1200 series lc system

¹H and ¹³C NMR
spectra were recorded on a Bruker Avance 400 spectrometer (400.1 MHz)
without an additional internal standard. Chemical shifts are reported
as δ in parts per million (ppm) and are calibrated against the
residual solvent signal.
Reverse-phase HPLC was performed on
an Agilent 1200 Series LC system equipped with a G1379A degasser,
a G1312A binary pump, a G1329 autosampler unit, a G1316A temperature-controlled
column compartment, and a G1315B diode array detector.

Biotransformation products of moxifloxacin were identified by liquid chromatography coupled by mass spectroscopy (LC–MS). The apparatus was an Agilent 1200 series LC system coupled with an Agilent 6520 quadrupole time of flight tandem MS instrument featured by an electrospray ion source (Agilent, Waldbronn, Germany). A 2.1–100 mm Nucleosil 100-3 C18 HD column (Macherey–Nagel, Düren, Germany) was applied with a flow rate of 0.35 mL min⁻¹. Deionized water + 0.1% formic acid (40%) and acetonitrile (60%) were used as the mobile phase.

An oscillating Vibromatic shaker and a Mixtasel-BL centrifuge from J.P. Selecta S.A.® (Barcelona, Spain), an Orto Alresa Digtor 21 centrifuge from Orto Alresa (Madrid, Spain) and a vortex shaker from IKA® (Wilmington, NC, USA) were used for sample preparation steps.
An Agilent 1200 series LC system (Palo Alto, CA, USA) with electrospray ionization (ESI) coupled to an Agilent 6410 triple quadrupole (QqQ) tandem mass spectrometer was used for the analysis of the prepared samples. For qualitative and quantitative analyses, MassHunter Workstation software (V—B.10, Agilent Technologies, Palo Alto, CA, USA) was used for data acquisition and processing.

Pooled protein (100 μg) from each of the 4 categories was subjected to reduction using tris(2-carboxyethyl) phosphine (TCEP) and cysteine block using methyl methanethiosulfonate (MMTS) followed by trypsin digestion at 37°C for 16 hours using 1 : 20 (w/w) sequencing grade trypsin (Promega, USA). The tryptic digests were labelled with 4 different iTRAQ reagents: premenopause low BMD with 114, premenopause high BMD with 115, postmenopause low BMD with 116, and postmenopause high BMD with 117 according to the manufacturer's instructions (Applied Biosystems, USA). The peptides with the four labels were pooled, dried, and reconstituted in solvent A (5 mM KH₂PO₄ pH 2.7, 30% ACN) and subjected to fractionation by a SCX column (PolySULFOETHYL A column, 100 × 2.1 mm, 5 μm particles with 300 Å pores; PolyLC, Columbia, MD) using an Agilent 1200 series LC system. A gradient of 50 min from 5 to 40% solvent B (350 mM KCl in solvent A) with flow rate of 300 μL/min was applied. A total of 15 SCX fractions were desalted using C₁₈ microtips, vacuum-dried, and stored at −80°C until mass spectrometric analysis. The sample labelling and fractionation were performed twice using the same pool of samples to generate an experimental replicate.