Qualitative and quantitative analysis of the major constituents in Acorus tatarinowii Schott by HPLC/ESI‐QTOF‐MS/MS

Acorus tatarinowii Schott (ATS) is a well‐known traditional Chinese medicine (TCM) for the treatment of epilepsy, amnesia and insomnia. In this study, a methodology utilizing high‐performance liquid chromatography (HPLC) coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐QTOF‐MS/MS) was established for the separation and structural identification of the major chemical constituents in ATS for the first time. Overall, 46 major constituents including flavonoid glycosides, phenylpropane derivatives, amides and lignans were identified or tentatively characterized. Seven major constituents, including four phenylpropane derivatives and three lignans, were further quantified as marker substances, which showed good linearity within the test ranges. These results indicated that the developed quantitative method was linear, sensitive, and precise for quality control of ATS. Copyright © 2014 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.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous Determination Baicalin and Forsythin in Shuanghuanglian Oral Liquid by HPLC/DAD and LC‐ESI‐MS

Rapid, simple and reliable HPLC/DAD and LC‐ESI‐MS methods for the simultaneous determination of baicalin and forsythin in the traditional Chinese medicinal preparation Shuanghuanglian oral liquid were described and validated. The separation condition for HPLC/DAD was optimized using a BDS hypersil C18 column (Thermo, 2.1 × 150 mm, particle size 5 μm) by gradient elution using methanol‐0.2 % ammonium acetate as the mobile phase. The suitable detection wavelength was set at 277 nm for the quantitative analysis of baicalin and forsythin in this method. Some operational parameters of the ESI interface were optimized, negative m/z 445[M?H]^? for baicalin and negative m/z 593[M+CH₃COO]^? for forsythin, positive m/z 447[M+H]⁺ for baicalin and positive m/z 552[M+NH 4]⁺ for forsythin, respectively. These HPLC/DAD and LC‐ESI‐MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). These methods can be used as a complementary method for the commercial quality control of Shuanghuanglian oral liquid and its pharmaceutical preparations.     

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.     

Characterization of protoberberine alkaloids in Coptidis Rhizoma (Huanglian) by HPLC with ESI‐MS/MS

This study aims to qualitatively analyze protoberberine alkaloids in crude extract of Coptidis Rhizoma using HPLC with ESI‐MS/MS. Possible specific molecular weights of protoberberine alkaloids were firstly deduced according to literatures and were adopted to screen the alkaloids in the HPLC with ESI‐MS of crude extract of Coptidis Rhizoma. As a result, 21 protoberberine alkaloids were found, including compounds of very low concentration and compounds coeluted in one peak. Among these, two compounds were positively identified and verified by comparison with standards. Ten of these compounds were first reported in this study for Coptidis Rhizoma. In addition, chromatographic retention parameters a and c of all compounds were obtained using their retention times under five gradient conditions and were applied to confirm the deduction about the structures of protoberberine alkaloids by tandem mass data.     

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.     

HPLC‐ESI‐MS/MS of brain neurotransmitter modulator lobeline and related piperidine alkaloids in Lobelia inflata L

There is a renewed interest in lobelia alkaloids because of their activity on the central nervous system. Lobeline, the most active of them, a nicotinic receptor ligand and neurotransmitter transporter inhibitor, is a candidate pharmacotherapy for metamphetamine abuse. In the present work, high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry in positive ion mode was used for investigating the alkaloid profile in Lobelia inflata L. Chromatographic separations were achieved on a Gemini C6‐phenyl reversed‐phase column providing good peak shape and improved selectivity. Being mostly 2,6‐disubstituted piperidines, lobelia alkaloids presented abundant [M + H]⁺ ions with typical fragmentation. Identification was possible from a few specific ions, especially those resulting from excision of one of the substituents. Based on fragmentation pattern of lobeline as reference compound, 52 alkaloids were identified in the aqueous methanolic extract of L. inflata in contrast to the previously known some 20. Structural variability of these alkaloids identified arises basically from their substituents which can be phenyl‐2‐ketoethyl‐ or phenyl‐2‐hydroxyethyl units as well as their methyl‐, ethyl‐ or propyl‐ homologues attached in different combinations. Several propyl homologue lobelia alkaloids and five hydroxypiperidine derivatives were found in the plant at the first time. In addition to 8‐O‐esters of 2‐monosubstituted piperidine alkaloids previously reported by us in L. inflata, a 3‐hydroxy‐3‐phenylpropanoic acid ester of hydroxyallosedamine ring‐substituted was also identified as a new natural product. High‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry can be successfully applied to Lobeliacae plant samples in the routine screening for new and known bioactive constituents, quality control of the crude drug, lobelia herba, alkaloid production studies, breeding and chemotaxonomy. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous analysis of 11 main active components in Cirsium setosum based on HPLC‐ESI‐MS/MS and combined with statistical methods

A novel method based on high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the 11 major active components including ten flavonoids and one phenolic acid in Cirsium setosum. Separation was performed on a reversed‐phase C₁₈ column with gradient elution of methanol and 0.1‰ acetic acid (v/v). The identification and quantification of the analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple‐reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the assay was carried out including linearity, precision, accuracy, stability, limits of detection and quantification. The results demonstrated that the method developed was reliable, rapid, and specific. The 25 batches of C. setosum samples from different sources were first determined using the developed method and the total contents of 11 analytes ranged from 1717.460 to 23028.258 μg/g. Among them, the content of linarin was highest, and its mean value was 7340.967 μg/g. Principal component analysis and hierarchical clustering analysis were performed to differentiate and classify the samples, which is helpful for comprehensive evaluation of the quality of C. setosum.     

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.     

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.     

Temperature‐controlled ionic liquid dispersive liquid‐phase microextraction for the sensitive determination of triclosan and triclocarban in environmental water samples prior to HPLC‐ESI‐MS/MS

A novel dispersive liquid‐phase microextraction method without dispersive solvents has been developed for the enrichment and sensitive determination of triclosan and triclocarban in environmental water samples prior to HPLC‐ESI‐MS/MS. This method used only green solvent 1‐hexyl‐3‐methylimidazolium hexafluorophosphate as extraction solvent and overcame the demerits of the use of toxic solvents and the instability of the suspending drop in single drop liquid‐phase microextraction. Important factors that may influence the enrichment efficiencies, such as volume of ionic liquid, pH of solutions, extraction time, centrifuging time and temperature, were systematically investigated and optimized. Under optimum conditions, linearity of the method was observed in the range of 0.1–20 μg/L for triclocarban and 0.5–100 μg/L for triclosan, respectively, with adequate correlation coefficients (R>0.9990). The proposed method has been found to have excellent detection sensitivity with LODs of 0.04 and 0.3 μg/L, and precisions of 4.7 and 6.0% (RSDs, n=5) for triclocarban and triclosan, respectively. This method has been successfully applied to analyze real water samples and satisfactory results were achieved.     

LC/ESI‐MS/MS characterisation of lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain

Lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain showed an interesting anti‐adhesion activity against biofilm formation of human pathogenic bacterial strains. The chemical characterisation of the crude extract of V9T14 strain was first developed through electrospray ionisation mass spectrometry (ESI‐MS) and ESI‐MS/MS direct infusions: two sets of molecular ion species belonging to the fengycin and surfactin families were revealed and their structures defined, interpreting their product ion spectra. The LC/ESI‐MS analysis of the crude extract allowed to separate in different chromatogram ranges the homologues and the isoforms of the two lipopeptide families. The extract was then fractionated by silica gel chromatography in two main fractions, I and II. The purified biosurfactants were analysed through a new, rapid and suitable LC/ESI‐MS/MS method, which allowed characterising the composition and the structures of the produced lipopeptides. LC/ESI‐MS/MS analysis of fraction I showed the presence of C₁₃, C₁₄ and C₁₅ surfactin homologues, whose structures were confirmed by the product ion spectra of the sodiated molecules [M + Na]⁺ at m/z 1030, 1044 and 1058. LC/ESI‐MS/MS analysis of fraction II confirmed the presence of two main fengycin isoforms, with the protonated molecules [M + H]⁺ at m/z 1478 and 1506 corresponding to C₁₇ fengycin A and C₁₇ fengycin B, respectively. Other homologues (C₁₄ to C₁₆) were revealed and confirmed as belonging to fengycin A or B according to the retention times and the product ions generated, although with the same nominal mass. Finally, a relative percentage content of each homologue for both lipopeptides families in the whole extract was proposed. Copyright © 2010 John Wiley & Sons, Ltd.     

Highly sensitive and selective separation of intact parathyroid hormone and variants by sheathless CE‐ESI‐MS/MS

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE‐ESI‐MS, combining CE resolution power and low‐flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral‐coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field‐amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused‐silica capillary with UV detection. An acidic BGE was used to separate 1–84 PTH (full length), 7–84 PTH, and 1–34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE‐ESI‐MS instrument. When using a fused silica capillary, CE‐MS was limited to μg/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1–84 PTH, 7–84 PTH, and 1–34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range.     

HPLC‐ESI‐MS/MS analysis and pharmacokinetics of luteoloside,a potential anticarcinogenic component isolated from Lonicera japonica,in beagle dogs

Luteoloside is a potential anticarcinogenic component isolated from Lonicera japonica, a traditional Chinese medicine (TCM). This study details the development and validation of a sensitive and accurate HPLC‐ESI‐MS/MS method for the quantification of luteoloside in dog plasma. Sample pretreatment includes simple protein precipitation using methanol–acetonitrile (1:1, v/v). A Phenomenex Gemini C₁₈ column (2.0 × 50 mm, i.d., 3.5 µm) was used to separate luteoloside and internal standard by gradient mode with mobile phase consisting of water containing 0.1% formic acid and methanol containing 0.1% formic acid at a flow rate of 0.40 mL/min with a column temperature of 25°C. The detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode. The calibration curves were linear (R > 0.995) over the concentration range 1.0–2000 ng/mL and the lower limit of quantification was 1.0 ng/mL. The intra‐day and inter‐day precisions (RSD) were all <15%, accuracies (RE) were within the range of ±15%, and recoveries were between 85.0 and 115%. The validated HPLC‐ESI‐MS/MS method was successfully applied to determine plasma concentrations of luteoloside after intravenous administration of luteoloside at a dose level of 20 mg/kg. Copyright © 2012 John Wiley & Sons, Ltd.     

Challenges in the identification process of phenidate analogues in LC‐ESI‐MS/MS analysis: Information enhancement by derivatization with isobutyl chloroformate

A new analytical technique for the structural elucidation of four representative phenidate analogues possessing a secondary amine residue, which leads to a major/single amine‐representative fragment/product ion at m/z 84 both in their GC‐EI‐MS and LC‐ESI‐MS/MS spectra, making their identification ambiguous, was developed. The method is based on “in vial” chemical derivatization with isobutyl chloroformate in both aqueous and organic solutions, followed by liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS/MS). The resulting carbamate derivatives promote rich fragmentation patterns with full coverage of all substructures of the molecule, enabling detailed structural elucidation and unambiguous identification of the original compounds at low ng/mL levels.     

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。     

Determination of asperosaponin VI in rat plasma by HPLC‐ESI‐MS and its application to preliminary pharmacokinetic studies

Asperosaponin VI (also named akebia saponin D) is a typical bioactive triterpenoid saponin isolated from the rhizome of Dipsacus asper Wall (Dipsacaceae). In this work, a sensitive high‐performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC‐ESI‐MS) assay has been established for determination of asperosaponin VI in rat plasma. With losartan as the internal standard (IS), plasma samples were prepared by protein precipitation with methanol. Chromatographic separation was performed on a C₁₈ column with a mobile phase of 10 mm ammonium acetate buffer containing 0.05% formic acid–methanol (32 : 68, v/v). The analysis was performed on an ESI in the selected ion monitoring mode using target ions at m/z 951.4 for asperosaponin VI and m/z 423.2 for the IS. The calibration curve was linear over the range 3–1000 ng/mL and the lower limit of quantification was 3.0 ng/mL. The intra‐ and inter‐assay variability values were less than 9.5 and 7.8%, respectively. The accuracies determined at the concentrations of 3.0, 100.0, 300.0 and 1000 ng/mL for asperosaponin VI were within ±15.0%. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of asperosaponin VI. Copyright © 2009 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.     

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.