The plasma ibrutinib and its active metabolite, PCI‐45227, were quantified using a Qtrap5500 liquid chromatography‐tandem mass spectrometry system (AB Sciex, Toronto, Canada). The mobile phase consisted of 0.1% formic acid and acetonitrile, and the chromatography was performed on a Luna C18 analytical column (2.1 × 100 mm inner diameter, 1.6 µm particle size; Phenomenex) operating at 30°C. The separation was achieved in 5 minutes using a linear gradient from 20 to 77% of acetonitrile at constant 300 µL/minute flow rate. The mass spectrometer was operated in positive multiple reaction‐monitoring mode with electrospray ionization. Ibrutinib and PCI‐45227 were monitored at mass‐to‐charge ratio (m/z) 441 to 304 and m/z 475 to 304, and the lower limits of quantification were 0.05 ng/mL and 0.1 ng/mL, respectively. The inter‐day precisions (coefficient of variation (CV)) were below 9% at relevant plasma drug concentrations for both analytes.
Luna c18 analytical column
Manufactured by Phenomenex
Sourced in United States
The Luna C18 analytical column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a C18 stationary phase, which provides excellent retention and selectivity for a variety of analytes.
Automatically generated - may contain errors
Lab products found in correlation
Empower software, by Waters Corporation (4 mentions)
2010 lcms system, by Shimadzu (3 mentions)
C18 guard column, by Phenomenex (3 mentions)
Lc 20ad binary solvent pumps, by Shimadzu (3 mentions)
Lcms solutions software version 3, by Shimadzu (3 mentions)
Sil 20a ht, by Shimadzu (2 mentions)
Spd m20a diode array detector, by Shimadzu (2 mentions)
Waters alliance e2695 system, by Waters Corporation (2 mentions)
Qtrap 5500, by AB Sciex (1 mentions)
Waters alliance e2695 hplc system, by Waters Corporation (1 mentions)
Superdex 75 10 300 gl column, by GE Healthcare (1 mentions)
Tyrosol, by Merck Group (1 mentions)
Strata c18 cartridge, by Phenomenex (1 mentions)
Lc 30ad pump, by Shimadzu (1 mentions)
Dgu 20a5 degasser, by Shimadzu (1 mentions)
Lc ms grade acetonitrile, by Merck Group (1 mentions)
E2695 system, by Waters Corporation (1 mentions)
14 protocols using luna c18 analytical column
1
Quantification of Ibrutinib and Metabolite in Plasma
2
Quantitative LC-MS/MS Analysis Protocol
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The LC–MS/MS system consisted of a Shimadzu Nexera X2 UHPLC liquid chromatograph (Shimadzu, Japan) coupled with an 8050 triple quadrupole detector (Shimadzu, Japan), controlled by LabSolution 5.60 SP2 software. The experiments were conducted in the positive electrospray mode using Luna C18 analytical column (50 × 2.0 mm, 3 µm) (Phenomenex, USA) with a C18 guard cartridge operated at 40°C. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid (B). The gradient was: 50% A (0 to 1.5 min), 80% A (1.51 to 2.5 min), 100% A (2.51 to 4 min, maximum to 5 min), 50% A (5 to 5.01 min, maximum to 7 min). The flow rate was 400 µL/min and the injection volume was 10 µL. The mass spectrometer working parameters (ionisation mode, capillary voltage, source temperature, fragmentary voltage, and collision energy) were optimised. The fragmentation reactions used for monitoring were selected on the basis of their significance in the production spectra. The analytes were quantified using the selected reactions monitoring (SRM) mode. For each analyte, at least two of the most abundant transitions were monitored.
3
HPLC Analysis of Organic Compounds
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The HPLC system was a LC Jasco consisting of a ternary gradient system (PU 980), in line degasser (DG‐2080‐53), autosampler (AS2055), and an UV multiple wavelength detector (MD‐1510). The chromatographic separation assay was performed with a Luna C18 analytical column (150 × 4.6 mm inner diameter, 3 μm particle size, Phenomenex) maintained at 30°C using a Peltier system (CO4062) (Jasco). The mobile phases were 0.1% v/v formic acid in water:ACN 95:5 (v/v) (phase A) and ACN (phase B). The column was eluted isocratically using 38% A and 62% B at a flow rate of 1 ml/min. The optimal wavelength for the quantification was set at 275 nm.
4
LCMS Compound Purity Verification
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The purity and identity
of all compounds
was verified using a Shimadzu 2010 LCMS system, consisting of a LC-20AD
binary solvent pumps, a DGU-20A degasser unit, a CTO-20A column oven,
and a SIL-20A HT auto sampler. A Shimadzu SPD-M20A diode array detector
was used for detections. A full spectra range of 190–600 nm
was obtained during analysis. Chromatographic separations were obtained
using a Phenomenex Luna C18 analytical column (5 μm, 50 ×
4.6 mm i.d.) The column was protected by a Phenomenex C18 column guard
(5 μm, 4 × 3.0 mm i.d.). All equipment was controlled and
integrated by Shimadzu LCMS solutions software version 3. Mobile phases
for LCMS analysis were HPLC grade or LCMS grade obtained from Sigma-Aldrich
and Fisher Scientific. The mobile phases consisted of a mixture LCMS
grade Acetonitrile/water (both with 0.1% formic acid for a pH of 2.7).
The initial setting for analysis was set at 5% acetonitrile (v/v),
then was linearly increased to 95% acetonitrile over 6 min. The gradient
was then held at 95% acetonitrile for 2 min and then linearly decreased
to 5% over 0.10 min and held until stop for an additional 1.90 min.
The total run time was equal to 12 min. The total flow rate was set
to 0.5 mL/min. The column oven and flow cell temperature for the diode
array detector was set at 30 °C. The auto sampler temperature
was held at 15 °C. 5uL was injected for analysis.
of all compounds
was verified using a Shimadzu 2010 LCMS system, consisting of a LC-20AD
binary solvent pumps, a DGU-20A degasser unit, a CTO-20A column oven,
and a SIL-20A HT auto sampler. A Shimadzu SPD-M20A diode array detector
was used for detections. A full spectra range of 190–600 nm
was obtained during analysis. Chromatographic separations were obtained
using a Phenomenex Luna C18 analytical column (5 μm, 50 ×
4.6 mm i.d.) The column was protected by a Phenomenex C18 column guard
(5 μm, 4 × 3.0 mm i.d.). All equipment was controlled and
integrated by Shimadzu LCMS solutions software version 3. Mobile phases
for LCMS analysis were HPLC grade or LCMS grade obtained from Sigma-Aldrich
and Fisher Scientific. The mobile phases consisted of a mixture LCMS
grade Acetonitrile/water (both with 0.1% formic acid for a pH of 2.7).
The initial setting for analysis was set at 5% acetonitrile (v/v),
then was linearly increased to 95% acetonitrile over 6 min. The gradient
was then held at 95% acetonitrile for 2 min and then linearly decreased
to 5% over 0.10 min and held until stop for an additional 1.90 min.
The total run time was equal to 12 min. The total flow rate was set
to 0.5 mL/min. The column oven and flow cell temperature for the diode
array detector was set at 30 °C. The auto sampler temperature
was held at 15 °C. 5uL was injected for analysis.
5
HPLC Analysis of Organic Compounds
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HPLC analysis was used Waters Alliance e2695 HPLC system (Waters Corp., Milford, MA, USA) equipped with a pump, degasser, column oven, automatic sample injector, and photodiode array (PDA) detector (#2998; Waters Corp.). Chromatographic separation for the two standard compounds was carried out with a Luna C18 analytical column (250 × 4.6 mm, 5 µm, Phenomenex, Torrance, CA, USA) maintained at 40 °C. The mobile phases consisted of 0.1% (v/v) aqueous TFA (A) and acetonitrile (B). The gradient conditions were as follows; 10% B for 0–10 min, 10–30% B for 10–40 min, and 100% B for 40–50 min. The flow rate was 1.0 mL/min, and the injection volume was 10 µL. The data were acquired and processed using Empower software (version 3; Waters Corp).
6
HPLC Quantification of Analytes
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The HPLC system was a LC Jasco consisting of a ternary gradient system (PU 980), in line degasser (DG‐2080‐53), autosampler (AS‐2055) and an UV multiple wavelength detector (MD‐1510). The chromatographic separation assay was performed with a Luna C18 analytical column (250 × 4.6 mm inner diameter, 5 μm particle size, Phenomenex) maintained at 30 °C using a Peltier system (CO‐4062) (Jasco). The mobile phase consisted of acetonitrile:0.1% TFA (21:79% v:v) in water with a flow rate of 1 ml/minutes. The optimal wavelength for the quantification was set at 350 nm.
7
Stabilizing Asp28 Peptide Formulation
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Asp28 was dissolved in 20 mM tris buffer at pH 8.0 at a concentration of 1.5 mg/ml. Excipients were added to the formulation as follows: buffer alone, 0.1% Tween 80, 0.1% SDS, 0.1% HSA, 5% Sucrose, 5% PPG, 5% PEG, and 0.1% F68. Samples were divided in agitated and non-agitated conditions and incubated at either 4 °C (data not shown), ambient temperature, or 37 °C for the duration of the study. Analysis was performed via RP-HPLC and SEC-HPLC at select time points. The RP-HPLC assay was performed on a Phenomenex Luna C18 analytical column (4.6×250 mm2) (Torrance, CA) employing a linear gradient from 30% to 53% B (A=0.025 M NH4HCO3, pH 8.0, B=90% ACN aq) over 15 min. The SEC assay was performed on a GE Healthcare Superdex 75 10/300 GL column (10×300 mm2) employing a 25 mM ammonium bicarbonate mobile phase over 75 min.
8
Quantification of Tyrosol in C. albicans
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C. albicans was grown in YPD at 30 °C, 110 rpm for 24 h. The cells were harvested by centrifugation at 9000× g for 25 min. The supernatant was filtered through a 0.22 µm membrane. tyrosol was isolated from culture supernatants by solid-phase extraction, according to Alem et al. [26 (link)], with slight modifications. tyrosol was quantified by reverse phase high-pressure liquid chromatography (RP-HPLC). The analytical standard of tyrosol was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Prior to extraction, 100 mL of cell-free supernatant was acidified by adding 0.4 mL of 0.1 M H2SO4. The extraction was performed with a Strata C-18 cartridge (Phenomenex, Torrance, CA, USA).
To quantify the amount of tyrosol in the extracts, the analytical RP-HPLC was performed using Luna C-18 analytical column (4.6 × 250 mm, 5 µm, Phenomenex, Torrance, CA, USA). The elution was carried out using solvent B (80% acetonitrile in ultrapure water with 0.1% TFA) in a linear gradient according to the following scheme: 0–5% for 5 min, 5–10% for 5 min, 10% for 5 min, 10–20% for 20 min, 20–70% for 5 min at a flow rate of 1.0 mL/min. The absorbance was detected at 225 nm. OpenLab CDS ChemStation software was used for processing the HPLC chromatograms. The calibration curve of tyrosol concentrations was plotted with 10, 20, 30, 50, and 100 µM of tyrosol.
Prior to extraction, 100 mL of cell-free supernatant was acidified by adding 0.4 mL of 0.1 M H2SO4. The extraction was performed with a Strata C-18 cartridge (Phenomenex, Torrance, CA, USA).
To quantify the amount of tyrosol in the extracts, the analytical RP-HPLC was performed using Luna C-18 analytical column (4.6 × 250 mm, 5 µm, Phenomenex, Torrance, CA, USA). The elution was carried out using solvent B (80% acetonitrile in ultrapure water with 0.1% TFA) in a linear gradient according to the following scheme: 0–5% for 5 min, 5–10% for 5 min, 10% for 5 min, 10–20% for 20 min, 20–70% for 5 min at a flow rate of 1.0 mL/min. The absorbance was detected at 225 nm. OpenLab CDS ChemStation software was used for processing the HPLC chromatograms. The calibration curve of tyrosol concentrations was plotted with 10, 20, 30, 50, and 100 µM of tyrosol.
9
RP-HPLC-FLD Analysis of Derivatives
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For EC determination, reversed-phase high-performance liquid chromatography coupled to uorescence detection (RP-HPLC-FLD) was utilized. The system was equipped with two LC-30AD pumps, a DGU-20A 5 degasser, an RF-20A XS uorescence detector (all Shimadzu, Kyoto, Japan), a six-port injection valve with a 20 mL external loop (Valco-Vici, Schenkon, Switzerland), and an LCO 102 column thermostat (Ecom, Prague, Czech Republic).
The optimised separation of derivatives was performed on a Luna C18 analytical column (150 × 3 mm; 3 mm particle size; Phenomenex, Torrance, USA) using a binary mobile phase consisting of sodium acetate (c = 20 mmol L -1 ) at pH 7.2 and 100% acetonitrile. The ow rate was 0.8 mL min -1 , the column temperature 35 °C, and the injection volume 20 mL. The nal mobile phase gradient was as follows: 0 min -62% B, 4 min -70% B, and 5 min -100% B. The excitation and emission wavelengths of the uorescence detector were set at 233 nm and 600 nm, respectively.
The optimised separation of derivatives was performed on a Luna C18 analytical column (150 × 3 mm; 3 mm particle size; Phenomenex, Torrance, USA) using a binary mobile phase consisting of sodium acetate (c = 20 mmol L -1 ) at pH 7.2 and 100% acetonitrile. The ow rate was 0.8 mL min -1 , the column temperature 35 °C, and the injection volume 20 mL. The nal mobile phase gradient was as follows: 0 min -62% B, 4 min -70% B, and 5 min -100% B. The excitation and emission wavelengths of the uorescence detector were set at 233 nm and 600 nm, respectively.
10
Identification and Purity of Compounds via LCMS
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