The concentrations of 6β-hydroxytestosterone was analyzed using an LC-MS/MS system consisting of an Agilent 1200 HPLC (Palo Alto, CA, United States) and an Applied Biosystem 3200 Q-Trap (Foster City, CA, United States) equipped with an electrospray ionization interface. The 10-µL aliquots of the samples were separated on a RP-C18 column (2.1 × 50 mm i. d, 3.5-µM particle size; Agilent, United States) maintained at 40°C. The mobile phase consisted of 0.1% formic acid in water (A) and in acetonitrile (B) with a flow rate of 0.4 ml/min. Phase A was linearly increased from 5 to 90% over a period of 0.1 min, maintained at 90% for an additional 4 min, and then decreased to 5% for re-equilibration; the total run time was 7 min. The TurboIonSpray interface was operated separately in the positive ion mode at 5500 V. The operating conditions were the following: ion source temperature, 400°C; curtain gas, 20 psi; ion source gas 1, 60 psi; ion source gas 2, 60 psi. The quantification was performed by multiple reaction monitoring (MRM) of the molecular ion and the related product ion. The MRM transitions (collision energy) of 6β-hydroxytestosterone, and propranolol, (IS) were m/z 305.1→269.3 (25 eV), and 261.3→116.1 (32 eV), respectively.
Rp c18 column
Manufactured by Agilent Technologies
Sourced in United States, Germany
The RP-C18 column is a reversed-phase liquid chromatography column. It is designed for the separation and analysis of a wide range of non-polar or moderately polar compounds. The column features a silica-based stationary phase with chemically bonded C18 alkyl chains, providing a hydrophobic surface for the retention of analytes.
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Lab products found in correlation
Silica gel, by Qingdao Haiyang Chemical (3 mentions)
Pda plus detector, by Thermo Fisher Scientific (2 mentions)
Lc pump plus, by Thermo Fisher Scientific (2 mentions)
Esi msq plus, by Thermo Fisher Scientific (2 mentions)
Sb aq, by Agilent Technologies (2 mentions)
Agilent 1200, by Agilent Technologies (1 mentions)
Dip 1000 polarimeter, by Jasco (1 mentions)
Kieselgel 60 f254, by Merck Group (1 mentions)
Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Kieselgel 60, by Merck Group (1 mentions)
J 815 cd spectrometer, by Jasco (1 mentions)
1260 series instrument, by Agilent Technologies (1 mentions)
6529b q tof mass instrument, by Agilent Technologies (1 mentions)
Aviii 500 nmr instrument, by Bruker (1 mentions)
P 1020 polarimeter, by Jasco (1 mentions)
Tensor 27 spectrometer, by Bruker (1 mentions)
Lh 20, by Mitsubishi (1 mentions)
Intelligent hplc pump, by Jasco (1 mentions)
Intelligent uv vis detector, by Jasco (1 mentions)
Pu 2080 plus, by Jasco (1 mentions)
12 protocols using rp c18 column
1
LC-MS/MS Quantification of 6β-Hydroxytestosterone
2
HPLC-MS Analysis of Compounds
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Analysis was carried out using an LC-PDA-MS Thermo Finnigan system (LC Pump Plus, Autosampler, Surveyor PDA Plus Detector) interfaced with an ESI MSQ Plus (Thermo Finnigan) and equipped with an Xcalibur software. The same column, timetable and flow rate were used during the HPLC-MS analyses. The mass spectrometer operated in both negative and positive ionization modes, scan spectra were from m/z 100 to 1000, gas temperature was at 350 °C, nitrogen flow rate at 10 L/min, and capillary voltage 3200 V. The cone voltage was in the range of 60–100 V. The column was a SB-Aq (Agilent) RP-C18 column (150 mm × 3 mm) with a particle size of 5 µm maintained at 30°C. The eluents were H2O at pH 2.8 by formic acid (0.05% v/v) (A) and acetonitrile (B) and with a flow rate of 0.4 mL/min. Gradient program was as follows: 0–15 min, 85–79% A; 15–25 min 79–77% A; 25–45min, 77%–65% A; 45–53 min, 65%–35% A; 53–56 min, 35%–85% A; 56–60 min, 85% A. Injected volume of the samples was 5 μL of solution. The UV–vis spectra were recorded between 220 and 600 nm and the chromatographic profiles were registered at 315, 330 and 350 nm.
3
LC-MS/MS Quantification of LPV and RTV
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The concentrations of LPV and RTV were analyzed using an LC-MS/MS system consisting of an Agilent 1260 HPLC system (Palo Alto, CA, United States) and an Applied Biosystem 4500 Q-Trap system (Foster City, CA, United States). The chromatographic separation was performed on an RP-C18 column (2.1 mm × 50 mm, i. d, 3.5 µM particle size; Agilent, United States) maintained at 30°C. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B); it was run according to the following gradient programs at a flow rate of 0.4 ml/min: 85% A (0–0.1 min), 85–10% A (0.1–2.1 min), and 10–85% A (2.1–4.0 min) for the LPV and RTV assays. The mass detector with electrospray ionization interface was operated in positive ion mode and set according to the following conditions: spray voltage, 5,500 V; spray temperature, 450 °C; curtain gas, 20 psi; source gas 1, 60 psi; and source gas 2, 60 psi. The quantification was performed through MRM of the molecular ion to related product ion for each compound. The MRM transitions (collision energy) of LPV, RTV, and indinavir (IS) were m/z 629.5→155.1 (28 eV), 721.3→268.2 (32 eV), and 614.4→421.1 (32 eV), respectively. The peak area ratio of the analyzed LPV or RTV versus IS was used for calculating the concentration.
4
Radiolabeling and Purification of 125I-Benzoquinazoline
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A drop of the reaction mixture (1–2 µl) was placed 2 cm above the edge of TLC strip (1 cm width, 13 cm length) and allowed to evaporate spontaneously. Using a freshly prepared mixture of CH2Cl2: EtOAc (2:1 v/v) to develop the strips in order to determine radiochemical yield and purity of 125I-benzoquinazoline. The radiochemical yield was further confirmed by reversed phase-HPLC (RP-HPLC). The compound was purified by HPLC for in vivo biodistribution studies. An aliquot of 25 μl of 125I-benzoquinazoline at the optimum conditions was injected into a RP C18 column (Agilent, 250 × 4.6 mm; 5 μm) kept at room temperature using a mobile phase consisting of 0.1 M sodium phosphate buffer and acetonitrile (60:40, v/v) adjusted to pH 2.5 at a flow rate of 1.00 mL/min. The radiolabeling yield and selectivity/specificity of the radioiodinated compound were evaluated after formulation and purification by the same procedure.
5
Quantification of Creatine Kinase by HPLC
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The concentration of CK was determined by HPLC on an Agi-lent 1200 (Agilent Co. Ltd, Waldbronn, Germany) equipped with an Agilent™ RP-C18 column (150 mm × 4.6 mm, 5 μm). The mobile phase of methanol and water (60:40, v:v) was used at a flow rate of 1.0 mL/min. The UV detector was set at 230 nm to analyze the column effluent and the column temperature was set at 30°C. The standard curve of CK was A = 3.0408C+3.624. The R2 (link) for the calibration was 0.998 and the linearity range was 2.3–57.5 μg/mL.
6
Characterization of Compound 1b
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ECD and UV spectra were recorded on a JASCO J-815 CD spectrometer. The ECD spectrum of 1b was recorded in the region from 225 to 470 nm at a concentration of 0.2 mg/mL (2.88 × 10−4 M) in CH2Cl2 in a 1 mm cell, totaling five accumulations at a scan rate of 20 nm/min and a temperature of 25 °C. Specific optical rotation (OR) of 1b was recorded on a Jasco DIP-1000 polarimeter at 589 nm (Na lamp) at a concentration of 0.12 g/100 mL in CH2Cl2 in a 1 dm/SiO2 cuvette (25 °C). NMR spectra were recorded on either a 950, 900, 800, or a 700 MHz Bruker, all the signals being referenced to 13C (54.00 ppm) and 1H (5.320 ppm) signals of CD2Cl2. HR-MS were obtained on a Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Semipreparative HPLC was performed on an Agilent column (RP-C18 column 10 × 100 mm; 4.6 mL/min). TLC was performed on silica gel (Merck, Kieselgel 60 F254) plates; the spots were visualized by exposure to UV light (254 nm). Column chromatography was carried on silica gel (Merck, Kieselgel 60).
7
HPLC-MS Characterization of Compounds
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Analysis was carried out using an HPLC-PDA-MS Thermo Finnigan system (LC Pump Plus, Autosampler, Surveyor PDA Plus Detector) interfaced with an ESI MSQ Plus (Thermo Finnigan, San Jose, CA, USA) and equipped with an Xcalibur 2.1 software. The same column, timetable and flow rate were used during the HPLC-MS analyses. The mass spectrometer operated in both negative and positive ionization modes, scan spectra ranged from m/z 100 to 1200, gas temperature was 350 °C, nitrogen flow rate was 10 L/min, and capillary voltage was 3000 V. The cone voltage was in the range 60–120 V. The column was a SB-Aq (Agilent, Santa Clara, CA, USA) RP-C18 column (150 mm × 3 mm) with a particle size of 3.5 µm maintained at 30 °C. The eluents were H2O at pH 2.8 by formic acid (0.05% v/v) (A) and acetonitrile (B), with a flow rate of 0.4 mL/min. The samples were analyzed using a gradient program, as follows: 0–5 min, 85%A; 5–15 min 85–78%A; 15–20 min 78%A; 20–22 min 78–75%A; 22–27 min 75%A; 27–37 min 75–60%A; 37–44 min 60%A; 44–48 min, 60–85%A; 48–53 min, 85%A. A total of 5 μL of solution was injected into the samples. The UV–vis spectra were recorded between 220 and 600 nm and the chromatographic profiles were registered at 288, 330 and 350 nm.
8
Spectroscopic Characterization of Compounds
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The optical rotations were measured on a JASCO P-1020 polarimeter at room temperature. The melting points were measured using an X-4 digital display micromelting apparatus and are uncorrected. The IR spectra were recorded on a Bruker Tensor 27 spectrometer using KBr pellets. The 1D- and 2D-NMR spectra were measured on a Bruker AVIII-500 NMR instrument (1H: 500 MHz, 13C: 125 MHz) using TMS as the internal standards. HRESIMS was performed on an Agilent 6529B Q-TOF mass instrument using electrospray ionization. All of the solvents used were of analytical grade (Jiangsu Hanbang Science and Technology. Co., Ltd.). Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd, China), Sephadex LH-20 (Pharmacia, Sweden), MCI (Mitsubishi, Japan) and RP-C18 silica (40–63 μm, Fuji, Japan) were used for the column chromatography. Preparative HPLC was conducted using an Agilent 1260 Series instrument with a Shim-Pak RP-C18 column (20 × 200 mm), at a flow rate of 10.0 mL/min, and detection by a binary channel UV detector. The fractions obtained from CC were monitored by TLC with precoated Silica gel GF254 (Qingdao Haiyang Chemical Co., Ltd, China) plates.
9
Validated RP-HPLC Method for Encapsulation Efficiency
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Encapsulation efficiency was analyzed by the validated RP-HPLC method reported earlier with slight modification [22 (link),23 (link)]. The RP-HPLC analysis was carried out at 22 °C (Jasco PU-2080 Plus, Intelligent HPLC pump, Japan, Detector of Jasco 2075, Intelligent UV–vis detector, Jasco, Tokyo, Japan, and RP-C18 column of Agilent, 250 × 4.6 mm, 5 μm particle size, Technologies, Mumbai, India). 20 μL of each sample was injected after suitable dilution with mobile phase and detected at 210 nm. The mobile phase consisted of phosphate buffer pH 5.5 and acetonitrile (25:75), and an isocratic flow of 1 mL/min was used [23 (link)].
10
UPLC-DAD Quantification of Polyphenols
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Among all the identified metabolites, 10 polyphenolic compounds viz. gallic acid, chlorogenic acid, 4-hydroxybenzoic acid, vanillic acid, caffeic acid, rutin, sinapic acid, ferulic acid, naringin, and quercetin were quantified from IA extract. Quantification was carried out in a UPLC-DAD system (Agilent, United States) with a polar RP C18 column (250 mm L, 4.6 mm ID, 5 μm particle size) using mobile phase 0.1% formic acid in HPLC grade water (A) and 100% acetonitrile (B). The column was thermostatically maintained at 40°C, flow rate was set at 1 mL/min and 15 μL was used as injection volume. The gradient program for separation was as follows: 90%B for 0 min, 70%B for 8 min, 50%B for 13 min, 40%B for 15 min, 90%B for 18 min and 20 min. DAD detector was set at 255 nm for 4-hydroxybenzoic acid, chlorogenic acid, and vanillic acid; 280 nm for cinnamic acid, gallic acid, naringin and p-coumaric acid; 320 nm for caffeic acid, sinapic acid and ferulic acid; 375 nm for rutin and quercetin.
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