HPLC‐DAD and HPLC‐ESI‐MS separation,determination and identification of the spin‐labeled diastereoisomers of podophyllotoxin

Spin‐labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2‐H configurations of these spin‐labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high‐performance liquid chromatography‐diode array detection (HPLC‐DAD) and a high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 position. In the HPLC‐ESI/MS spectra, each pair of diastereoisomers of the spin‐labeled derivatives in the mixture was directly confirmed and identified by [M+H]⁺ ions and ion ratios of relative abundance of [M‐ROH+H]⁺ (ion 397) to [M+H]⁺. When the [M‐ROH+H]⁺ ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M‐ROH+H]⁺ (ion 397) to [M+H]⁺, [A+H]⁺ (ion 313) to [M‐ROH+H]⁺, [A+H‐OCH₃]⁺ (ion 282) to [M‐ROH+H]⁺ and [M‐ROH‐ArH+H]⁺ (ion 229) to [M‐ROH+H]⁺ of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 in the mixture.     

Gas‐phase fragmentation of γ‐lactone derivatives by electrospray ionization tandem mass spectrometry

Fragmentation reactions of β‐hydroxymethyl‐, β‐acetoxymethyl‐ and β‐benzyloxymethyl‐butenolides and the corresponding γ‐butyrolactones were investigated by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) using collision‐induced dissociation (CID). This study revealed that loss of H₂O [M + H ?18]⁺ is the main fragmentation process for β‐hydroxymethylbutenolide (1) and β‐hydroxymethyl‐γ‐butyrolactone (2). Loss of ketene ([M + H ?42]⁺) is the major fragmentation process for protonated β‐acetoxymethyl‐γ‐butyrolactone (4), but not for β‐acetoxymethylbutenolide (3). The benzyl cation (m/z 91) is the major ion in the ESI‐MS/MS spectra of β‐benzyloxymethylbutenolide (5) and β‐benzyloxymethyl‐γ‐butyrolactone (6). The different side chain at the β‐position and the double bond presence afforded some product ions that can be important for the structural identification of each compound. The energetic aspects involved in the protonation and gas‐phase fragmentation processes were interpreted on the basis of thermochemical data obtained by computational quantum chemistry. Copyright © 2009 John Wiley & Sons, Ltd.     

One‐Electron Oxidation and Sensitivity of Uric Acid in On‐Line Electrochemistry and in Electrospray Ionization Mass Spectrometry

The sensitivity of detection of uric acid (H₂U) in positive ion mode electrospray ionization mass spectrometry (ESI MS) was enhanced by uric acid oxidation during electrospray ionization. With a carrier solution of pH 6.3>pK_a1=5.4 of H₂U, protonated unoxidized uric acid [H₂U+H]⁺ (m/z 169) was detected together with the protonated uric acid dimer [2H₂U+H]⁺ (m/z 337). The dimer likely forms by 1e^? oxidation of urate (HU^?) followed by rapid radical dimerization. A covalent structure of the dimer was verified by H/D exchange experiments. Efficiency of 2e^?, 2H⁺ oxidation of uric acid is low during ESI in pH 6.3 carrier solution and improves when a low on‐line electrochemical cell voltage is floated on the high voltage of the ES in on‐line electrochemistry ESI MS (EC/ESI MS). The intensity of the uric acid dimer decreases with an increase in the low applied voltage. In a carrier solution with 0.1 M KOH, pH 12.7>pK_a2=9.8 of H₂U, allantoin (Allnt) (MW 158.04), the final 2e^?, 2H⁺ oxidation product of uric acid, was detected as a potassium complex [K(Allnt)+K]⁺ (m/z 235) and the [2H₂U+H]⁺ dimer was not detected. In direct ESI MS analysis of 1000‐fold diluted urine [NaHU+H]⁺ (pK_sp NaHU=4.6) was detected in 40/60 (vol%) water/methanol, 1 mM NH₄Ac, pH ca. 6.3 carrier solution. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity in ESI and EC/ESI MS measurements of uric acid.     

Determination of diphenylarsinic acid and phenylarsonic acid,the degradation products of organoarsenic chemical warfare agents,in well water by HPLC–ICP‐MS

Diphenylarsinic acid (DPAA) and phenylarsonic acid (PAA), which were degradation products of organoarsenic chemical warfare agents used as sternutatory gas, were detected in the well water at Kamisu, Ibaraki Prefecture, Japan. The standard material of DPAA was synthesized with aqueous arsenic acid and phenylhydrazine in order to determine organic arsenic compounds in well water. The DPAA showed a protonated ion at m/z 263 [M + H]⁺ and a loss of H₂O ion at m/z 245 [M + H ? H₂O]⁺ from protonated ion by the electrospray ionization time‐of‐flight mass spectrometry. The quantitative analysis of DPAA and PAA was performed by high‐performance liquid chromatography inductively coupled plasma mass spectrometry and the system worked well for limpid liquid samples such as well water. Copyright © 2005 John Wiley & Sons, Ltd.     

Some PhCNO-containing molecules; mass spectral similarity and molecular diversity

Some diverse compounds possessing a PhC?NO unit cleave the N? O link upon electron impact to give [PhCN]⁺˙. Different and especially significant modes of N? O scission occur in metastable processes when the oxygen atom of the PhCNO group is exocyclic to a heterocyclic nitrogen atom. Upon electron impact, rupture of the N? O bond in PhCNO-containing molecules generally dominates over 1,3-dipolar cycloreversion, which generates the radical cation of benzonitrile oxide. Stable PhCNO-containing molecules survive competing fragmentations in the ion source to produce [M+H]⁺ ions of moderate relative intensity. Other ions, which are larger than [M]⁺ are implicated where M is PhCH?NOR. Several values of m/z for metastable ions are common to compounds which have a PhCNO moiety. These m/z values generally derive both from a parent ion with m/z?105 and from a PhC?NO moiety.     

Salicylaldehyde azine cluster formation observed by cold‐spray ionization mass spectrometry

We installed a cold‐spray ionization (CSI) source on a mass spectrometer to investigate the self‐assembly behavior of an aggregation‐induced emission enhancement system. Using a CSI source and the three‐dimensional platform, a self‐assembly system of a salicylaldehyde azine (SAA) was studied in mixture solution. This method permitted the determination of the structural information of the solution state, which cannot be detected by conventional mass spectrometry. In addition to the [M+H]⁺ ion (M is the SAA molecule), many major ion clusters such as [2M+Na]⁺ at m/z 503, [3M+Na]⁺ at m/z 743, [4M+Na]⁺ at m/z 983 and higher order aggregates were observed in the CSI mass spectra. However, many fragment ions, with the exception of cluster ions, appeared with high abundance when the ESI ion source was used due to the desolvation chamber temperature, suggesting that some aggregation can be detected at low temperatures. To investigate the effect of solvent on the aggregation, the CSI‐mass spectrometry (MS) experiments of SAA in absolute ethanol solution and ethanol/water (good/poor solvent) mixture solution were conducted. The most abundant ion peak was protonated SAA (m/z 241) in absolute ethanol, but many cluster ions and some multiple charged ion peaks were observed after adding a small amount of water into the ethanol solution. The results showed good agreement with that inferred by the combinational analysis of scanning electron microscope and fluorescence microscopy, indicating that CSI‐MS is capable of providing self‐assembly information of labile molecules in the solution state. Copyright © 2013 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.     

Estimation of peptide N–Cα bond cleavage efficiency during MALDI‐ISD using a cyclic peptide

Matrix‐assisted laser desorption/ionization in‐source decay (MALDI‐ISD) induces N–C_α bond cleavage via hydrogen transfer from the matrix to the peptide backbone, which produces a c′/z? fragment pair. Subsequently, the z? generates z′ and [z + matrix] fragments via further radical reactions because of the low stability of the z?. In the present study, we investigated MALDI‐ISD of a cyclic peptide. The N–C_α bond cleavage in the cyclic peptide by MALDI‐ISD produced the hydrogen‐abundant peptide radical [M + 2H]⁺? with a radical site on the α‐carbon atom, which then reacted with the matrix to give [M + 3H]⁺ and [M + H + matrix]⁺. For 1,5‐diaminonaphthalene (1,5‐DAN) adducts with z fragments, post‐source decay of [M + H + 1,5‐DAN]⁺ generated from the cyclic peptide showed predominant loss of an amino acid with 1,5‐DAN. Additionally, MALDI‐ISD with Fourier transform‐ion cyclotron resonance mass spectrometry allowed for the detection of both [M + 3H]⁺ and [M + H]⁺ with two ¹³C atoms. These results strongly suggested that [M + 3H]⁺ and [M + H + 1,5‐DAN]⁺ were formed by N–C_α bond cleavage with further radical reactions. As a consequence, the cleavage efficiency of the N–C_α bond during MALDI‐ISD could be estimated by the ratio of the intensity of [M + H]⁺ and [M + 3H]⁺ in the Fourier transform‐ion cyclotron resonance spectrum. Because the reduction efficiency of a matrix for the cyclic peptide cyclo(Arg‐Gly‐Asp‐D‐Phe‐Val) was correlated to its tendency to cleave the N–C_α bond in linear peptides, the present method could allow the evaluation of the efficiency of N–C_α bond cleavage for MALDI matrix development. Copyright © 2016 John Wiley & Sons, Ltd.     

Mass spectrometric monitoring of oxidation of aliphatic C6–C8 hydrocarbons and ethanol in low pressure oxygen and air plasmas

Experimental and theoretical studies on the oxidation of saturated hydrocarbons (n‐hexane, cyclohexane, n‐heptane, n‐octane and isooctane) and ethanol in 28 Torr O₂ or air plasma generated by a hollow cathode discharge ion source were made. Ions corresponding to [M + 15]⁺ and [M + 13]⁺ in addition to [M ? H]⁺ and [M ? 3H]⁺ were detected as major ions where M is the sample molecule. The ions [M + 15]⁺ and [M + 13]⁺ were assigned as oxidation products, [M ? H + O]⁺ and [M ? 3H + O]⁺, respectively. By the tandem mass spectrometry analysis of [M ? H + O]⁺ and [M ? 3H + O]⁺, H₂O, olefins (and/or cycloalkanes) and oxygen‐containing compounds were eliminated from these ions. Ozone as one of the terminal products in the O₂ plasma was postulated as the oxidizing reagent. As an example, the reactions of C₆H₁₄^+? with O₂ and of C₆H₁₃⁺ (CH₃CH₂CH⁺CH₂CH₂CH₃) with ozone were examined by density functional theory calculations. Nucleophilic interaction of ozone with C₆H₁₃⁺ leads to the formation of protonated ketone, CH₃CH₂C(=OH⁺)CH₂CH₂CH₃. In air plasma, [M ? H + O]⁺ became predominant over carbocations, [M ? H]⁺ and [M ? 3H]⁺. For ethanol, the protonated acetic acid CH₃C(OH)₂⁺ (m/z 61.03) was formed as the oxidation product. The peaks at m/z 75.04 and 75.08 are assigned as protonated ethyl formate and protonated diethyl ether, respectively, and that at m/z 89.06 as protonated ethyl acetate. Copyright © 2016 John Wiley & Sons, Ltd.     

Differentiation of lisinopril and its RSS diastereomer by liquid chromatography combined with collision‐induced dissociation mass spectrometry

A simple and sensitive liquid chromatography tandem multiple‐stage mass spectrometry (HPLC/MS/MS) method suitable for bulk lisinopril analysis was developed, by which lisinopril and its RSS isomer were separated and differentiated. In the collision‐induced dissociation (CID) mass spectra of the [M + H]⁺ ions, the abundance of the fragment ion of m/z 246 for lisinopril was about two times higher than the ion of m/z 245; however, the former fragment ion was noted to be a little lower than the latter for RSS isomer at all collision energies. In the CID mass spectra of the [M + Li]⁺ ion, the abundance of the rearrangement ion of m/z 315 for the RSS isomer was about three times higher than that for lisinopril. Furthermore, the difference was supported by the results of energy‐resolved mass spectrometry (ERMS) in the test range of collision energies. Similar differences were also observed between the CID mass spectra of lisinopril and RSS isomer methylester, which indicated that the RSS isomer could be rapidly characterized by the CID mass spectra of both the protonated and lithium adduct ion. Elemental compositions of all the ions were confirmed by Fourier Transform ion cyclotron resonance ESI mass spectrometry (FT‐ICR‐ESI/MS). In addition, theoretical computations were carried out to support the experimental results. Copyright © 2009 John Wiley & Sons, Ltd.     

Antioxidant Pathways and One‐Electron Oxidation of Dopamine and Cysteine in Electrospray and On‐Line Electrochemistry Electrospray Ionization Mass Spectrometry

Dopamine [DA]⁺ (m/z 154), DA dimer [2DA‐H]⁺ (m/z 307) and DA quinone [DAQ]⁺ (m/z 152) are detected in positive ion mode electrospray ionization mass spectrometry (ESI MS) of dopamine in 50/1/49 (vol%) water/acetic acid/methanol. H/D exchange experiments support a covalent structure of DA dimer. Thus, ESI of DA may involve 1e^?, 1H⁺ oxidation processes followed by rapid radical dimerization. The DA quinone signal is low in ESI MS, which indicates a low efficiency of the 2e^?, 2H⁺ oxidation reaction. On‐line electrochemistry ESI MS (EC/ESI MS) with low electrochemical cell voltage floated on high ES voltage increases electrospray current and improves sensitivity for DA. The DA quinone signal increases and DA dimer signal decreases. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity of ESI and EC/ESI MS measurements. A DA quinone‐cysteine adduct [DAQ+Cys]⁺ was detected in solutions of DA with cysteine (Cys). ESI MS and EC/ESI MS indicate formation of the DA quinone‐cysteine adduct by 1e^? pathway. Oxidation pathways in ESI MS are relevant to biological reactivity of DA and Cys.     

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.     

Direct analysis in real time mass spectrometry (DART‐MS) of highly non‐polar low molecular weight polyisobutylenes

Low molecular weight polyisobutylenes (PIB) with chlorine, olefin and succinic acid end‐groups were studied using direct analysis in real time mass spectrometry (DART‐MS). To facilitate the adduct ion formation under DART conditions, NH₄Cl as an auxiliary reagent was deposited onto the PIB surface. It was found that chlorinated adduct ions of olefin and chlorine telechelic PIBs, i.e. [M + Cl]^? up to m/z 1100, and the deprotonated polyisobutylene succinic acid [M? H]^? were formed as observed in the negative ion mode. In the positive ion mode formation of [M + NH₄]⁺, adduct ions were detected. In the tandem mass (MS/MS) spectra of [M + Cl]^?, product ions were absent, suggesting a simple dissociation of the precursor [M + Cl]^? into a Cl^? ion and a neutral M without fragmentation of the PIB backbones. However, structurally important product ions were produced from the corresponding [M + NH₄]⁺ ions, allowing us to obtain valuable information on the arm‐length distributions of the PIBs containing aromatic initiator moiety. In addition, a model was developed to interpret the oligomer distributions and the number average molecular weights observed in DART‐MS for PIBs and other polymers of low molecular weight. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of cyclotrimethylenetrinitramine (RDX) by pyrolysis H2O/D2O atmospheric-pressure chemical ionization tandem mass spectrometry

The oxidative pyrolysis-atmospheric-pressure chemical ionization tandem mass Spectrometry (Py-APCI MS/MS) of Cyclotrimethylenetrinitramine (RDX) was investigated under various sample introduction conditions. Subambient (0.97 atm) as opposed to ambient (0.98 atm) pressure (1 atm = 101325 kPa) facilitated the appearance of new pyrolysis mass spectral ions, including m/z 44. Deuterated decomposition products from [²H]RDX contained amide groups and, depending on the ion source pressure, significant differences in the degree of proton-deuterium exchange occurred on the amide groups. The D₂O Py-APCI MS/MS method also confirmed and extended the analogous H₂O APCI information from RDX, [²H]RDX and pure standards. The m/z 44 decompositon species was identified as protonated dimethylimine, [H₃CN?CH₂]⁺ as opposed to its primary amine isomer, [H₃CC(H)?NH]H⁺, which contains an acidic proton. It was determined that m/z 60 is due to protonated N-methylformamide and acetaldoxime, [H₃CC(H)?NOH]H⁺.     

Use of liquid chromatography/tandem mass spectrometry and online databases for identification of phosphocholines and lysophosphatidylcholines in human red blood cells

In this work a systematic strategy integrating liquid chromatography/tandem mass spectrometry (LC/MS/MS) and online databases was developed to identify phosphocholines (PC) and lysophosphatidylcholines (LPC) in human red blood cells (RBCs). First of all, the neutral loss scan of 59 and the precursor ion scan of m/z 184 were performed to find out the possible lipids with phosphocholine head‐group structure in RBCs. The acquired [M+H]⁺ and [M+Na]⁺ adduct ions were then identified online using the Human Metabolome Database (HMDB) and the LIPID MAPS, which were then further confirmed by their MS/MS fragmentation. Based on the comparison of chemical structures of the detected PC and LPC with their corresponding MS/MS fragmentation pathways, several new diagnostic fragments or fragmentation pathway were found. These include, (1) the neutral losses of 183 could be used as a diagnostic fragmentation to discriminate PC and LPC; (2) product ions at m/z 104 could be used to distinguish LPC and their sn‐2 isomers; (3) fragment ions at m/z 184 are characteristic fragmentation that could be used for discrimination of sodiated ions [M+Na]⁺ and protonated ions [M+H]⁺, especially for co‐eluted PC or LPC with a molecular weight difference of 22. The structures of the above‐mentioned fragment ions were confirmed by quadrupole time‐of‐flight (Q‐TOF) MS. Furthermore, a PC and LPC focused LC/MS semi‐quantification approach was also developed and validated. This approach could be useful for future lipidomic study. Copyright © 2009 John Wiley & Sons, Ltd.     

Positive chemical ionization triple‐quadrupole mass spectrometry and ab initio computational studies of the multi‐pathway fragmentation of phthalates

We report the first positive chemical ionization (PCI) fragmentation mechanisms of phthalates using triple‐quadrupole mass spectrometry and ab initio computational studies using density functional theories (DFT). Methane PCI spectra showed abundant [M + H]⁺, together with [M + C₂H₅]⁺ and [M + C₃H₅]⁺. Fragmentation of [M + H]⁺, [M + C₂H₅]⁺ and [M + C₃H₅]⁺ involved characteristic ions at m/z 149, 177 and 189, assigned as protonated phthalic anhydride and an adduct of phthalic anhydride with C₂H₅⁺ and C₃H₅⁺, respectively. Fragmentation of these ions provided more structural information from the PCI spectra. A multi‐pathway fragmentation was proposed for these ions leading to the protonated phthalic anhydride. DFT methods were used to calculate relative free energies and to determine structures of intermediate ions for these pathways. The first step of the fragmentation of [M + C₂H₅]⁺ and [M + C₃H₅]⁺ is the elimination of [R? H] from an ester group. The second ester group undergoes either a McLafferty rearrangement route or a neutral loss elimination of ROH. DFT calculations (B3LYP, B3PW91 and BPW91) using 6‐311G(d,p) basis sets showed that McLafferty rearrangement of dibutyl, di(‐n‐octyl) and di(2‐ethyl‐n‐hexyl) phthalates is an energetically more favorable pathway than loss of an alcohol moiety. Prominent ions in these pathways were confirmed with deuterium labeled phthalates. Copyright © 2010 John Wiley & Sons, Ltd.     

Direct evidence for preformed ions of porphyrins in the solvent matrix for fast atom bombardment mass spectrometry

The observation that protonated molecules are present in solvents utilized for fast atom bombardment (FAB) mass spectrometric studies has been demonstrated using visible absorption spectrometry. Addition of porphyrins to thioglycerol, a solvent used for FAB analyses, results in partial protonation of the molecule. This reaction can be monitored by observing the shift in visible absorption maxima associated with the molecular transition from free base to protonated structure. A good correlation is observed between the degree of protonation indicated by the appropriate absorption bands and the abundance of the [M + H]⁺ ion in the FAB spectrum of the corresponding solution. Addition of certain non-polar porphyrin molecules to thioglycerol does not result in the protonation of the molecule in solution; in these cases, analyses of the corresponding solutions by FAB do not yield [M + H]⁺ ions. Subsequent addition of trifluoroacetic acid to the solvent has proved sufficient to protonate the analyte molecule, as indicated by the visible absorption spectrum; FAB analyses of these non-polar porphyins in acidified solvent result in the observation of [M + H]⁺ ions. These experiments demonstrate that analyses of these analyte molecules requires that they be present as ions in solution prior to analysis by FAB. This study provides experimental evidence for the presence of ions in solutions employed for FAB analysis, suggesting that these ions are essential for the generation of the protonated molecules observed during FAB mass spectrometric analyses.     

Structural determination of glucosylceramides isolated from marine sponge by fast atom bombardment collision‐induced dissociation linked scan at constant B/E

Five glucosylceramides (GlcCers) were isolated by reversed phase high‐performance liquid chromatography from the MeOH extracts of a marine sponge, Haliclona (Reniera) sp., collected from the coast of Ulleung Island, Korea, and analyzed by fast atom bombardment mass spectrometry (FAB–MS) in positive‐ion mode. FAB‐mass spectra of these compounds included protonated molecules [M + H]⁺ and abundant sodiated molecules [M + Na]⁺ from a mixture of m‐NBA and NaI. The structures of these GlcCers, which were similar, were elucidated by FAB‐linked scan at constant B/E. To find diagnostic ions for their characterization, the GlcCers were analyzed by collision‐induced dissociation (CID) linked scan at constant B/E. The CID‐linked scan at constant B/E of [M + H]⁺ and [M + Na]⁺ precursor ions resulted in the formation of numerous characteristic product ions via a series of dissociative processes. The product ions formed by charge‐remote fragmentation provided important information for the characterization of the fatty N‐acyl chain moiety and the sphingoid base, commonly referred to as the long‐chain base. The product ions at m/z 203 and 502 were diagnostic for the presence of a sodiated sugar ring and β‐D ‐glucosylsphinganine, respectively. For further confirmation of the structure of the fatty N‐acyl chain moiety in each GlcCer, fatty acid methyl esters were obtained from the five GlcCers by methanolysis and analyzed by FAB–MS in positive‐ion mode. On the basis of these dissociation patterns, the structures of the five GlcCers from marine sponge were elucidated. In addition, the accurate mass measurement was performed to obtain the elemental composition of the GlcCers isolated from marine sponge. Copyright © 2009 John Wiley & Sons, Ltd.     

Sodiation as a tool for enhancing the diagnostic value of MALDI‐TOF/TOF‐MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono‐ and diesters. For rapid fingerprinting of these esters, matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF/TOF‐MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono‐ and diester palmitate standards in MALDI‐TOF/TOF‐MS showed that sodium adduct parent masses [M + Na]⁺ gave much simpler MS² spectra than radical / protonated [M]^+● / [M + H]⁺ parents. [M + Na]⁺ fragments yielded diagnostic polyene‐specific eliminations and fatty acid neutral losses, whereas [M]^+● / [M + H]⁺ fragmentation resulted in a multitude of non‐diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [M + Na]⁺ ionization by addition of sodium acetate, and best signal‐to‐noise ratios were obtained in the 0.1 to 1.0 mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI‐TOF/TOF‐MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono‐ and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all‐trans esterified esters found in LC were identified with MALDI‐TOF/TOF‐MS, with the exception of two minor monoesters. Copyright © 2013 John Wiley & Sons, Ltd.     

The application of gas chromatography/atmospheric pressure chemical ionisation time‐of‐flight mass spectrometry to impurity identification in Pharmaceutical Development

Accurate mass measurement (used to determine elemental formulae) is an essential tool for impurity identification in pharmaceutical development for process understanding. Accurate mass liquid chromatography/mass spectrometry (LC/MS) is used widely for these types of analyses; however, there are still many occasions when gas chromatography (GC)/MS is the appropriate technique. Therefore, the provision of robust technology to provide accurate mass GC/MS (and GC/MS/MS) for this type of activity is essential. In this report we describe the optimisation and application of a newly available atmospheric pressure chemical ionisation (APCI) interface to couple GC to time‐of‐flight (TOF) MS. To fully test the potential of the new interface the APCI source conditions were optimised, using a number of standard compounds, with a variety of structures, as used in synthesis at AstraZeneca. These compounds were subsequently analysed by GC/APCI‐TOF MS. This study was carried out to evaluate the range of compounds that are amenable to analysis using this technique. The range of compounds that can be detected and characterised using the technique was found to be extremely broad and include apolar hydrocarbons such as toluene. Both protonated molecules ([M + H]⁺) and radical cations (M^+.) were observed in the mass spectra produced by APCI, along with additional ion signals such as [M + H + O]⁺. The technique has been successfully applied to the identification of impurities in reaction mixtures from organic synthesis in process development. A typical mass accuracy of 1–2 mm/zunits (m/z 80–500) was achieved allowing the reaction impurities to be identified based on their elemental formulae. These results clearly demonstrate the potential of the technique as a tool for problem solving and process understanding in pharmaceutical development. The reaction mixtures were also analysed by GC/electron ionisation (EI)‐MS and GC/chemical ionisation (CI)‐MS to understand the capability of GC/APCI‐MS relative to these two firmly established techniques. Copyright © 2010 John Wiley & Sons, Ltd.