1100 series hplc instrument

Non-targeted HPLC-UV and HPLC-FLD chromatographic fingerprints were obtained with an Agilent 1100 Series HPLC instrument (Waldbronn, Germany) equipped with an automatic injector, a binary pump, and both diode-array and fluorescence detectors (connected in series). Agilent Chemstation software was employed for instrument control and data processing. Reversed-phase separation using a Kinetex^® C18 (100 × 4.6 mm i.d., 2.6 µm partially porous particle size) column (Phenomenex, Torrance, CA, USA) was employed. Separation was carried out by gradient elution employing as mobile phase components a 0.1% formic acid aqueous solution and methanol. The gradient elution conditions are shown in Table 1. An injection volume of 5 µL was used. Acquisition was carried out at 280 nm for UV detection and at 280 nm (excitation) and 350 nm (emission) for FLD detection.

An Agilent 1100 Series HPLC instrument equipped with a quaternary pump (G1311A), a degasser (G1322A), an autosampler (G1329A), a diode array detector (G1315B), and a PC with the Agilent Chemstation software, all of them from Agilent Technologies (Waldbronn, Germany), was employed. HPLC-UV fingerprints were obtained by reversed-phase mode using a Kinetex C18 porous-shell column (100 mm × 4.6 mm I.D., 2.6 µm particle size) from Phenomenex (Torrance, CA, USA) at room temperature. Chromatographic separation was performed under gradient elution mode, using 0.1% (v/v) formic acid aqueous solution (solvent A) and methanol (solvent B) as mobile phase components, following the next elution program: 0–20 min, linear gradient from 15% to 95% solvent B; 20–30 min, isocratic elution at 95% solvent B; 30–30.1 min, back to initial conditions; and from 30.1–35 min, at 15% solvent B for column re-equilibration. The mobile phase flow rate was 0.4 mL/min, and the injection volume was 5 µL. The HPLC-UV fingerprints were registered at 250 nm.

The polyphenols in different leaves extracts were evaluated by chromatographic technique, using the earlier reported method [32 ]. The chromatographic system consists of an Agilent 1100 series HPLC instrument (Santa Clara, USA) equipped with an MS detector. Analytical separation was carried out in a C18 column (4.6 mm × 100 mm × 5 μm, Agilent Technology) with a flow rate of 0.8/min with two mobile solvent phases (eluent A = 10 mM ammonium acetate and 1% acetic acid in water; eluent B = 1% acetic acid in methanol). The gradient elution was performed as follows: 0.3 min, 15–50% A; 3–5.5 min, 50–90% A; 5.5–9 min, 90% A; 9–9.5 min, 90–15% A, 9.5–10 min, 15% A. The sample injection volume was 20 μL and the temperature of the column was fixed at 40 °C. Compounds were identified with the mass spectra and Rt with the NIST library of standard compounds.

HPLC analyses were performed on an Agilent 1100 series HPLC instrument, equipped with a quaternary pump, an autosampler, a column oven, and a photodiode array detector (Agilent, Waldbronn, Germany). Optimum separation was achieved on a YMS-Pack Pro C18 RS (150 × 4.6 mm, 3 µm) column from YMC, guarded with an in-line filter and using a mobile phase consisting of 0.9% (v/v) formic acid and 0.1% (v/v) acetic acid in water (A) and methanol (B). The applied gradient was as follows: 0% B at 0 min, 0% B at 15 min, 10% B at 23 min, 15% B at 30 min, and 98% B at 35 min, and held at this composition for 5 min (total runtime of 40 min); for the next 15 min the column was equilibrated under initial conditions. Flow rate, temperature, and injection volume were adjusted to 0.55 mL· min⁻¹, 25 °C, and 4.0 μL. The detection wavelength was set to 330 nm.

For quantitative analysis, five of the reference compounds solutions, glycyrrhizin and liquiritin (1,000 μg/mL), were prepared in 100% methanol and stored at 4°C. The standard solutions were prepared by six concentrations of diluted solutions (methanol). All calibration curves were attained by assessing the peak areas at six concentrations in the range of 10–500 μg/mL for all reference compounds. The linearity of the peak area (y) versus concentration (x, μg/mL) curve for each component was used to calculate the contents of the main Sagunja-tang. 102.8 mg of Sagunja-tang extract powder was subsequently resuspended in 10 mL distilled water for high-performance liquid chromatography (HPLC) analysis.
The contents of glycyrrhizin and liquiritin in the SGJT water extract were analyzed using a 1100 series HPLC instrument (Agilent Technologies, USA) with a Gemini C18 column (4.6 × 250 mm, 5 μm; Phenomenex, USA). The mobile phase consisted of the solvents, distilled water (A) and acetonitrile with 0.1% formic acid (B). The following gradient was used: 0 min, A : B 80 : 20 (v/v); 10 min, A : B 60 : 40; 15 min, A : B 40 : 60; 30 min, A : B 15 : 85; and 40 min, A : B 0 : 100. The mobile phase flow rate was 1.0 mL/min, the column temperature was 30°C, the injection volume was 10 uL, and UV detection was at 254 nm (glycyrrhizin) and 280 nm (liquiritin).

Sample reactions were analyzed to quantitate metabolites by absorbance. Analytes were separated on a Waters XBridge BEH C18 3.5 μM column (4.6 mm × 100 mm) using an Agilent 1100 series HPLC instrument equipped with a VWD detector (280 nm). The mobile phase consisted of solvent A (10% solvent B, 90% water) and solvent B (0.1% formic acid/acetonitrile). A gradient method started at 80% solvent A for the first minute, then decreased to 70% for the next 6 min. The method held 70% A for 3 min before decreasing to 15% A over 11 min, then returning to 80% over 3 min and holding for the final 2 min. The flow rate was 1.2 mL/min for a total run time of 25 min. Parent drug and metabolite responses were normalized to the internal standard acenocoumarol and quantitated using a standard curve generated with 5OH-APINACA. The absorbance response corresponds to the indazole ring, which remained unmodified in these reactions [28 (link)], so that the relative absorbance response for all analytes were approximately the same, making inference for quantitation purposes possible.