L 2200 autosampler

Two hundred microliters of plasma (baseline and standing samples) were diluted with double-distilled water (1:2, vol/vol) and loaded on pre-conditioned anion exchange columns (Chromabond SB, Macherey-Nagel, Düren, Germany). After a washing step with double-distilled water nitrite and nitrate were eluted with 1 mL 0.5 mol/L sodium chloride. In the eluates, nitrite and nitrate were determined simultaneously by means of HPLC analysis according to a previously published method (Romitelli et al., 2007 (link)) but with some modifications. Briefly, the HPLC consisted of a L-2200 autosampler, two L-2130 HTA pumps, and a L-2450 diode array detector (all: VWR Hitachi, VWR, Vienna, Austria). Separation was performed on a Hypersil ODS column (5 μm; 250 × 4 mm I.D.) with 10.0 min isocratic elution (buffer A: 0.1 mol/L NaH₂PO₄, pH = 5.5, containing 5.9 mmol/L tetrabutylammonium hydrogensulphate) followed by a linear gradient to 20% buffer B (buffer B: 0.1 mol/L NaH₂PO4, pH = 5.5, containing 5.9 mmol/L tetrabutylammonium hydrogensulphate/acetonitrile, 3:1, vol/vol) within another 10 min. The injection volume of standard and sample solutions was 40 μL. The absorbance at 205 nm was recorded. Data acquisition and subsequent analysis was done with the EZchrom Elite (VWR) program. Retention time was ~7.80 min for nitrite and ~14.5 min for nitrate.

The HPLC system consists of: Elite LaChrom with an L2200 autosampler, L2130 pump, L2350 column oven, L2444 DAD, and EZChrom Elite 3.1.7 software (Hitachi, Tokyo, Japan); column: Kinetex^® Biphenyl, 100 Å, 250 × 4.6 mm 5 µm (same material pre-column; Phenomenex, Torrance, CA, USA); injection volume 20 µL; oven temp. 25 °C; auto sampler temp.: 10 °C; detection wavelength: 340 nm; flow: 1.2 mL/min; A = H₂O + 0.1% TFA, B = acetonitrile + 0.1% TFA; gradient: 0–2 min 10% B, 2–15 min 10% B → 70% B, 15–16 min 70% B → 10% B, 16–20 min 10% B. All samples were filtered (Phenex RC Membrane 0.2 um, Phenomenex, USA) before injection.

Solubility studies were conducted to select the appropriate components of ME formulation with high drug loading capacity. In the process, an excess amount of acacetin was separately added into 0.1 g of solvents such as ethanol, PEG300, PEG400; oils such as Lipoid MCT, mineral oil, IMP; surfactants like CrEL, Tween 80, Labra; co-surfactants like Trans. Then samples were vortexed for 5 min thoroughly mixed and followed by constantly shaken at 37 °C for 24 h to achieve the dissolution equilibrium. After that, the samples were centrifuged at 14 000 rpm for 30 min to precipitate the un-dissolved particles. The concentration of acacetin in the supernatants was qualified by HPLC analysis. All experiments were performed in triplicate.
The HPLC system was equipped with a C-18 column (Grace, 4.6 × 150 mm, 5 μm) and mobile phase was composed of a mixture of water and methanol (v/v, 3:7, with 0.2% phosphate acid) using an isocratic elution (Hitachi L2130 pump, L2200 Autosampler and L2400 Detector, Hitachi, Japan). The flow rate was 1.0 mL/min. The UV absorbance detector was set at 330 nm.