1200 series autosampler

Polymer samples were dissolved
in HPLC-grade tetrahydrofuran (THF)
(∼1 mg/mL), filtered through alumina to remove the Cu catalyst,
and then filtered through a 0.45 μm PTFE filter. Size exclusion
chromatography was then performed on a PL-Gel 300 × 7.5 mm (5
μm) mixed C column using an Agilent 1200 series autosampler,
an inline degasser, and a diode array detector set to monitor the
absorbance at 254 nm. The column and detector temperatures were 35
°C. HPLC-grade THF was used as the eluent (1 mL/min). Linear
polystyrene standards were used for calibration.

DKT concentrations in the samples were measured by HPLC-UV (Hewlett Packard series 1100 HPLC Pump combined with Agilent Technologies 1200 Series Autosampler). A volume of 75 µL was injected into the HPLC system (Waters 515 HPLC Pump with Waters 717 Autosampler). DKT was detected with an UV lamp at 262 nm (Water 996 Photodiode Array Detector). The mobile phase consisted of 60:40 mixture of acetonitrile and purified water A (both containing 0.1% TFA). Stationary phase was a C-18 Agilent Eclipse XDB (4.6 × 150 mm; 3.5 µm). Elution flow was 1 mL/min and retention time for DKT was 3.95 min. Calibration curves were made in mobile phase based on a stock solution of DKT in methanol. Linearity was observed between 1.5 µg/mL and 300 µg/mL covering all the experimental sample values. The observed peaks were integrated using Millenium software (Agilent Technologies, County of Santa Clara, CA, USA). The developed analytical method met the standards for precision and accuracy.

All samples derived from the GIS studies were analyzed for loratadine by HPLC-UV (Hewlett Packard series 1100 HPLC Pump combined with Agilent Technologies 1200 Series Autosampler (Santa Clara, CA, USA). A volume of 100 µL was injected into the HPLC system connected to a UV-lamp that was able to detect loratadine at a wavelength of 248 nm (Agilent 1100 Series UV lamp). An isocratic run with a mixture of 70% acetonitrile and 30% purified water was used to detect loratadine using a C-18 column (Kinetex C18 HPLC column, 250 × 4.60 mm^‒5 micron, Phenomenex, Golden, CO, USA) and a 1 mL/min flow rate. Calibration curves were made in methanol, based on a stock solution of loratadine in methanol (0.1 mg/mL). Linearity was observed between 40 µg/mL and 0.156 µg/mL with a regression coefficient of at least 0.995 between the AUC of the obtained peaks versus the spiked concentrations. The peaks were integrated using ChemStation software (Agilent Technologies, B.04.03 version, Santa Clara, CA, USA).

Molecular weights of Fab fractions were determined by electrospray ionization mass spectrometry (ESI-MS) conducted at the Center for Metabolomics and Mass Spectrometry. Samples were run through an Agilent PLRP-S 100 Å 5 µm column, number 0006140735-10. A column guard was used to protect the column from overloading and from particulates. The autosampler used was an Agilent Technologies 1200 series autosampler, and the instrument an Agilent Technologies 6230 TOF LC/MS with a Dual AJS ESI ion source. The LC/MS gradient makes use of two solvents: Solvent A being 0.1% Formic Acid in H₂O, and Solvent B being 0.1% Formic Acid in ACN. The gradient consisted of three steps: the first flowing 95% Solvent A and 5% B for 5 min, the second ramping up the concentration of B to 90% for 10 more min, and finally returning the concentration of B to 5%, flowing for another 1 min. The gradient was run at a flow of 300 µL/min at a pressure of 400 bar (40 MPa).

Solubility samples were analyzed by HPLC–UV (Hewlett Packard series 1100 HPLC Pump, Santa Clara, CA, USA), combined with Agilent Technologies 1200 Series Autosampler (Santa Clara, CA, USA). A volume of 5 µL was injected into the HPLC system connected to a UV lamp that was able to detect ibuprofen at a wavelength of 220 nm (Agilent 1100 Series UV lamp, Santa Clara, CA, USA). An isocratic run containing 70% acetonitrile (VWR International, West Chester, PA, USA) and 30% purified water (both containing 0.1% TFA) was used to detect ibuprofen at a retention time of 2.9 min using a reversed-phase C-18 column (Eclipse Plus C18, 4.6 × 150 mm, 5.5 µm, Agilent Technologies) and a 1 mL/min flow rate. The calibration curve was made in methanol based on a stock solution of ibuprofen in methanol (1 mM). Linearity was observed between 10.32 µg/mL and 0.32 µg/mL. The observed peaks were integrated using ChemStation software (Agilent Technologies, B.04.03 version). The developed analytical method met the FDA requirements for bioanalytical method validation [21 ].

For qualification and peak identification purposes, an HPLC-ESI-MS system consisting of a binary HPLC pump (1100 series) and autosampler (1200 series) from Agilent Technologies (Waldbronn, Germany) equipped with an LC-ESI-MS/MS ion-trap system (HCT Ultra ETD II, Bruker Daltonics, Bremen, Germany) was used. Mass spectra were recorded in the negative ionization mode with the capillary voltage set at 3500 V, end plate −500 V, and capillary exit −115.0 V. Drying gas was nitrogen at 330 °C, and 10.0 L/min flow rate with nebulizer pressure of 50 psi, target mass setting m/z 350, scan range from m/z 100–2000 in Ultra Scan mode, fragmentation amplitude 1 V. Compass Hystar Software (version V. 3.2, Bruker Daltonics) was used for analysis and data collection. HPLC separation was adapted from Kiene and co-workers and performed on a C18-column (Aqua 3u, 100 Å, 3 μm, 150 mm × 2.0 mm i.d.) from Phenomenex (Aschaffenburg, Germany) with a guard column of the same material at a flow rate of 0.20 mL/min [35 (link)]. The mobile phase consisted of 2% aqueous acetic acid (v/v) (A) and acetonitrile (B). HPLC conditions for ESI-MS/MS analysis were 0 min (20% B), 20 min (30% B), 40 min (50% B), 50 min (80% B), 55 min (80% B), 60 min (20% B), 70 min (20% B).