Synthesis of benzofurazan derivatization reagents for short chain carboxylic acids in liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI‐MS/MS)

Benzofurazan derivatization reagents, 4‐[2‐(N,N‐dimethylamino)ethylaminosulfonyl]‐7‐(2‐aminopentylamino)‐2,1,3‐benzoxadiazole (DAABD‐AP) and 4‐[2‐(N,N‐dimethylamino) ethylaminosulfonyl]‐7‐(2‐aminobutylamino)‐2,1,3‐benzoxadiazole (DAABD‐AB), for short‐chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60°C for 60 min. The generated derivatives were separated on the reversed‐phase column and detected by ESI‐MS/MS with the detection limits of 0.1–0.12 pmol on column. Upon collision‐induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD‐AP and DAABD‐AB are suitable as the derivatization reagents in LC/ESI‐MS/MS analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Isothiocyanates as derivatization reagents for amines in liquid chromatography/electrospray ionization–tandem mass spectrometry

The applicability of 3‐pyridyl isothiocyanate, p‐(dimethylamino)phenyl isothiocyanate and m‐nitrophenyl isothiocyanate as the derivatization reagents for amines in high‐performance liquid chromatography/electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS) was examined. The generated derivatives of amines with these reagents were favorably separated on the reversed‐phase column and detected by ESI‐MS/MS. The C–N bond of the generated thiourea structure was efficiently cleaved by collision‐induced dissociation and gave the single and intense product ion. Among the three reagents, 3‐pyridyl isothiocyanate was the most suitable as the derivatization reagent with regard to the reactivity to amines and the detection sensitivity. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of benzofurazan derivatization reagents for carboxylic acids in liquid chromatography/electrospray ionization-tandem mass spectrometry

The applicability of benzofurazan derivatization regents to carboxylic acids analysis in LC/ESI-MS/MS (high-performance liquid chromatography/electrospray ionization tandem mass spectrometry) was examined. The product ion spectra of DAABD-AE {4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole}, DAABD-PZ {4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-N-piperazino-2,1,3-benzoxadiazole}, DAABD-PiCZ {4-[4-carbazoylpiperidin-1-yl]-7-[2-(N,N-dimethylamino)ethylaminosulfonyl]-2,1,3-benzoxadiazole}, DAABD-ProCZ {4-[2-carbazoylpyrrolidin-1-yl]-7-[2-(N,N-dimethylamino) ethylaminosulfonyl]-2,1,3-benzoxadiazole} and DAABD-Apy {4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole}, and their acetylated compounds were obtained. An intense fragment ion at m/z 151 corresponding to (dimethylamino)ethylaminosulfonyl moiety was observed in each spectra, suggesting that these reagents were suitable for ESI-MS/MS analysis. DAABD-AE, DAABD-APy and DAABD-PZ were applied to the analysis of octanoic acid and it was found that DAABD-AE and DAABD-APy gave high signal intensity suitable for LC/ESI-MS/MS.     

Comparative metabolite profiling of carboxylic acids in rat urine by CE‐ESI MS/MS through positively pre‐charged and 2H‐coded derivatization

A new approach to the selective comparative metabolite profiling of carboxylic acids in rat urine was established using CE‐MS and a method for positively pre‐charged and ²H‐coded derivatization. Novel derivatizing reagents, N‐alkyl‐4‐aminomethyl‐pyridinum iodide (alkyl=butyl, butyl‐d9 or hexyl), containing quaternary amine and stable‐isotope atoms (deuterium), were introduced for the derivatization of carboxylic acids. CE separation in positive polarity showed high reproducibility (0.99–1.32% RSD of migration time) and eliminated problems with capillary coating known in CE‐MS anion analyses. Essentially complete ionization and increased hydrophobicity after the derivatization also enhanced MS detection sensitivity (e.g. formic acid was detected at 0.5 pg). Simultaneous derivatization of one sample using two structurally similar reagents, N‐butyl‐4‐aminomethyl‐pyridinum iodide (BAMP) and N‐hexyl‐4‐aminomethyl‐pyridinum iodide, provided additional information for recognizing a carboxylic acid in an unknown sample. Moreover, characteristic fragmentation acquired by online CE‐MS/MS allowed for identification and categorization of carboxylic acids. Applying this method on rat urine, we found 59 ions matching the characteristic patterns of carboxylic acids. From these 59, 32 ions were positively identified and confirmed with standards. For comparative analysis, 24 standard carboxylic acids were derivatized by chemically identical but isotopically distinct BAMP and N‐butyl‐d9‐4‐aminomethyl‐pyridinium iodide, and their derivatization limits and linearity ranges were determined. Comparative analysis was also performed on two individual urine samples derivatized with BAMP and N‐butyl‐d9‐4‐aminomethyl‐pyridinium iodide. The metabolite profiling variation between these two samples was clearly visualized.     

A novel derivatization reagent possessing a bromoquinolinium structure for biological carboxylic acids in HPLC‐ESI‐MS/MS

A novel bromoquinolinium reagent, i.e. 1‐(3‐aminopropyl)‐3‐bromoquinolinium bromide (APBQ), was synthesized for the analysis of carboxylic acids. A simple and practical precolumn derivatization procedure using the APBQ in RP chromatography and MS (HPLC‐MS) has been developed using bile acids and free fatty acids, as the representative carboxylic acids in biological samples. The APBQ efficiently reacted with carboxylic acids at 60°C for 60 min in the presence of N,N‐dicyclohexylcarbodiimide and pyridine as the activation reagents. Because the APBQ possesses a bromine atom in the structure, the identification of a series of carboxylic acids was easily achieved due to the characteristic bromine isotope pattern in the mass spectra. The APBQ also has a quaternary amine structure, thus the positively charged derivatives are predominate for the highly sensitive detection of carboxylic acids. The APBQ was successfully applied to the selective determination of biological carboxylic acids in human plasma. The bile acids (chenodeoxycholic acid and deoxycholic acid) and several saturated (stearic acid and palmitic acid) and unsaturated free fatty acids (oleic acid and linoleic acid) were reasonably determined by HPLC‐MS under the proposed procedure. Based on the results of analyses of human plasma and saliva, the proposed procedure using APBQ seems to be applicable for the qualitative and quantitative analyses of a series of carboxylic acids in biological samples.     

Derivatization of chiral carboxylic acids with (S)‐anabasine for increasing detectability and enantiomeric separation in LC/ESI‐MS/MS

A simple and practical derivatization procedure for increasing the detectability and enantiomeric separation of chiral carboxylic acids in LC/ESI‐MS/MS has been developed. (S)‐Anabasine (ANA) was used as the derivatization reagent and rapidly reacted with carboxylic acids [3‐hydroxypalmitic acid (3‐OH‐PA), 2‐(β‐carboxyethyl)‐6‐hydroxy‐2,7,8‐trimethylchroman (γ‐CEHC), and etodolac] in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholium chloride. The resulting ANA‐derivatives were highly responsive in ESI‐MS operating in the positive‐ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using selected reaction monitoring; the detection responses of the ANA‐derivatives were increased by 20–160‐fold over those of the intact carboxylic acids and the limits of detection were in the low femtomole range (1.8–11 fmol on the column). The ANA‐derivatization was also effective for the enatiomeric separation of the chiral carboxylic acids; the resolution was 1.92, 1.75, and 2.03 for 3‐OH‐PA, γ‐CHEC, and etodolac, respectively. The derivatization procedure was successfully applied to a biological sample analysis; the derivatization followed by LC/ESI‐MS/MS enabled the separation and detection of trace amounts of 3‐OH‐PA in neonatal dried blood spot and γ‐CEHC in human saliva with a simple pretreatment and small sample volume.     

A quantitative LC–MS/MS method for determination of a small molecule agonist of EphA2 in mouse plasma and brain tissue

Compound 27 {1, 12‐bis[4‐(4‐amino‐6,7‐dimethoxyquinazolin‐2‐yl)piperazin‐1‐yl]dodecane‐1,12‐dione} is a novel small molecule agonist of EphA2 receptor tyrosine kinase. It showed much improved activity for the activation of EphA2 receptor compared with the parental compound doxazosin. To support further pharmacological and toxicological studies of the compound, a method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC–MS/MS) has been developed for the quantification of this compound. Liquid–liquid extraction was used to extract the compound from mouse plasma and brain tissue homogenate. Reverse‐phase chromatography with gradient elution was performed to separate compound 27 from the endogenous molecules in the matrix, followed by MS detection using positive ion multiple reaction monitoring mode. Multiple reaction monitoring transitions m/z 387.3 → 290.1 and m/z 384.1 → 247.1 were selected for monitoring compound 27 and internal standard prazosin, respectively. The linear calibration range was 2–200 ng/mL with the intra‐ and inter‐day precision and accuracy within the acceptable range. This method was successfully applied to the quantitative analysis of compound 27 in mouse plasma and brain tissue with different drug administration routes.     

Comparative evaluation of new Cookson‐type reagents for LC/ESI‐MS/MS assay of 25‐hydroxyvitamin D3 in neonatal blood samples

The screening of vitamin D deficiency in neonatal infants, which is based on the blood 25‐hydroxyvitamin D₃ [25(OH)D₃] quantification, is important for the early detection, diagnosis and health risk assessment of several diseases. In this study, two new Cookson‐type reagents, 4‐(4‐diethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DEAPTAD) and 4‐(6‐quinolyl)‐1,2,4‐triazoline‐3,5‐dione, were designed and synthesized, then compared with the previous reagents, 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) and 4‐(4‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DAPTAD), in terms of sensitivity and specificity in the assay of 25(OH)D₃ in neonatal blood samples by liquid chromatography/electrospray ionization–tandem mass spectrometry. Among the reagents, DEAPTAD was found to be the most promising. The limit of detection (0.38 fmol on the column) of the DEAPTAD‐derivatized 25(OH)D₃ was 60 and 2 times lower than those of the intact 25(OH)D₃ and the PTAD derivative, respectively. 25(OH)D₃ was more clearly detected in the plasma sample as the DEAPTAD derivative than the DAPTAD derivative owing to the lower background noise. DEAPTAD derivatization was also useful for the separation of 25(OH)D₃ from a potent interfering metabolite, 3‐epi‐25‐hydroxyvitamin D₃. By using DEAPTAD, a trace amount of 25(OH)D₃ in dried blood spots was reproducibly determined without interference from coexisting compounds. Thus, DEAPTAD was proved useful in the measurement of 25(OH)D₃ in neonatal blood samples. Copyright © 2015 John Wiley & Sons, Ltd.     

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

Rapid qualitative analysis of 2 flavonoids,rutin and silybin,in medical pills by direct analysis in real‐time mass spectrometry (DART‐MS) combined with in situ derivatization

Direct analysis in real‐time mass spectrometry (DART‐MS) with in situ silylation was used for the rapid analysis of the flavonoids silybin ((2R,3R)‐3,5,7‐trihydroxy‐2‐[3‐(4‐hydroxy‐3‐methoxyphenyl)‐2‐hydroxymethyl‐2,3‐dihydrobenzo[1,4]dioxin‐6‐yl]chroman‐4‐one) and rutin (quercetin‐3‐O‐rutinoside). Three different derivatization reagents, hexamethyldisilazane/trimethylchlorosilane/pyridine (HMDS/TMCS/pyridine), N,O‐bis(trimethylsilyl)acetamide/trimethylchlorosilane/N‐trimethylsilyimidazole (BSA/TMCS/TMSI), and N,O‐bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (BSTFA/TMCS), were applied. Silybin and rutin were detected with various degrees of silylation, and the formation of dimers with pyridine and imidazole was also observed. HMDS/TMCS/pyridine was the best choice for the DART‐MS analysis of silybin, and BSA/TMCS/TMSI was the most effective for the detection of rutin. The effects of the DART source temperature on desorption, ionization, in‐source fragmentation, dimer formation, and hydrolysis of the trimethylsilyl groups were also studied. In addition, the collision‐induced dissociation properties of the derivatized silybin and rutin were explored. With our in situ silylation method, the derivatized bioactive compounds in intact medical pills could also be detected by DART‐MS.     

Development of a validated UPLC–MS/MS method for PK/PD analysis of SYL930 and its two major metabolites in dogs

A sensitive and specific UPLC‐MS/MS method was developed and validated for the simultaneous determination of 2‐amino‐2‐(2‐(4′‐(2‐propyloxazol‐4‐yl)‐[1,1′‐biphenyl]‐4‐yl)ethyl)propane‐1,3‐diol (SYL930), phosphorylated metabolite (SYL930‐P) and hydroxylated metabolite (SYL930‐M) in dog blood using SYL927 and SYL927‐P, analogues of SYL930, as the internal standards. Analytes were extracted with protein precipitation followed by chromatographic separation on a ZorbaxSB‐C₁₈ column (3.5 μm, 2.1 × 100 mm) with a gradient elution of methanol–water containing 0.1% formic acid (v /v). A triple quadrupole tandem mass spectrometer operating in the positive electrospray ionization mode was used to detect SYL930, SYL930‐P, SYL930‐M and IS transitions of 381.2 → 364.2, 461.2 → 334.2, 397.3 → 380.3, 367.1 → 350.4 and 447.5 → 320.2, respectively. The linear calibration curves for SYL930, SYL930‐P and SYL930‐M were 0.5–500, 0.2–100 and 0.5–100 ng/mL, respectively (r ² > 0.99). The intra‐day and inter‐day precisions (RSD, %) of analytes did not exceed 9.16% except for low QCs (≤16.22%), and the accuracy (RE, %) ranged from −14 to 11.4%. The mean recoveries for SYL930, SYL930‐P and SYL930‐M in dog blood were 85.13–107.94, 73.84–80.08 and 85.64–95.44%, respectively. The validated method was successfully applied to pharmacokinetic and PK/PD studies of SYL930 and its two major metabolites in dogs after an oral administration of SYL930.     

An isomer-specific high-energy collision-induced dissociation MS/MS database for forensic applications: a proof-of-concept on chemical warfare agent markers

Spectra database search has become the most popular technique for the identification of unknown chemicals, minimizing the need for authentic reference chemicals. In the present study, an isomer‐specific high‐energy collision‐induced dissociation (CID) MS/MS spectra database of 12 isomeric O‐hexyl methylphosphonic acids (degradation markers of nerve agents) was created. Phosphonate anions were produced by the electrospray ionization of phosphonic acids or negative‐ion chemical ionization of their fluorinated derivatives and were analysed in a hybrid magnetic‐sector–time‐of‐flight tandem mass spectrometer. A centre‐of‐mass energy (E_com) of 65 eV led to an optimal sequential carbon–carbon bond breakage, which was interpreted in terms of charge remote fragmentation. The proposed mechanism is discussed in comparison with the routinely used low‐energy CID MS/MS. Even‐mass (odd‐electron) charge remote fragmentation ion series were diagnostic of the O‐alkyl chain structure and can be used to interpret unknown spectra. Together with the odd‐mass ion series, they formed highly reproducible, isomer‐specific spectra that gave significantly higher database matches and probability factors (by 1.5 times) than did the EI MS spectra of the trimethylsilyl derivatives of the same isomers. In addition, ionization by negative‐ion chemical ionization and electrospray ionization resulted in similar spectra, which further highlights the general potential of the high‐energy CID MS/MS technique. Copyright © 2011 John Wiley & Sons, Ltd.     

ESI‐MS studies of palladium (II) complexes with 1‐(p‐toluenesulfonyl)cytosine/cytosinato ligands

The mononuclear complex Pd(1‐TosC‐N3)₂Cl₂ (2) containing 1‐(p‐toluenesulfonyl)cytosine (1) as a ligand, as well as dinuclear complexes Pd₂(1‐TosC^?‐N3,N4)₄ (3) and Pd₂(1‐TosC^?‐N3,N4)₂DMSO₂Cl₂ (4) containing the ligand anion (1‐TosC^?), was mass analyzed by electrospray ionization ion trap MS/MS and high resolution MS. Complexes 3 and 4 were obtained by recrystallization of 2 from DMF and DMSO, respectively. The behavior of complex 2 in different solutions was monitored by electrospray ionization mass spectrometry (ESI‐MS). Under the applied ESI‐MS conditions, complex 2 in methanol reorganized itself dominantly as new complex 3 and the solvent did not coordinate the formed species. In H₂O/DMSO, CH₃CN/DMSO and CH₃OH/DMSO solutions, complex 2 formed several new species with solvent molecules involved in their structure, e.g. complex 4 was formed as the major product. The newly formed species were also examined by LC‐MS‐DAD, confirming the solvent induced reorganization and the solution instability of complex 2. Copyright © 2009 John Wiley & Sons, Ltd.     

A convenient route to pyridones,pyrazolo[2,3‐a]pyrimidines and pyrazolo[5,1‐c]triazines incorporating antipyrine moiety

Condensation of 4‐aminoantipyrine with ethyl acetoacetate, ethyl benzoylacetate, and ethyl cyanoacetate furnished the corresponding ethyl 3‐(1,2‐dihydro‐1,5‐dimethyl‐2‐phenyl‐3‐oxo‐3H‐pyrazol‐4‐yl)aminoacrylate and 2‐cyano‐N‐[(1,2‐dihydro‐1,5‐dimethyl‐2‐phenyl‐3‐oxo‐3H‐pyrazol‐4‐yl)]acetamide derivatives. The aminoacrylates derivatives react with acetonitrile and sodium hydride to give 2‐amino‐6‐methyl‐1‐(1,2‐dihydro‐1,5‐dimethyl‐2‐phenyl‐3‐oxo‐3H‐pyrazol‐4‐yl)‐4‐pyridone. Reaction of the cyanoacetamide derivative with dimethylformamide‐dimethylacetal (DMF‐DMA) afforded 2‐cyano‐N‐[1,2‐dihydro‐1,5‐dimethyl‐2‐phenyl‐3‐oxo‐pyrazol‐4‐yl]‐2‐(N,N‐dimethylamino)methylene acetamide in high yield. Treatment of the latter with 5‐aminopyrazole derivatives afforded the corresponding pyrazolo[2,3‐a]pyrimidines. 2‐cyano‐N‐[(1,2‐dihydro‐1,5‐dimethyl‐2‐phenyl‐3‐oxo‐3H‐pyrazol‐4‐yl)]acetamide also reacts with heterocyclic diazonium salts to give the corresponding pyrazolo[5,1‐c]‐1,2,4‐triazine derivatives. © 2004 Wiley Periodicals, Inc. Heteroatom Chem 15:508–514, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.20046     

Development and validation of an LC‐ESI‐MS/MS quantification method for a potential γ‐aminobutyric acid transporter 3 (GAT3) marker and its application in preliminary MS binding assays

Binding assays for the γ‐aminobutyric acid (GABA) transporter GAT3 can be assumed to significantly facilitate screening for respective inhibitors. As appropriate labeled ligands for this promising drug target are not available so far, we started efforts to set up mass spectrometry‐based binding assays (MS binding assays), for which labeled markers are not required. Therefore, we developed a sensitive and rapid LC‐ESI‐MS/MS quantification method for DDPM‐1007 {(RS)‐1‐[4,4,4‐Tris(4‐methoxyphenyl)but‐2‐en‐1‐yl]piperidine‐3‐carboxylic acid}, one of the most potent GAT3 inhibitors yet known, as a potential GAT3 marker. Using a 50 × 2 mm C₈ column in combination with a mobile phase composed of 10 mm ammonium bicarbonate buffer pH 8.0 and acetonitrile (60:40, v/v) at a flow rate of 450 μL/min DDPM‐1007 could be analyzed in the positive multiple reaction monitoring mode [(m/z) 502.5 → 265.4] within a chromatographic cycle time of 3 min. Deuterated DDPM‐1007 [(²H₉)DDPM‐1007] was synthesized and employed as internal standard. This way DDPM‐1007 could be quantified in a range from 100 pm to10 nm in the matrix resulting from respective binding experiments without any sample preparation. The established quantification method met the requirements of the FDA guidance for bioanalytical method validation concerning linearity and intra‐ and inter‐batch accuracy. Based on this LC‐ESI‐MS/MS quantification preliminary MS binding assays employing membrane preparations obtained from a stably GAT3 expressing HEK293 cell line and DDPM‐1007 as nonlabeled GAT3 marker could be performed. In these experiments specific binding of DDPM‐1007 at GAT3 could be unambiguously detected. Additionally, the established LC‐MS method provides a suitable analytical tool for further pharmacokinetic characterization of DDPM‐1007, as exemplified for its logD determination. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Formation of cyclic acylphosphoramidates in mass spectra of N‐monoalkyloxyphosphoryl amino acids using electrospray ionization tandem mass spectrometry

The fragmentation reactions of N‐monoalkyloxyphosphoryl amino acids (N‐MAP‐AAs) were studied by electrospray ionization tandem mass spectrometry (ESI‐MS). The sodiated cyclic acylphosphoramidates (CAPAs) were formed through a characteristic pentacoordinate phosphate participated rearrangement reaction in the positive‐ion ESI‐MS/MS and HR‐MS/MS of N‐MAP‐AAs, in which the fragmentation patterns were clearly different from those observed in the corresponding ESI‐MS/MS of N‐dialkyloxyphosphoryl amino acids/peptides and N‐phosphono amino acids. The formation of CAPAs depended on the chemical structures of N‐terminal phosphoryl groups, such as alkyloxy group, negative charge and alkali metal ion. A possible integrated rearrangement mechanism for both P‐N to P‐O phosphoryl group migration and formation of CAPAs was proposed. The fragmentation patterns of CAPAs as novel intermediates in gas phase were also investigated. In addition, it was found that the formation of α‐amino acid CAPAs was more favorable than β‐ or γ‐CAPAs in gas phase, which was consistent with previous solution‐phase experiments. Copyright © 2010 John Wiley & Sons, Ltd.     

Fluorous derivatization combined with liquid chromatography/tandem mass spectrometry: a method for the selective and sensitive determination of sialic acids in biological samples

We have developed a novel method for selective and sensitive analysis of sialic acids (N‐acetylneuraminic, N‐glycolylneuraminic, and 2‐keto‐3‐deoxy‐D ‐glycero‐D ‐galactonononic acid) utilizing liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with a fluorous derivatization technique. In this method, the carboxylic groups in the sialic acids are derivatized via amidation with heptadecafluoroundecylamine, a commercially available perfluoroalkylamine reagent. This reaction proceeds rapidly and readily at room temperature in the presence of a condensation reagent. Subsequently, the derivatives are retained specifically on an LC column with a perfluoroalkyl stationary phase by means of a fluorophilic or 'fluorous' interaction, and detected by positive electrospray ionization MS/MS. The detection limits of the examined sialic acids are in the range of 60–750 amol on column. We show that the proposed method can be used to analyze trace amounts of sialic acids in biological samples. Copyright © 2010 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.     

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.