Structural characterization of sulfadiazine metabolites using H/D exchange combined with various MS/MS experiments

Abstract

Two major metabolites and one minor metabolite of sulfadiazine were found in pig manure, using a special combination of different MS techniques like parent and product ion scans, H/D exchange, accurate mass measurement, and MS/MS experiments with substructures. N4-acetylsulfadiazine and 4-hydroxysulfadiazine were identified as major metabolites. N4-acetylsulfadiazine could be verified by H/D exchange and comparison with product ion spectra of a synthetic reference compound. In the case of 4-hydroxysulfadiazine, the majority of possible isomers could be discounted after H/D exchange. Substructure-specific MS/MS experiments with fragment ions and comparison with product ion spectra of two references revealed the presence of 4-hydroxysulfadiazine. The minor metabolite was characterized to some degree using H/D exchange and tandem mass spectrometry in combination with a high-resolution time of flight mass spectrometer. The aminopyrimidine moiety contained an additional modification with a likely elemental composition of . Antibiotics and antimicrobial agents are used for both therapeutic and prophylactic purposes in addition to their application as growth promoters. The antimicrobial sulfonamides are one class of pharmaceuticals that is widely used. As a consequence, these compounds have been found in surface and ground water, liquid manure, and soil [3][4][5][6][7][8][9][10][11][12]. After administration to animals, the majority of the pharmaceuticals used are excreted unchanged or as phase I and phase II metabolites. As part of an environmental risk assessment, these metabolites also have to be characterized because phase I metabolites can still be active against bacteria and phase II metabolites can be converted back to the parent drug or phase I metabolites. Moreover, very little is known about the capacity of microbiological decay and the occurrence of new metabolites after incubation for several months in a manure tank.There are only a few references describing the metabolism of sulfadiazine in pigs [13,14]. After intravenous administration of 40 to 60 mg sulfadiazine per kg body weight to male pigs without coadministration of trimethoprim, only low levels of metabolites have been observed. The N-acetyl sulfadiazine was the main metabolite in plasma. Traces of 4-hydroxysulfadiazine were also detected. N-acetyl sulfadiazine and 4-hydroxysulfadiazine were the major metabolites in urine collected up to three hours after application. No further metabolites were identified and investigations in feces were not carried out.The aim of the present work was to use different mass spectrometric strategies to identify possible metabolites of the widely used drug sulfadiazine in manure originating from a pig farm. The metabolites of interest should still have a similar structure as the parent drug and, therefore, retain to a greater or lesser extent their bioactive profile against micro-organisms. Earlier investigations about the biological activity of metabolites showed action of hydroxylated metabolites of sulfadiazine ag...

Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

“…In electrospray mass spectrometry, hydrogen/deuterium exchange is a commonly used method for structure elucidation for confirming the presence of active hydrogens in organic compounds (i.e. hydrogens attached to heteroatoms O, N, and S) 11–14. After switching the mobile phases of the HPLC system to D 2 O and CH 3 OD and extensive flushing with these deuterated solvents, an exchange rate of greater than 96% was achieved.…”

Simultaneous Determination of Selected Veterinary Antibiotics and their Main Metabolites in Fish and Mussel Samples by High-Performance Liquid Chromatography with Diode Array-Fluorescence (HPLC-DAD-FLD) Detection

“…The NMR, NMZ, NDI, AMA, and APA were synthesized in our laboratory according to the protocols described by Fuchs 2005 andBaker 1989, and the purity was checked by mass spectrometry and thin layer chromatography [using silica gel plates eluted with mixtures dichloromethane:methanol (30:10 v=v) and sprayed with ninhydrine 0.1% ethanolic solution; the plate was dryed at 100 C and the visualization of spots was realized under a blacklight (UV 254nm )]. Methanol, acetonitrile, ethyl acetate, diethyl ether, 2-propanol (HPLC grade), formic acid (98% purity), hydrochloric acid (37% purity), and sodium hydroxide, all analytical grade, were purchased from VWR (Barcelona, Spain).…”

‘On‐the‐fly’ hydrogen/deuterium exchange liquid chromatography/mass spectrometry using a dual‐sprayer atmospheric pressure ionisation source

“…distinguishing between R‐NH‐C 2 H 5 and R‐N‐(CH 3 ) 2 , which would not necessarily fragment differently in a tandem MS experiment. For H/D exchange experiments, samples are most commonly prepared in deuterated solvents and then infused into the mass spectrometer, or deuterated eluents are used with liquid chromatography/mass spectrometry (LC/MS) 11–17. In these instances H/D exchange occurs during the chromatographic separation, while the analytes of interest interact with the stationary phase of the column and the deuterated mobile phase.…”