1200 series lc

An isocratic RP-HPLC was adopted to quantify RU in all in vitro samples according to Kuntić et al. (2007 (link)) using Agilent Technologies (Santa Clara, CA, USA) 1200 series LC equipped with G 1311 A solvent delivery pump and G1315D diode array detector. A Kromasil^® C18 reverse-phase analytical column (5 μm particle size; 250 × 4.6 mm ID) was used and maintained at temperature =40 °C. The mobile phase constituted of a binary mixture of methanol–water 1:1 (v/v), pH 2.8 (adjusted with phosphoric acid) adjusted at flow rate of 1 mL/min. The wavelength of UV detector was set at λ_max 360 nm. The method was first validated according to the International Conference on Harmonization guidelines (ICH, Topic Q2A, Validation of Analytical Procedures: Methodology, PMP/ICH/281/95). The data was analyzed using ChemStation B.04.01 software (Santa Clara, CA, USA).

For IR spectroscopy, the sample was first dissolved in methanol at 0.05 g mL⁻¹. A drop of this solution is deposited on surface of KBr cell. The solution was then evaporated to dryness and the film formed on the cell is analyzed directly on a Nicolet IN 10 Micro FTIR spectrometer by transmission mode (Supplementary Data 2). For UV spectroscopy, the same solution was analyzed by the DAD detector of an Agilent 1200 series LC and scanned from 210 to 600 nm (Supplementary Data 2). For optical rotation, the same solution was analyzed on a Anton Paar MCP100 polarimeter and the specific rotation was [α]²⁵_D = + 7.25 (c = 0.035, CHCl₃). Values represent means ± s.d. of three technical replicates.

LC–MS/MS (Agilent) was performed as previously described (8 (link)) using an Agilent 1200 series LC and 6470 triple quadrupole tandem mass spectrometer. In brief, DMP was quantified using m/z 97.4→56.2 and qualified using m/z 97.4→70.2, and 3,4-HDMP used m/z 99.2→57.2. The same Phenomenex Kinetex biphenyl column (50 × 3 mm, 2.6 µm) was used, and a second Phenomenex Kinetex C18 column (50 × 3 mm, 2.6 µm) was also used to identify whether the stationary phase played a specific role in the chromatography. Perfluorooctanoic acid (0.01%) in water (phase A) and methanol (phase B) was used for quantification and the flow rate was optimized to 0.4 mL/min, with an injection volume of 1 µL. The gradient started with 20% phase B for 0.5 min and then increased to 80% by 4 min, held until 7.5 min and then returned to 20%.

All ¹H spectra were recorded
at 25 °C with a Bruker Avance spectrometer operating at 300.13
MHz and referenced to the protonated solvent residual. Mass spectra
(ESI⁺) were obtained with a Bruker micrOTOF II mass spectrometer
with an Agilent Technologies 1200 Series LC. The UV–vis spectra
were acquired on a Hewlett-Packard 8752A diode array spectrometer.
Elemental analyses were performed by Galbraith Laboratories, Inc.
of Nashville, TN, or by Kolbe Microanalytical Laboratory in Oberhausen,
Germany. Electrochemical measurements were performed using a CHI 620C
electrochemical analyzer workstation with a Ag/AgCl reference electrode,
glassy carbon working electrode, Pt wire as the auxiliary electrode,
and [ⁿBu₄N][PF₆]
as the supporting electrolyte. Under these conditions, the Cp₂Fe⁺/Cp₂Fe couple consistently occurred
at +0.50 V. Spectroelectrochemical measurements were made with an
Ocean Optics HR2000 spectrophotometer along with a Pine Research Instruments
platinum honeycomb working electrode and a Ag/AgCl reference electrode.
Procedural details regarding crystal growth, X-ray diffraction data
collection, data processing, and structure solution and refinement
are deferred to the Supporting Information (SI). Unit cell data and selected refinement statistics for the
compounds that have been structurally identified are presented in Table 1; more complete crystallographic
data are summarized in Table S1.