Xselect hss t3

1290 Infinity UHPLC system (Agilent, United States), XSelect HSS T3 (2.1 × 100 mm, 2.5 μm, Waters, United States), 6530 QTOF/MS (Agilent, United States), Trace GC ultra-gas chromatograph (Thermo Fisher Scientific, United States), an Agilent DB-5MS column DSQ II mass spectrometer (Thermo Fisher Scientific, United States), Electronic balance (Switzerland Mettler company, Japan), high-speed freezing centrifuge (Thermo Fisher Scientific, United States), DZG-6020 vacuum drying oven (Shanghai Yiheng experimental instrument company, China). ZH-Mm Tis water maze equipment (Zhenghua biological instrument equipment company, China), Tecan infinite M200 pro multifunctional microplate reader (Switzerland), Vortex-6 vortex instrument (Macao slinberg Instrument Manufacturing Co., Ltd.); RVC2-18CD plus vacuum concentrator (Beijing gaodetong Technology Co., Ltd.).

A Waters 2690SM (Waters Corp., Milford, MA, USA) HPLC with a Waters Xselect HSS T3 (3.5 µm, 4.6 × 100 mm) column and a Waters 2996 photodiode array detector were used for thiamine quantification in samples containing crystalline TMN, physical blends of crystalline TMN with polymers, and thiamine:polymer solid dispersions. The amount of thiamine remaining was quantified by the standard curve prepared at a concentration range 0.005–1 mg/mL prior to analysis (r² = 0.9997–1.0000). Samples were diluted with solvent to a final thiamine concentration of 0.25–0.5 mg/mL and filtered through a 0.2 µm syringe filter. Acetonitrile (solvent A) and water with 0.1% TFA (solvent B) were used as mobile phases. The following gradient method was adapted from Xia, et al. [33 ] and performed as follows: 0/100 at 0 min (immediate), 3/97 at 4 min (linear), 10/90 at 6 min (linear), 0/100 10 min (linear), and 0/100 from 10 to 15 min (immediate). The flow rate and the injection volume used were 1 mL/min and 10 μL, respectively. Samples were scanned between 235–400 nm, and the integration was conducted at 254 nm using Masslynx V4.1 software (Waters Corp.).

In-line filter disks (0.5 µm × 3.0 mm, Phenomenex^®, Torrance, CA, USA) and X select HSS T3 (2.5 µm, 2.1 × 150 mm, Waters^®, Milford, MA, USA, 40 °C) were employed as a pre-column and analytical column, respectively. The mobile phases were composed of buffer A (5 mM ammonium formate buffer pH 3 with 1% methanol), buffer B (5 mM ammonium formate buffer pH 8 with 1% methanol), and buffer C (100% acetonitrile). The flow rate was set at 0.3 mL/min. The composition of the mobile phase began with 90 (A or B) to 10 (C) for the first 20 min, then inversed from minute 21 to 25, and lastly returned back for the last three minutes until the end at 28 min. Furthermore, the instrument was coupled with the Triple TOF 5600+ (Sciex, Framingham, MA, USA) for IDA acquisition and Analyst TF 1.7.1 (Sciex^®) for LC-Triple TOF control.

Best separation and sensitivity were obtained using a mobile phase consisting of a mixture of 2.5 mM ammonium acetate buffer at pH 5.3 and acetonitrile. The chromatographic column was an XSelect HSS T3 3.5 µm 75 × 2.1 mm from Waters (Milford, MA, USA) used with a SecurityGuard precolumn holder and Polar C18 cartridges 4 × 2.0 mm from Phenomenex (Torrance, CA, USA), both thermostated at 40 °C. The gradient program was as follows: 15 to 35 % ACN from 0 to 0.4 min, 35 to 52 % from 0.4 to 2.7 min, 52 to 95 % ACN from 2.7 to 3.5 min, isocratic step at 95 % ACN between 3.5 and 4.7 min, back to initial condition (15 % ACN) at 4.8 min and column equilibration to 6 min. The flow rate and injection volume were 0.6 mL/min and 5 µL, respectively. The injection needle was rinsed before and after drawing each sample with ACN/MeOH/water (4:4:2). The autosampler was kept at 4 °C to enhance the sample stability.

TClHCl was quantified in the absence and presence of polymers throughout the study using a Waters 2690SM HPLC with a Waters XSelect HSS T3 (3.5 µm, 4.6 × 100 mm) column and a Waters 2996 photodiode array detector (Waters Corp., Milford, MA, USA). Prior to each analysis, standard curves of TClHCl at a concentration range from 0.005 to 1 mg/mL were prepared (R² = 0.9997−1.0000). Samples were diluted with solvent and filtered through a 0.2 µm syringe filter. The mobile phase containing solvent A (acetonitrile) and solvent B (water and 0.1% TFA) was used with the following gradient procedure adapted from Xia et al. [31 ]: 0/100 at 0 min (immediate), 3/97 at 4 min (linear), 10/90 at 6 min (linear), 0/100 at 10 min (linear), and 0/100 from 10 to 15 min (immediate), for a total chromatographic run time of 15 min. The flow rate was 1 mL/min, and the samples were scanned between 235–400 nm. Integration was conducted at 247 nm using MassLynx software (V4.1, Waters Corp., Milford, MA, USA).

Palbociclib plasma concentrations were measured using high-performance liquid chromatography coupled to tandem mass spectrometry at the Laboratory of Clinical Pharmacology of the Lausanne University Hospital. The chromatographic separation was performed on a column XSelect HSS T3 2.1 × 75 mm, 3.5 µm (Waters^®, Milford, MA, USA) using a mobile phase composed of a gradient of ammonium acetate 2 mM (+0.1 formic acid) and acetonitrile (+0.1 formic acid). Palbociclib was detected by electrospray triple-stage quadrupole mass spectrometry and quantified using the calibration curves with stable isotope-labeled internal standard palbociclib-D8 (Alsachim, Illkirch-Graffenstaden, France). The assay showed appropriate inter-day and intra-day precision for the quantification of palbociclib (coefficient of variation [CV] 0.6–5.9% and 5.7–8.4%, respectively) and accuracy (−1.2% to 5.4%). The method was validated over the clinically relevant concentration range, i.e., from 0.5 to 500 ng/mL.
ANC was measured either by the Laboratory of Clinical Chemistry or by point-of-care tests (POCTs) in the service of Medical Oncology. Both results were integrated in the dataset, and POCT measures were flagged.

Analysis was carried out in the Proteomics and Metabolomics Unit, Children’s Cancer Hospital (57357), Basic Research Department, Cairo, Egypt. Adopting the methods previously reported by Negm et al. [21 (link)]. In-line filter disks (0.5 µm × 3.0 mm, Phenomenex^®, Torrance, CA, USA) and X select HSS T3 (2.5 µm, 2.1 mm × 150 mm, Waters^®, 40 °C, Milford, MA, USA) were used as a pre-column and analytical column, respectively. The mobile phases consisted of buffer A (5 mM ammonium formate buffer pH 3 containing 1% methanol), buffer B (5 mM ammonium formate buffer pH 8 containing 1% methanol), and buffer C (100 % acetonitrile). The flow rate was adjusted at 0.3 mL/min. The liquid chromatography (ExionLC -High flow LC, Sciex^®) was programmed to use a mobile phase composition of buffer A and C in positive mode, and buffer B and C in negative mode. The mobile phase composition started with 90 (A or B): 10 (C) for the first 20 min, which was inversed from 21–25 min, and finally returning to the starting values for the last 3 min until the end of the protocol at 28 min. In addition, the instrument (ExionLC -High flow LC-, Sciex®, Framingham, MA, USA) was coupled with Triple TOF 5600+ (Sciex^®) for IDA acquisition and Analyst TF 1.7.1 (Sciex^®) for LC-Triple TOF control.