Waters breeze 2

A total of 500 mg of fresh colon tissue was homogenized at 24,000 rpm/min with 1.15 percent KCl. After combining the homogenate with 250 uL of 6M NaOH and incubating at 67 °C for 43 min, the mixture was vortexed with the same amount of acetonitrile. When the samples had been hydrolyzed, they were centrifuged for ten minutes at a speed of 15,000× g. The supernatant (240 μL) was combined with 2,4-dinitrophenylhydrazine (DNPH) (24 μL). For another 10 min, it was kept in the dark. The resultant samples were evaluated by HPLC (Waters Breeze-2, USA) via the ODS2 reverse phase column (Waters Breeze-2, USA). The mobile phase was 38: 62 acetonitrile: 0.2% acetic acid HPLC grade water. MDA in the sample was measured at 310 nm using isocratic HPLC and a UV detector. As per procedure, a standard curve was generated using 20 nmol/mL of MDA standard solution (TCI, Japan) diluted with 1% H₂SO₄ [26 (link),29 ,30 (link)].

Sugar consumption and fermentation end-products were measured as previously reported52 (link) using a Waters Breeze 2 high-performance liquid chromatography (HPLC) system (Waters Corp., Milford, MA) equipped with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) and a refractive index detector. Headspace hydrogen was measured using an Agilent Technologies 6850 Series II gas chromatograph equipped with a Carboxen 1010 plot column and a thermal conductivity detector. Total H₂ production calculations (headspace and liquid fractions) were done as previously described53 (link).

Docetaxel was quantified using high-pressure liquid chromatography (HPLC) in a Waters Breeze 2 (Waters Technologies, Milford, MA, USA) and a C18 Gemini, 5 µM, 150 × 4.60 mm column, at 35 °C. The mobile phase was a mixture of methanol:water 70:30 (v/v). The flow rate, injection volume and wavelength detection were set at 1 mL.min⁻¹, 20 µL and 270 nm, respectively [79 (link)]. The encapsulation efficiency (% EE) of DTX by the nanoparticles was determined by the ultrafiltration–centrifugation method, using cellulose filters (10 kDa, Millipore). Briefly, the total amount (100%) of DTX in the NLC was determined (DTX_total) by diluting the samples in the mobile phase (n = 3). The amount of DTX in the filtrate (DTX_free) was quantified by HPLC and the percentage of encapsulated DTX was calculated according to equation 2. Drug loading, the amount of loaded DTX in relation to the total weight of the nanoparticles, was also calculated according to Equation (3) [10 (link)]: $$\%\mathrm{EE}=\frac{\mathrm{DTXtotal}-\mathrm{DTX}\mathrm{free}}{\mathrm{DTX}\mathrm{total}}\times 100$$
$$\% Drug Loading=\frac{weight of encapsulated DTX }{weight of nanoparticles}\times 100$$