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.     

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.     

Mechanistic Insights into the Palladium(II)-catalyzed Intramolecular Cyclization of o-Alkynylphenylphosphonamide and N-(o-Alkynylphenyl)acetamide by Electrospray Ionization Mass Spectrometry

The intramolecular cyclization of o‐alkynylphenylphosphonamide and N‐(o‐alkynylphenyl)acetamide was monitored by electrospray ionization mass spectrometry (ESI‐MS) and its tandem version (ESI‐MS/MS). The proposed intermediates were successfully intercepted and characterized. In addition, the intermediates composed of the substrate coordinated to the palladium(II) center in the reaction of o‐alkynylphenylphosphonamide were unexpected before, and this interesting phenomenon of the substrate coordination seems associated with the unique structure of substrates.     

Rapid screening and determination of 4‐chloroamphetamine in saliva by paper spray‐mass spectrometry and capillary electrophoresis‐mass spectrometry

A novel drug‐screening system, consisting of paper spray‐MS (PS‐MS) and a CE‐ESI‐MS method was developed. This system can be easily switched either to PS‐MS for rapidly screening samples or to the traditional CE‐ESI‐MS method for separation and to obtain detailed mass spectral information, while sharing the same mass spectrometer. In the former case, when a sharp (15°‐tip) chromatography paper was used, the optimized distance from the paper tip to the mass inlet was 7.7 mm, whereas the optimized distance for the CE‐ESI tip was ～13.5 mm. Using 4‐chloroamphetamine as a model compound, the LODs for PS‐MS and CE‐ESI‐MS were determined to ～0.1 and 0.25 ppm, respectively. Comparisons of results obtained using PS‐MS and CE‐ESI‐MS and the experimental conditions are described.     

Combined use of tandem mass spectrometry and computational chemistry to study 2H‐chromenes from Piper aduncum

Natural 2H‐chromenes were isolated from the crude extract of Piper aduncum (Piperaceae) and analyzed by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) applying collision‐induced dissociation. Density functional theory (DFT) calculations were used to explain the preferred protonation sites of the 2H‐chromenes based on thermochemical parameters, including atomic charges, proton affinity, and gas‐phase basicity. After identifying the nucleophilic sites, the pathways were proposed to justify the formation of the diagnostic ions under ESI‐MS/MS conditions. The calculated relative energy for each pathway was in good agreement with the energy‐resolved plot obtained from ESI‐MS/MS data. Moreover, the 2H‐chromene underwent proton attachment on the prenyl moiety via a six‐membered transition state. This behavior resulted in the formation of a diagnostic ion due to 2‐methylpropene loss. These studies provide novel insights into gas‐phase dissociation for natural benzopyran compounds, indicating how reactivity is correlated to the intrinsic acid‐base equilibrium and structural aspects, including the substitution pattern on the aromatic moiety. Therefore, these results can be applied in the identification of benzopyran derivatives in a variety of biological samples.     

Structural analysis of glycosphingolipid analogues obtained by the saccharide primer method using CE‐ESI‐MS

A glycosphingolipid analogue (12‐azidododecyl β‐lactoside) as a saccharide primer has been shown to be useful for the synthesis of oligosaccharide libraries by mammalian cells. In the present study, CE‐ESI‐MS was employed to elucidate the structure of glycosphingolipid analogues derived from 12‐azidododecyl β‐lactoside (Lac‐C12N3) by mammalian cells. MDCK cells and COLO201 cells were cultured with Lac‐C12N3, and the glycosylated products secreted into the medium were collected and separated into acidic and neutral products by column chromatography. The acidic products could be directly analyzed by CE‐ESI‐MS, while the neutral products were converted to anionic derivatives via a reaction with propiolic acid. With this method, it was possible to analyze both acidic and neutral products glycosylated by MDCK cells and COLO201 cells at high sensitivity.     

Metabolite analysis in Curcuma domestica using various GC‐MS and LC‐MS separation and detection techniques

The metabolic profile of polar (methanol) and non‐polar (hexane) extracts of Curcuma domestica, a widely used medicinal plant, was established using various different analytical techniques, including GC‐FID, GC‐MS, HR‐GC‐MS and analytical HPLC‐ESI‐MS/MS by means of LTQ‐Orbitrap technology. The major non‐volatile curcuminoids curcumin, demethoxycurcumin and bisdemethoxycurcumin were identified when their chromatographic and precursor ion masses were compared with those of authentic standard compounds. In this paper we describe for the first time a GC/MS‐based method for metabolic profiling of the hydrophilic extract. We also identified 61 polar metabolites as TMS derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Study of the Transient Reactive Pd(IV) Intermediate in the Pd(OAc)2‐Catalyzed Oxidative Coupling Reaction System by Electrospray Ionization Tandem Mass Spectrometry

Pd‐catalyzed oxidative coupling reaction was of great importance in the aromatic C? H activation and the formation of new C? O and C? C bonds. Sanford has pioneered practical, directed C? H activation reactions employing Pd(OAc)₂ as catalyst since 2004. However, until now, the speculated reactive Pd(IV) transient intermediates in these reactions have not been isolated or directly detected from reaction solution. Electrospray ionization tandem mass spectrometry (ESI‐MS/MS) was used to intercept and characterize the reactive Pd(IV) transient intermediates in the solutions of Pd(OAc)₂‐catalyzed oxidative coupling reactions. In this study, the Pd(IV) transient intermediates were detected from the solution of Pd(OAc)₂‐catalyzed oxidative coupling reactions by ESI‐MS and the MS/MS of the intercepted Pd(IV) transient intermediate in reaction system was the same with the synthesized authentic Pd(IV) complex. Our ESI‐MS(/MS) studies confirmed the presence of Pd(IV) reaction transient intermediates. Most interestingly, the MS/MS of Pd(IV) transient intermediates showed the reductive elimination reactivity to Pd(II) complexes with new C? O bond formation into product in gas phase, which was consistent with the proposed reactivity of the Pd(IV) transient intermediates in solution.     

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.     

Comparison of ESI‐MS/MS and APCI‐MS methods for the quantification of folic acid analogs in C. elegans

Folic acid (FA) plays a vital role in central metabolism, including the one carbon cycle, nucleotide, and amino acid biosynthesis. The development of sensitive, accurate analytical methods to measure FA intermediates in tissues is critical to understand their biological roles in diverse physiological and pathological contexts. Here, we developed a highly sensitive method for the simultaneous quantification of FA intermediates in the nematode Caenorhabditis elegans as a model to dissect metabolic networks. The method was further validated by analyzing the worm folate pool upon RNAi knockdown of the dihydrofolate reductase gene dhfr‐1. Comparative mass spectrometry behavior of the FA analogs using two different ion sources, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), revealed ESI‐MS/MS to be more sensitive, but APCI‐MS provided more detailed structure inferences, which can elucidate chemical investigation and synthesis of FA analogs. Finally, we report on the use of in vitro oxidation coupled with high‐resolution mass spectrometry as a tool to discover new endogenous FA derivatives in the nematode.     

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.     

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.     

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.     

Structural elucidation of V‐type nerve agents by liquid chromatography/electrospray ionization mass spectrometry

V‐nerve agents present information‐poor spectra, both in GC‐EI‐MS and LC‐ESI‐MS/MS, with dominant fragments/product ions corresponding to the amine‐containing residue. Hence, derivatives/isomers with the same amine residue exhibit similar mass spectral patterns, leading to ambiguity in the phosphonate structure. We present a simple approach for their structural elucidation based on two complementary experiments: ESI‐MS/MS of the original compound, which provides information about the amine moiety, and ESI‐MS/MS of the phosphonic acid hydrolysis products generated by N‐iodosuccinimide, which provides ions' characteristic of the phosphonate structure. This approach enables the structural elucidation of the original V‐agents with a higher degree of certainty.     

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.     

Quantification and application of a liquid chromatography–tandem mass spectrometric method for the determination of WKYMVm peptide in rat using solid‐phase extraction

A liquid chromatographic–electrospray ionization–time‐of‐flight/mass spectrometric (LC‐ESI‐TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro‐elution solid‐phase extraction (SPE) for sample preparation and LC‐ESI‐TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2 m hydrogen chloride to plasma before SPE for stability of WKYMVm peptide. Then sample preparation using micro‐elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration²), with the equation y = ax² + bx + c was used to fit calibration curves over the concentration range of 3.02–2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25% accuracy and precision values. For quality control samples at 15, 165 and 1820 ng/mL from the quantification experiment, the within‐run and the between‐run accuracy ranged from 92.5 to 123.4% with precision values ≤15.1% for WKYMVm peptide from the nominal values. This novel LC‐ESI‐TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.     

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.     

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.