The UPLC platform Agilent 1290 Infinity included a binary pumping system (1290 Infinity), a compartment thermostat column (1290 Infinity) autosampler (1290 Infinity) coupled with Diode-Array Detector (1290 Infinity). An Eclipse Plus C18 column (2.1 × 100 mm, 1.8 µm) was employed for the chromatographic separation. UV detection was carried out at 220 and 270 nm. The mobile phases employed were solvent A (H2O) and B (MeOH), the rate of flow was 300 μL/min, and eluent B varied as follows: 0 min,1%; 3 min, 5%; 7 min, 8%; 25 min, 12.5%; 30 min, 15%; 35 min, 20%; 40 min, 99%; 42 min, 99%; 46 min, 1%. The injection amount was 20 μL. This UPLC was connected with an HRMS, described below.
1290 infinity
Manufactured by Agilent Technologies
Sourced in United States, Germany, France
The 1290 Infinity is a high-performance liquid chromatography (HPLC) system designed for analytical applications. It offers precise and reliable separation and detection of a wide range of samples. The 1290 Infinity features advanced technology to ensure accurate and reproducible results.
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Lab products found in correlation
Zorbax eclipse plus c18 column, by Agilent Technologies (5 mentions)
Eclipse plus c18 column, by Agilent Technologies (4 mentions)
Eclipse plus c18, by Agilent Technologies (4 mentions)
Openlab cds chemstation, by Agilent Technologies (3 mentions)
Acquity uplc beh c18 column, by Waters Corporation (3 mentions)
Low mass calibrant solution, by Agilent Technologies (3 mentions)
Zorbax eclipse plus c18, by Agilent Technologies (3 mentions)
Zorbax eclipse xdb c8 reverse phase column, by Agilent Technologies (3 mentions)
Qtrap 5500, by AB Sciex (3 mentions)
Qtrap 6500, by AB Sciex (3 mentions)
Eclipse xdb c18 column, by Agilent Technologies (2 mentions)
Agilent open lab a 02.02 cds chemstation, by Agilent Technologies (2 mentions)
Poroshell 120 sb c18 column, by Agilent Technologies (2 mentions)
Masshunter software, by Agilent Technologies (2 mentions)
Masshunter workstation, by Agilent Technologies (2 mentions)
6495c triple quadrupole mass spectrometer, by Agilent Technologies (2 mentions)
Masshunter quantitative analysis software, by Agilent Technologies (2 mentions)
Dataanalysis version 5, by Bruker (2 mentions)
Impact 2, by Bruker (2 mentions)
6490 triple quad lc ms, by Agilent Technologies (2 mentions)
160 protocols using 1290 infinity
1
UPLC-HRMS Analysis of Chemical Compounds
2
Pigment Extraction and HPLC Analysis
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Freeze-dried pellet samples were weighed and re-suspended in a solvent ratio of between 2–3 mg of sample (DW) per mL of ethanol and sonicated with an ultrasonic homogenizer (Qsonica Q125) at 100% amplitude for 3 × 3-s pulses before being stored at −20 °C overnight once blanching of pellets was confirmed using a centrifuge. They were then filtered using 0.2 µm PTFE 13 mm syringe filters and stored in −80 °C until analysis. High Performance Liquid Chromatography (HPLC) was conducted using an Agilent Technologies 1290 Infinity, equipped with a binary pump with integrated vacuum degasser, thermostatted column compartment modules, Infinity 1290 auto-sampler, and PDA detector. Column separation was performed using a 4.6 mm × 150 mm Zorbax Eclipse XDB-C8 reverse-phase column (Agilent Technologies, Santa Clara, CA, USA) and guard column using a gradient of TBAA (tert-Butyl acetoacetate): Methanol mix (30:70) (solvent A) and Methanol (Solvent B) as follows: 0–22 min, from 5 to 95% B; 22–29 min, 95% B; 29–31 min, 5% B; and 31–40 min, column equilibration with 5% B. Column temperature was maintained at 55 °C. A complete pigment profile from 270 to 700 nm was recorded using PDA detector with 3.4 nm bandwidth. Example chromatograms can be found in supplementary files .
3
Analytical Characterization of Synthesized Compounds
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All general chemicals were
purchased from Acros Organics (Belgium), Merck (Germany), Sigma-Aldrich
(USA), Guangdong Guanghua (China), and Chemsol (Vietnam) and used
without further purification unless otherwise stated.
Thin-layer
chromatography was conducted on silica gel 60 F254, and
the spots were located under UV light (254 nm). The uncorrected melting
points were conducted in open capillaries on a Krüss Optronic
M5000 melting point meter (Germany). The UV–vis spectra were
recorded on a UV–vis Metash UV-5100 spectrophotometer or JASCO
V-630 UV–vis spectrophotometer. The NMR spectra were measured
using either a Bruker Advanced 500 or 600 MHz NMR spectrometer in
(CD3)2SO. The chemical shifts (δ) were
expressed in ppm and referred to the residual peak of tetramethylsilane
as an internal standard. The IR spectra were recorded on a Bruker
Tensor 27 FTIR spectrometer or PerkinElmer Frontier FTIR spectrometer
by using KBr pellets. The high-resolution mass spectra were measured
on the Agilent 6200 series TOF and 6500 series Q-TOF LC/MS system.
The purity of all tested compounds was >95% according to HPLC performed
on the Shimadzu SPD-20A HPLC system (Shimadzu, Japan) equipped with
a BDS Hypersil C18 column (250 × 4.6 mm, 5 μm) or the Agilent
1290 Infinity equipped with a Zorbax Eclipse Plus C18 column (250
× 4.6 mm, 5 μm).
purchased from Acros Organics (Belgium), Merck (Germany), Sigma-Aldrich
(USA), Guangdong Guanghua (China), and Chemsol (Vietnam) and used
without further purification unless otherwise stated.
Thin-layer
chromatography was conducted on silica gel 60 F254, and
the spots were located under UV light (254 nm). The uncorrected melting
points were conducted in open capillaries on a Krüss Optronic
M5000 melting point meter (Germany). The UV–vis spectra were
recorded on a UV–vis Metash UV-5100 spectrophotometer or JASCO
V-630 UV–vis spectrophotometer. The NMR spectra were measured
using either a Bruker Advanced 500 or 600 MHz NMR spectrometer in
(CD3)2SO. The chemical shifts (δ) were
expressed in ppm and referred to the residual peak of tetramethylsilane
as an internal standard. The IR spectra were recorded on a Bruker
Tensor 27 FTIR spectrometer or PerkinElmer Frontier FTIR spectrometer
by using KBr pellets. The high-resolution mass spectra were measured
on the Agilent 6200 series TOF and 6500 series Q-TOF LC/MS system.
The purity of all tested compounds was >95% according to HPLC performed
on the Shimadzu SPD-20A HPLC system (Shimadzu, Japan) equipped with
a BDS Hypersil C18 column (250 × 4.6 mm, 5 μm) or the Agilent
1290 Infinity equipped with a Zorbax Eclipse Plus C18 column (250
× 4.6 mm, 5 μm).
4
Analytical Characterization of Samples
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Five microliters of the collected samples were applied to an Ultra Performance Liquid Chromatographic system (UPLC, Agilent 1290 Infinity) on a Zorbax XDB C18 reverse phase column (4.6 × 150 mm, temperature-controlled at 30 °C), and eluted with methanol-water (80:20, v/v) at a flow rate of 0.4 ml/min in a diode array detector (Agilent G4212A). 1H, 13C, 1H-1H COSY, and heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra in CH3DO solution were obtained using a Bruker Avance III 600 MHz.
5
Thiol Quantification by UPLC-FLD
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For Cys, GSH and PCs measurement, they were extracted by 1 ml TFA (0.1%) and 6.3 mM diethylene triaminepentaacetic acid, put the homogenate on ice for 15 min. Following centrifugation (13200 g, 30 min, 4°C), the supernatant were derivatized based on the methods of Kühnlenz et al. (2015) (link). UPLC system (Agilent 1290 Infinity, Germany) was equipped with a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm) used for the separation of the mBBr-labeled thiols. Thiols were detected using the fluorescence detector set at excitation and emission wavelengths of 380 and 470 nm. Quantification was performed via authentic Cys, GSH, PC2, PC3, and PC4 standards.
6
HPLC Quantification of Danazol and Fenofibrate
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Analysis was conducted using an HPLC
(Agilent Technologies 1290 Infinity) with a Zorbax Eclipse XDB-C18
column (4.6 mm × 100 mm) at 40 °C. The injection volume
was 20 μL. The mobile phase consisted of acetonitrile:sodium
acetate buffer (pH 5) 70:30 (v/v) for danazol and 80:20 (v/v) for
fenofibrate; an isocratic flow rate was used at 1 mL/min. UV absorbance
was monitored at a wavelength of 286 nm for danazol and 287 nm for
fenofibrate. The retention times were 2.45 min for danazol and 3.04
min for fenofibrate. Calibration curves were used over a range between
0.78 and 100 μg/mL. Intraday validation with quality control
samples (12.5–50 μg/mL) resulted in inaccuracy ranging
from 2.44 to 4.79% and 3.59–4.94% and a repeatability (coefficient
of variation, CV) of 0.77–1.10% and 0.14–0.37% for danazol
and fenofibrate, respectively. The interday inaccuracy for the respective
compounds was −2.11–5.99% and −1.79–2.27%,
while interassay CV was 3.67–4.57% and 1.04–1.54%.
(Agilent Technologies 1290 Infinity) with a Zorbax Eclipse XDB-C18
column (4.6 mm × 100 mm) at 40 °C. The injection volume
was 20 μL. The mobile phase consisted of acetonitrile:sodium
acetate buffer (pH 5) 70:30 (v/v) for danazol and 80:20 (v/v) for
fenofibrate; an isocratic flow rate was used at 1 mL/min. UV absorbance
was monitored at a wavelength of 286 nm for danazol and 287 nm for
fenofibrate. The retention times were 2.45 min for danazol and 3.04
min for fenofibrate. Calibration curves were used over a range between
0.78 and 100 μg/mL. Intraday validation with quality control
samples (12.5–50 μg/mL) resulted in inaccuracy ranging
from 2.44 to 4.79% and 3.59–4.94% and a repeatability (coefficient
of variation, CV) of 0.77–1.10% and 0.14–0.37% for danazol
and fenofibrate, respectively. The interday inaccuracy for the respective
compounds was −2.11–5.99% and −1.79–2.27%,
while interassay CV was 3.67–4.57% and 1.04–1.54%.
7
Quantitative Analysis of Fungal Metabolite TL1-1
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Fungus growth of D. eschscholzii was determined by dry cell weight (DCW) (Yang et al. 2016 (link)), and residual sugar (mannitol) was determined by the colorimetric assay (Bok and Demain 1977 (link)).
The whole fermentation broth was pretreated as report (Zhu et al. 2014 (link)). The qualitative and quantitative measurements of TL1-1 were analyzed by HPLC (Agilent 1290 Infinity, DAD-G4212B, USA) with a ZORBAX Eclipse SB-C18 column (4.6 × 250 mm, 5 µm). Each injected sample (20 µL) was eluted with a mobile phase made up of methanol/water (60:40) for 25 min. The flow rate was set as 1.0 mL/min, the operating temperature 25 °C, and the detection wavelength 272 nm, respectively. The standard calibration curve equation was Y = 42.95X − 341.77 with high linearity in the range of 62.5–750.0 mg/L (linear relative coefficients up to 0.9999), where Y is the peak area and X is the concentration of standard TL1-1 (mg/L) (data not shown and detailed data in Additional file1 : Figure S2).
The whole fermentation broth was pretreated as report (Zhu et al. 2014 (link)). The qualitative and quantitative measurements of TL1-1 were analyzed by HPLC (Agilent 1290 Infinity, DAD-G4212B, USA) with a ZORBAX Eclipse SB-C18 column (4.6 × 250 mm, 5 µm). Each injected sample (20 µL) was eluted with a mobile phase made up of methanol/water (60:40) for 25 min. The flow rate was set as 1.0 mL/min, the operating temperature 25 °C, and the detection wavelength 272 nm, respectively. The standard calibration curve equation was Y = 42.95X − 341.77 with high linearity in the range of 62.5–750.0 mg/L (linear relative coefficients up to 0.9999), where Y is the peak area and X is the concentration of standard TL1-1 (mg/L) (data not shown and detailed data in Additional file
8
Quantitative Analysis of Artemisinin in QHP
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The contents of artemisinin in QHP were determined by HPLC. Quantitative analysis of artemisinin in QHP was performed on the Agilent 1290 Infinity apparatus comprising two solvent delivery systems and a photodiode array detector (Agilent, USA). The column was an Agilent ZORBAX SB-C18 chromatographic column (4.6 mm × 250 mm, 5.0 μm). The mobile phase consisted of acetonitrile and H2O (60: 40), and the pH value was 6.8–7.2. Reagents were filtered through a Millipore 0.45 mm filter and degassed prior to use. The entire run was carried out by gradient elution at a flow rate of 1.0 mL/min; the detection wavelength was set at 210 nm; the column was maintained at 35°C, and the injection volume was 10 μL. Data collection and quantification were performed with Agilent Open LAB A.02.02 CDS ChemStation (Agilent, USA). The peak of artemisinin was identified by comparison with chemical standards.
9
HPLC-MS Protocol for Compound Purity
10
Analytical Method for Acylcarnitines and Fatty Acid Amides
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To analyse the samples a new methodology was developed, based on reversed-phase UHPLC (1290 Infinity, Agilent Technologies) coupled to an ESI(AJS)-QQQ-MS (6460, Agilent Technologies) mass spectrometer. During the method development, standards of acylcarnitines (acetylcarnitine, butyrylcarnitine, hexanoylcarnitine, octanoylcarnitine, decanoylcarnitine, palmitoylcarnitine) and fatty acid amides (dodecanamide, hexadecanamide, oleamide, linoleamide) were used. Detailed description of developed method can be found in Supplementary Information . Analyses were controlled with QC samples consisted of equal volumes of serum from each investigated sample. QCs were prepared following the same procedure as the rest of samples for QQQ analysis and were injected after every 10th sample.
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