neuropeptide levels in sexually immature sea lamprey. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analyses revealed sex differences in GnIH-related neuropeptide levels in the brain and plasma of immature sea lamprey. Exposure to 3kPZS and 3kACA exerted differential effects on GnIH-related neuropeptide levels in both sexes, but the effects were more prominent in female brains. We conclude that sea lamprey pheromones regulate GnIH-related neuropeptide levels in a sexually dimorphic manner.

American eels produce and release bile acid profiles that vary across life stage.

The American eel (Anguilla rostrata) is an imperilled fish hypothesized to use conspecific cues, in part, to coordinate long-distance migration during their multistage life history. Here, holding water and tissue from multiple American eel life stages was collected and analysed for the presence, profile and concentration of bile acids. Distinct bile acid profiles were identified in glass, elver, yellow eel and silver eel holding waters using ultraperformance liquid chromatography high-resolution mass spectrometry and principal component analysis. Taurochenodeoxycholic acid, taurodeoxycholic acid, cholic acid, deoxycholic acid, taurolithocholic acid and taurocholic acid were detected in whole tissue of American glass eels and elvers, and in liver, intestine and gallbladder samples of late-stage yellow eels. Bile acids were not a major component of silver eel washings or tissue. This study is novel because little was previously known about bile acids produced and emitted into the environment by American eels. Future behavioural studies could evaluate whether any bile acids produced by American eels influence conspecific migratory behaviour.

Metabolism of a sea lamprey pesticide by fish liver enzymes part B: method development and application in quantification of TFM metabolites formed in vivo.

The sea lamprey (Petromyzon marinus) is a destructive invasive species in the Great Lakes. Since the 1960s, tons of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) has been applied to selected tributaries each year to eliminate or reduce sea lamprey larval populations. Therefore, the environmental impact of TFM needs to be evaluated. However, the metabolism of TFM and its mechanism of selective toxicity in sea lamprey is not yet fully understood. Based upon our previous report on the identification, synthesis, and characterization of TFM metabolites observed in liver incubates from sea lamprey and non-target fishes, we now provide a robust assay for quantifying TFM and its metabolites in fish liver tissue. This method is important for assessing bioaccumulation of TFM in the ecosystems. The compounds purified in our previous report were used to develop and validate a quantitative ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for TFM and TFM metabolites formed in vivo. Several sample preparation techniques were compared, and a protein precipitation method was selected. The unavailability of stable isotopic internal standards was overcome by using a matrix matching method. After a thorough validation, this method was applied to determine the concentrations of TFM and its metabolites in fish liver tissues from animals exposed to TFM, and in the comparison between dead animals and survivors. Seven of eight expected metabolites were observed, some for the first time in vivo. Our results indicate that in vivo nitroreduction, glucuronidation, sulfation, and glutathione conjugation are involved in TFM metabolism in sea lamprey.

Metabolism of a sea lamprey pesticide by fish liver enzymes part A: identification and synthesis of TFM metabolites.

The sea lamprey (Petromyzon marinus) is a destructive invasive species in the Great Lakes that contributed to the collapse of native fish populations in the mid-1900s. 3-Trifluoromethyl-4-nitrophenol (TFM) is a selective pesticide that has been applied to sea lamprey infested tributaries of the Great Lakes to kill larvae since the 1960s and has reduced the populations by as much as 90%. However, the metabolism of TFM by sea lamprey and non-target species is not fully illuminated. Elucidation of TFM metabolism is critical for understanding its mode of action and possible environmental impact. Here, we describe the screening, identification, synthesis and structural characterization of TFM metabolites in livers from sea lamprey and three non-target species that differ in their ability to survive TFM exposure. We identified glucuronidation, sulfation, N-acetylation, glutathione conjugation, and aromatic nitro group reduction as potential detoxification mechanisms. Seven metabolites were synthesized for use as markers of TFM metabolism in fish. Quantitative 1H NMR was used to assay synthesized metabolite stock solutions that were then used as standard material to develop a quantitative LC-MS/MS method for TFM metabolites.

Quantification of Oxidized and Unsaturated Bile Alcohols in Sea Lamprey Tissues by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

A sensitive and reliable method was developed and validated for the determination of unsaturated bile alcohols in sea lamprey tissues using liquid-liquid extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The liver, kidney, and intestine samples were extracted with acetonitrile and defatted by n-hexane. Gradient UHPLC separation was performed using an Acquity BEH C18 column with a mobile phase of water and methanol containing 20 mM triethylamine. Multiple reaction monitoring modes of precursor-product ion transitions for each analyte was used. This method displayed good linearity, with correlation coefficients greater than 0.99, and was validated. Precision and accuracy (RSD %) were in the range of 0.31%-5.28%, while mean recoveries were between 84.3%-96.3%. With this technique, sea lamprey tissue samples were analyzed for unsaturated bile alcohol analytes. This method is practical and particularly suitable for widespread putative pheromone residue analysis.

Hsp90 and hepatobiliary transformation during sea lamprey metamorphosis.

BACKGROUND: Biliary atresia (BA) is a human infant disease with inflammatory fibrous obstructions in the bile ducts and is the most common cause for pediatric liver transplantation. In contrast, the sea lamprey undergoes developmental BA with transient cholestasis and fibrosis during metamorphosis, but emerges as a fecund adult. Therefore, sea lamprey liver metamorphosis may serve as an etiological model for human BA and provide pivotal information for hepatobiliary transformation and possible therapeutics. RESULTS: We hypothesized that liver metamorphosis in sea lamprey is due to transcriptional reprogramming that dictates cellular remodeling during metamorphosis. We determined global gene expressions in liver at several metamorphic landmark stages by integrating mRNA-Seq and gene ontology analyses, and validated the results with real-time quantitative PCR, histological and immunohistochemical staining. These analyses revealed that gene expressions of protein folding chaperones, membrane transporters and extracellular matrices were altered and shifted during liver metamorphosis. HSP90, important in protein folding and invertebrate metamorphosis, was identified as a candidate key factor during liver metamorphosis in sea lamprey. Blocking HSP90 with geldanamycin facilitated liver metamorphosis and decreased the gene expressions of the rate limiting enzyme for cholesterol biosynthesis, HMGCoA reductase (hmgcr), and bile acid biosynthesis, cyp7a1. Injection of hsp90 siRNA for 4 days altered gene expressions of met, hmgcr, cyp27a1, and slc10a1. Bile acid concentrations were increased while bile duct and gall bladder degeneration was facilitated and synchronized after hsp90 siRNA injection. CONCLUSIONS: HSP90 appears to play crucial roles in hepatobiliary transformation during sea lamprey metamorphosis. Sea lamprey is a useful animal model to study postembryonic development and mechanisms for hsp90-induced hepatobiliary transformation.

In situ ultrafast 2D NMR spectroelectrochemistry for real-time monitoring of redox reactions.

The in situ implementation of an electrochemical cell (EC) inside a nuclear magnetic resonance (NMR) spectrometer is extremely powerful to study redox reactions in real time and identify unstable reaction intermediates. Unfortunately, the implementation of an electrochemical device near the sensitive volume of an NMR probe significantly affects the quality of the NMR signal, inducing significant line broadening resulting in peak overlap and partial loss of the multiplet structures. Two-dimensional (2D) NMR spectroscopy allows one to bypass signal overlapping by spreading the peaks along two orthogonal dimensions, while providing precious information in terms of structural elucidation. Nevertheless, the acquisition of 2D NMR data suffers from long acquisition durations which are incompatible with fast redox processes taking place in solution. Here, we present a new approach to deal with this issue, consisting of coupling EC-NMR with ultrafast 2D spectroscopy, capable of recording 2D spectra much faster than conventional 2D NMR. This approach is applied to the real-time monitoring of a model reaction. Fast correlation spectroscopy (COSY) spectra are recorded every 3 min in the course of the 80 min reaction, leading to the unambiguous identification of one reaction intermediate and two reaction products. The evolution of 2D NMR peak volumes in the course of time provides further insight into the mechanism of this reaction involving an unstable intermediate. This study demonstrates the feasibility and the relevance of coupling in situ spectroelectrochemistry with ultrafast 2D spectroscopy to monitor real-time electrochemical reactions in the NMR tube.

Unexpected benzimidazole ring formation from a quinoneimide species in the presence of ammonium acetate as supporting electrolyte used in the coupling of electrochemistry with mass spectrometry.

RATIONALE: Electrochemistry (EC) coupled to mass spectrometry (MS) has been used to study different phase-I reactions. Despite of the versatility of EC/MS, the effect of the nature of the supporting electrolyte on the formation of oxidation products has seldom been discussed during EC/MS experiments. Here, we present a comparison of two different supporting electrolytes and their effect on the identification of unstable intermediate oxidation species is discussed. METHODS: The oxidation of acebutolol was performed with a coulometric cell in the presence of two supporting electrolytes namely ammonium acetate and lithium acetate. Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/QTOFMS) using a binary gradient (water/acetonitrile) with positive electrospray ionization was used to identify the oxidation products in the presence and absence of glutathione. Chemical structure elucidations of the oxidation products were performed by high-resolution mass spectrometry (HRMS) and were also supported by nuclear magnetic resonance (NMR) measurements. RESULTS: From the electrochemical study and HRMS measurements, we demonstrate that the quinoneimide species resulting from the oxidative hydrolyses of acebutolol gives a benzimidazole ring product in the presence of ammonium acetate. Through the example of the oxidation of acebutolol, a correlation between the supporting electrolyte nature and oxidation product formation was established. The obtained results were supported by quantum mechanical calculations. CONCLUSIONS: We present here evidence of the side reactions induced by the presence of ammonia as supporting electrolyte during EC/MS measurements. Acebutolol was used as a model to postulate an uncommon and unexpected side reaction leading to benzimidazole ring formation. The findings may help to understand the identification of the intermediate species in the oxidative degradation process.

A quantitative assay for reductive metabolism of a pesticide in fish using electrochemistry coupled with liquid chromatography tandem mass spectrometry.

This is the first study to use electrochemistry to generate a nitro reduction metabolite as a standard for a liquid chromatography-mass spectrometry-based quantitative assay. This approach is further used to quantify 3-trifluoromethyl-4-nitrophenol (TFM) reductive metabolism. TFM is a widely used pesticide for the population control of sea lamprey (Petromyzon marinus), an invasive species of the Laurentian Great Lakes. Three animal models, sea lamprey, lake sturgeon (Acipenser fulvescens), and rainbow trout (Oncorhynchus mykiss), were selected to evaluate TFM reductive metabolism because they have been known to show differential susceptibilities to TFM toxicity. Amino-TFM (aTFM; 3-trifluoromethyl-4-aminophenol) was the only reductive metabolite identified through liquid chromatography-high-resolution mass spectrometry screening of liver extracts incubated with TFM and was targeted for electrochemical synthesis. After synthesis and purification, aTFM was used to develop a quantitative assay of the reductive metabolism of TFM through liquid chromatography and tandem mass spectrometry. The concentrations of aTFM were measured from TFM-treated cellular fractions, including cytosolic, nuclear, membrane, and mitochondrial protein extracts. Sea lamprey extracts produced the highest concentrations (500 ng/mL) of aTFM. In addition, sea lamprey and sturgeon cytosolic extracts showed concentrations of aTFM substantially higher than those of rainbow trout. However, other fractions of lake sturgeon extracts tend to show aTFM concentrations similar to those of rainbow trout but not with sea lamprey. These data suggest that the level of reductive metabolism of TFM may be associated with the sensitivities of the animals to this particular pesticide.

Simultaneous determination of gonadotropin-inhibitory and gonadotropin-releasing hormones using ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry.

Gonadotropin-inhibitory hormones (GnIH) and gonadotropin-releasing hormones (GnRH) are neuropeptides essential for the regulation of reproduction in all vertebrate animals examined. Determination of neuropeptides in the biological sample is highly challenging due to their complex matrix and weak stability. The wide variety of peptides or protein degradation products often interferes with the determination of the target peptide. This study aims to develop a specific ultra-high performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of nine critical neuropeptides in biological samples. A separation method by ultra-performance liquid chromatography coupled to a multiple reaction monitoring (MRM) by tandem mass spectrometry allows the selective determination of the neuropeptides in brain and plasma matrices after solid-phase extraction. Specific MSMS transitions were optimized using MRM of multiple-charged peptides generated by electrospray ionization in positive mode. The resulting analytical method was fully validated with thorough evaluation of stability, recovery, matrix effect, and intra- and interday accuracy and precision in sea lamprey brain and plasma. The optimized method shows linearity in a wide range of concentrations with limit of quantification ranging from 0.1 to 0.75 ng/mL. With slight modification, this method can be applied to other biological samples.

Recent advances and applications of experimental technologies in marine natural product research.

Marine natural products are a rich source of novel and biologically active compounds. The number of identified marine natural compounds has grown 20% over the last five years from 2009 to 2013. Several challenges, including sample collection and structure elucidation, have limited the development of this research field. Nonetheless, new approaches, such as sampling strategies for organisms from extreme ocean environments, nanoscale NMR and computational chemistry for structural determination, are now available to overcome the barriers. In this review, we highlight the experimental technology innovations in the field of marine natural products, which in our view will lead to the development of many new drugs in the future.

Iso-petromyroxols: novel dihydroxylated tetrahydrofuran enantiomers from sea lamprey (Petromyzon marinus).

An enantiomeric pair of new fatty acid-derived hydroxylated tetrahydrofurans, here named iso-petromyroxols, were isolated from sea lamprey larvae-conditioned water. The relative configuration of iso-petromyroxol was elucidated with 1D and 2D NMR spectroscopic analyses. The ratio of enantiomers (er) in the natural sample was measured by chiral-HPLC-MS/MS to be ca. 3:1 of (-)- to (+)-antipodes.

Advances in the electrochemical simulation of oxidation reactions mediated by cytochrome p450.

Combining electrochemistry with mass spectrometry constitutes an increasingly useful approach for simulating reactions catalyzed by cytochrome P450 (CYP450). In this review, we discuss the ability of the electrochemical cell to act as a reliable tool to mimic CYP450. The electrochemical oxidation process and CYP450-catalyzed reactions are compared in terms of mechanistic pathways, chemical structures of reactive intermediate metabolites, and final chemical structures of oxidation products. The oxidation reactions mediated by CYP450 are known to occur by either a single electron transfer (SET) or a hydrogen atom transfer (HAT) mechanism. The similarities between the reactions mediated electrochemically or by CYP450 are discussed in terms of SET and HAT mechanisms.

Phase I and phase II reductive metabolism simulation of nitro aromatic xenobiotics with electrochemistry coupled with high resolution mass spectrometry.

Electrochemistry combined with (liquid chromatography) high resolution mass spectrometry was used to simulate the general reductive metabolism of three biologically important nitro aromatic molecules: 3-trifluoromethyl-4-nitrophenol (TFM), niclosamide, and nilutamide. TFM is a pesticide used in the Laurential Great Lakes while niclosamide and nilutamide are used in cancer therapy. At first, a flow-through electrochemical cell was directly connected to a high resolution mass spectrometer to evaluate the ability of electrochemistry to produce the main reduction metabolites of nitro aromatic, nitroso, hydroxylamine, and amine functional groups. Electrochemical experiments were then carried out at a constant potential of -2.5 V before analysis of the reduction products by LC-HRMS, which confirmed the presence of the nitroso, hydroxylamine, and amine species as well as dimers. Dimer identification illustrates the reactivity of the nitroso species with amine and hydroxylamine species. To investigate xenobiotic metabolism, the reactivity of nitroso species to biomolecules was also examined. Binding of the nitroso metabolite to glutathione was demonstrated by the observation of adducts by LC-ESI(+)-HRMS and the characteristics of their MSMS fragmentation. In conclusion, electrochemistry produces the main reductive metabolites of nitro aromatics and supports the observation of nitroso reactivity through dimer or glutathione adduct formation.

In situ NMR spectroelectrochemistry for the structure elucidation of unstable intermediate metabolites.

In situ NMR spectroelectrochemistry is presented in this study as a useful hybrid technique for the chemical structure elucidation of unstable intermediate species. An experimental setting was designed to follow the reaction in real time during the experimental electrochemical process. The analysis of (1)H NMR spectra recorded in situ permitted us (1) to elucidate the reaction pathway of the electrochemical oxidation of phenacetin and (2) to reveal the quinone imine as a reactive intermediate species without using any trapping reaction. Phenacetin has been considered as hepatotoxic at high therapeutic amounts, which is why it was chosen as a model to prove the applicability of the analytical method. The use of 1D and 2D NMR experiments led to the elucidation of the major species produced from the oxidation process. We demonstrated that in situ NMR spectroelectrochemistry constitutes a fast way for monitoring unstable quinone imines and elucidating their chemical structures.