Hplc dad

Andrographis paniculata aqueous extract capsules were analyzed for the levels of the four major active diterpenoids using the previously described HPLC-DAD method (Pholphana et al., 2013 (link)). Briefly, 50 mg of A. paniculata aqueous extract in capsule (20 capsules) was accurately weighed in a 100 mL volumetric flask and extracted with methanol in an ultrasonic bath (Elma, Germany) for 30 min. The extracted solution was filtered through a 0.2 µm PVDF membrane (Chrom Tech, MN, United States). The four major active diterpenoids were simultaneously analyzed by HPLC-DAD (Agilent Technologies, Waldbronn, Germany) on a reverse phase column (Zorbax SB-C18; 4.6 × 75 mm, 3.5 μm) (Agilent Technologies, CA, United States), using 28% acetonitrile in water as the mobile phase delivered at a flow rate of 1.2 mL/min. The diode array detector was set at 205 nm. The contents of andrographolide; 14-deoxy-11, 12-didehydroandrographolide; neoandrographolide; and 14-deoxyandrographolide in A. paniculata aqueous extract capsule are presented in Table 1. The HPLC chromatograms of the four standard diterpenoids and A. paniculata aqueous extract are shown in Supplementary Figure S1.

The HPLC analysis of FXE was performed using HPLC-DAD (Agilent 1200, Germany) equipped with a Zorbax RXC8 (Agilent, USA) analytical column with a 5 μm particle size and a 25 ml capacity using the previously reported method by Zu et al.^{23 (link)} Each sample was diluted with HPLC grade methanol. The mobile phase consisted eluent A, (acetonitrile–methanol–water–acetic acid/5 : 10 : 85 : 1)-and eluent B (acetonitrile–methanol–acetic acid/40 : 60 : 1). The gradient (A : B) utilized was as follows: 0–20 min (0 to 50% B), 21–25 min (50 to 100% B), 26–30 min (100% B) and 31–40 (100 to 0% B) at a flow rate of 1 ml min⁻¹. The standards and samples were prepared in HPLC grade methanol (1 mg ml⁻¹), filtered through a 0.45 μm-membrane filter and 20 μl was injected for the analysis. Myricetin, quercetin, and kaempferol were investigated at the wavelength of 368 nm, gallic acid and rutin acid were analyzed at 257 nm, catechin was examined at 279 nm and caffeic acid was evaluated at 325 nm. Before each column was run the column was reconditioned for 10 min and the analysis was performed in triplicate for each one of the FXE samples. By using the external standard method, we have fully assimilated the peaks for the quantification of samples. All work was carried out at ambient temperature.

Chromatographic analysis was carried out by using HPLC-DAD (Agilent Germany) attached with Sorbex RX-C8 (Agilent USA) analytical column. Briefly, mobile phase A was acetonitrile-methanol-water acetic acid (5: 10: 85: 1) and mobile phase B was acetonitrile methanol- acetic acid (40: 60: 1). A gradient of time 0–20 min for 0 to 50 % B, 20–25 min for 50 to 100 % B, and then isocratic 100 % B till 40 min was used. The flow rate was 1 ml/min and injection volume was 20 μl. Rutin and gallic acid were analyzed at 257 nm, catechin at 279 nm, caffeic acid at 325 nm and quercetin, myricetin, kaempferol were analyzed at 368 nm. Every time column was reconditioned for 10 min before the next analysis. All the samples were assayed in triplicate at ambient temperature. Quantification was carried out by the integration of peak using the external standard method using following formula: $\mathrm{Conc}.\mathrm{of}\mathrm{S}\mathrm{C}\mathrm{in}\mathrm{sample}=\left[\frac{\mathrm{Peak}\mathrm{area}\left(\mathrm{m}\mathrm{A}\mathrm{U}\ast \mathrm{s}\right)\mathrm{of}\mathrm{S}\mathrm{C}\mathrm{in}\mathrm{sample}}{\mathrm{Peak}\mathrm{area}\left(\mathrm{m}\mathrm{A}\mathrm{U}\ast \mathrm{s}\right)\mathrm{of}\mathrm{S}\mathrm{C}}\right]\times \mathrm{Conc}.\mathrm{Of}\mathrm{S}\mathrm{C}$
SC is for standard compound
The concentration of standard compound in each fraction was expressed as μg/100 of dry plant powder.

Phenolic compounds were extracted from SCWBOE previous to their analysis by HPLC-DAD (Agilent Technologies, Santa Clara, CA, USA). Then, 2.0 g of SCWBOE was extracted twice with 2 mL of methanol by vortex agitation for 2 min. The two methanol extracts were mixed and centrifuged at 3214× g for 30 min, the supernatant was separated and evaporated under vacuum at 40 °C. The dry residue was suspended in 2 mL of water: methanol (80:20), filtered (20 µm) and analyzed by a HPLC-DAD system according to the method previously reported by Pérez-Magariño et al. [20 (link)] Chromatographic separation was performed in a Spherisorb ODS2-3µm column (250 × 4.6 mm) at a flow rate of 0.6 mL/min with (A) water/acetic acid (98:2) and (B) water/acetonitrile/acetic acid (78:20:2) and the following linear gradient: from 0% to 25% solvent B in 25 min, from 25% to 70% B in 35 min, from 70% to 100% B in 40 min and then isocratic for 20 min. Diode array detection was performed from 200 to 400 nm. The injection volume was 200 µL. The phenolic compounds analyzed were identified by comparing their retention times and UV-Vis spectra with their respective standard according to previously published data [20 (link)]. Quantification was performed by using the calibration curves obtained with the corresponding standard compound.

HPLC analysis of ANM and selected plant fractions (ANE and ANA) was performed using HPLC-DAD (Agilent 1200, Germany) equipped with Zorbex RXC8 (Agilent, USA) analytical column with 5 μm particle size and 25 ml capacity using previously reported method [28 (link)]. Each sample was diluted with HPLC grade methanol. Mobile phase was consisted of eluent A, (acetonitrile- methanol--water- acetic acid-/5: 10: 85: 1)-and eluent B (acetonitrile-methanol-acetic acid/40: 60:-1). The gradient (A: B) utilized was the following: 0–20 min (0 to 50 % B), 21–25 min (50 to 100 % B), 26–30 min (100 % B) and 31–40 (100 to 0 % B) at flow rate ofL1 ml/min. The standards and samples were prepared in HPLC grade methanol (1 mg/ml), filtered through 0.45 μm-membrane filter andL20 μl was injected for the analysis. Among the standards rutin was investigated at 257 nm, catechin and gallic acid at 279 nm, caffeic acid and apigenin at 325 nm while quercetin, myricetin and kampferol were analyzed atL368 nm [29 (link)]. The analysis was performed in triplicate and the column was reconditioned for 10 min after each run. Quantification was done by the integration of the peak by using the external standard method.

The extract with the highest antioxidant activity was analyzed on a high-performance liquid chromatograph coupled to a diode array detector, HPLC-DAD, Agilent Technologies (Palo Alto, CA, USA). The separation of the compounds was performed on a GEMINI C18 column (Phenomenex, Torrance, CA, USA), 250 mm, L × 4.6 mm, I.D. × 5 µm particle size, 25 °C, using as mobile phase (A): H₂O (0.5% CH₂O₂) and (B): acetonitrile, at a flow rate of 1 mL/min, with an elution gradient 0 min: 98% A, 15 min: 88% A, 15–23 min: 88% A, 46 min: 60% A, 71 min: 10% A, 71 min: 10% A, 71–75 min: 10% A, 80 min: 98% A, 80–85 min: 98% A. The injection volume was 20 µL and the phenolic compounds were detected at 290 nm. For the identification of the phenolic compounds, 13 certified standards were used (Figure S10).

The Pf content was determined via HPLC-DAD (Agilent/Thermo) using a Kromasil C18 250 × 4.6 mm (5 µm) column (Labtec research site method). The mobile phase A consisted of a mixture of 0.02 M KH₂PO₄ buffer/mobile phase B (95:5, v/v), and the mobile phase B consisted of acetonitrile/methanol (22:13, v/v). The percentage of mobile phase B was gradually increased to 30% over 10 min, held at 30% for 12 min, increased to 75% over a period of 5 min, and subsequently increased to 95% over 10 min. It was then decreased gradually to 0% for 3 min and held for another 5 min (total run time 45 min).
The injection volume was 15 µL, with a column oven temperature of 40 °C, a flow rate of 1.0 mL/min, and a detection wavelength of 220 nm.