Openlab cds software

0.3 mL samples were taken every 20 min from 1 to 3 hr after inoculation. To separate the cells from the medium, the samples were centrifuged at maximum speed for 3 min, the supernatant transferred onto a filter column (SpinX, pore size 0.22 µm), again centrifuged at maximum spend and the flow through was further analyzed. The glucose, pyruvate, glycerol, acetate and ethanol concentration was detected using an Agilent 1290 LC HPLC system equipped with a Hi-Plex H column and 5 mM H₂SO₄ as eluent at a constant flow rate of 0.6 mL min⁻¹. The injection volume was 10 µL and the column temperature was kept constant at 60°C. Glucose, glycerol, ethanol and acetate were detected by refractive index and pyruvate by UV (constant wave length of 210 nm) and the respective concentrations were determined using an external standard with known concentrations. The data were analyzed using the Agilent Open Lab CDS software.

Organic acids as well as fructose and maltotriose were detected and quantified by HPLC, according to the method described by Marsili et al. (1981) (link) with the following modifications. Samples (1.00 g) were mixed with water (MilliQ), 0.5 M H₂SO₄ and acetonitrile in a MultiRS-60 BIOSAN turner (Montebello Diagnostics A/S, Oslo, Norway) operated at 30 rpm for 30 min. Samples were centrifuged for 15 min at 1470 × g using a Kubota 2010 centrifuge (Kubota Corporation, Tokyo, Japan) prior to filtration through 0.2 μm PTFE membrane (Acrodisc CR 13 mm Syringe Filter, PALL, United Kingdom). Organic acids were separated on an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, United States) with 0.05 M H₂SO₄ as mobile phase and a flow rate of 0.4 mL/min. The column, operated at 30°C, was connected to a 1260 Infinity HPLC instrument (Agilent Technologies, Singapore) with pump, autosampler, column oven, RI-detector (refractive index, used for acetic acid, fructose and maltotriose) and diode array detector-ultra violet (DAD-UV) detector, used for the other organic acids. Openlab CDS software (Agilent Technologies) was used to operate the system and detection and quantification were done according to calibration with external standards. Maltose, sucrose and glucose were quantified by the K-MASUG enzymatic kit (Megazyme, Wicklow, Ireland), used according to the instructions.

A validated reverse phase HPLC methodology was used to determine the content of the API in the acceptor medium. Detection was achieved using a spectrophotometer at λ = 264 nm. The flow rate of the eluent was set to 1.0 mL min⁻¹ and all injections of 50 μl. Data was collected and analyzed using Agilent Technologies OpenLAB CDS software (version C.01.07 [27 (link)]). Mobile phase solvents used were HPLC grade and consisted of mobile phase (a) 100% v/v water, 0.1% TFA; mobile phase (b) 100% v/v MeCN. The calibration curve for ibuprofen was linear at concentrations ranging from 1 to 50 μg mL⁻¹. The accuracy of the assay was tested by triplicate injection of five samples within 50–200% range of nominal concentration (50 μg mL⁻¹), and subsequent calculation of the recovery percent. Estimated average values of recovery and RSD were found to meet accuracy criteria of 100 ± 2% and < 1%, respectively. The limit of detection (LOD) was defined as lowest amount of ibuprofen which can be reproducibly detected as the signal which is three times higher than the baseline and was found to be 200 ng mL⁻¹. The limit of quantification (LOQ) was defined as the lowest amount of analyte which reproducibly gives signal-to-noise ratio (SNR) of 10/1 and was found to be 600 ng mL⁻¹.

Size exclusion high-performance liquid chromatography (HPLC) analysis was performed on an Agilent 1260 HPLC system controlled using OpenLab CDS software (Agilent Technologies). The analysis was performed using an AdvanceBio SEC column (Agilent Technologies, 4.6 × 300 mm, 300 Å, 2.7 µm) and AdvanceBio SEC guard column (Agilent Technologies, 4.6 × 50 mm, 300 Å, 2.7 µm). The column was operated at a flow rate of 0.25 mL/min and ambient temperature. The mobile phase buffer was 150 mM sodium phosphate (Sigma-Aldrich), pH 7.0. Total method run time was 30 min and sample injection volumes were 10 µL. A diode array detector was set for absorbance detection at 214 nm. Data analysis was completed using Agilent’s OpenLab CDS Data Analysis.

A Chromaster High Performance Liquid Chromatograph (HPLC) (Hitachi, Tokyo, Japan) with EZChrom Elite Ver. 3.2.1 software, an autosampler, and a BIOshell™ A400 Protein column C18 (L × I.D. 15 cm × 3 mm, 3.4 μm particle size, Supelco, Bellefonte, PA, USA) were used. The mobile phase consisted of trifluoroacetic acid (TFA) (0.1%, v/v) in water (A) and TFA (0.1%, v/v) in acetonitrile (B).
The extraction of albumins/globulins, gliadins, and glutenins was carried out according to Pronin et al. [11 (link)]. For the determination of wheat protein fractions, the gradient profile was 0 min, 0% B; 0.5 min, 20% B; 7 min, 60% B; 7.1–11 min, 90% B; and 11.1–17 min, 0% B for albumins/globulins and 0 min, 0% B; 0.5 min, 24% B; 20 min, 56% B; 20.1–24.1 for gliadins and glutenins. Flow and temperature were 0.2 mL/min and 60 °C, respectively. The injection volume was 10 μL for gliadins and 20 μL for albumins/globulins and glutenins. Detection was performed at 210 nm using a diode array detector (HPLC-DAD 300). Data were processed using OpenLAB CDS software (Agilent Technologies, Santa Clara, CA, USA). Prolamin Working Group (PWG)-gliadin (2.5 mg/mL in 60% ethanol) was used for external calibration and calculation of protein content [23 (link)].

The peptide fingerprint was obtained via reversed-phase High-Performance Liquid Chromatography (HPLC), as described by Teng et al. [13 (link)]. Chromatograms were generated using Agilent OpenLab CDS software.