High performance liquid chromatography (hplc)

The release experiment was carried out in vitro as follows: Paclitaxel-incorporated nanoparticle solution was prepared as described above. After the dialysis procedure, the volume of nanoparticle solution was adjusted to 50 ml and then 5 ml of this solution was introduced into a dialysis tube (MWCO = 12,000 g/mol) and then dialysis tube was introduced into a 200-ml bottle with 95 ml of phosphate-buffered saline (PBS, 0.1 M, pH 7.4, 0.1% (w/v) Tween 80). This bottle was placed in a shaking incubator with stirring speed of 100 rpm at 37°C. At specific time intervals, the media were taken for analysis of drug concentration. After that, the whole media was replaced with fresh PBS to prevent drug saturation. The concentration of the paclitaxel released into PBS was evaluated using a high-performance liquid chromatography (HPLC, PerkinElmer, Waltham, MA, USA) using Phenomenex Sphereclone 5 micro ODS(2) 250 mm × 4.6 mm column and 75% methanol as a moving phase. The Flexar HPLC system (PerkinElmer, Waltham, MA, USA) was equipped with a Solvent Manager 5-CH degasser, an autosampler, a quaternary LC pump, a column oven, and a UV-visible detector. The flow rate was set to 1.5 ml/min and the temperature at 50°C. The UV detector (226 nm) was used for the measurement. To prepare the sample for the measurement of HPLC, paclitaxel was dissolved in ethanol, making 20 μl for injection.

Single colonies of freshly grown bacteria were inoculated into the LB broth (100 ml) with or without L-tryptophan (100 mg L⁻¹) following incubation for 2 days at 28 ± 2°C with continuous shaking. After centrifugation for 10 min at 13,000 g, the 100 μL supernatant was collected and mixed with Salkowski's reagent (100 μl) following incubation for 30 min at room temperature. The supernatant was further acidified at pH=2.8 and extracted using the same quantity of ethyl acetate for quantification. The extract was then evaporated until it dried completely and was collected in methanol following filtration using a nylon filter (0.2 μm). High-performance liquid chromatography (HPLC) (Perkin Elmer, USA) was used to analyze purified extract, equipped with a C-18 column. The mobile phase used was acetic acid, methanol, and water (1:30:70 v/v/v) at a 1 ml/min flow rate (Tien et al., 1979 (link)).

Analyses were performed with a Perkin Elmer HPLC with UV–Vis detector of the 200 series equipped with a solvent delivery unit with gradient of elution, a KNAUER Vertex Plus column (250 mm × 4.6 mm, 5 μm) Eurospher II 100–5 C18 P and TotalChrom software. The UV detector wavelength was set at 205 nm, the column temperature was maintained at 30 °C, and the flowrate was 1 mL/min. For chromatographic runs, a stepwise method was used: 100% of methanol in 0 min, 50% of methanol and 50% of 5:4 2-propanol/n-hexane in 10 min, maintained with isocratic elution for 10 min [68 (link)]. All reactions were performed at least in duplicate and the results were expressed as mean values, being percentage differences between them and the mean always less than 5%.

A standard stock solution of squalene at 10 mg/mL was prepared with pure squalene in absolute ethanol (≥ 99.8%). The standard calibration curve was prepared using squalene concentrations ranging from 0.001 to 10 mg/mL in acetonitrile. The analysis was performed by HPLC (PerkinElmer, Waltham, MA, USA) using a C18 reverse-phase end-capped chromatography column (NUCLEOSIL^® 100-5 C18, 5 µm particles, 100 Å pores, 50 mm, MACHEREY–NAGEL GmbH & Co. KG, Düren, Germany) operating at 30 °C with HPLC-grade acetonitrile:water (9:1, v/v) as mobile phase at a flow rate of 1.5 mL/min with 20 μL of injection volume. The squalene was detected with a UV detector at 210 nm. Samples of lipid-containing squalene were dissolved in 100% acetonitrile.

The supernatant solutions of each group of SESDs of ARTM were withdrawn and then filtered through the cellulose acetates filters of 0.22 μm in pore size. The amount of drug dissolved was then analyzed by using HPLC (Perkin Elmer, USA) at 215 nm after suitable dilution. This assay was determined by using reverse phase C18 column (4.6 mm × 250 mm, 5 μ), while UV detector was set at a wavelength of about 215 nm. Mobile phase consisted of a mixture of acetonitrile and water (75 : 25, v/v) operated at a flow rate of 1 mL/min. The injection volume was 20 μL.
The validation data shows that the used HPLC method follows linearity in the range of 0.078 to 2.5 mg, as evident from the value of R² = 0.999 with Y = 462.5X − 21.32. It relates to the closeness of the test results to true values, that is, measure of exactness of analytical method. It is expressed as percentage recovery by the assay of known amount of analyte in the linearity range. For the determination of accuracy, the ARTM percentage recovery was 99.98, 100.34, 101.64, 101.75, 101.83, and 101.94% for dilutions 0.078, 0.1562, 0.3125, 0.625, 1.25, and 2.5 mg/mL, respectively. The accuracy and precision of method were 99.31 ± 2.94 and 98.72 ± 2.02, respectively [11 ].