Agilent 1200 hplc instrument

HPLC analysis was performed with an Agilent 1200 HPLC instrument (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump, vacuum degasser, autosampler, column compartment, and diode array detector (DAD). The Kinetex C18 column was used (100 × 4.6 mm, 5.0 μm, Phenomenex, Torrance, CA, USA). The mobile phase was a mixture of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile). A linear gradient elution was performed from 5% to 20% B in 20 min and from 20% to 25% B in 10 min, followed by washing and reconditioning the column. Column temperature was maintained at 35 °C. Analysis was performed at a flow rate of 1.0 mL/min and monitored at 250 nm. Standard compound ecdysterone was purchased from Acade Chemical (Kowloon, Hong Kong). Additionally, 25R-inokosterone, and 25S-inokosterone were purchased from Chengdu Biopurity Phytochemicals (Wenjiang, Chengdu, China)

HPLC analyses were performed using an Agilent 1200 HPLC instrument (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump (G1312A), vacuum degasser (G1322A), auto-sampler (G1329A), column compartment (G1316A), and diode array detector (DAD, 1365B). Data were collected and analyzed using the Agilent ChemStation software. Chromatographic separation was conducted using a Luna C18(2) (250 × 4.6 mm, 5.0 μm; Phenomenex, Torrance, CA, USA), and the column temperature was maintained at 40 °C. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B) with gradient elution for better separation. The gradient solvent system was optimized as follows: 95–55% A (0–40 min), 55–0% A (40–41 min), 100% B (41–45 min), and 95% A (45–55 min). The flow rate was 1 mL/min. The detection was conducted at 240 nm, and the injection volume of each sample was 10 μL. To test for linearity, standard solutions at five levels were prepared by serially diluting the stock solution. Each analysis was repeated three times, and the calibration curves were fitted by linear regression. The limit of detection (LOD) and limit of quantification (LOQ) data obtained under the optimal chromatographic conditions were determined using signal-to-noise (S/N) ratios of 3 and 10, respectively.

NIC was analysed by HPLC using an Agilent 1200 HPLC instrument (Agilent Technologies, Cheshire, UK) with an auto sampler. The stationary phase used was a C-18 reverse-phase column, 4.6 × 250 mm (Phenomenex, Cheshire, UK). Sodium acetate solution, methanol and trimethylamine, (88:12:0.5 v/v) were used as mobile phase with pH adjusted to 4.2 using glacial acetic acid, at a flow rate of 1 mL/min and UV detection at 259 nm [39 (link)]. The retention time of NIC was detected at approximately 4.5 min. A calibration curve was plotted from NIC standards ranging from 40 µg/mL to 400 µg/mL (R² = 0.9994).

Dried Panax ginseng C. A. Mey. was obtained from Wooshin Industrial Co. Ltd. (Geumsan, Korea). Red ginseng extract was incubated with 10% pectin lyase (Novozyme, Denmark) at 50 °C for 5 days. The extract was then heated at 95 °C for 10 min, freeze-dried, and stored at 4 °C until use. The contents of ginsenosides in the extract were determined by HPLC analysis. HPLC analysis was performed with an Agilent 1200 HPLC instrument (Agilent Technologies, Santa Clara, CA, USA). The pectin-lyase-modified ginseng extract contained 1.55% ginsenoside Rb1, 0.95% ginsenoside Rb2, 0.99% ginsenoside Rc, 1.56% ginsenoside Rd, 0.64% ginsenoside Re, 0.22% ginsenoside Rf, and 0.33% ginsenoside Rg1.

Samples were analyzed by high performance liquid chromatography (HPLC, Agilent 1200 HPLC instrument, Agilent Technologies) on Dionex Acclaim surfactant column (3 µm, 120Å, 2.1×150 mm, DIONEX) and MS (Esquire 6000 electrospray ionization: ESI, Bruker Daltonics). The mobile phases were H₂O with 0.1% formic acid (eluent A) and acetonitril with 0.1% formic acid (eluent B). They were delivered at a flow rate of 0.2 ml/min and the column was operated at 25°C. The gradient was as follows: 0–3 min. 20% B, 3–12 min. 20–100% B, 12–70 min. 100% B. The injection volume to the system was fixed at 10 µl. The column eluent was connected to MS. The ESI-MSⁿ spectrum conditions were optimized in the negative-ion mode with the conditions as follows: nebulizer gas, 30.0 psi; drying gas, flow 8 l/min; dry temperature 330°C; high voltage (HV) capillary, 4500 V; HV end plate offset, −500 V; target ion trap, 30000; scan range 100–3000 m/z. The width for targeted precursor ions was set at 4 m/z.

O. schwerinae was collected from Shrubs (2000 m), Kunming, Fuming County, China, in September 2013 and identified by Prof. J.-H. Kim, Gachon University, Republic of Korea. A voucher specimen (No. DiAB-2006-141) was deposited in the Herbarium of Korea Institute of Oriental Medicine, Republic of Korea. Dried leaves and twigs of O. schwerinae (1.0 kg) extract and its major compounds [2′-O-acetylvitexin (4.2 mg, t_R 18.3 min), quercitrin (12 mg, t_R 22.8 min) and hyperoside (7 mg, t_R 28.5 min)] were further purified, as previously described [15 (link)]. To check the its major compounds, high-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1200 HPLC instrument chromatogram of OSSC. The column temperature was maintained at 30 °C. The analysis was performed at a flow rate of 1.0 mL/min and monitored at UV254 nm [15 (link)].

HPLC analyses were performed using an Agilent 1200 HPLC instrument (Agilent Technologies, Santa Clara, CA). The instrument consisted of an auto-sampler, binary pump, column compartment, diode array detector, and vacuum degasser. The Agilent ChemStation software was used as the data processor. The analytical column was used a Zorbax Eclipse Plus column (250 × 4.6 mm, 5.0 μm; Agilent) and its temperature was kept at 40°C. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B) with gradient elution. The gradient solvent system was optimized as follows: 95–50% A (0–45 min), 50–0% A (45–50 min), 100% B (50–60 min), and 95% A (61–70 min) at a flow rate of 1.0 ml/min. The detection was performed at 260 nm and the injection volume was 5 μL. Standard solutions at 5 levels were arranged by serially diluting the stock solution to evaluate for linearity. Each analysis was repeated 3 times, and the calibration curves were fitted by linear regression. The limit of detection (LOD) and limit of quantification (LOQ) data collected under the optimal chromatographic conditions were accessed using signal-to-noise (S/N) ratios of 3 and 10, respectively.