RPLC-MS experiments were performed on an Acquity UPLC I-Class PLUS system, coupled to an FL detector (λexcitation = 280 nm and λemission = 350 nm) and a high-resolution BioAccord ToF mass spectrometer from Waters. A prototype BioResolve RP mAb Polyphenyl column (2.7 μm, 10 × 2.1 mm, 450 Å) from Waters was used. The column temperature was set at 70 °C. Mobile phase A was 0.05% (v/v) DFA in water, and mobile phase B was 0.05% (v/v) DFA in ACN. The injection volume was 0.5 µL. Optimized linear gradients were run in 1 min (without re-equilibration) from 30% to 36%, 26% to 54%, 29% to 37% and 25% to 38% of mobile phase B, for bsAb1, bsAb2, bsAb3 and bsAb4, respectively. The LC flow rate was set at 1000 µL/min and split with a PEEK T-junction so that the flow rate entering the MS was equal to 275 µL/min. The MS was used in ESI-positive mode with an acquisition range of 400 to 7000 m/z. A 200 pg/µL sodium iodide solution diluted in a mixture of 50/50 water/IPA (v/v) with 0.1% formic acid was used as a mass spectrometer calibrant. The desolvation temperature was set at 550 °C, the source temperature at 120 °C, the cone voltage at 30 V, and the capillary voltage at 1.5 kV. Data acquisition and instrument control were performed with Unifi software (Waters, Milford, MA, USA) and mass spectra data treatment was performed with MassLynx software (Waters, Milford, MA, USA).
Uplc 1 class plus system
Manufactured by Waters Corporation
Sourced in United States
The UPLC I-Class Plus system is a high-performance liquid chromatography (HPLC) instrument designed to provide efficient and accurate separation and analysis of complex samples. It utilizes ultra-high pressure liquid chromatography (UPLC) technology to deliver rapid and sensitive results. The system is capable of operating at pressures up to 15,000 psi, enabling the use of smaller particle size columns for improved resolution and faster analysis times.
Automatically generated - may contain errors
Lab products found in correlation
Masslynx software, by Waters Corporation (1 mentions)
Unifi software, by Waters Corporation (1 mentions)
Bioresolve rp mab polyphenyl column, by Waters Corporation (1 mentions)
Targetlynx, by Waters Corporation (1 mentions)
Masslynx, by Waters Corporation (1 mentions)
Xevo tq s micro, by Waters Corporation (1 mentions)
Q exactive high resolution mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Compound discoverer 3, by Thermo Fisher Scientific (1 mentions)
Qtrap 5500 system, by AB Sciex (1 mentions)
5 protocols using uplc 1 class plus system
1
RPLC-MS Analysis of Bispecific Antibodies
2
Quantitative UPLC-MS/MS Analysis Protocol
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Analyses were performed on an Acquity UPLC I-Class Plus system coupled to a Xevo TQ-S micro mass spectrometer (Waters, Milford, MA, USA). Chromatographic separation was performed on an ACQUITY UPLC BEH C18 2.1 × 100 mm column (i.d. 1.7 μm). Mobile phases consisted of water (A) and acetonitrile:methanol (8:2, v/v) (B), both containing 0.1% formic acid, by applying the following gradient program: 0–10 min, 5–95% B; 10–11 min, 95% B; 11–11.5 min, 95–5% B; 11.5–13 min, 5% B. Other chromatographic conditions were as follows: column temperature, 40 °C; sample temperature, 10 °C; injection volume, 2 μL; and flow rate, 0.45 mL/min. Mass spectrometry detection was performed under positive and negative ionisation in one run, using the multiple reaction monitoring (MRM) mode. General MS parameters were as follows: capillary voltage, 2.5 kV; desolvation temperature, 350 °C; desolvation gas, 650 L/h. The optimised MRM transitions, cone voltages, collision energies, and retention times for the analytes were listed in Table S2 . MassLynx and TargetLynx software (Waters, Milford, MA, USA) were used for data acquisition and processing, respectively.
3
Cotinine Quantification in Urine
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Quantification of cotinine in human urine was conducted according to Landmesser et al. (2019) (link) with minor modifications. In brief, 20 μL of 50 ng/mL cotinine-d3 as internal standard (IS) and 4 μL of 6 M sodium hydroxide (NaOH) were added to 100 μL urine sample. 1 mL ethyl acetate was added for liquid-liquid extraction and the sample was extracted for 30 min on a multi-vortex mixer. The mixture was subsequently centrifuged for 15 min (3000 rpm, 4 °C). 850 μL of the organic supernatant were transferred into a vial and evaporated to dryness for 7 min 200 μL acetonitrile (ACN) were added to reconstitute the sample. Samples were analyzed by means of LC-MS/MS with an Acquity UPLC I-Class PLUS System coupled to a Xevo TQ-S Triple Quadrupol (Waters GmbH, Eschborn, Germany). Details on the analytical method applied can be found in the Appendix. The method was fully validated according to FDA guidelines (Bioanalytical Method Validation Guidance for Industry) (FDA, 2018 ). The linear calibration ranged from 0.2 to 1000 μg/L (LLOQ 0.2 μg/L), intra- and inter-day precisions were found to be <8% (CV < 17% for levels < 3x LLOQ) and the method accuracy rates were between 93 and 108% throughout the calibration range. For cotinine in urine, a mean matrix suppression of 33% was observed, which was fully compensated by the deuterated internal standard.
4
Metabolite Profiling Using UPLC-HRMS
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For this experiment, we used the Waters UPLC I-Class Plus system (Waters, Milford, MA, USA) coupled with the Q Exactive high-resolution mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) for the separation and detection of metabolites. After transferring the off-line mass spectrometry data to the Compound Discoverer 3.3 software (Thermo Fisher Scientific, Waltham, MA, USA) and conducting analysis on the mass spectrometry data in conjunction with BMDB (BGI metabolome database), the mzCloud database, and the online ChemSpider database, a data matrix was generated. This matrix contained details such as the metabolite peak area and the identification outcomes. The detailed experiment on metabolite profiling is presented in the Supplementary Material
5
Amniotic Eicosanoid Profiling in Human Parturition
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The amnion within the artificial rupture site from TNL (n = 8) and the spontaneous rupture site from TL (n = 10) was used to screen AA-derived eicosanoids pertinent to human parturition using AA-targeted metabolomics. The amnion tissue was homogenized in precipitating solvent (1:4 water: acetonitrile) followed by sonication on ice for 30 min. After centrifugation, the supernatant was collected for extraction of AA analogs with the Oasis HLB elution system (Waters, Milford, MA) which was preactivated and equilibrated with methanol and water, respectively. After elution with methanol, the eluted AA analogs were lyophilized and reconstituted in 1% formic acid and 80% acetonitrile solution for analysis with high-performance LC-MS on a UPLC I-Class PLUS System (Waters, Milford, MA) coupled with the 5500 QTRAP system (AB SCIEX, Framingham, MA). Quantitative control samples were used to monitor the stability and repeatability of the system. MultiQuant software was used to extract chromatographic peak area and retention time. The relative quantitative analysis of each AA metabolite was based on the peak area.
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