Measurement of MPP+ level was conducted as previously described with modifications (Hu et al., 2019 (link)). Briefly, mice were sacrificed 90 min after a single 30 mg/kg i.p. administration of MPTP, and the bilateral striatum were dissected and weight. Tissues were homogenized and sonicated in lysis buffer which contained 5% trichloroacetic acid (Sigma, China) with 4 μg/ml 4-phenylphridine (Sigma, China) as an internal standard, followed by centrifugation for 15 min at 15,000 g. Chromatographic separation of supernatant was performed on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm, Waters). The mobile phase consisted of 50 mM potassium phosphate and 0.1% with ultrapure phosphoric acid (pH = 2.5, solvent A, 92%) and acetonitrile (solvent B, 8%). MPP+ and 4-phenylpyridine signals were detected under PDA ultraviolet detection by Acquity Ultra Performance Liquid Chromatography H-Class system (Waters, Milford, MA, USA).
Acquity ultra performance liquid chromatography h class system
Manufactured by Waters Corporation
Sourced in United States
The Acquity Ultra-performance Liquid Chromatography (UPLC) H-Class system is a laboratory instrument designed for high-performance liquid chromatography. It utilizes advanced technology to enable efficient separation, identification, and quantification of chemical compounds in complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Aeris peptide xb c18 column, by Phenomenex (1 mentions)
Carboxypeptidase a, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Acquity beh c18, by Waters Corporation (1 mentions)
Accq tag ultra derivatization kit, by Waters Corporation (1 mentions)
Accq tag ultra c18 1.7 μm 2 1 100 mm column, by Waters Corporation (1 mentions)
Uplc optimized fluorescence detector, by Waters Corporation (1 mentions)
Transstart fastpfu dna polymerase, by Transgene (1 mentions)
Rtaq dna polymerase, by Vazyme (1 mentions)
Acquity uplc ben c18 column, by Waters Corporation (1 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Beh glycan column, by Waters Corporation (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
10 protocols using acquity ultra performance liquid chromatography h class system
1
MPTP-Induced Striatal MPP+ Quantification
2
Stability Assays for Vaccatide vH2
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The thermal, acidic, endopeptidase, and exopeptidase stability assays were performed as previously described (Wong et al., 2016 (link)). Trypsin and carboxypeptidase A were acquired from Sigma–Aldrich (MO, United States). Briefly, the purified vaccatide vH2 was incubated with or without enzyme at the optimal temperature and with a buffer solution as recommended by the manufacturer. At each specific time point, the sample was aliquoted and analyzed by an Aeris peptide XB-C18 column (particle size 3.6 μm, 100 mm × 2.7 mm; Phenomenex, CA, United States) attached to an Acquity ultra-performance liquid chromatography H-class system (Waters, Milford, Japan). The areas under the peaks before and after treatment were determined to evaluate the stability.
3
ESI-MS Analysis of Aqueous Samples
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Samples for
ESI-MS analyses were prepared by using Milli-Q water to make the concentration
of 0.1 mg/mL. Mass spectra were collected using a Waters Acquity ultra-performance
liquid chromatography H-Class system in negative mode.
ESI-MS analyses were prepared by using Milli-Q water to make the concentration
of 0.1 mg/mL. Mass spectra were collected using a Waters Acquity ultra-performance
liquid chromatography H-Class system in negative mode.
4
UPLC Analysis of Chemical Compounds
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UPLC analyses were performed on a Waters Acquity Ultra-performance Liquid Chromatography (UPLC) H-Class system (Waters, Milford, MA). An Acquity BEH C18 (2.1 mm × 100 mm, 1.7 μm; Waters, Milford, MA) analytical column coupled with a column filter were used to test samples, and column temperature was set at 30 °C. Mobile phase A consisted of 0.05% (v/v) phosphoric acid in distilled water, while mobile phase B was acetonitrile (75 : 25) at a flow rate of 0.2 mL min−1 for 4 min. Samples were detected at 225 nm. The autosampler temperature was kept at 4 °C and 1 μL of the samples was injected into the UPLC system.
5
Quantification of Cabotegravir in Macrophages
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Human peripheral blood monocytes were obtained and cultured as described [21 (link)]. Briefly, peripheral blood monocytes were obtained by leukapheresis from HIV-1/2 and hepatitis B seronegative donors and purified by countercurrent centrifugal elutriation. Monocytes were cultured in Dulbecco's modified eagle's medium supplemented with 10% heat-inactivated pooled human serum, 10 μg/ml ciprofloxacin, 50 μg/ml gentamicin and 1000 U/ml recombinant macrophage colony-stimulating factor for 7 days to promote differentiation into macrophages. Macrophages were treated with 100 μM CAB nanoformulations. At predetermined time points, monocyte-derived macrophages (MDM) were washed three-times with sterile phosphate-buffered saline (PBS) and scraped into 1 ml PBS. Cell pellets were collected by centrifugation at 1000 × g for 8 min, followed by probe sonication in 200 μl of HPLC grade methanol to extract CAB. CAB concentrations were determined using a Waters ACQUITY ultra performance liquid chromatography (UPLC) H-Class System with TUV detector and Empower 3 software (Milford, MA, USA) [22 (link)].
6
Amino Acid Profiling in Plasma and Liver
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Amino acids in plasma and liver samples were derivatized by using an AccQ•Tag Ultra Derivatization Kit (Waters, Milford, MA, USA), and concentrations were determined by an ACQUITY Ultra Performance Liquid Chromatography (UPLC) H-Class system (Waters) equipped with an AccQ•Tag Ultra C18 1.7-μm 2.1 × 100-mm column (Waters) and tunable UV detector (Waters). Amino acid data were processed by using Empower 3 Personal Single System software (Waters). Pathway maps were created by using Visualization and Analysis of Networks software containing Experimental Data V2.6.5 and referenced KEGG pathway databases. The colors in the pathway maps indicated the log2-transformed ratios of each amino acid between groups.
7
UPLC-Based Aflatoxin Identification
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AF identification was carried out according to the technique proposed by Jardon-Xicontencatl et al. [12 (link)] using a Waters ACQUITY Ultra Performance Liquid Chromatography (UPLC) H-Class System equipped with a quaternary solvent manager and an ACQUITY UPLC BEH C18 phase reverse column (2.1 × 100 mm, 1.7 μm). Standards, as well as samples collected from the IAC (1 μL) were injected and eluted with a single ternary mixture of 64:18:18 water/methanol/acetonitrile (all HPLC grade) at a flow rate of 400 μL/min. AF were fluorometrically detected and identified using an UPLC-optimized fluorescence detector (Waters, Milford, MA, USA). The excitation and emission wavelengths were 365 and 429 nm, respectively. AF were identified by their retention time (Rt) and compared with those for a pure AF standard solution under identical conditions. The estimated detection limits are 0.58 and 2.01 ng/L for AFB2 and AFB1, respectively.
8
Optimized Cloning and Analysis Protocol
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Primer and gene synthesis was conducted at
Generay Biotech Co. Ltd. DNA sequencing was performed at RuiBiotech
Company. ClonExpress II One Step Cloning Kit was purchased from Vazyme
Biotech. PCR to obtain DNA fragments was carried out using standard
thermos cycling protocols with TransStart FastPfu DNA polymerase (Transgene
Biotech) and MEGAWHOP PCR was conducted using KOD-Plus-Neo polymerase
purchased from TOYOBO. Error-prone PCR was performed using rTaq DNA
polymerase (Vazyme Biotech). All protein quantification was performed
using a NanoDrop 2000 Spectrophotometer (Thermo). The analysis of
enzymatic reaction products was performed on an ACQUITY ultraperformance
liquid chromatography (UPLC) H-class system (Waters) coupled with
an SQ Detector 2 equipped with an electrospray ionization source.
All the UPLC/mass spectrometry (MS) analysis was conducted on an ACQUITY
UPLC BEN C18 column (length, 50 mm; inner diameter, 2.1 mm; particle
size, 1.7 μm; Waters) at a flow rate of 0.3 mL per minute at
40 °C. The gradient elution consists of 0.1% formic acid (A)
and MeCN (B). The gradient programs were 100% A, 0–0.5 min,
100–98% A, 0.5–1 min, 98–95% A, 1–2.5
min, 95–60% A, 2.5- 4.5 min, 60–10% A, 4.5–6.5
min, 10% A, 6.5–8.0 min.
Generay Biotech Co. Ltd. DNA sequencing was performed at RuiBiotech
Company. ClonExpress II One Step Cloning Kit was purchased from Vazyme
Biotech. PCR to obtain DNA fragments was carried out using standard
thermos cycling protocols with TransStart FastPfu DNA polymerase (Transgene
Biotech) and MEGAWHOP PCR was conducted using KOD-Plus-Neo polymerase
purchased from TOYOBO. Error-prone PCR was performed using rTaq DNA
polymerase (Vazyme Biotech). All protein quantification was performed
using a NanoDrop 2000 Spectrophotometer (Thermo). The analysis of
enzymatic reaction products was performed on an ACQUITY ultraperformance
liquid chromatography (UPLC) H-class system (Waters) coupled with
an SQ Detector 2 equipped with an electrospray ionization source.
All the UPLC/mass spectrometry (MS) analysis was conducted on an ACQUITY
UPLC BEN C18 column (length, 50 mm; inner diameter, 2.1 mm; particle
size, 1.7 μm; Waters) at a flow rate of 0.3 mL per minute at
40 °C. The gradient elution consists of 0.1% formic acid (A)
and MeCN (B). The gradient programs were 100% A, 0–0.5 min,
100–98% A, 0.5–1 min, 98–95% A, 1–2.5
min, 95–60% A, 2.5- 4.5 min, 60–10% A, 4.5–6.5
min, 10% A, 6.5–8.0 min.
9
Glycan Analysis of Monoclonal Antibodies
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The 2‐AA‐derivatized mAb1 samples and controls described earlier were profiled with a Waters Acquity Ultra‐Performance Liquid Chromatography (UPLC) H class System (Milford, MA, USA) equipped with a Waters BEH Glycan column (1.7 µm, 2.1 × 150 mm) (Milford, MA, USA). Solvent A consisted of 100 mm ammonium formate prepared with formic acid (1116701000; Fisher Scientific) and brought to pH 3.0 with ammonium hydroxide (338818; Sigma). Solvent B consisted of 100% acetonitrile (MX0486‐6; Fisher Scientific). The column temperature was maintained at 35°C, and the fluorescence detection was set at λex = 360 nm and λem = 425 nm. The flow rate was 0.25 mL·min−1 with an initial 22% solvent A ramped up to 40% solvent A in 111 min, followed by a 6.5‐min wash at 90% solvent A, then a 5‐min stepdown to 22% solvent A and a 25‐min final reequilibration. The flow rate was decreased by 20% for the wash step to avoid approaching the pressure limit of the system.
10
UPLC-PDA Analysis of Compounds
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The concentrations of compounds 1 , 3 , and 4 were determined using a Waters Acquity ultra performance liquid chromatography UPLC H-Class system with photodiode array detector. All data were processed using Empower software. An Acquity UPLC BEH C18 column (130 Å, 1.7 μm, 2.1 mm × 50 mm) was used. A mixture of water: MeCN: 5% TFA in H2O with a gradient of 93:2:5 changing to 0:95:5 over 5 min was used as the mobile phase. All peaks were analyzed at a wavelength of 330 nm (Supplementary Fig. 19 ).
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