Disposition and metabolism of rifapentine, a rifamycin antibiotic, in mice, rats, and monkeys.

Rifapentine is a cyclopentyl derivative of rifampin under development for the treatment of Mycobacterium tuberculosis and Mycobacterium avium complex infections. These studies were designed to investigate the disposition and biotransformation of single iv and oral doses of 14C-rifapentine in mice, bile duct-cannulated and uncannulated rats, and monkeys. Mass balance studies included 14C analyses of urine, feces, bile, cage wash, carcasses, and cage air collected for up to 120 hr postdose. Separation of radioactive compounds extracted from urine, bile, and feces was conducted using high-performance liquid chromatography and radioisotope detection. The mass spectra of selected chromatographic peaks were obtained. Disposition results were similar for all three species. Less than 5% of the radioactive dose of 14C-rifapentine was recovered in urine, indicating that renal excretion is a minor route of elimination in these species. The major route of elimination of radioactivity was into the feces, where more than 75% of the radioactivity was recovered. Biliary excretion was the major route of elimination of radioactivity in bile duct-cannulated rats dosed either po or IV. Radiochromatograms were similar for fecal samples from animals dosed by IV or orally. Ten regions of radioactivity were observed in mouse and rat fecal sample radiochromatograms, and seven regions of radioactivity were observed in monkey fecal sample radiochromatograms. The most abundant compound identified in feces was usually intact rifapentine (27%-41% of dose in mouse, 3%-35% of dose in rat, and 17%-29% of dose in monkey). Other peaks identified or characterized in feces based on liquid chromatography/ultraviolet/14C and/or liquid chromatography/mass spectrometry methods included 25-desacetyl-rifapentine, 3-formyl-25-desacetyl-rifapentine, and 3-formyl-rifapentine. The compounds rifapentine, 25-desacetyl-rifapentine, and 3-formyl-rifapentine were present in rat bile samples. These studies show that the metabolism and disposition of rifapentine in mice, rats, and monkeys were similar.

Preparation and characterization of biologically active 6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A and its conjugated derivative.

N-tert-butyloxycarbonyl (t-Boc) protected 6-aminocaproic (Cap) anhydride was reacted with unprotected hexaacyl-4'-O-monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Escherichia coli J5 to yield t-Boc-Cap-MLA. After a column purification step, the t-Boc group was removed by incubating the sample at low temperature in the presence of acid to yield Cap-MLA. This product was analyzed by californium plasma desorption mass spectrometry (PDMS). Purified t-Boc-Cap-MLA was further fractionated by reverse-phase high-performance liquid chromatography as its methyl ester and characterized by laser desorption mass spectrometry, PDMS, and proton nuclear magnetic resonance spectroscopy. These analyses revealed that the Cap group was selectively introduced into the 6'-position of MLA. To demonstrate that Cap-MLA can be conjugated to other compounds, it was reacted with biotin-Cap N-hydroxysuccinimide ester to yield biotin-(Cap)2-MLA. Analysis of this product by PDMS confirmed its expected molecular weight of 2171 and showed the presence of fragments containing the biotin and Cap groups. Monoclonal antibodies and streptavidin were used to show the presence of both lipid A and biotin in this conjugated product. These two novel lipid A derivatives were then tested for their bioactivities. Although both Cap-MLA and biotin-(Cap)2-MLA showed mitogenic activity using murine splenocytes, they were about 4-8 times less active than MLA at 20 micrograms/mL or less and only one-half as active at 100 micrograms/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatography-time-of-flight mass spectrometry and its application to peptide analyses.

High-performance liquid chromatography (HPLC) has been successfully interfaced on-line with liquid secondary-ion time-of-flight mass spectrometry, utilizing a continuous-flow interface. Time-of-flight mass spectrometry (TOF-MS) is a low-resolution, high-mass-range technique, compatible with extremely rapid data acquisition rates. Thus a TOF-MS system is extremely well suited for coupling with HPLC. This paper describes the interface used to couple the HPLC and TOF-MS as well as the basic operating principles of such a system. Using both standard and packed-capillary reversed-phase HPLC columns, the HPLC-TOF-MS system has been successfully used to separate and detect peptides, providing molecular weight information for the peptide analytes. Experimental data, including chromatograms (UV, reconstructed ion and selected ion) and mass spectra, are presented to demonstrate the ability of the HPLC-liquid secondary-ion TOF-MS system to resolve chromatographically analytes as well as to resolve mass spectrometrically analytes which are unresolved on the chromatographic column.

Liquid chromatography/time-of-flight mass spectrometry with high-speed integrated transient recording.

High-performance liquid chromatography (HPLC) has been interfaced to a time-of-flight mass spectrometer. The interface is a continuous flow probe and ions are desorbed from the liquid matrix by energetic ion bombardment. Pulsed and delayed ion extraction from the source permits the use of broad ionization times, results in the production of analog signals in each time-of-flight cycle, and provides both energy and spatial focusing. A high-speed integrated transient recording system has been developed and is also reported. This instrument is the prototype for development of a high-speed, high-mass range LC detector with high duty cycle. Its performance is demonstrated for the separation of several mixtures of small peptides.

Laser desorption electron impact: application to a study of the mechanism of conjugation of glutathione and cyclophosphamide.

Toward the objective of producing ion radical species from involatile and thermally labile samples, we have combined laser desorption of neutral molecules with electron impact ionization on a time-of-flight mass analyzer with a delayed draw-out pulse. The analytical capabilities of this method are tested in the analysis of isotope labels in the involatile product in a mechanistic study of both the chemical and the enzyme catalyzed reactions of cyclophosphamide with glutathione.

Pulsed gas introduction into quadrupole ion traps.

Two different Paul-type quadrupole ion traps were equipped with pulsed-valve gas inlets. The duration of a gas pulse inside the trap is variable, and pulses as short as 50 ms (FWHH) have been measured, allowing the use of several gas pulses during one experiment. The benefits of pulsed valves are outlined and demonstrated for chemical ionization experiments and for the use of selective ion-molecule reactions in structure determination of ions and neutral molecules.

Structural characterization of two isomeric dimethoxyindoles of biological interest, using high- and low-energy collisional spectroscopy.

It is not possible to distinguish isomers of biologically important dimethoxyindoles using electron-ionization mass spectra, but they may be distinguished by collisionally activated dissociation. In particular, energy-resolved mass spectrometry yields the best data for distinguishing between these isomers.

Nucleophilic reactions between biological conjugates and tetraalkylammonium ion pair reagents during desorption chemical ionization.

Tetraalkylammonium salts, commonly used as ion pair reagents in chromatography, were found to react with biological conjugates under desorption chemical ionization conditions in a mass spectrometer. The reactions occur for aromatic glucuronide and glucoside conjugates using solid samples loaded on the direct exposure probe. Evidence is presented that several mechanisms contribute to the degradative alkylation of benzo(a)pyrene glucuronide. This undesirable process can be prevented by using ammonium or trialkylammonium instead of tetraalkylammonium salts in the chromatographic separation. Nucleophilic attack on the tetraalkylammonium cations in the energized condensed phase was found to occur also for some simpler aromatic compounds.

Desorption ionization/tandem mass spectrometry with a caesium ion source and a triple quadrupole mass spectrometer.

The coupling of a caesium ion source to a triple quadrupole mass spectrometer is described. The potential of this combination for examining thermally labile, non-volatile molecules, such as thiamine hydrochloride, is examined. Emphasis is placed on the advantages the various scanning modes of tandem mass spectrometry provide in ion structure elucidation and the investigation of desorption ionization mechanisms. Use of the caesium ion source for desorption of neutral molecules which are chemically ionized by an auxiliary gas is demonstrated. This procedure may be especially useful for examining non-volatile, non-ionic samples.