For the analysis 4 mg of lyophilized protein were diluted in ultrapure water. The profile of the proteins were analyzed by RP-HPLC. Separation was performed on a reversed-phase using a column Hypersil™ ODS C18 (250 mm � 4.6 mm, 5 μm particle size), operating at 35 � C, installed in a UHPLC chromatograph, equipped with an intelligent HPLC pump LC-30AD, NexeraX2, (Shimadzu, Japan). The elution flow rate was 2 mL/ min with the following gradient of HPLC grade solvents (A: 0.1% trifluoroacetic acid (TFA) in water; B: 0.1% TFA in acetonitrile): 0-30 min, 0-50% B; 30-35 min, 50-80% B; 35-40 min, 80% B. Monitoring was made at 250 nm and 35 � C by diode array detector SPD-M20A, NexeraX2 (Shimadzu, Japan).
Nexera x2
Manufactured by Shimadzu
Sourced in Japan, Germany, China, United States
The Nexera X2 is a high-performance liquid chromatography (HPLC) system designed for analytical applications. It features a compact and modular design, providing efficient and precise separation of complex samples. The Nexera X2 offers reliable and reproducible results, making it a suitable choice for a wide range of analytical tasks.
Automatically generated - may contain errors
Lab products found in correlation
4500 q trap, by Thermo Fisher Scientific (31 mentions)
Sb c18, by Agilent Technologies (13 mentions)
Applied biosystems 4500 q trap, by Thermo Fisher Scientific (11 mentions)
Qtrap 5500, by AB Sciex (11 mentions)
Scaa 104, by ANPEL (9 mentions)
Agilent sb c18, by Agilent Technologies (9 mentions)
Kinetex c18 column, by Phenomenex (4 mentions)
Scientz 100f, by Ningbo Scientz Biotechnology (4 mentions)
6500 q trap, by Thermo Fisher Scientific (3 mentions)
Spd m20a, by Shimadzu (3 mentions)
Lcms 8050, by Shimadzu (3 mentions)
Rf 20axs, by Shimadzu (3 mentions)
Lc 30ad pump, by Shimadzu (3 mentions)
Tripletof 5600, by AB Sciex (3 mentions)
Lcms 8045, by Shimadzu (2 mentions)
V 630, by Jasco (2 mentions)
Acquity beh c18 column, by Waters Corporation (2 mentions)
Qtrap 5500 ms, by AB Sciex (2 mentions)
Kinetex c18, by Phenomenex (2 mentions)
Kinetex xb c18 column, by Phenomenex (2 mentions)
174 protocols using nexera x2
1
Reversed-Phase HPLC Protein Profiling
2
Metabolomic Profiling of Rambutan Pulp
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Fifteen rambutan pulps were mixed by a blender for two min. Six hundred microliters of methanol solution (70 %) was added to a 50 mg fruit sample, and the tube vortexed was vortexed three min for extraction. The tube was centrifuged at 12,000 r/min for ten min for separation. The supernatant of the tube was collected after treatment with a 0.22 μm filter.
An UPLC-MS/MS system (UPLC, Nexera X2, Shimadzu; MS, Applied Biosystems 6500 Q TRAP, Japan) was used to identify and quantify all the metabolites. Briefly, a 2 mL filtered sample was loaded onto the UPLC. The mobile phase was solvent A (ultrapure water with 0.1 % formic acid) and solvent B (acetonitrile with 0.1 % formic acid). The mobile phase was programmed as follows: 0–9 min, 5 %–95 % B; 9–10 min, 95 % B; 10–11.1 min, 95 % B-5 % B; 11.1–14 min, 5 % B. Multiple reaction monitoring (MRM) transitions were monitored for effluent metabolites. Five crucial MS parameters, including DP (declustering potential), CE (collision energy), RT (retention time), Q1 (precursor ion), and Q3 (product ion) were used to identify the metabolite from the metware database (Wuhan Metware Biotechnology Co. Ltd). The peak area of the metabolite was used for quantification.
An UPLC-MS/MS system (UPLC, Nexera X2, Shimadzu; MS, Applied Biosystems 6500 Q TRAP, Japan) was used to identify and quantify all the metabolites. Briefly, a 2 mL filtered sample was loaded onto the UPLC. The mobile phase was solvent A (ultrapure water with 0.1 % formic acid) and solvent B (acetonitrile with 0.1 % formic acid). The mobile phase was programmed as follows: 0–9 min, 5 %–95 % B; 9–10 min, 95 % B; 10–11.1 min, 95 % B-5 % B; 11.1–14 min, 5 % B. Multiple reaction monitoring (MRM) transitions were monitored for effluent metabolites. Five crucial MS parameters, including DP (declustering potential), CE (collision energy), RT (retention time), Q1 (precursor ion), and Q3 (product ion) were used to identify the metabolite from the metware database (Wuhan Metware Biotechnology Co. Ltd). The peak area of the metabolite was used for quantification.
3
CPC Sustained Release Evaluation
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To evaluate the CPC reuptake capacity, the three specimens were immersed in a 10.0 wt% CPC solution for 24 h at 37.0 °C for reuptake after 14 d of sustained release. After rinsing and drying, the specimens were immersed in 3.0 mL of distilled water for 24 h at 37 °C. Thereafter, the specimens were removed and immersed in 3.0 mL of fresh distilled water. This cycle was repeated continuously for 7 d. The absorption spectra of the supernatant solution obtained after 7 d were measured using liquid chromatography (Nexera X2, Shimadzu Corporation, Kyoto, Japan) to evaluate the sustained release of CPC.
4
Multi-residue LC-MS/MS for Pesticide Detection
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All samples were analyzed using Shimadzu LC–MS-8045 with UHPLC Nexera X2 (Kyoto, Japan). Chromatographic separation was performed using a Kinetex C18 column (2.1 × 150 mm; 2.6 μm particle size; Phenomenex, USA) maintained at 40 °C. Information regarding the mobile phase, gradient program, and injection volume for all analytes are presented in Supplementary Tables S3 a and S3 b. A triple quadrupole (QqQ) mass spectrometer (Shimadzu) with a positive electrospray ionization (ESI) source was used for all the analytes, except fludioxonil (negative ESI; Supplementary Table S4 ). Supplementary Table S4 shows the multi reaction monitoring conditions for all analytes.
5
Quantitative Analysis of Bacterial Chlorophyll a
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Cells grown in Basal_YE (pH6.0) with air/CO2 (90:10, v/v) at 30 °C for 14 days (exponential growth phase) under continuous light (250 μmol m–2 s–1) and continuous dark were used for the pigment assays. The absorption spectrum was determined in a cell suspension in 60% (w/v) sucrose and in a 100% methanol extract using a spectrophotometer (V-630; JASCO, Tokyo, Japan) at 350–1100 nm. The BChl a concentration was determined spectroscopically in 100% methanol [47 (link)]. Dry cell weight was measured after harvested cells were washed twice with Milli-Q water and dried at 80 °C for 3 days. The extract was also analyzed by HPLC (NEXERA X2; Shimadzu; Kyoto, Japan) equipped with a 4.6 × 250 mm COSMOSIL 5C18-AR (Nakarai Taque; Tokyo, Japan) with isocratic elution of 92.5% (v/v) methanol in water at a flow rate of 1.0 mL/min. BChl a was monitored at 766 nm using a diode-array spectrophotometer detector (SPD-M20A; Shimadzu; Kyoto, Japan).
6
UPLC-ESI-MS/MS Metabolite Quantification Protocol
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The sample extracts were analyzed using a UPLC-ESI-MS/MS system (UPLC, SHIMADZU Nexera X2, www.shimadzu.com.cn/ , accessed on 8 June 2021; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/ accessed on 8 June 2021). The analytical conditions were as follows: UPLC: column, Agilent SB-C18 (1.8 µm, 2.1 × 100 mm). The mobile phase consisted of solvent A, pure water with 0.1% formic acid, and solvent B, acetonitrile with 0.1% formic acid. Sample measurements were performed with a gradient program that employed the starting conditions of 95% A, 5% B. Within 9 min, a linear gradient to 5% A, 95% B was programmed, and a composition of 5% A, 95% B was kept for 1 min. Subsequently, a composition of 95% A, 5.0% B was adjusted within 1.10 min and kept for 2.9 min. The flow velocity was set as 0.35 mL per min. The column oven was set to 40 °C, and the injection volume was 4 μL. The effluent was connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS [21 (link)]. The analytical conditions were adapted from Chen et al. [17 (link)]. Metabolite quantification was conducted using multiple-reaction monitoring (MRM) [22 (link)] and the self-built MetWare database (MWDB) based on their standard metabolic operating procedures [15 (link),17 (link)].
7
Quantitative Bioanalysis of Compounds
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Chromatographic separation was performed using Shimadzu Nexera X2 (Shimadzu, Japan) equipped with an autosampler, a column heater, and a binary pump. Utilizing the Cadenza CD-C18 column (150 × 3.0 mm, 3 µm) (Imtakt, Japan) at 40℃, the analytes were separated using the isocratic mobile phase of 10 mM ammonium acetate:100% methanol (5:95, v/v) at a flow rate of 0.4 mL/min. The column was eluted into the Applied Biosystems MDS SCIEX API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Canada) with an ESI source, operating in the multiple reaction monitoring (MRM) mode under unit mass resolution conditions in mass analyzers. The optimized MS condition was as follows: curtain gas flow 20 L/h, collision gas flow 6 L/h, collision energy 45 V, and declustering potential 51 V. Data analysis was performed using Analyst 1.6 software (Applied Biosystems, Canada).
8
Phthalate Metabolite Analysis in Urine
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We collected urine samples between 10:00 AM and 12:00 PM and stored them at −20 °C until analysis. To reduce the possibility of contamination, monoester phthalate metabolites, such as MEOHP, MEHHP, and MnBP, were measured instead of their parent compounds. Urinary phthalate metabolites were analyzed using ultra-high performance liquid chromatography tandem mass spectrometry (Nexera X2; Shimadzu, Kyoto, Japan) according to a previously reported procedure [6 (link)]. The limits of detection (LODs) for MEOHP, MEHHP, and MnBP were 0.32 μg/L, 0.20 μg/L, and 0.35 μg/L, respectively. We substituted urinary phthalate metabolite levels below the LOD with the LOD divided by the square root of 2 [23 (link)]. Because MEOHP and MEHHP are metabolized from the same parent compound, DEHP, a summed measure (∑DEHP) was calculated by adding the molar sums of MEOHP and MEHHP. We adjusted the urinary phthalate metabolite concentrations by dividing them by the creatinine concentration from the same urine sample to consider the different urinary excretion rates of study participants. The urinary creatinine level was determined using the kinetic Jaffe method (Cobas 8000 C702; Roche Diagnostics, Mannheim, Germany).
9
UHPLC-MS/MS Analysis of Target Compounds
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UHPLC-MS/MS analysis was carried out using a UHPLC system (Nexera X2, Shimadzu, Japan) coupled with a Shimadzu LC-MS 8060 triple quadrupole mass spectrometer (Kyoto, Japan). UHPLC separation was performed on a Waters ACQUITY BEH C18 column (2.1 mm × 100 mm; 1.7 mm; MA, USA). The column temperature was set at 40°C with a flow rate of 0.3 mL/min, and the injection volume was 5 μL. The mobile phases consist of methanol and water. The initial gradient conditions were 30% methanol, followed by a linear increase to 100% methanol in 6 min, and then changed to isocratic conditions with 100% methanol for 2 min. Subsequently, the mobile phase was decreased to 30% methanol in 1 min, held for 3 min before the next injection. The MS/MS detection was operated in the negative ionization mode with multiple reaction monitoring. N2 was used as the nebulizing gas at flow rate of 3 L/min; heating gas and drying gas flow rate were 10 L/min and 10 L/min, respectively. Interface temperature, desolvation line temperature, and heat block temperature were 300°C, 250°C, and 400°C, respectively. The optimized MS/MS parameters for the target compounds are given in Table S1 .
10
Metabolomics Profiling of Plant Samples
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Widely targeted metabolome was made by the Wuhan MetWare Biotechnology Co., Ltd. (Wuhan, China) with an ultra-performance liquid chromatography electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS) system (UPLC, SHIMADZU Nexera X2, www.shimadzu.com.cn/ , accessed on 15 January 2022; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/ , accessed on 15 January 2022). Each treatment contained three biological replicates. A metabolite with both a variable importance in projection (VIP) > 1 in the OPLS-DA and a |log2(fold change)| > 1 was considered differentially abundant. DAMs were annotated using the MetWare metabolite database and KEGG compound databases [6 (link),31 (link)].
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