Micromass zq spectrometer

Three Dba/2J mice were given i.p. injections of 0.8 mg/kg SPHINX31 and sacrificed after 24 h when blood was taken by cardiac puncture into EDTA tubes. Plasma was isolated by centrifugation, and an equal volume (100 µl) acetonitrile added. An internal standard of 100 µg/ml of a related compound (compound 3 from Batson et al) was added to samples to account for any loss of material during preparation. The solutions were centrifuged for 15 min at 4 °C and the supernatant taken for analysis. Solutions were evaporated at 37 °C for eight hours and resuspended in 30 µl acetonitrile ready for analysis by LC MS, using a Waters 2795 HPLC system. Detection was achieved by positive ion electrospray (ESI + ) mass spectrometry using a Waters Micromass ZQ spectrometer in single ion monitoring (SIM) mode, at 352 m/z units ([M+H]⁺). Chromatography (flow rate 1 mL·min⁻¹) was achieved using a Phenomenex Kinetex column (2.6 μ, C₁₈, 100 Å, 4.6 × 50 mm) equipped with a Phenomenex Security Guard precolumn (Luna C₅ 300 Å). Peaks occurring at these times in the SIM chromatograms per compound were integrated using Water MassLynx software. The chromatograms produced clear peaks at the expected molecular weights. The integrated area under the peaks and read from a standard curve led to quantification of the circulating concentration of SPHINX31.

PTM-B was obtained by dissolving PTM-I in distilled water and adding a 25% w/w NH₄OH solution at 4 °C. The obtained precipitate was filtered, washed with a 5% NH₄OH solution and dried under vacuum conditions overnight. The conversion of PTM-I into PTM-B was confirmed by mass spectrometry analysis using electrospray ionization or by atmospheric pressure chemical ionization, in positive ion mode, on a Micromass ZQ spectrometer (Waters, Milan, Italy), as previously reported [56 (link)].

All organic solvents used in this study were purchased from Sigma–Aldrich (St. Louis, MO, USA), Alfa Aesar (Haverhill, MA, USA), or TCI (Tokyo, Japan). Prior to use, acetonitrile was dried with molecular sieves with an effective pore diameter of 4 Å. Column chromatography purifications were performed under ‘‘flash” conditions using Merck (Darmstadt, Germany) 230–400 mesh silica gel. Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel plates (silica gel 60 F254), which were visualized by exposure to ultraviolet light and an aqueous solution of cerium ammonium molybdate (CAM). ESI-MS spectra were recorded with a Waters (Milford, MA, USA) Micromass ZQ spectrometer. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker (Billerica, MA, USA) AC 400 or 100, respectively, spectrometer and analyzed using the TopSpin 1.3 (2013) software package. Chemical shifts were measured by using the central peak of the solvent.

General procedures: Reactions were monitored either by TLC and/or analytic HPLC (UV, detection, 220 nm). Flash columns, filled with silica gel Merck 60 (230–400) were used for chromatographic separations. A reversed-phase column, Sunfire C18 (4.6 × 50 mm, 3.5 µm), a flux of 1 mL/min, and mixtures of CH₃CN, phase A, and H₂O, phase B, both containing 0.01% formic acid, were used for analytical HPLCs. Mass spectra, in electrospray, positive mode, were obtained on a Waters Micromass ZQ spectrometer. High resolution mass spectrum (ESI-HRMS) was recorded on an Agilent 6520 Q-TOF instrument. Optical rotation for final compound RGM8-51 was measured in a polarimeter Perkin Elmer 141 apparatus. NMR spectra were recorded in a Varian INOVA-400 (400 MHz) spectrometer, operating at 400 and 100 MHz for ¹H and ¹³C experiments, respectively (with chemical shifts expressed in ppm and coupling constants in Hz).

Pentamidine base (PTM-B) was obtained by precipitation of pentamidine isethionate (PTM-S) solution in alkaline medium as previously reported with minor modifications [41 (link)]. Briefly, PTM-S (0.2 g, 0.34 mmol) was dissolved in the minimum volume of MilliQ^® water and the solution was cooled at 4 °C under magnetic stirring. Little aliquots of NH₄OH solution (25% w/w) were added, until no more precipitation occurred. The precipitate was filtered under vacuum and washed twice with diluted NH₄OH solution (5% w/w) and the obtained powder was dried under vacuum.
To confirm the molecular structure of the obtained PTM-B, mass spectrometry analysis was carried out using electrospray ionization (ESI) or by atmospheric pressure chemical ionization (APCI), in positive ion mode, on a Micromass ZQ spectrometer (Waters). An absorption peak at m/z = 467.35 was detected only for PTM-S. The difference between m/z values of this peak and of the pseudo-molecular ion (m/z = 341.27) was 126.08, which corresponds to the molecular weight of isethionate. Since the absorption peak at m/z = 467.35 was not detected in PTM-B sample, it was concluded that isethionate was not present and that PTM-B was successfully obtained from PTM-S.
Melting points of PTM-S and PTM-B were determined using a BUCHI Melting Point B-450 (set point: 165 °C, heating rate: 2 °C/min).