The collisionally activated decomposition of [M + H]+ ions, generated by fast atom bombardment (FAB) of glutathione conjugates, has been studied by tandem mass spectrometry (MS/MS) using hybrid sector/quadrupole instruments. Abundant fragments of diagnostic utility were present in the daughter ion spectra. Common fragmentation modes were observed but their relative importance was strongly dependent on the nature of the conjugated species. As an example of a general approach to the characterization of glutathione conjugates in biological samples, the acetaminophen-glutathione conjugate was identified in rat bile, following coadministration of (2H0)- and (2H3)acetaminophen, using the experimental sequence: (i) conventional FAB mass spectrometric analysis, (ii) MS/MS using constant neutral loss (129 u) scanning to identify parent ions corresponding to glutathione conjugates, (iii) MS/MS to yield daughter ion spectra of parents so identified and corresponding to (2H0)- and (2H3)-labeled conjugates.

The clinical use of amifampridine phosphate for neuromuscular junction disorders is increasing. The metabolism of amifampridine occurs via polymorphic aryl N-acetyltransferase (NAT), yet its pharmacokinetic (PK) and safety profiles, as influenced by this enzyme system, have not been investigated. The objective of this study was to assess the effect of NAT phenotype and genotype on the PK and safety profiles of amifampridine in healthy volunteers (N = 26). A caffeine challenge test and NAT2 genotyping were used to delineate subjects into slow and fast acetylators for PK and tolerability assessment of single, escalating doses of amifampridine (up to 30 mg) and in multiple daily doses (20 mg QID) of amifampridine. The results showed that fast acetylator phenotypes displayed significantly lower Cmax, AUC, and shorter t1/2 for amifampridine than slow acetylators. Plasma concentrations of the N-acetyl metabolite were approximately twofold higher in fast acetylators. Gender differences were not observed. Single doses of amifampridine demonstrated dose linear PKs. Amifampridine achieved steady state plasma levels within 1 day of dosing four times daily. No accumulation or time-dependent changes in amifampridine PK parameters occurred. Overall, slow acetylators reported 73 drug-related treatment-emergent adverse events versus 6 in fast acetylators. Variations in polymorphic NAT corresponding with fast and slow acetylator phenotypes significantly affects the PK and safety profiles of amifampridine.

Analyses are described of small peptides and related compounds using a tandem hybrid mass spectrometer of BEQQ geometry. Collisionally activated decomposition of [M + H]+ ions, generated by fast atom bombardment, was performed in the radio frequency (rf)-only quadrupole. Interpretation of fragmentation was greatly facilitated by analysis of labeled analogs, obtained by 18O exchange of carboxyl oxygens. N-Acetylation was also valuable although significant changes in fragmentation resulted from derivatization. Daughter ion spectra of [M + H]+ ions of angiotensin III showed diagnostic fragmentations throughout the peptide chain.

BackgroundLate-onset Pompe disease is a rare genetic neuromuscular disorder caused by lysosomal acid alpha-glucosidase (GAA) deficiency that ultimately results in mobility loss and respiratory failure. Current enzyme replacement therapy with recombinant human (rh)GAA has demonstrated efficacy in subjects with late-onset Pompe disease. However, long-term effects of rhGAA on pulmonary function have not been observed, likely related to inefficient delivery of rhGAA to skeletal muscle lysosomes and associated deficits in the central nervous system. To address this limitation, reveglucosidase alfa, a novel insulin-like growth factor 2 (IGF2)-tagged GAA analogue with improved lysosomal uptake, was developed. This study evaluated the pharmacokinetics, safety, and exploratory efficacy of reveglucosidase alfa in 22 subjects with late-onset Pompe disease who were previously untreated with rhGAA.ResultsReveglucosidase alfa plasma concentrations increased linearly with dose, and the elimination half-life was <1.2 h. Eighteen of 22 subjects completed 72 weeks of treatment. The most common adverse events were hypoglycemia (63%), dizziness, fall, headache, and nausea (55% for each). Serious adverse events included hypersensitivity (n = 1), symptomatic hypoglycemia (n = 2), presyncope (n = 1), and acute cardiac failure (n = 1). In the dose-escalation study, all treated subjects tested positive for anti-reveglucosidase alfa, anti-rhGAA, anti-IGF1, and anti-IGF2 antibodies at least once. Subjects receiving 20 mg/kg of reveglucosidase alfa demonstrated increases in predicted maximum inspiratory pressure (13.9%), predicted maximum expiratory pressure (8.0%), forced vital capacity (−0.4%), maximum voluntary ventilation (7.4 L/min), and mean absolute walking distance (22.3 m on the 6-min walk test) at 72 weeks.ConclusionsAdditional studies are needed to further assess the safety and efficacy of this approach. Improvements in respiratory muscle strength, lung function, and walking endurance in subjects with LOPD may make up for the risk of hypersensitivity reactions and hypoglycemia. Reveglucosidase alfa may provide a new treatment option for patients with late-onset Pompe disease.Trial registration ISRCTN01435772 and ISRCTN01230801, registered 27 October 2011.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-017-0693-2) contains supplementary material, which is available to authorized users.

A procedure is described for the quantitative determination of platelet activating factor (PAF) using stable isotope dilution and fast atom bombardment/tandem mass spectrometry. Low-energy collisional activation of the [M + H]+ ion of PAF yields a single daughter ion of m/z 184, characteristic of phosphocholine derivatives. For precise and accurate quantification, the internal standard is (2H3)acetyl-hexadecyl PAF, which yields an analogous daughter ion of m/z 185. Quantitative analyses are based on limited mass-range parent ion scanning with transmission of daughters of m/z 184 and 185 during alternate scans; all scans are accumulated into a single data file to facilitate determination of the analyte/internal standard response ratio. Analysis of authentic hexadecyl PAF indicates a low-picogram detection limit. The method has been applied to the determination of PAF in preparations of human neutrophils stimulated by addition of a calcium ionophore. Concentrations of PAF of 7-17 ng/10(6) cells were observed, in keeping with earlier reports. The method has been validated by standard addition and dilution experiments. Comparison of data obtained by the new procedure and those obtained by a method involving gas chromatography/electron capture mass spectrometry of dephosphorylated and derivatized PAF showed excellent agreement.