Waters 2695 separation module

Plasma free AA concentrations (μmol/L) were analyzed by HPLC according to the Waters Pico Tag method for free AA [29 ] with pre-column derivatization with phenylisothiocyanate, using a Waters 2695 separation module (Waters Cromatografía, SA, Madrid, Spain) as described previously [30 (link)]. Before derivatization, 500 μL of plasma was deproteinized by diluting 1:1 with 20% trichloroacetic acid and 2 mM norleucine as an internal standard, and centrifuged at 11,000× g and 4 °C for 15 min. A Millennium 32 chromatography manager system was used for gradient control and data processing.

A Waters 2695 Separation module (Waters GmbH, Eschborn, Germany) equipped with a Waters 2487 Dual λ Absorbance Detector (Waters GmbH, Eschborn, Germany) at 214 and 280 nm was used. Isocratic elution with a 25 mM sodium phosphate running buffer containing 200 mM sodium chloride (pH 7.0) was performed.
For mAb1-formulations (F1–F3), 10 µL of a reconstituted solution corresponding to a loading of 50 µg protein were loaded on a Tosoh TSKgel G3000SWxl, 7.8 × 300 mm, 5 µm column (Tosoh Bioscience, Griesheim, Germany) and separated with a flow rate of 0.7 mL/min.
For mAb2 (F4), samples were diluted with 10 mM histidine buffer (pH 5.5) to 1 g/L protein concentration, and 25 µL was injected, corresponding to a load of 25 µg protein. A YMC-Pack Diol-300, 300 × 8.0 mm, 5 µm column (YMC Europe GmbH, Dinslaken, Germany) with a flow rate of 0.8 mL/min was used for separation. Samples were measured in triplicates with three individual injections. Data integration of relative areas at 280 nm was performed using Chromeleon 6.80 (Thermo Scientific, Wilmington, DE, USA), provided that every peak eluting before the monomer corresponded to high molecular weight (HMW) species. No peaks could be detected after the monomer. For verification of equipment performance, an internal standard of thawed mAb formulation was injected at the beginning and end of a sequence.

The amino acid content of olive pomace and the LMW peptides (<3 kDa) extracted from olive pomace was determined after hydrolysis in 6 N HCl plus 1% phenol in sealed Pyrex tubes (Corning, NY, USA) at 110 °C for 24 h by high-performance liquid chromatograph (HPLC) according to the Waters Pico Tag method [47 ] as described in [48 (link)], using a Waters 2695 separation module (Waters Corporation, Mildford, MA, USA). L-alpha-amino adipic acid and DL-norleucine were used as internal standards. Pre-column derivatization was carried out with phenyl isothiocyanate. The cysteine and methionine contents were determined as cysteic acid and methionine sulphone, respectively, after oxidation of samples with performic acid prior to the protein hydrolysis step, as described in [49 (link)]. For quantification, seventeen amino acid standards (Amino Acid Standard H, Thermo Fisher Scientific, Waltham, MA, USA), L-Cysteic acid, and L-Methionine sulfone (Sigma-Aldrich, St. Louis, MO, USA) were used at concentrations of 0.1, 0.2, and 0.3 mM. Tryptophan was not determined. A Millennium 32 chromatography manager system was used for gradient control and data processing. Each sample was analysed three times with two technical replicates.

For determination of drug encapsulation efficiency and drug loading content, the lyophilized PLA NPs/MMC-SPC were dissolved in DMF. The organic solution was filtered and submitted to HPLC analysis. The amount of MMC was assayed using a HPLC (Waters Corporation, Milford, MA, USA) system consisting of a Waters 2695 Separation Module (Waters Corporation, Milford, MA, USA) and a Waters 2996 Photodiode Array Detector (Waters Corporation, Milford, MA, USA) with the following conditions: chromatographic column, symmetry C₁₈ column (250 × 4.6 mm, 5 μm); temperature, 25°C; and elution flow rate, 1.0 mL/min. The mobile phase for the determination of MMC was water-methanol (65/35, v/v). The detection wavelength for the determination of MMC was 362 nm. The drug encapsulation efficiency and drug loading content were calculated according to the formula
$\begin{array}{l}\mathrm{Drug}\mathrm{encapsulation}\mathrm{efficiency}\left(\%\right)\\ =(\mathrm{the}\mathrm{weight}\mathrm{of}\mathrm{the}\mathrm{loaded}\mathrm{drug}\\ /\mathrm{the}\mathrm{weight}\mathrm{of}\mathrm{the}\mathrm{input}\mathrm{drug})\times 100\%\end{array}$
$\begin{array}{l}\mathrm{Drug}\mathrm{loading}\mathrm{content}\left(\%\right)\\ =(\mathrm{the}\mathrm{weight}\mathrm{of}\mathrm{the}\mathrm{loaded}\mathrm{drug}\\ /\mathrm{the}\mathrm{weight}\mathrm{of}\mathrm{the}\mathrm{N}\mathrm{P}\mathrm{s})\times 100\%\end{array}$

The flavonoid compounds; nicotiflorin and rutin were purchased from Extrasynthese (Genay, France) whereas clitorin and manghaslin were isolated from C. papaya leaf extract and the structural elucidation were compared to previous publish data [19 (link)–21 (link)]. Analysis of HPLC was performed to monitor these flavonoid compounds in C. papaya leaf samples on a Waters 2695 HPLC system (Milford, USA) equipped with a Waters 996 photodiode array detector and a Waters 2695 separation module with empower 2.0 software. A Waters Symmetry C18 Column, 300 Å, 5 μm, 3.9 mm X 150 mm, (Milford, USA) was used as stationary phase and the mobile phase consisted of water containing 0.01% Trifluoroacetic acid (TFA) (Solvent A) and acetonitrile containing 0.01% TFA (Solvent B). The gradient solvent system of solvent A and solvent B as follows: 90% solvent A until 5 min, followed by 90–750% solvent A over 20 min, then 75–5% A over 5 min, then going back to 90% solvent A until 5 min, and finally reconditioning the column with 90% solvent A isocratic for 5 min. The flow rate for this analysis was maintain in 1.0 ml/min and the injection volume were 20 μl. The chromatogram was monitored using a wavelength range of 210–400 nm.

HPLC analyses were performed on a Waters 2695 separation module (Waters Company, USA) equipped with a Waters 2996 photo-diode array (PDA) detector. HSE and standard compound PPD were dissolved in 50% acetonitrile. After centrifugation at 12,000 rpm for 15 minutes, supernatants of both samples were transferred into sample vials and subjected to HPLC elution. HPLC separations were obtained on an Xterra MS C18 column (4.6 × 150 mm, 2.5 μM, Waters) at a temperature of 30°C. The mobile phase consisted of 0.1% formic acid in acetonitrile (mobile phase A) and 0.1% formic acid in ultrapure water (mobile phase B). The program for gradient elution was set as follows: 0–30 min, 15%–60% A, with a flow rate of 0.2 mL per minute. The volume for each HPLC analysis was set as 10 μL, while the monitoring wavelength was 220 nm.

A high-performance liquid chromatography (HPLC) system Waters 2695 Separation Module, (Waters Inc., Milford, MA, USA). with a Waters UV 2996 Photodiode Array UV Detector, (Waters Inc., Milford, MA, USA) was used for determining in vitro activation of the different conjugates by PLA₂. Identification of the complex was carried out with a RP-18 column and confirmed by UV and co-injection with an authentic sample. The HPLC conditions were as follows: Thermo SCIENTIFIC Part No. 25005-254630 Hypersil gold Dim column 250 × 4.6, particle Size 5 µm, SN: 08830609, LOT: 9278 (Thermo Fisher Scientific, Waltham, MA, USA), an isocratic mobile phase containing isopropanol:water:methanol (70/22/8 v/v), and acetic acid 0.1% in 20 min at the flow rate of 1 mL/min, and the detection wavelength was 265 nm.