Fragmentation pathways of deprotonated amide‐sulfonamide CXCR4 inhibitors investigated by ESI‐IT‐MSn,ESI‐Q‐TOF‐MS/MS and DFT calculations

Amide‐sulfonamides provide a potent anti‐inflammatory scaffold targeting the CXCR4 receptor. A series of novel amide‐sulfonamide derivatives were investigated for their gas‐phase fragmentation behaviors using electrospray ionization ion trap mass spectrometry and quadrupole time‐of‐flight mass spectrometry in negative ion mode. Upon collision‐induced dissociation (CID), deprotonated amide‐sulfonamides mainly underwent either an elimination of the amine to form the sulfonyl anion and amide anion or a benzoylamide derivative to provide sulfonamide anion bearing respective substituent groups. Based on the characteristic fragment ions and the deuterium–hydrogen exchange experiments, three possible fragmentation mechanisms corresponding to ion‐neutral complexes including [sulfonyl anion/amine] complex ( INC‐1 ), [sulfonamide anion/benzoylamide derivative] complex ( INC‐2 ) and [amide anion/sulfonamide] complex ( INC‐3 ), respectively, were proposed. These three ion‐neutral complexes might be produced by the cleavages of S–N and C–N bond from the amide‐sulfonamides, which generated the sulfonyl anion (Route 1), sulfonamide anion (Route 2) and the amide anion (Route 3). DFT calculations suggested that Route 1, which generated the sulfonyl anion (ion c ) is more favorable. In addition, the elimination of SO₂ through a three‐membered‐ring transition state followed by the formation of C–N was observed for all the amide‐sulfonamides.     

Metabolism studies of casticin in rats using HPLC‐ESI‐MSn

Casticin (3′,5‐dihydroxy‐3, 4′,6,7‐tetramethoxyflavone) has been revealed to possess various kinds of pharmacological activities, including immunomodulatory, anti‐hyperprolactinemia, anti‐tumor and neuroprotetective activities. In order to gain an understanding of the biotransformation of casticin in vivo, a systematic method based on liquid chromatography–electrospray ionization tandem mass spectrometry (LC‐ESI‐MSⁿ) was developed to identify the metabolites of casticin in rats after oral administration of single dose of casticin at 200 mg/kg. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug, the parent compound and 25 metabolites were identified in rat plasma, urine and six selected tissues. This is the first systematic metabolism study of casticin in vivo. The results indicated that methylation, demethylation, glucuronidation and sulfation were the main biotransformation pathways of casticin in vivo. Copyright © 2012 John Wiley & Sons, Ltd.     

The investigation of substituent effects on the fragmentation pathways of pentacoordinated phenoxyspirophosphoranes by ESI‐MSn

The fragmentation pathways of pentacoordinated phenoxyspirophosphoranes were investigated in the positive mode by electrospray ionization multistage mass spectrometry. The results demonstrate that the sodium adducts of the title compounds undergo two competitive fragmentation pathways, and the fragmentation patterns are heavily dependent on the various substituent patterns at the phenolic group. An electron‐withdrawing substituent at the ortho‐position always results in the removal of a corresponding phenol analogue, while cleavage by spiroring opening becomes the predominant fragmentation pathway if an electron‐donating substituent is at the phenolic group. The substituent effects on the competitive fragmentation pathways were further elucidated by theoretical calculations, single crystal structure analysis, and high‐resolution mass spectrometry. The results contribute to the understanding of the gas‐phase fragmentation reactions and the structure identification of spirophosphorane analogues by electrospray ionization multistage mass spectrometry.     

Studies on the metabolism and toxicological detection of glaucine,an isoquinoline alkaloid from Glaucium flavum (Papaveraceae), in rat urine using GC‐MS,LC‐MSn and LC‐high‐resolution MSn

Glaucine ((S)‐5,6,6a,7‐tetrahydro‐1,2,9,10‐tetramethoxy‐6‐methyl‐4H‐dibenzo [de,g]quinoline) is an isoquinoline alkaloid and main component of Glaucium flavum (Papaveraceae). It was described to be consumed as recreational drug alone or in combination with other drugs. Besides this, glaucine is used as therapeutic drug in Bulgaria and other countries as cough suppressant. Currently, there are no data available concerning metabolism and toxicological analysis of glaucine. To study both, glaucine was orally administered to Wistar rats and urine was collected. For metabolism studies, work‐up of urine samples consisted of protein precipitation or enzymatic cleavage followed by solid‐phase extraction. Samples were afterwards measured by liquid chromatography (LC) coupled to low or high‐resolution mass spectrometry (HR‐MS). The phase I and II metabolites were identified by detailed interpretation of the corresponding fragmentations, which were further confirmed by determination of their elemental composition using HR‐MS. From these data, the following metabolic pathways could be proposed: O‐demethylation at position 2, 9 and 10, N‐demethylation, hydroxylation, N‐oxidation and combinations of them as well as glucuronidation and/or sulfation of the phenolic metabolites. For monitoring a glaucine intake in case of abuse or poisoning, the O‐ and N‐demethylated metabolites were the main targets for the gas chromatography‐MS and LC‐MSⁿ screening approaches described by the authors. Both allowed confirming an intake of glaucine in rat urine after a dose of 2 mg/kg body mass corresponding to a common abuser's dose. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of piperaquine and its N‐oxidated metabolite in rat plasma using LC‐MS/MS

A sensitive and efficient liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of piperaquine (PQ) and its N ‐oxidated metabolite (PQ‐M) in plasma. A simple protein precipitation procedure was used for sample preparation. Adequate chromatographic retention was achieved on a C₁₈ column under gradient elution with acetonitrile and 2 mm aqueous ammonium acetate containing 0.15% formic acid and 0.05% trifluoroacetic acid. A triple‐quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode. The method was linear in the range of 2.0–400.0 ng/mL for PQ and 1.0–50.0 ng/mL for PQ‐M with suitable accuracy, precision and extraction recovery. The lower limits of detection (LLOD) were established at 0.4 and 0.2 ng/mL for PQ and PQ‐M, respectively, using 40 μL of plasma sample. The matrix effect was negligible under the current conditions. No effect was found for co‐administrated artemisinin drugs or hemolysis on the quantification of PQ and PQ‐M. Stability testing showed that two analytes remained stable under all relevant analytical conditions. The validated method was successfully applied to a pharmacokinetic study performed in rats after a single oral administration of PQ (60 mg/kg).     

Fragmentation pathways and differentiation of positional isomers of sorafenib and structural analogues by ESI‐IT‐MSn and ESI‐Q‐TOF‐MS/MS coupled with DFT calculations

Sorafenib is an orally active multikinase inhibitor for the treatment of renal cell carcinoma. A series of sorafenib structural analogues were investigated in this work for their gas‐phase fragmentation behaviors using electrospray ionization ion trap mass spectrometry and quadrupole time‐of‐flight mass spectrometry in the positive mode. The possible fragmentation pathways were proposed based on ESI‐MS/MS data and theoretical calculation. Different from the typical α‐cleavage of amide, consecutive reactions that involved elimination of H₂O and CH₃NC were observed for 2‐pyridinecarboxamide derivatives, which were followed by the formation of a stabilized 7‐membered ring carbocation by loss of CO. Two possible protonation sites occurred at carbonyl oxygen atoms for aryl‐urea derivatives and the α‐cleavage of urea was the main fragmentation pathways, which was followed by the formation of stable benzo [d] oxazole ring characteristic to aryl‐urea derivatives. The relative abundance of characteristic fragment ions and the energy‐resolved breakdown curves were used to distinguish the 4 sets of positional isomers of sorafenib and analogues. The methodology and results of the present work would contribute to the chemical structure identification of other structural analogues and the potential impurities presented in active pharmaceutical ingredients and drug formulations.     

Fragmentation features of intermolecular cross‐linked peptides using N‐hydroxy‐ succinimide esters by MALDI‐ and ESI‐MS/MS for use in structural proteomics

The use of mass spectrometry coupled with chemical cross‐linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross‐linked peptides. The interpretation of the MS/MS data generated in cross‐linking experiments using N‐hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross‐linked peptides occur when two different peptides are connected by the cross‐linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross‐linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross‐linked peptides present in cross‐linking experiments and be further implemented in search engine's algorithms. Copyright © 2011 John Wiley & Sons, Ltd.     

ESI‐MS Studies on Alcoholysis Mechanism of Electron‐deficient Cyclopropane Derivatives

Electrospray ionization mass spectrometry (ESI‐MS) was utilized to perform monitoring of the intermediates in the reaction of 1,2,3‐trisubstituted electron‐deficient cyclopropane derivatives, cis‐1‐thien‐2′‐oyl‐2‐(p‐subustituted phenyl‐6,6‐dimethyl)‐5,7‐dioxaspiro[2.5]‐4,8‐octadiones, with methanol. Key intermediates, either cationic or protonated forms of neutral species, were intercepted and characterized by ESI‐MS and its tandem version (ESI‐MS/MS). Therefore, the mechanism of the ring‐opening process for electron‐deficient cyclopropane derivatives was fully confirmed by the intermediates monitored.     

In vivo metabolism study of vaccarin in rat using HPLC‐LTQ‐MSn

In order to illustrate the main biotransformation pathways of vaccarin in vivo, metabolites of vaccarin in rats were identified using a specific and sensitive high‐performance liquid chromatography–electrospray ionization linear ion trap mass spectrometry (LTQ XL?) method. The rats were administered a single dose (200 mg/kg) of vaccarin by oral gavage. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug, the parent compound and six metabolites were found in rat urine after oral administration of vaccarin. The parent compound and five metabolites were detected in rat plasma. In heart, liver and kidney samples, respectively, one, four and three metabolites were identified, in addition to the parent compound. Three metabolites, but no trace of parent drug, were found in the rat feces. This is the first systematic metabolism study of vaccarin in vivo. The biotransformation pathways of vaccarin involved methylation, hydroxylation, glycosylation and deglycosylation. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification and structural characterization by LC‐ESI‐IONTRAP and LC‐ESI‐TOF of some metabolic conjugation products of homovanillic acid in urine of neuroblastoma patients

The levels of urinary catecholamine metabolites, such as homovanillic acid (HVA) and vanillylmandelic acid, are routinely used as a clinical tool in the diagnosis and follow‐up of neuroblastoma (NB) patients. Recently, in the Clinical Pathology Laboratory Unit of G. Gaslini Children Hospital, a commercial method that employs liquid chromatography coupled to electrochemical detection (LC‐EC) has been introduced for the measurement of these metabolites in the routine laboratory practice. Using this LC‐EC method, an unknown peak could be observed only in samples derived from NB patients. To investigate the nature of this peak, we used a combination of liquid chromatography‐time‐of‐flight mass spectrometry (LC‐TOF‐MS) and liquid chromatography‐ion trap tandem mass spectrometry (LC‐IT‐MS). The first approach was used to obtain the elemental composition of the ions present in this new signal. To get additional structural information useful for the elucidation of unknown compounds, the ion trap analyzer was exploited. We were able to identify not just one, but three unknown signals in urine samples from NB patients which corresponded to three conjugated products of HVA: HVA sulfate and two glucuronoconjugate isomers. The enzymatic hydrolysis with β‐glucuronidase confirmed the proposed structures, while the selective alkaline hydrolysis allowed us to distinguish the difference between phenol‐ and acyl‐glucuronide of HVA. The latter was the unknown peak observed in LC‐EC separations of urine samples from NB patients. Copyright © 2012 John Wiley & Sons, Ltd.     

Measurement of caffeine and its three primary metabolites in human plasma by HPLC‐ESI‐MS/MS and clinical application

Caffeine is a mild stimulant with significant potential for abuse, being consumed in larger doses with the widespread availability of energy drinks and by novel routes of administration such as inspired powder, oral sprays and electronic cigarettes. How these recent changes in caffeine consumption affecting caffeine disposition and abuse potential is of growing concern. In the study of caffeine disposition in humans, it is common to only measure the caffeine concentration; however, caffeine's three major metabolites (paraxanthine, theobromine and theophylline) retain central nervous system stimulant activity that may contribute to the overall pharmacological activity and toxicity. Therefore, it would be scientifically more rigorous to measure caffeine and its major metabolites in the evaluation of caffeine disposition in human subjects. Herein, we report a method for the simultaneous quantification of caffeine and its three major metabolites in human plasma by high‐performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC‐ESI‐MS/MS). Human plasma samples were treated by simple protein precipitation and the analytes were separated using a 6 min gradient program. Precision and accuracy were well within in the 15% acceptance range. The simple sample preparation, short runtime, sensitivity and the inclusion of caffeine's major metabolites make this assay methodology optimal for the study of caffeine's pharmacokinetics and pharmacodynamics in human subjects.     

Detection and Sequence Identification of Dinucleotides Produced from N‐Phosphoryl Alanine and Four Nucleosides by HPLC‐ESI‐MS/MS

本文利用液相色谱质谱联用技术研究了N－磷酰化丙氨酸和四种核苷（腺苷,尿苷,胞苷和鸟苷）的模板反应产物。结果表明生成了不同类型的单核苷酸和二核苷酸,并且生成的二核苷酸序列也得到了确证。研究结果揭示,二核苷酸骨架裂解形成的c离子可以作为确证二核苷酸序列的诊断离子。本文首次证明不论是在正离子模式还是在负离子模式下,c离子都可以用来确定此反应体系中生成的二核苷酸产物的序列。     

Analysis of hydroxyl radical‐induced oxidation process of glucagon by reversed‐phase HPLC and ESI‐MS/MS

Structural modification of a polypeptide hormone, glucagon, by a hydroxyl radical in vitro was investigated by reversed‐phase high‐performance liquid chromatography (RP‐HPLC), and the oxidized site of glucagon was detected by electrospray ionization tandem mass spectrometry (ESI‐MS/MS). It was shown that ²⁷methionine (Met) was oxidized to ²⁷Met sulfoxide by hydroxyl radical, and the production rate of ²⁷Met sulfoxide was faster than that by hydrogen peroxide. In addition, production of ²⁷Met sulfoxide enantiomer was confirmed by RP‐HPLC analysis. cAMP production in a HepG2 cell induced by ²⁷Met sulfoxide glucagon was reduced to approximately 75% as compared with that induced by the native form of glucagon. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC‐ESI‐MS/MS and stopped‐flow HPLC‐NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid–liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C₁₈ reversed‐phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI‐MS/MS detection in an on‐line mode or ¹H‐NMR (500 MHz) spectroscopic detection in a stopped‐flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12‐hydroxy‐ilaprazole sulfide (M2), 11,12‐dihydroxy‐ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC‐ESI‐MS/MS and HPLC‐NMR could be widely applied in detection and identification of novel metabolites. Copyright © 2010 John Wiley & Sons, Ltd.     

ESI‐QTof‐MS characterization of hirsutinolide and glaucolide sesquiterpene lactones: Fragmentation mechanisms and differentiation based on Na+/H+ adducts interactions in complex mixture

Sesquiterpene lactones (SL) have been reported with various biological effects. Among the described SL skeletons, hirsutinolide and glaucolide have not been extensively studied by mass spectrometry (MS), especially how to distinguish them in organic matrices. Thus, this paper reports (1) a strategy of their differentiation based on MS behavior during the ionization and (2) a proposal of the fragmentation pattern for both SL‐subtypes. ESI(+)‐HRMS data of four isolated SL (hirsutinolides 1 and 3 ; glaucolides 2 and 4 ) were recorded by direct and UPLC water‐sample combined injections. These analyses revealed that hirsutinolides and glaucolides formed [M+Na]⁺ ion during the operation of the direct MS injection, and ([M+Na]⁺ and [M+H‐H₂O]⁺) and [M+H]⁺ ions were respectively observed for hirsutinolides and glaucolides during the operation of combined UPLC water and sample MS injection. Computational simulations showed that the complex hirsutinolide ( 1 )‐Na⁺ formed with a lower preparation energy compared with the complex glaucolide ( 2 )‐Na⁺. However, despite their different behavior during the ionization process, ESI(+)‐HRMS/MS analyses of 1 ‐ 4 gave similar fragmentation patterns at m/z 277, 259, 241, and 231 that can be used as diagnostic ions for both skeletons. Moreover, the differentiation strategy based on the nature of the complex SL‐adducts and their MS/MS fragmentation pattern were successfully applied for the chemical characterization of the extract from Vernonanthura tweedieana using UPLC‐ESI‐HRMS/MS. Among the characterized metabolites, SL with hirsutinolide and glaucolide skeletons showed the aforementioned diagnostic fragments and an ionization behavior that was similar to those observed during the water‐sample combined injection.     

Determination of spiramycin and neospiramycin antibiotic residues in raw milk using LC/ESI‐MS/MS and solid‐phase extraction

A simple confirmatory method for the determination of spiramycin and its metabolite neospiramycin in raw milk using LC ESI MS/MS is presented. Macrolide residues in raw milk were extracted by ACN, and sample extracts were further cleaned up and concentrated using SPE cartridges. Both spiramycin and neospiramycin were protonated in electrospray positive ion mode to form singly and/or doubly charged pseudomolecular ions. Data acquisition was achieved using multiple reaction monitoring, i.e., two transitions, for quantification and confirmation. Matrix‐matched standard calibration curves were utilized to achieve the best accuracy for the method. The method performance was evaluated according to both a conventional validation procedure and a designed experimental result. The measurement uncertainty arising from accuracy and precision was estimated. The method accuracy, expressed as a percentage of an overall recovery, was from 82.1 to 108.8%, and its intermediate precision was less than 20%. LC/ESI‐MS/MS method LODs (S/N ? 3:1) of spiramycin and neospiramycin were less than 1.0 μg/kg.     

A quantitative analysis of the polyamine in lung cancer patient fingernails by LC‐ESI‐MS/MS

A quantitative analysis of polyamines in lung cancer patient fingernails by the combination of 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole derivatives and liquid chromatography–electrospray ionization tandem mass spectrometry is described. The reaction of the reagent with eight kinds of polyamines, that is, N¹‐acetylputrescine (N¹‐actPUT), N⁸‐acetylspermidine, N¹‐acetylspermine, 1,3‐diaminopropane, putrescine (PUT), cadaverine, spermidine and spermine (SPM) effectively occurs at 60 °C for 30 min. The detection limits (signal‐to‐noise ratio 5) were 5–100 fmol. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), that is, 1,6‐diaminohexane, vs the injected amounts of polyamines (r² > 0.996), and the intra‐day and inter‐day assay precisions were <9.84%. Furthermore, the recoveries (%) of the polyamines spiked in the human fingernails were 89.14–110.64. The present method was applied to human fingernail samples from 17 lung cancer patients and 39 healthy volunteers. The polyamine concentration was different based on the gender, that is, the N¹‐actPUT and PUT contents were 3.10 times and 2.56 times higher in healthy men than in women, respectively. Additionally, in the lung cancer patient group, as compared with the healthy volunteers, the concentrations of SPM had a statistically significant (p < 0.05) correlation. Therefore, because the proposed method provides a good mass accuracy and the trace detection of the polyamines in human fingernails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in lung cancer patients. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of eight components in Angelica sinensis based on UHPLC‐ESI‐MS/MS method for quality evaluation

A method employing ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) for determination of eight components including ferulic acid, senkyunolide A, butylphthalide, ligustilide, butylidenephalide, senkyunolide I, senkyunolide H and levistolide A in Angelica sinensis was established. The separation was carried out using a Waters ACQUITY UHPLC BEH C₁₈ column with gradient elution with 0.1% formic acid aqueous and acetonitrile at a flow rate of 0.4 mL/min. Good linearity was attained with R² of 0.9983–0.9998 in wide concentration ranges. The method had limit of detection (LOD) and quantitation (LOQ) in the range of 0.42–6.98 ng/mL and 1.39–23.28 ng/mL, respectively. Intra‐ and inter‐day precisions varied with relative standard deviations (RSDs) from 0.33% to 0.88% and 0.37% to 1.04%, respectively. Moreover, the average recoveries were in a satisfactory range of 92.7%–102.1% with RSDs of less than 3.60%. Finally, the method was successfully applied to analyze 19 batches of A. sinensis samples grown in Min County, Gansu province, China, as well as that collected in other regions. The findings indicated that the established method is reliable and may thus be applied as a powerful tool for qualitative and quantitative analysis of components in A. sinensis, which has its implications in quality control of A. sinensis.     

Stability‐indicating validation of acitretin and isoacitretin in human plasma by LC‐ESI‐MS/MS bioanalytical method and its application to pharmacokinetic analysis

LC‐ ESI‐ MS/MS simultaneous bioanalytical method was developed to determine acitretin and its metabolite isoacitretin in human plasma using acitretin‐d3 used as the internal standard for both analytes. The compounds were extracted using protein precipitation coupled with liquid–liquid extraction with flash freezing technique. Negative mass transitions (m/z) of acitretin, isoacitretin and acitretin‐d3 were detected in multiple reactions monitoring (MRM) mode at 325.4 → 266.3, 325.2 → 266.1 and 328.3 → 266.3, respectively, with a turbo ion spray interface. The chromatographic separation was achieved on an Ascentis‐RP amide column (4.6 × 150 mm, 5 µm) with mobile phase delivered in isocratic mode. The method was validated over a concentration range of 1.025–753.217 ng/mL for acitretin and 0.394–289.234 ng/mL for isoacitretin with a limit of quantification of 1.025 and 0.394 ng/mL. The intra‐day and inter‐day precisions were below 8.1% for acitretin and below 13.8% for isoacitretin, while accuracy was within ±7.0 and ±10.6% respectively. For the first time, the best possible conditions for plasma stability of acitretin and isoacitretin are presented and discussed with application to clinical samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Fragmentation pathways of protonated coumarin by ESI‐QE‐Orbitrap‐MS/MS coupled with DFT calculations

Coumarin is one of the basic structures of naturally oxygen heterocyclic compound, which was investigated in this paper for its gas‐phase fragmentation behaviors using electrospray quadrupole extractive orbitrap mass spectrometry in the positive mode. The possible fragmentation pathways were proposed based on electrospray ionization (ESI)‐ mass spectrometry (MS)/MS data and theory calculation. The elimination of two CO and CO₂ was observed for protonated coumarin, which was followed by the formation of a stabilized seven‐, six‐, and five‐membered ring carbocation by loss of C2H2. The possible protonation sites occurred at Oxygen 11 atom of coumarin were the main fragmentation pathways. The relative abundance of characteristic fragment ions and the energy‐resolved breakdown curves were used to confirm the cleavage mechanism of protonated coumarin. The methodology and results of present work would contribute to the chemical structure identification of other coumarins.