Bulb tissues [3 replicates × 10 genotypes (FF, AA, and eight MALs)] were freeze-dried using a rotary evaporator (BUCHI, Rotavapor R-3) coupled with a vacuum pump (BUCHI, v-700) under reduced pressure at 10 °C ± 1. The sample preparation process was performed automatically by a liquid handling system (Microlab Star Plus, Hamilton) as described by Sawada et al. (2017)51 (link). Briefly, 4 mg dry weight of bulb tissues was accurately weighted and transferred into a 2 ml tube with a 5 mm Zirconia bead. The metabolites were extracted using a proportional volume of 4 mg ml−1 extraction solvent (80% methanol, 0.1% formic acid, 16.8 nmol L−1 lidocaine, and 105 nmol L−1 10-camphorsulfonic acid as internal standards) using a multi-bead shocker (Shake Master NEO, Bio Medical Science) at 1000 rpm for 5 min. After centrifugation, the extracts were diluted to 40 µg ml−1 using an extraction solvent. Then 250 µL of the extract was transferred to a 96-well plate, dried, redissolved in 250 µL of ultra-pure water, and filtered using Whatman® UNIFILTER® plates 384 (GE Healthcare). One microliter of the solution extract at a final concentration of 40 µg ml−1 was subjected to widely targeted metabolomics using LC-QqQ-MS (UPLC-TQS, Waters). A KEGG ID and name, as well as our internal standard ID, were assigned to each metabolite. For detailed information, see Supplementary Tables S1 –S4 .
Uplc tq
Manufactured by Waters Corporation
Sourced in United States
The UPLC-TQ is a high-performance liquid chromatography system coupled with a triple quadrupole mass spectrometer. It is designed for the separation, identification, and quantification of a wide range of analytes in complex samples.
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5 protocols using uplc tq
1
Metabolite Profiling of Onion Bulb Tissues
2
UPLC-TQS Analysis of Liquid Samples
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A liquid sample (1 μL) was analyzed using the UPLC-TQS (Waters Corp., Watertwon, MA, USA). Analytical conditions were as follows. LC: column, ACQUITY UPLC HSS T3 (1.8 μm, 1.0 mm ×100 mm, Waters Corp.); solvent system, solvent A (water with 0.1%formic acid) and solvent B (acetonitrile with 0.1% formic acid); gradient program, 99.9% A/0.1% B at 0 min, 99.9% A/0.1% B at 0.25 min, 91.0% A/0.9% B at 0.40 min, 83.0% A/17.0% B at 0.80 min, and 0.1% A/99.9% B at 1.90 min, 0.1% A/99.9% B at 2.10 min; 99.9% A/0.1% B at 2.11 min flow rate, 0.240 mL/min.
3
Plasma Bioanalysis of Octreotide and Internal Standard
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Some of the coming conditions were adopted from the authors’ previous LC-MS/MS work (published repositioning study on OTN and IS using direct precipitation for rats’ plasma [11 (link)]). LC-MS/MS was performed via Waters® UPLC-TQ with Electro Spray Ionization (ESI) (USA), Mass Lynx software (4.1 version) and Agilent C18 column (1.8 µm, 50 × 2.1 mm). An isocratic mobile phase of acetonitrile/0.3% formic acid (90:10, v/v), filtered via a 0.2 μm filter membrane degassed for 25 min, ten microliters as the injection volume, 0.3 mL/min as the selected flow rate to decrease the retention time and provide a faster method and 1.2 min as the run time for OTN bioassay, were applied. The column temperature was set at 25 °C. The mass spectrometer parameters included the adjustment of cone voltage values to 40 V and 30 V and collision energy values to 50 eV and 55 eV for OTN and IS, respectively. Multiple Reaction Monitoring (MRM) of m/z 399.2 to 153.0 for OTN and m/z 340.2 to 116.0 for IS in the ESI positive mode was implemented. Further MS parameters were adjusted including turbo ions spray at 400 °C, capillary temperature at 275 °C, sheath and auxiliary gas at 15 and 2 psi, respectively, ion spray voltage of 3800 V, capillary voltage of 4 KV, capillary offset of 35 and desolvating line temperature at 400 °C.
4
UPLC-MS/MS Quantification of Alogliptin
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TLN (99.0%), Alogliptin as IS (99.2%), ZAFATEK® (50 mg) tablets, TBME, and DEE were thankfully contributed by the Center for Drug Research and Development, at the British University in Egypt, BUE (Cairo, Egypt). HPLC-grade methanol and acetonitrile were purchased from (Sigma, USA). The Phenomenex C18 column (1.6 μm, 150 × 2.1 mm), Waters® UPLC-TQ, and Mass Lynx software, with a flow rate of 0.3 mL/min, mixture of acetonitrile/0.3 formic acid (90 : 10, v/v) as an isocratic mobile phase, and injection V of 10 μL, were used. Cone voltage values of 25 V and 30 V and collision energy values of 60 eV and 55 eV were applied for TLN and IS (Alogliptin), respectively. MRM of m/z 358.2 to 133.9 for TLN and m/z 340.2 to 116.0 for IS in the ESI positive mode were applied.
5
Ultra-Sensitive LC-MS/MS Method for OMR Quantification
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The following conditions were implemented based on the previously published work by Ayoub et al. after minor modifications to increase the sensitivity (published preliminary repositioning study on OMR using direct precipitation as an extraction technique for rats’ plasma [17 (link)] by Ayoub et al.). LC-MS/MS was performed via Waters® UPLC-TQ with ESI, Mass Lynx software (4.2 version) and ACQUITY UPLC® BEH shield RP C18 column (1.7 µm, 150 × 2.1 mm), (WATERS, Ireland, serial 01853004518304). Mobile phase of acetonitrile/0.3 formic acid (90:10, v/v) in the isocratic mode, 10 µL as the injection volume, 0.3 mL/min as the flow rate and 1.5 min as the run time for OMR bio-assay, were adopted. The mass spectrometer parameters included the adjustment of cone voltage values to 40 V and 30 V and collision energy values to 50 eV and 55 eV for OMR and alogliptin (IS), respectively. MRM of m/z equals 399.2 to 153.0 for OMR and m/z equals 340.2 to 116.0 for alogliptin IS in the ESI positive mode was applied (Figure 1 ). MS tuning was implemented and the stated parameters were adopted: turbo ion spray at 400 °C, capillary temperature at 275 °C, sheath gas at 15 psi, auxiliary gas at 2 psi, ion spray voltage at 5500 V, capillary voltage at 4 kV, capillary offset at 35, desolvating line temperature at 400 °C, source temperature at 130 °C, and desolvation gas flow at 600 L/h.
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