Ptfe syringe filter

For extract preparation of the 19 Trichoderma isolates, the culture media (60 x 15 mm) containing the fungal colonies, previously cultivated on PDA at 28°C for four days, were cut into small pieces and extracted with methanol (15 mL), vortexed for 1 min, maintained at rest for 5 min, and vortexed again for 1 min. After filtration, the supernatants were concentrated to approximately 1 mL, lyophilized, and stored at -47°C until use. For analyses by ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS), lyophilized samples were reconstituted in a solution containing water/methanol/acetonitrile (1:2:2 v/v/v, 1 mL). The samples were vortexed for 1 min, sonicated for 30 min at room temperature, filtered using a 0.22 μm PTFE syringe filter (Millipore, USA), and transferred to vials for LC–MS analysis [35 (link)].

The gel sample, placebo matrix, working standards of ADP, and BPO were provided by Dr. Reddy’s Lab, India. Benzoic acid (BA), benzaldehyde (BZ), and ethyl benzoate (EB) were provided by SD Fine Chemicals, India. Impurity-A, Impurity-C, and Impurity-D were used from the United States Pharmacopeia, European Pharmacopeia, and Toronto Research Lab Reference Standard, respectively. HPLC grade acetonitrile, HPLC grade methanol, and glacial acetic acid were used (Rankem, Delhi, India). A nylon membrane filter (0.22 µm), PTFE syringe filter (0.45 µm), and nylon syringe filter (0.45 µm) were from Millipore, Mumbai, India. Water for HPLC was generated using the Milli-Q Plus water purification system (Millipore, Milford, MA, USA).

The freshly prepared formulation was diluted in milli-Q water (1:5), filtered through a 5 µm nitrocellulose membrane filter (Millipore, Ireland), and diluted 1:10 in ethanol to extract UA from NLC. The mixture was centrifuged at 4620× g at 25 °C for 15 min (Thermo Scientific Heraeus Multifuge X1R Refrigerated Benchtop Centrifuge, Indianapolis, IN, USA), the supernatant collected, and filtered through a 0.45 µm PTFE syringe filter (Millipore, Ireland). The supernatant was diluted (1:6) in eluent solution and applied to the HPLC column and the amount of UA released quantified. The encapsulation efficiency (EE) was calculated according to the following equation: $EE\%=\frac{AmountofUAinthefilteredformulation}{TotalamountofUA}\times 100$

Standard solution of trans-p-hydroxycinnamic acid: the stock solution of trans-p-hydroxycinnamic acid was diluted with 40% (v/v) methanol to obtain 6 standard solutions at 11.0, 22.0, 44.0, 88.0, 176.0, and 352.0 μg·mL⁻¹ and filtered through a 0.45 μm PTFE syringe filter (Millipore, Billerica, MA, USA) before HPLC analysis.
Test solution of original extract: the original extracting solution of L. robustum was diluted with 40% (v/v) methanol and percolated using a 0.45 μm PTFE syringe filter prior to HPLC measurement.
Test solution of hydrolyzed extract: 1 mL sodium hydroxide aqueous solution (1 M) and 1 mL the extracting solution of L. robustum were mixed and incubated at 80 °C for 2 h [26 (link)], and then, 1 mL hydrochloride (1 M) solution was added. After it cooled down to room temperature, the above mixture solution was transferred to 10 mL volumetric flask, and the volume was filled with 40% (v/v) methanol. The diluted solution was filtered with a 0.45 μm PTFE syringe filter prior to HPLC determination.