Elucidation of the dissociation pathways of electro‐ionized estrone

With the future aim of using gas chromatography coupled with mass spectrometry to characterize the transformation products of estrone submitted to UV‐photolysis or to waste water treatment plants, an interpretation of the electron impact mass spectrum of estrone is presented. Fragmentation mechanisms are proposed on the basis of high‐resolution measurements performed with a magnetic sector analyzer. Multiple‐stage mass spectrometry experiments were carried out using an ion trap mass spectrometer. The structures proposed for product ions were confirmed by the m/z shifts observed in the estrone‐d₄ and estrone methyl ether electron ionization mass spectra. If the formation of some of the most abundant ions may easily be explained by α‐cleavages and retro‐Diels‐Alder type rearrangements, complex mechanisms need to be considered to rationalize the formation of others. Isotope labelling allows discrimination of isobaric ions. Copyright © 2010 John Wiley & Sons, Ltd.     

Study of the forced degradation behavior of prasugrel hydrochloride by liquid chromatography with mass spectrometry and liquid chromatography with NMR detection and prediction of the toxicity of the characterized degradation products

Prasugrel was subjected to forced degradation studies under conditions of hydrolysis (acid, base, and neutral), photolysis, oxidation, and thermal stress. The drug showed liability in hydrolytic as well as oxidative conditions, resulting in a total of four degradation products. In order to characterize the latter, initially mass fragmentation pathway of the drug was established with the help of mass spectrometry/time‐of‐flight, multiple stage mass spectrometry and hydrogen/deuterium exchange data. The degradation products were then separated on a C₁₈ column using a stability‐indicating volatile buffer method, which was later extended to liquid chromatography‐mass spectrometry studies. The latter highlighted that three degradation products had the same molecular mass, while one was different. To characterize all, their mass fragmentation pathways were established in the same manner as the drug. Subsequently, liquid chromatography‐nuclear magnetic resonance (NMR) spectroscopy data were collected. Proton and correlation liquid chromatography with NMR spectroscopy studies highlighted existence of diastereomeric behavior in one pair of degradation products. Lastly, toxicity prediction by computer‐assisted technology (TOPKAT) and deductive estimation of risk from existing knowledge (DEREK) software were employed to assess in silico toxicity of the characterized degradation products.     

Photolysis and photocatalysis of ibuprofen in aqueous medium: characterization of by‐products via liquid chromatography coupled to high‐resolution mass spectrometry and assessment of their toxicities against Artemia Salina

The degradation of the pharmaceutical compound ibuprofen (IBP) in aqueous solution induced by direct photolysis (UV‐A and UV‐C radiation) and photocatalysis (TiO₂/UV‐A and TiO₂/UV‐C systems) was evaluated. Initially, we observed that whereas photocatalysis (both systems) and direct photolysis with UV‐C radiation were able to cause an almost complete removal of IBP, the mineralization rates achieved for all the photodegradation processes were much smaller (the highest value being obtained for the TiO₂/UV‐C system: 37.7%), even after an exposure time as long as 120 min. Chemical structures for the by‐products formed under these oxidative conditions (11 of them were detected) were proposed based on the data from liquid chromatography coupled to high‐resolution mass spectrometry (LC‐HRMS) analyses. Taking into account these results, an unprecedented route for the photodegradation of IBP could thus be proposed. Moreover, a fortunate result was achieved herein: tests against Artemia salina showed that the degradation products had no higher ecotoxicities than IBP, which possibly indicates that the photocatalytic (TiO₂/UV‐A and TiO₂/UV‐C systems) and photolytic (UV‐C radiation) processes can be conveniently employed to deplete IBP in aqueous media. Copyright © 2014 John Wiley & Sons, Ltd.     

Study of degradation behavior of besifloxacin,characterization of its degradation products by LC–ESI–QTOF–MS and their in silico toxicity prediction

Knowledge and understanding of the stability profile of a drug is important as it affects its safety and efficacy. In the present work, besifloxacin, a new, fourth‐generation fluoroquinolone antibiotic, was subjected to different forced‐degradation conditions as per International Conference on Harmonization (ICH) guidelines such as hydrolysis (acid, base and neutral), oxidation, thermal and photolysis. The drug degraded under acidic, basic, oxidative and photolytic conditions while it was found to be stable under dry heat and neutral hydrolytic conditions. In total, five degradation products (DPs) were formed under different conditions—DP1 and DP2 (photolysis), DP3 (oxidation), DP4 (acidic), DP3 and DP5 (basic). The chromatographic separation of besifloxacin and its degradation products was achieved on a Sunfire C₁₈ (250 mm × 4.6 mm, 5 μm) column with 0.1% aqueous formic acid–acetonitrile as a mobile phase. The gradient RP‐HPLC method was developed and validated as per ICH guidelines. The degradation products were characterized with the help of LC–ESI–QTOF mass spectrometric studies and the most likely degradation pathway of the drug was proposed. In silico toxicity assessment of the drug and its degradation products was carried out, which indicated that DP3 and DP4 carry a mutagenicity alert.     

Photochimie d'hétérocycles azotés. IV. Transposition de type photo-fries des N-acétyl benzimidazolones

The photolysis of benzene or alcoholic solutions of N-acetyl-and N,N-diacetylbenzimidazolones gave mixtures of ortho- and para-rearrangements products accompanied to some extent with de-N-acylation. The different products have been identified by means of nmr, mass spectrometry and unambiguous synthesis.     

Characterisation of captopril photolysis and photocatalysis by-products in water by direct infusion,electrospray ionisation,high-resolution mass spectrometry and the assessment of their toxicities

Pharmaceuticals of different therapeutic classes are found in the environment. Captopril is used worldwide as an antihypertensive drug, and it has been found in the influent, effluent and secondary sludge of wastewater treatment plants. Advanced oxidation processes, such as direct photolysis (UV-C) and heterogeneous photocatalysis (TiO₂/UV-C), are alternatives to enhance mineralisation of pharmaceuticals and their removal during water treatment. In this article, it was evaluated the degradation of captopril in aqueous solution induced by UV-C and TiO₂/UV-C systems. The process focused on the identification and monitoring of the by-products formed under these conditions by applying direct-infusion electrospray ionisation high-resolution mass spectrometry in the negative ion mode (ESI(-)-HRMS) and high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC/HRMS). To evaluate the by-products toxicity, acute ecotoxicity tests were performed with the crustacean Artemia salina, and the cytotoxicity was evaluated with (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using HepG2 cells. It was observed by ESI(-)-HRMS that after 120 min of light exposure, there was almost complete removal of captopril, with 93.5% removal efficiency during photolysis and 99.9% during photocatalysis. At these conditions, the rate of mineralisation, by total organic carbon (TOC), was only 2.92% for photolysis and 9.09% for photocatalysis, evidencing the formation of degradation by-products. Nine by-products of captopril photodegradation were identified, and their respective chemical structure elucidations were proposed. The treated samples were nontoxic to A. salina and HepG2 cells, indicating that both oxidative treatments (photocatalytic or photolytic processes) can be conveniently employed to remove captopril from aqueous media.     

Time‐of‐flight mass spectrometry for time‐resolved measurements: Some developments and applications

In this paper, the time resolution for kinetic studies of reactions with mass spectrometric detection is characterized in detail, and it is shown how this allows faster kinetic processes to be determined. The time‐resolved technique used pulsed laser photolysis to initiate reaction and a time‐of‐flight mass spectrometer (TOFMS) to monitor progress, where the reactant gas was sampled by a sampling orifice and photoionized using pulsed, laser vacuum ultraviolet light before being analyzed by the TOFMS. Characterization of this setup has been carried out to identify the parameters that affect the time for “sampling,” which limits the fastest reactions that can be measured. A simple mathematical equation has been developed to correct for “sampling” delays (k_sampling～25, 000 s^?1), which extends the range of rate coefficients to be measured in a kinetic mass spectrometry reactor to k′ < 7000 s^?1. This method could be applied to any other kinetic mass spectrometry system where k_sampling can be measured; an important advantage since it allows the study of reactions over a wider range of conditions (e.g., larger concentrations of reagents/products can be used to minimize the contribution from wall losses). The system can produce reliable kinetic data whether monitoring reactant decay or product growth even when the reaction and sampling processes are occurring on a similar timescale (k′ < 7000 s^?1). Reproducible and reliable kinetic data have been obtained for the following reactions: SO + NO₂ → products (R1), ClSO + NO₂ → products (R2), where SO and ClSO were monitored under pseudo‐first‐order conditions, and HCO + O₂ → CO + HO₂ (R3), where CO was monitored by a [1+1] resonance enhanced ionization multiphoton ionization (REMPI) scheme with HCO reacting under pseudo–first‐order conditions. The limitations and potential developments of this setup are described. © 2011 Wiley Periodicals, Inc. Int J Chem Kinet 44: 532–545, 2012     

Reactivity of Tyr–Leu and Leu–Tyr dipeptides: identification of oxidation products by liquid chromatography–tandem mass spectrometry

The exposure of peptides and proteins to reactive hydroxyl radicals results in covalent modifications of amino acid side‐chains and protein backbone. In this study we have investigated the oxidation the isomeric peptides tyrosine–leucine (YL) and leucine–tyrosine (LY), by the hydroxyl radical formed under Fenton reaction (Fe²⁺/H₂O₂). Through mass spectrometry (MS), high‐performance liquid chromatography (HPLC‐MS) and electrospray tandem mass spectrometry (HPLC‐MSⁿ) measurements, we have identified and characterized the oxidation products of these two dipeptides. This approach allowed observing and identifying a wide variety of oxidation products, including isomeric forms of the oxidized dipeptides. We detected oxidation products with 1, 2, 3 and 4 oxygen atoms for both peptides; however, oxidation products with 5 oxygen atoms were only present in LY. LY dipeptide oxidation leads to more isomers with 1 and 2 oxygen atoms than YL (3 vs 5 and 4 vs 5, respectively). Formation of the peroxy group occurred preferentially in the C‐terminal residue. We have also detected oxidation products with double bonds or keto groups, dimers (YL–YL and LY–LY) and other products as a result of cross‐linking. Both amino acids in the dipeptides were oxidized although the peptides showed different oxidation products. Also, amino acid residues have shown different oxidation products depending on the relative position on the dipeptide. Results suggest that amino acids in the C‐terminal position are more prone to oxidation. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of bioactive components and multi‐component pharmacokinetics of saponins from the leaves of Panax notoginseng in rat plasma after oral administration by LC–MS/MS

In this study, simple ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass tandem mass spectrometry is used to characterize the absorbed components in rat plasma after the oral administration of saponins from the leaves of Panax notoginseng. Seventeen prototype compounds are structurally characterized. Furthermore, a simple and sensitive liquid chromatography with tandem mass spectrometry method is also used for the simultaneous determination of notoginsenoside Fc, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb3, ginsenoside Rd, and notoginsenoside Fe in rat plasma within 5 min. After n‐butanol mediated liquid–liquid extraction, all analytes were separated on a C₁₈ column and monitored in negative ion mode. Linearity, sensitivity, intra‐ and inter‐assay precision, accuracy, recovery, matrix effect, and stability were all within acceptable ranges. The validated liquid chromatography with tandem mass spectrometry method is successfully applied to the pharmacokinetic study of saponins from the leaves of Panax notoginseng in rats after oral administration. The results suggest that notoginsenoside Fc and ginsenoside Rb3 showed relatively higher exposure compared with other saponins. All saponins showed a long duration in plasma with a t_1/2 longer than 15 h, except notoginsenoside Fe (t_1/2 = 2.78 h). This study provides important information about the metabolism of saponins from the leaves of Panax notoginseng, which is useful for completely understanding its mechanism of action.     

Quantification of peptides using N‐terminal isotope coding and C‐terminal derivatization for sensitive analysis by micro liquid chromatography‐tandem mass spectrometry

Stable isotope‐coding coupled with mass spectrometry is a popular method for quantitative proteomics and peptide quantification. However, the efficiency of the derivatization reaction at a particular functional group, especially in complex structures, can affect accuracy. Here, we present a dual functional‐group derivatization of bioactive peptides followed by micro liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). By separating the sensitivity‐enhancement and isotope‐coding derivatization reactions, suitable chemistries can be chosen. The peptide amino groups were reductively alkylated with acetaldehyde or acetaldehyde‐d₄ to afford N‐alkylated products with different masses. This process is simple, quick and high‐yield, and accurate comparative analysis can be achieved for the mass‐differentiated peptides. Then, the carboxyl groups were derivatized with 1‐(2‐pyrimidinyl)piperazine to increase MS/MS sensitivity. Angiotensins I–IV, bradykinin and neurotensin were analyzed after online solid phase extraction by micro LC‐MS/MS. In all instances, a greater than 17‐fold increase in sensitivity was achieved, compared with the analyses of the underivatized peptides. Furthermore, the values obtained from the present method were in agreement with the result from isotope dilution quantification using isotopically labeled angiotensin I [Asp‐Arg‐(Val‐d₈)‐Tyr‐Ile‐His‐Pro‐(Phe‐d₈)‐His‐Leu]. Copyright © 2016 John Wiley & Sons, Ltd.     

Benzoin type photoinitiator for free radical polymerization

Benzoin, a popular photoinitiator for free radical polymerization of vinyl monomers, was improved by introduction of two methyl thioether substituents. This new benzoin derivative showed an about 50 times higher light absorption in the near‐UV spectral region and performed better than the unsubstituted benzoin in polymerization experiments in bulk solutions or films of acrylate monomers when low initiator concentrations are used. Laser flash photolysis, low temperature luminescence experiments and photoproduct studies by mass spectrometry suggest that a slow α‐cleavage mechanism (k_α = 2.2 × 10⁵ s^?1) from the electronic triplet state with a quantum yield of 0.1 is the primary photoreaction to generate the initiating free radicals. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Investigation of the photochemical behaviour of pyrethroids lacking the cyclopropane ring by photo‐solid‐phase microextraction and gas chromatography/mass spectrometry

The characterization of by‐products arising from the UV photodegradation of two insecticide pyrethroids lacking the cyclopropane ring (flucythrinate and fenvalerate) has been investigated by gas chromatography coupled with mass spectrometry (GC/MS). Twenty photoproducts were tentatively identified mainly based on the interpretation of the experimental mass spectra or by using reference mass spectra. Some of these compounds had not previously been detected. Furthermore, the generation of some of the photoproducts might be a matter for concern due to their potential toxicity. The corresponding photodegradation routes, including postulation of the intermediate radicals, have also been proposed. These photodegradation studies were performed by photo‐solid‐phase microextraction (photo‐SPME) in which the SPME fibre was exposed to light after extraction of the target analytes from aqueous solutions. The degradation kinetics of the investigated pyrethroids and the photoformation‐photodegradation curves of the photoproducts generated in situ were also monitored through the ion chromatograms obtained for different irradiation times and the corresponding mass spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

A photo‐degradable helix: Synthesis,structure, and photolysis of optically active poly[2,7‐bis(4‐t‐butylphenyl)‐9‐methylfluoren‐9‐yl acrylate]

2,7‐Bis(4‐t‐butylphenyl)‐9‐methylfluoren‐9‐yl acrylate ( BBPMFA ) was synthesized and polymerized using α,α′‐azobisisobutyronitrile or n‐Bu₃B‐air as a radical initiator and using the complex of 9‐fluorenyllithium with (S)‐(+)‐1‐(2‐pyrrolidinylmethyl)pyrrolidine as an optically active anionic initiator. Although the radical polymerization led to rather low‐molecular‐weight products at low yields, the anionic polymerization afforded polymers with higher molecular weights in higher yields. The poly( BBPMFA ) obtained by the anionic polymerization was slightly rich in isotacticity (meso diad 57%) and showed an intense circular dichroism (CD) spectrum and large dextrorotation. The intensity of the CD spectrum and magnitude of optical activity increased with an increase in M_n, suggesting that the polymer possesses a preferred‐handed helical conformation. The CD spectrum disappeared within 1 s on irradiation to the polymer in a CHCl₃ solution using a 500‐W Hg‐Xe lamp. This was ascribed to fast photolysis of the ester linkage leading to a loss of helical conformation of the entire chain. Photolysis products of poly( BBPMFA ) were poly(acrylic acid) and 2,7‐bis(4‐t‐butylphenyl)‐9‐methylenefluorene (2,7‐bis(4‐t‐butylphenyl)dibenzofulvene). The photolysis reaction seemed to proceed through the “unzipping” mechanism. The rate constant of photolysis of poly( BBPMFA ) under irradiation at monochromated 325 nm was around 0.01 s^?1 independent of molecular weight. Photolysis at 325 nm was approximately 2400 times faster than that for chemical ester solvolysis under a neutral condition in the dark. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Rate constants for the reactions of HCCCO and NCCO radicals with molecular oxygen

Reactions of HCCCO and NCCO radicals with O₂ have been studied by a combination of pulsed laser photolysis and photoionization mass spectrometry. HCCCO was produced by 193‐nm photolysis of methylpropiolate or 3‐butyn‐2‐one, and NCCO was formed by 193‐nm photolysis of acetylcyanide. The rate constants obtained at 298 ± 3 K were (6.5 ± 0.7) × 10^?12 cm³ molecule^?1 s^?1 for the HCCCO + O₂ reaction, and no pressure dependence was observed between 1.5 and 16 Torr of N₂ as a bath gas. Because HCO and HCCO radicals were observed as reaction products, it was confirmed that the reaction proceeds by a two‐body reaction. On the other hand, the rate constants of NCCO with O₂ depended on the total pressure and were (5.4–8.8) × 10^?13 cm³ molecule^?1 s^?1 for total pressures 2.0–15.5 Torr of N₂, confirming that the reaction proceeds by a three‐body process. © 2001 John Wiley & Sons, Inc. Int J Chem Kinet 33: 440–448, 2001     

Formation of oligomeric alkenylperoxides during the oxidation of unsaturated fatty acids: an electrospray ionization tandem mass spectrometry study

This study reports the identification of oligomeric alkenylperoxides by electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (ESI‐MS²), during the oxidation of oleic, linoleic and linolenic acids with Fenton's (Fe²⁺/H₂O₂) and Fe²⁺/O₂ systems. The reactions were followed by ferrous oxidation‐xylenol orange method together with GC‐MS and GC‐FID, allowing to observe that both oxidation systems are different in terms of hydroperoxide evolution, probably due to the presence of different intermediate reactive species: perferryl ion and OH^· radical responsible for the decomposition of lipid hydroperoxides and formation of new compounds. The analysis of ESI‐MS in the negative mode, obtained after oxidation of each fatty acid, confirmed the presence of the monomeric oxidation products together with other compounds at high mass region above m/z 550. These new ions were attributed to oligomeric structures, identified by the fragmentation pathways observed in the tandem mass spectra. Copyright © 2012 John Wiley & Sons, Ltd.     

Separation and fragmentation study of isocoproporphyrin derivatives by UHPLC‐ESI‐exact mass MS/MS and identification of a new isocoproporphyrin sulfonic acid metabolite

Isocoproporphyrin and its derivatives are commonly used as biomarkers of porphyria cutanea tarda, heavy metal toxicity and hexachlorobenzene (HCB) intoxication in humans and animals. However, most are isobaric with other porphyrins and reference materials are unavailable commercially. The structural characterisation of these porphyrins is important but very little data is available. We report here the separation and characterisation of isocoproporphyrin, deethylisocoproporphyrin, hydroxyisocoproporphyrin and ketoisocoproporphyrin, isolated in the faeces of rats fed with a diet containing HCB, by ultra high performance liquid chromatography‐exact mass tandem mass spectrometry (UHPLC‐MS/MS). Furthermore, we report the identification and characterisation of a previously unreported porphyrin metabolite, isocoproporphyrin sulfonic acid isolated in the rat faeces. The measured mass‐to‐charge ratio (m/z) of the precursor ion was m/z 735.2338, corresponding to a molecular formula of C₃₆H₃₉N₄O₁₁S with an error of 0.3 ppm from the calculated m/z 735.2336. The MS/MS data was consistent with an isocoproporphyrin sulfonic acid structure, derived from dehydroisocoproporphyrinogen by sulfonation of the vinyl group. The metabolite was present in a greater abundance than other isocoproporphyrin derivatives and may be a more useful biomarker for HCB intoxication. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of hydroxyl radical oxidation products of N‐hexanoyl‐homoserine lactone by reversed‐phase high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A reversed‐phase high‐performance liquid chromatography/electrospray tandem mass spectrometry method was developed for the characterization of hydroxyl radical oxidation products of N‐hexanoyl‐homoserine lactone (C6‐HSL), a member of the N‐acylhomoserine lactone (AHL) class of microbial quorum‐sensing signaling molecules identified in many Gram‐negative strains of bacteria. Six products were identified: four with molecular weight (MW) of 213 and two with MW of 260. The characteristic product ions formed through collision‐induced dissociation (CID) provided diagnostic structural information. One of the photolysis products was determined to be N‐(3‐oxohexanoyl)homoserine lactone (3OC6‐HSL), a highly active quorum‐sensing signal, by comparison with a reference standard. Three structural isomers with the same mass as 3OC6‐HSL were identified as acyl side chain oxidized C6‐HSL (keto/enol functionalized) by accurate mass measurement and the structures of these products were proposed from CID spectral interpretation. Two structural isomers formed from concurrent oxidation and nitration of C6‐HSL were also observed and their structures were postulated based on CID spectra. In addition to the six hydroxyl radical oxidation products formed from the C6‐HSL precursor, five additional compounds generated from combined oxidation and lactonolysis of C6‐HSL were identified and structures were postulated. Copyright © 2009 John Wiley & Sons, Ltd.     

Mass‐Spectrometric Observation of Counter Anion Production in SU‐8 Exposed to UV Light and its Use for Dill C Parameter Determination

Photopolymerization is a phenomenon that is the basis of much of today's microfabrication technology and intense research is conducted to improve its control and the characteristics of end products for a variety of applications. The design of microscopic structures often relies on the accurate knowledge and modeling of photopolymer's behavior upon exposure, i.e. the Dill parameters, for each radiation species of interest and therefore the development of flexible characterization techniques is of great importance. SU‐8 is a popular compound that is representative of a whole class that relies on cationic polymerization, where an acid is obtained via photolysis of an onium salt during exposure. Here we report on the observation of SbF₆^? via laser desorption mass spectrometry on SU‐8 exposed to UV light at the wavelength of 365 nm and demonstrate that the yield of this counter‐anion as a function of exposure is consistent with the Dill C parameter value available in the literature. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2019 , 57, 967–972     

Preparation and Spectral Characterization of Novel Species in Matrices

A review of matrix‐isolation experiments performed in this laboratory for the past thirteen years (1992–2004) is given; the work emphasizes the preparation and spectral characterization of novel species of atmospheric interest, and the significance of detection of some species is discussed. A previous review (J. Chin. Chem. Soc. 1992 , 39, 503) covers work from 1981 to 1991. Various methods for production of novel species, including reaction in the gaseous phase, laser photolysis of a single precursor in situ, reaction of a molecule with an atom or radical in a matrix cage and reaction of a molecule with an electronically excited molecule, are discussed. The effect of secondary photolysis on products is illustrated with formation of various conformers of HOONO from photolysis of HNO₃. The advantage of investigating electronic transitions that are difficult to observe in the gaseous phase and the mode and site selectivity of photodissociation for species in a matrix cage are also discussed. New developments involving use of p‐H₂ as a matrix host, including the introduction of a pulse‐deposition method for use at ?5 K and the exploration of internal rotation of larger molecules in solid p‐H₂, are also presented.     

Online H/D exchange liquid chromatography as a support for the mass spectrometric identification of the oxidation products of melatonin

The hydrogen–deuterium exchange of protonated melatonin and its in vitro oxidation end‐products have been examined by liquid chromatography coupled with ion‐trap mass spectrometry. Specific H/D scrambling of protons in the C2 and C4 positions of the indole ring during gas‐phase fragmentation process was observed for both melatonin and its oxidation products. Collision‐induced dissociation spectra showed losses of variably deuterated NH₃, H₂O and CH₃CONH₂. In addition, a similar H/D scrambling behaviour was observed for the oxidation products, obtained from the opening of the indole ring by oxidative attack. Fragmentation pathways are proposed and H/D scrambling has been employed as a fingerprint, allowing identification of N¹‐acetyl‐5‐methoxykynurenin (AMK), N¹‐acetyl‐N²‐formyl‐5‐methoxykynurenin (AFMK), dehydro‐AFMK and hydroxymelatonin as the oxidation products of melatonin in vitro. Copyright © 2008 John Wiley & Sons, Ltd.