Lachrom elite

The experimental setup consisted of a standard VWR-Hitachi LaChrom Elite^® HPLC system (VWR international, Darmstadt, Germany) with a quaternary gradient pump L-2130, Autosampler L-2200, and diode array detector L-2455.

Drug release was investigated under in vitro conditions at 37 °C in PBS at pH 7.4 (or pH = 6.8) for 84 days. After sampling at the predetermined intervals, the buffer was replaced to maintain the sink condition. The samples were collected for the evaluation of the amount of released drug. High-performance liquid chromatograph (VWR-Hitachi/LaChromElite^®) equipped with a LiChrospher^® RP-18 column (250 mm × 4 mm, 5 μm) and LiChrospher^® RP-18 guard column (4 mm × 4 mm, 5 μm) was employed. The mobile phase consisted of acetonitrile and water (60:40, v/v) and was delivered at a flow rate of 1 mL/min. Docetaxel and cabazitaxel were detected at a wavelength of 227 nm.

Determination of organic acids (acetic acid, propionic acid, lactic acid, butyric acid) was carried out using a UHPLC (VWR-HITACHI LaChrom Elite) system consisting of an autosampler (model L-2200), pump (model L-2130) and a UV detector (L-2400) connected in a series (Primec et al., 2017 (link)). Analyses were performed isocratically at a flow rate of 0.6 mL/min at 40 °C, on Rezex ROA—Organic Acid H+, 300 × 7.8 mm (Phenomenex) column. Standards (lactic acid—1.1, 0.55, 0.275, 0.11 g/L; acetic acid—1.0, 0.5, 0.25, 0.1 g/L; propionic acid—1.0, 0.5, 0.25, 0.1 g/L, and butyric acid- 0.55, 0.275, 0.1375, 0.055 g/L) were used.

High Performance Liquid Chromatography (HPLC) analysis was performed, for each molecule under study, based on previous reported methods^{24 ,25 (link)}. CLV and IMP were measured using a VWR Hitachi LaChromElite equipped with column oven, auto sampler, and UV-DAD detector. Samples were diluted with distilled water before analysis. Reversed phase runs for CLV analysis comprised a mobile phase of 85% 20 mM potassium bi-acid phosphate adjusted to pH 7 and 15% methanol (Merck), at a flow rate of 0.7 ml/min with a Kromasil® C18 3.5 µm 150 mm × 4.6 mm column (AkzoNovel). For IMP analysis, the chromatographic conditions consider the mobile phase described earlier with 5% methanol adjusted to pH 5 with ortho-phosphoric acid. A flow rate of 0.8 ml/min on a C18 5 µm 150 × 4.6 mm ACE Generix® column was used. Stability studies were made considering the areas of the chromatographic peaks, due to its direct relation with the quantity of the intact drug. Final measures considered the following formula: $$\%\mathit{remaining}\mathit{drug}=\frac{Are{a}_t}{Are{a}_0}\times 100$$ where Area_t is the area of the chromatographic signal measured, which is directly related to the intact drug content in each sample for a specific sampling time. Area₀ is the signal measured initially in each sample just after its dissolution in the stability study.

The experimental setup consisted of a standard VWR-Hitachi LaChrom Elite^® HPLC system with a quaternary gradient pump L-2130, Autosampler L-2200 and diode array detector L-2455 (VWR International, Radnor, PA, USA). The later was not used for the ICM but for comparative measurements. For the ICM measurements, a Smartline DAD 2600 with 10 mm, 10 µL flow cell from Knauer Wissenschaftliche Geräte GmbH, Berlin, Germany was used. Experimental validations were based on SEC analysis after online fractionation with a Foxy Jr.^® from Teledyn Isco, Lincoln, NE, USA.

Reverse-phase HPLC analysis was performed on the Nova-Pak C18 (125 × 3.9 mm, 5 µm) from VWR (Milano, Italy). The HPLC apparatus was a LaChrom Elite (L-2130 constant-flow quaternary pump, L-2400 UV-Vis detector, L-2200 autosampler, and data acquisition system EZChrom Elite 3.1) from VWR Hitachi (Milan, Italy). The mobile phase was a 55:45 (v/v) acetonitrile–0.02 mole L⁻¹ sodium phosphate buffer with a pH of 4.6. The flow rate was set at 1.0 mL/min, the injection volume was 5 µL, and the detection wavelength was 278 nm.
Diclofenac and mefenamic acid standard solutions at concentrations ranging from 2 to 200 µg/mL were prepared in the mobile phase immediately before use. The standards were analyzed three times consecutively and peak areas were plotted against the concentration. The calibration plot was drawn by using a weighted linear regression (weight = 1/conc, r² = 0.998). The limit of quantification (LOQ_DIC = 2.8 µg/mL, LOQ_MEF = 2.6 µg/mL) was calculated as LOQ = 10 Sy/b, where Sy is the standard error of the response and b is the slope of the calibration plot.