G1379a degasser

An Agilent 1100 Series HPLC instrument equipped with a G1311A quaternary pump, a G1379A degasser, a G1392A autosampler, a G1315B diode-array detector, and a computer with the Agilent Chemstation software (Rev. A 10.02), all from Agilent Technologies (Waldbronn, Germany), was employed to obtain the HPLC-UV chromatographic fingerprints for the PCA and PLS studies. Chromatographic separation was carried out in reversed-phase mode by using a Zorbax Eclipse XDB-C8 column (150 × 4.6 mm i.d., 5 µm particle size) also provided by Agilent Technologies. Formic acid (0.1%, v/v) aqueous solution (solvent A) and methanol (solvent B) were used as mobile phase to stablish the gradient elution as follows: 0–2 min at 10% B (initial conditions); 2–4.5 min linear gradient from 10% B to 25% B; 4.5–7 min at 25% B; 7–22 min linear gradient from 25% B to 90% B; 22–24 min at 90% B; 24–25 min back to initial conditions at 10% B; and 25–30 min at 10% B for column equilibration. A mobile phase flow-rate of 1 mL min⁻¹ and an injection volume of 10 µL were employed. Photodiode array (PDA) acquisition from 190 to 600 nm was performed to register UV spectra and to guarantee peak purity when necessary. HPLC-UV fingerprints for PCA and PLS analysis were obtained by direct UV absorption detection at 257, 280, and 316 nm.

The HPLC-ESI-MS system consisted of an Agilent 1100 series HPLC system equipped with a G1312A Bin pump, a G1379A Degasser (Agilent, San Jose, CA, USA) and a G1316A automatic column temperature control box. Chromatographic separation was performed on a HiQ sil-C18 reversed-phase column (4.6 mm×250 mm, 5 µm, KYA TECH). An API3000 Triple tandem quadrupole mass spectrometer with a Turbolon-Spray interface from Applied Biosystems (Foster City, CA, USA) was operated in the positive electrospray ionization (ESI+) source mode. All mass spectra were acquired in multiple reaction monitoring transitions.
For HPLC-ESI-MS analysis, acetonitrile-water-acetic acid (18∶82∶0.1, v/v/v) was used at a flow rate of 1.0 ml/min with a run time of 65 min. The injection volume was 10 µl and the column temperature was maintained at 25°C. The ion source was operated at a temperature of 250°C. The nebulizing gas, curtain gas and collision gas were set at 12, 10 and 6 a.u., respectively. The ion spray voltage was 5500 V. The entrance potential and focusing potential were set at 10 and 400 V, respectively. Analyst software (version 1.4, Ab Sciex, Framingham, MA, USA) installed on a Dell computer was used for data acquisition and processing. The LC-ESI-MS chromatogram of DHQ is shown in Figure 1(d).

The chromatographic separation was performed on an Agilent 1100 HPLC system equipped with a G1379A degasser, G1312A binary gradient pump, G1329A autosampler, G1316A column thermostat and G1315C diode array detector (DAD) (Agilent Technologies, Waldbronn, Germany). Samples were separated on a Zorbax SB-C18 (Agilent Technologies, Santa Clara, CA, USA) (150 mm length, 3.0 mm i.d., 3.5 μm particle diameter) column, maintained at 25 °C. The mobile phase was composed of 0.3% acetic acid in water (v/v) (A) and methanol (B). The following gradient program was applied, at a flow rate of 0.3 mL/min with the composition of the mobile phase changing from 5% B to 100% B in 30 min, maintaining 100% B for 5 min and returning to 5% B in 1 min. All aqueous solvents were filtered through MF-Millipore (Millipore, Billerica, MA, USA) (0.45 μm, mixed cellulose esters) membrane filters. Chromatograms were acquired at 280 nm. Injection volume was 5 μL. Prior to injection, all samples were filtered through Sartorius (Goettingen, Germany) Minisart RC15 (0.2 μm) syringe filters.