Mingkun Fu

Phenyl radical-induced damage to dipeptides.

Laser-induced acoustic desorption (LIAD) incorporated with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR) has been utilized to investigate phenyl radical-induced damage to dipeptides in the gas phase. On the basis of the product branching ratios measured for the reactions of two different positively charged phenyl radicals with 17 different dipeptides, the overall order of susceptibility to attack of the different sites in the dipeptides was determined to be heteroaromatic side chain approximately = S atom in SCH(3) group > H atom in SH group > H atom in CH group > aromatic side chain > S atom in SH group > NH(2) in side chain > N-terminal NH(2) > COOH in side chain approximately = C-terminal COOH. The amino acid sequence also influences the selectivity of these reactions. As expected, the ability of a phenyl radical to damage dipeptides increases as the electrophilicity of the phenyl radical increases.

Identification of Epoxide Functionalities in Protonated Monofunctional Analytes by Using Ion/Molecule Reactions and Collision-Activated Dissociation in Different Ion Trap Tandem Mass Spectrometers

A mass spectrometric method has been delineated for the identification of the epoxide functionalities in unknown monofunctional analytes. This method utilizes gas-phase ion/molecule reactions of protonated analytes with neutral trimethyl borate (TMB) followed by collision-activated dissociation (CAD) in an ion trapping mass spectrometer (tested for a Fourier-transform ion cyclotron resonance and a linear quadrupole ion trap). The ion/molecule reaction involves proton transfer from the protonated analyte to TMB, followed by addition of the analyte to TMB and elimination of methanol. Based on literature, this reaction allows the general identification of oxygen-containing analytes. Vinyl and phenyl epoxides can be differentiated from other oxygen-containing analytes, including other epoxides, based on the loss of a second methanol molecule upon CAD of the addition/methanol elimination product. The only other analytes found to undergo this elimination are some amides but they also lose O = B-R (R = group bound to carbonyl), which allows their identification. On the other hand, other epoxides can be differentiated from vinyl and phenyl epoxides and from other monofunctional analytes based on the loss of (CH3O)2BOH or formation of protonated (CH3O)2BOH upon CAD of the addition/methanol elimination product. For propylene oxide and 2,3-dimethyloxirane, the (CH3O)2BOH fragment is more basic than the hydrocarbon fragment, and the diagnostic ion (CH3O)2BOH2+ is formed. These reactions involve opening of the epoxide ring. The only other analytes found to undergo (CH3O)2BOH elimination are carboxylic acids, but they can be differentiated from the rest based on several published ion/molecule reaction methods. Similar results were obtained in the Fourier-transform ion cyclotron resonance and linear quadrupole ion trap mass spectrometer.

Differentiation of Regioisomeric Aromatic Ketocarboxylic Acids by Positive Mode Atmospheric Pressure Chemical Ionization Collision-Activated Dissociation Tandem Mass Spectrometry in a Linear Quadrupole Ion Trap Mass Spectrometer

Positive-mode atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MSn) was tested for the differentiation of regioisomeric aromatic ketocarboxylic acids. Each analyte forms exclusively an abundant protonated molecule upon ionization via positive-mode APCI in a commercial linear quadrupole ion trap (LQIT) mass spectrometer. Energy-resolved collision-activated dissociation (CAD) experiments carried out on the protonated analytes revealed fragmentation patterns that varied based on the location of the functional groups. Unambiguous differentiation between the regioisomers was achieved in each case by observing different fragmentation patterns, different relative abundances of ion-molecule reaction products, or different relative abundances of fragment ions formed at different collision energies. The mechanisms of some of the reactions were examined by H/D exchange reactions and molecular orbital calculations.

Reactions of An Aromatic σ,σ-Biradical with Amino Acids and Dipeptides in the Gas Phase

Gas-phase reactivity of a positively charged aromatic σ,σ-biradical (N-methyl-6,8-didehydroquinolinium) was examined toward six aliphatic amino acids and 15 dipeptides by using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR) and laser-induced acoustic desorption (LIAD). While previous studies have revealed that H-atom and NH2 abstractions dominate the reactions of related monoradicals with aliphatic amino acids and small peptides, several additional, unprecedented reaction pathways were observed for the reactions of the biradical. For amino acids, these are 2H-atom abstraction, H2O abstraction, addition — CO2, addition — HCOOH, and formation of a stable adduct. The biradical reacts with aliphatic dipeptides similarly as with aliphatic amino acids, but undergoes also one additional reaction pathway, addition/C-terminal amino acid elimination (addition — CO — NHCHRC). These reactions are initiated by H-atom abstraction by the biradical from the amino acid or peptide, or nucleophilic addition of an NH2 or a HO group of the amino acid or peptide at the radical site at C-6 in the biradical. Reactions of the unquenched C-8 radical site then yield the products not observed for related monoradicals. The biradical reacts with aromatic dipeptides with an aromatic ring in N-terminus (i.e., Tyr-Leu, Phe-Val, and Phe-Pro) similarly as with aliphatic dipeptides. However, for those aromatic dipeptides that contain an aromatic ring in the C-terminus (i.e., Leu-Tyr and Ala-Phe), one additional pathway, addition/N-terminal amino acid elimination (addition — CO — NHCHRN), was observed. This reaction is likely initiated by radical addition of the biradical at the aromatic ring in the C-terminus. Related monoradicals add to aromatic amino acids and small peptides, which is followed by Cα-Cβ bond cleavage, resulting in side-chain abstraction by the radical. For biradicals, with one unquenched radical site after the initial addition, the reaction ultimately results in the loss of the N-terminal amino acid. Similar to monoradicals, the C-S bond in amino acids and dipeptides was found to be especially susceptible to biradical attack.

Ion–molecule reactions for the differentiation of primary, secondary and tertiary hydroxyl functionalities in protonated analytes in a tandem mass spectrometer

A mass spectrometric method utilizing gas-phase ion-molecule reactions of 1-butanethiol and di-tert-butyl peroxide has been developed for the differentiation of primary, secondary and tertiary hydroxyl functionalities in protonated analytes in a FT-ICR mass spectrometer.

Liquid chromatography/tandem mass spectrometry utilizing ion-molecule reactions and collision-activated dissociation for the identification of N-oxide drug metabolites.

A liquid chromatography/tandem mass spectrometry (LC/MS(3)) method based on ion-molecule reactions and collision-activated dissociation (CAD) is presented for the identification of analytes with the N-oxide functional group directly in mixtures. Tri(dimethylamino)borane (TDMAB) rapidly and selectively derivatizes protonated N-oxides in a modified commercial linear quadrupole ion trap (LQIT) mass spectrometer to yield a distinct product ion (adduct-(CH(3))(2)NH). The LQIT was outfitted with an external reagent-mixing manifold that allows TDMAB to be mixed with the helium buffer gas used in the trap. The derivatized analytes are readily identified on the basis of a shift of 98 Th (Thomson) relative to the m/z value of the protonated analyte. Further probing of the derivatized analytes via isolation followed by CAD can be used to confirm the presence of an N-oxide, and distinguish between aliphatic and aromatic tertiary N-oxides. Since the ion-molecule reaction is fast, these experiments can be accomplished on the same time scale as typical CAD-based MS(n) experiments, thus maintaining the duty cycle of the instrument for this type of experiment. To demonstrate real world applicability, the method was tested on real active pharmaceutical ingredients and their derivatives.

An Ion/Molecule Reaction for the Identification of Analytes with Two Basic Functional Groups

A mass spectrometric method is presented for the identification of analytes with two basic functionalities and PA between 222 and 245 kcal/mol, including diamines. This method utilizes gas-phase ion-molecule reactions of protonated analytes with neutral 1,1-diethoxyethene (DEE) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR). A variety of protonated mono-, bi-, and trifunctional analytes containing different functional groups, namely, amido, amino, N-oxide, hydroxy, carboxylic acid, keto, thio, thioether, alkene, phosphite, and phosphonate, were tested in the FT-ICR. The results demonstrate that basic protonated bifunctional compounds (PA between 222 and 245 kcal/mol) react selectively with DEE by forming a specific addition/elimination product ion (adduct - EtOH) (this product was also observed for lysine with three functionalities). The diagnostic reaction sequence involves proton transfer from the protonated analyte to the basic vinyl group in DEE, followed by addition of one of the functional groups of the analyte to the electrophilic α-carbon in protonated DEE. The next step involves proton transfer from this functionality to the other analyte functionality, followed by proton transfer to DEE and elimination of ethanol. Since the mechanism involves proton transfer between two functional groups of the analyte, the reaction does not occur for analytes where the two functionalities cannot be in close proximity (i.e., meta-phenylenediamine), and where no proton is available (i.e., dimethylaminoketone).

A novel chemical ionization reagent ion for organic analytes: the aquachloromanganese(II) cation [ClMn(H2O)+]

The reactivity of ClMn(H(2)O)(+) towards small organic compounds (L) was examined in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The organic compounds studied are aliphatic and aromatic alcohols, aliphatic amines, ketones, an epoxide, an ether, a thiol and a phosphine. All the reactions lead to the formation of the ClMn(H(2)O)(L)(+) complex, which dissociates by loss of the H(2)O molecule. In general, the reactions were found to occur with high efficiencies (>85%), indicating them to be exothermic. Electron transfer was also observed between ClMn(H(2)O)(+) and compounds with low ionization energies (IE), to form the molecular ion (L(+•)) of the analyte. Based on these observations, the IE of ClMn(H(2)O)(+) is approximated to be 8.1 ± 0.1 eV. Thus, the utility of ClMn(H(2)O)(+) as a chemical ionization reagent in mass spectrometry is expected to be limited to organic compounds with IEs greater than 8 eV.

Data-Dependent Neutral Gain MS3: Toward Automated Identification of the N-Oxide Functional Group in Drug Metabolites

We report here an automated method for the identification of N-oxide functional groups in drug metabolites by using the combination of liquid chromatography/tandem mass spectrometry (LC/MSn) based on ion-molecule reactions and collision-activated dissociation (CAD). Data-dependent acquisition, which has been readily utilized for metabolite characterization using CAD-based methods, is adapted for use with ion-molecule reaction-based tandem mass spectrometry by careful choice of select experimental parameters. Two different experiments utilizing ion-molecule reactions are demonstrated, data-dependent neutral gain MS3 and data-dependent neutral gain pseudo-MS3, both of which generate functional group selective mass spectral data in a single experiment and facilitate increased throughput in structural elucidation of unknown mixture components. Initial results have been generated by using an LC/MSn method based on ion-molecule reactions developed earlier for the identification of the N-oxide functional group in pharmaceutical samples, a notoriously difficult functional group to identify via CAD alone. Since commercial software and straightforward, external instrument modification are used, these experiments are readily adaptable to the industrial pharmaceutical laboratory.