An HPLC Agilent® (Agilent® 1290 Infinity Quaternary LC System, Les Ulis, France) equipped with a binary pump with integrated vacuum degasser, a thermostated column compartment, an autosampler and a diode array detector was used for analysis of melatonin content. The results were collected and evaluated statistically using HP ChemStation® software. Attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR) spectra of powdered samples (pure raw materials and microcrystalline cellulose and melatonin mixtures) were recorded in Nicolet® iS 50 FT-IR Spectrometer (Thermo Scientific®, Thermo Nicolet Corp.®, Waltham, MA, USA) applying ATR method with diamond crystal and having a standard deuterated triglycine sulfate (DTGS) detector (optical path: 10 cm). For each analysis, a total number of scans was 32 with spectral resolution of 4 cm−1. Omnic Spectra® software was used to process simplified and multi-component spectra. Dissolution testing was performed with a USP dissolution tester Pharmatest DT 70® with paddles (Pharmatest Apparatebau AG®, Hainburg, Germany) equipped with seven stirred positions. For sampling, guiding tubes were placed into the seven holes in the top cover of the instrument. The system ensured that all samples were taken from the same position in the testing vessel.
Agilent 1290 infinity quaternary lc system
Manufactured by Agilent Technologies
Sourced in France, United States
The Agilent 1290 Infinity Quaternary LC System is a high-performance liquid chromatography (HPLC) instrument designed for analytical and preparative applications. It features a quaternary pump capable of delivering up to four different solvents, allowing for flexible solvent composition and gradient formation. The system is equipped with a variety of detectors, including diode-array and refractive index, enabling the analysis of a wide range of sample types.
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5 protocols using agilent 1290 infinity quaternary lc system
1
HPLC Analysis of Melatonin Content
2
Lipidomic analysis of C. glutamicum
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IM and OM lipids of C. glutamicum were extracted and analysed as previously described [27 (link)]. In brief, four replicates of WT C. glutamicum and mutant strain were grown to the exponential phase (OD600nm = 2.5–3) in BHI medium and the cells were harvested by centrifugation. The cell pellet was extracted with water-saturated 1-butanol to selectively remove OM lipids. After another centrifugation step, the same pellet was sequentially extracted in chloroform:methanol (2:1, v/v) and chloroform:methanol:water (1:2:0.8 v/v) to extract the remaining lipids of the IM. Both IM and OM lipids were separated on an Agilent 1290 Infinity Quaternary LC System (Agilent Technologies) using a C18 column (Phenomenex Kinetex, 2.6 um EVO C18 100A) eluted with an isopropanol mobile phase binary solvent system at a flow rate of 260 µL/min. Eluted lipids were analysed on a 6550 iFunnel Q-TOF LC/MS instrument (Agilent Technologies) in positive ionization mode. Lipids were identified based on their mass-to-charge ratio and fragmentation pattern using a lipid library [27 (link)]. Statistical analyses were performed using MetaboAnalyst.
3
Intact Mass Measurement of Proteins
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The pooled fraction at a 0.4 mg ml−1 concentration was buffer exchanged into 0.1% formic acid using 10 kDa MWCO centricon (Pall Corporation, USA). Intact mass measurements on these fractions were made on AdvanceBio RP mAb C4 (4.6 × 100 mm, 5 μm, Agilent Technologies) column operated at 80 °C using Agilent 1290 Infinity Quaternary LC system coupled online to Agilent 6230 ESI-TOF–MS. 5 μg sample was loaded on the column and separated using a 35 min linear gradient from 2 to 60% B at a flow rate of 0.5 ml min−1. Detection was performed by monitoring UV absorption at 280 nm and TIC was recorded for 1000–6000 m/z. MS spectra were calibrated in the positive ion mode before analysis. The capillary gas temperature/voltage (Vcap) was set to 350 °C and 5500 V, respectively, and the fragmentor voltage (Vfrag) was 400 V. The MS spectra were deconvoluted using the maximum entropy (MaxEnt) algorithm with resolution of 20,000 and 8 iterations.
4
Agilent 1290 Infinity Quaternary LC Analysis
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The Agilent 1290 Infinity Quaternary LC System (Agilent Technologies, Santa Clara, CA, USA) was used in the present study. It was composed of a binary pump, an integrated vacuum degasser, a thermostated column compartment (30 °C), an autosampler, and a mass spectrometer. A Kinetex 2.6 µm C18 100 Å, 100×2.1 mm LC Column (Phenomenex, Torrance, CA, USA) was used as the stationary phase. The mobile phase A was an 5 mM ammonium formate buffer (pH = 3), and the mobile phase B was acetonitrile with 10% of mobile phase A. The following gradient was applied: starting at 90% A / 10% B, increasing linearly to 10% A / 90% B over 2 min and then maintained for between 2 and 3 min, and then decrease to 90% A / 10% B between 3 and 4 min. The flow rate was 0.5 mL/min and the injection volume was 2 µL.
5
Characterization of Bevacizumab Charge Variants
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The subunit analysis of the bevacizumab was carried out by separating the light chain and heavy chain. The sample preparation involved solubilizing the isolated charge variants (50 µg) with 30 µl of 6 M guanidine followed by the addition of 3 µl of 0.5 M DTT to a final concentration of 50 mM. The samples were reduced by incubation at 55 °C for 1 h and finally equilibrated to room temperature before subjecting to LC–MS analysis43 (link). LC–MS analysis consisted of separating the light chain and the heavy chain of bevacizumab charge variants on an Agilent AdvanceBio RP mAb C4 (4.6 × 100 mm, 5 μm, Agilent Technologies) column operated at 60 °C using Agilent 1290 Infinity Quaternary LC system coupled online to Agilent 6230 ESI-TOF–MS. The mobile phase used for the separation consisted of buffer A (water + 0.1% formic acid) and buffer B (Acetonitrile + 0.1% formic acid) at a linear gradient from 2%B to 65%B in 30 min. MS spectrum was acquired in the mass range 500–3000 Da in positive mode. Other MS parameters included capillary gas temperature/voltage (Vcap) was set to 250 °C and 4500 V, respectively, and the fragmentor voltage (Vfrag) was 280 V. The MS spectra were deconvoluted using the maximum entropy (MaxEnt) algorithm with resolution 20,000 and 9 iterations.
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