Openlab cds chemstation edition

MX was quantified using high-pressure liquid chromatography (HPLC) with UV detection. The HPLC system included an Agilent 1100 Series liquid chromatograph (Agilent Technlogies, Santa Clara, CA, USA) and the Agilent Chemstation software (OpenLab CDS, ChemStation Edition, Rev. C.01.10, Agilent Technologies). A reversed-phase C18 column (YMC Triart C18 ExRS plus, 5 µm, 4.6 × 150 mm, YMC America Inc., Allentown, PA, USA) was used as the stationary phase. The column temperature was maintained at 30.0 ± 0.2 °C. The mobile phase composed of 1% phosphoric acid (A) and acetonitrile (B) at a flow rate of 1.0 mL/min. The gradient program is: 0 min, 35% B; 5.5 min, 75% B; 7.2 min, 35% B. The UV detector was set at a wavelength of 360 nm for MX. The retention time for MX was about 8.5 min. The method was linear at a concentration range of 0.05–50µg/mL with R² of 0.9995 for meloxicam. The limit of detection (LOD) was found to be 0.05 µg/mL and the limit of quantification (LOQ) was 0.22 µg/mL. The relative standard deviation for both intra-day and inter-day precision was less than 2%.

An advanced oligonucleotide C18 column was used for the purification of chemically modified DNA. The column was run with 0.1 M TEAA in water as solvent A and acetonitrile as solvent B. DNA was purified using the following method, 10% for 3 min followed by 10–35% solvent B gradient over 25 min at 0.5 mL/min flow rate. Tetrazine conjugated DNA was purified using a similar method but using a 10–60% solvent B over 25 min at 0.5 mL/min. Peptides were purified using a Grace C18 column which was eluted with water as solvent A and acetonitrile as B, both containing 0.05% TFA. A gradient of 10–40% solvent B over 30 min at 1 mL/min was used for peptide purification. Separated fractions were dried in a vacuum concentrator overnight. The purified oligonucleotide conjugates were reconstituted in 1× TE buffer and were stored at −30 °C. The concentration of oligonucleotides and peptides was determined using its absorbance at 260 nm and 214 nm, respectively. OpenLAB CDS chem station edition from Agilent Technologies was used for HPLC data collection.

OpenLab CDS ChemStation Edition (Agilent Technologies) was used for instrument control, electropherograms acquisition, and data analysis.
The screening phase was carried out by employing a symmetric screening matrix generated by NemrodW software (NemrodW, LPRAI sarl, Marseille, France), with which related data analysis and graphical analysis of effects were performed.
The optimization phase, including RSM and MODR identification using Monte Carlo Simulations [41 (link)], was carried out by MODDE 13 software (Sartorius Data Analytics AB, Göttingen, Germany), which allowed contour plots and probability maps to be drawn. The MODR was defined by setting the target for defect per million opportunities (DPMO) at 100,000, namely to a risk of error equal to 10% [40 ].

Sterols were measured by gas chromatography equipped with a flame ionization detector (GC-FID) (Hewlett Packard 6890 plus), and with a capillary column (DB-XLB 30 m × 0.25 mm i.d. × 0.25 μm; Agilent Technologies, Amstelveen, Netherlands). Extraction of cholesterol and non-cholesterol sterols was performed based on Mackay et al. [19 (link)]. Briefly, a 100 μL plasma sample was saponified with 1 ml of 90% ethanolic sodium hydroxide for 1 h at 60 °C. 5α-cholestane and epicoprostanol were used as internal standards. After two rounds of cyclohexane extraction, samples were derivatized with 30 μL of TMS reagent (pyridine, hexamethyldisilazane and trimethylchlorosilane (9:3:1, v/v/v)). Samples were injected into GC-FID; cholesterol and non-cholesterol sterol peaks were integrated (OpenLab CDS ChemStation Edition; Agilent Technologies, Santa Clara, CA, USA) and their concentrations were calculated relative to the internal standard 5α-cholestane. Non-cholesterol sterol concentrations were standardized for cholesterol concentrations, as determined within the same GC run and expressed as μmol/mmol cholesterol.

The assay methodology utilized an Agilent 1100 series high-performance liquid chromatograph (HPLC) coupled with UV detection (with diode array detector—DAD) and Agilent Chemstation software (OpenLab CDS, Chemstation Edition, Rev. C.01.10, Agilent Technologies, Santa Clara, CA, USA). A Phenomenex C18 column (150 mm × 4.6 mm, 5.0 µ particle size) was used as the stationary phase at 25 °C (Phenomenex, Torrance, CA, USA). The mobile phase methanol: water (with 0.02% formic acid) was used in a 50:50 ratio by volume at a flow rate of 1.0 mL/min with the sample injection volume of 20 μL with a run time of 10 min. The retention time for OXC was 6.1 min with UV detection at a wavelength of 229 nm. Linearity of the peak area vs. concentration was recorded with a standard concentration range from 0.25 µg/mL to 100 µg/mL and the coefficient of regression (R²) of 0.99 was obtained. The % RSD for intra-day and inter-day precision of the method was 0.1% and 0.9%, respectively. The limit of detection was 0.25 µg/mL and limit of quantification was 0.5 µg/mL.