Mass spectrometric analyses were performed using an AB SCIEX QTRAP 5500 instrument (AB SCIEX, Foster City, CA, USA) coupled with an ultrahigh‐performance liquid chromatography (UPLC) instrument (Waters, Milford, MA, USA). For U, T, DHU, and DHT quantification, one microliter of reconstituted sample was injected directly onto Synergi 4 µm Polar‐RP 80 A LC column (150 × 4.6 mm; Phenomenex, Torrance, CA, USA). The isocratic mobile phase consisted of high‐performance liquid chromatography (HPLC) grade water (solvent A) and acetonitrile (solvent B) at a flow rate of 0.6 ml/min. For uridine quantification, one microliter of supernatant was injected directly onto 2.1 mm × 100 mm ACQUITY 1.7 μm UPLC BEH C18 Column (Waters, USA). The isocratic mobile phase consisted of HPLC grade water with 0.1% formic acid (solvent A) and methanol with 0.1% formic acid (solvent B) at a flow rate of 0.3 ml/min. Mass spectrometric detection was conducted in negative electrospray ionization mode. Calibration curves were generated using linear regression after plotting the peak areas against the analyte concentrations within a dynamic range.
Uplc instrument
Manufactured by Waters Corporation
Sourced in United States
The UPLC (Ultra-Performance Liquid Chromatography) instrument is a high-performance liquid chromatography system designed for rapid, efficient separation and analysis of chemical compounds. It utilizes small particle size columns and high pressure to achieve superior resolution, sensitivity, and speed compared to conventional HPLC systems.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc beh c18 1.7 μm column, by Waters Corporation (1 mentions)
5500 qtrap ms system, by AB Sciex (1 mentions)
Analyst 1, by AB Sciex (1 mentions)
Phenytoin, by Fujifilm (1 mentions)
Oasis hlb elution plate, by Waters Corporation (1 mentions)
Precellys evolution, by Bertin Technologies (1 mentions)
Api 4000 triple quadrupole mass spectrometer, by AB Sciex (1 mentions)
9 protocols using uplc instrument
1
Quantification of Urinary Metabolites by UPLC-MS
2
Targeted Metabolomics of Amino Acids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At least 600 seedlings were pooled per sample and a total of four samples per genotype was analyzed. Approximately 50 mg to 100 mg fresh weight of each sample was weighed into an Eppendorf tube and frozen in liquid nitrogen. 10 μL of 2.5 mM 13C- and 15N-labeled amino acid internal standard was then added to each tube, followed by 600 μL extraction solution (3:5:12 water:chloroform:methanol) and two steel balls. These samples were then put onto a tissulizer to lyse cells and extract free amino acids. Samples were centrifuged to pellet tissue and the supernatant was transferred to a fresh Eppendorf tube. This extraction procedure was then repeated and combined with the first extraction to ensure thorough extraction of all amino acids. After the second extraction, 300 μL chloroform and 450 μL water were added to each tube, which were then mixed vigorously. Debris was separated by centrifugation, the supernatant was collected and dried with a Speedvac, and then the pellet was resuspended in 1.0 mL 80% methanol. The methanol-dissolved samples were transferred to a vial for analysis by LC/MS/MS with a Velos ion trap, and separation was accomplished using a HILIC column combined with a Waters UPLC instrument.
3
Isoflavone Profiling by UPLC-QTOF-MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isoflavone analysis was performed using a modification of a previously reported method [21 ]. Typically, a 100-mg aliquot of the lyophilized sample was extracted with 80 vol% aqueous ethanol (3 × 4 mL) in an ultrasonicator (3 × 15 min). The combined supernatants were filtered through 0.22-μm filters, diluted with methanol, and analyzed on a UPLC instrument (Waters, Milford, MA, USA) interfaced with a QTOF-MS spectrometer. An ACQUITY BEH C18 column (2.1 mm inner diameter × 100 mm length, 1.7 μm particle size) maintained at 35 °C and an injection volume of 2 μL were employed. The mobile phase corresponded to mixtures of 0.1 vol% aqueous formic acid (A) and 0.1 vol% formic acid in acetonitrile (B), and was supplied at 0.4 mL/min. The following gradient was used: 0 min, 90% A; 1 min, 90% A; 7 min, 85% A; 13 min, 70% A; 23 min, 20% A; 25 min, 0% A; 28 min, 0% A; and 30 min, 90% A. QTOF-MS analysis was performed in positive-ion mode over a range of m/z 100–1500 under the following conditions: capillary voltage = 2300 V; cone voltage = 50 V; ion source temperature = 110 °C; desolvation temperature = 450 °C; desolvation nitrogen gas flow rate = 800 L/h; and mass scan time = 0.25 s. Leucine enkephalin (m/z 556.2771) was used as a reference.
4
Phytohormone Quantification by UPLC-MS/MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All detection was performed on a UPLC instrument (Waters, Milford, MA, USA) combined with a 5500 Qtrap MS system equipped with an electrospray ionization (ESI) source (AB SCIEX, Foster City, CA). Instrument control and data acquisition and processing were performed using Analyst 1.6.2 software (AB SCIEX, Foster City, CA). The phytohormones were separated with a BEH C18 column (130 Å, 1.7 μm, 2.1 × 100 mm). The UPLC methods for different phytohormone groups are shown in Supplemental Tables 1–4 .
The optimized ESI operating parameters for negative mode were: IS, −4500 V; CUR, 30 psi; TEM, 600°C; GS1, 45 psi; GS2, 55 psi. For positive mode they were: IS, 5500 V; CUR, 30 psi; TEM, 600°C; GS1, 45 psi; GS2, 55 psi. All analytes were detected using scheduled multiple reaction monitoring (sMRM) mode, and the specific MRM parameters for each analyte and the corresponding isotope-labeled IS are given inSupplemental Tables 5–7 .
The optimized ESI operating parameters for negative mode were: IS, −4500 V; CUR, 30 psi; TEM, 600°C; GS1, 45 psi; GS2, 55 psi. For positive mode they were: IS, 5500 V; CUR, 30 psi; TEM, 600°C; GS1, 45 psi; GS2, 55 psi. All analytes were detected using scheduled multiple reaction monitoring (sMRM) mode, and the specific MRM parameters for each analyte and the corresponding isotope-labeled IS are given in
5
UPLC-MS Metabolite Profiling Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The analysis for metabolites was also performed by the Waters UPLC instrument through an ESI interface. Separations were carried out using an Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm), the mobile phase was 0.1% formic acid (A): Acetonitrile (B), the gradient elution condition was 0–3 min (99.8% → 98% A), 3–5 min (98% → 95% A), 5–8 min (95% → 90% A), 8–12 min (90% → 85% A), 12–17 min (85% → 70% A), 17–22 min (70% → 60% A), 22–23 min (60% → 58% A), 23–25 min (58% A), 25–32 min (58% → 45% A), and 32–37 min (45% → 35% A), 0.4 mL min−1 was the flow rate. The temperature for the column and sample room was set at 40 ℃ and 8 ℃ respectively. The mass spectrometry conditions mentioned above were used.
6
Medicinal Material Profiling by UPLC-QTOF-MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nine batches of representative medicinal materials were collected or purchased from Jilin, the main area producing PG and PQ, and from different areas producing PJ. The detailed sample information is presented in Table 1 . High-performance liquid chromatography-grade acetonitrile was provided by Oceanpak (Sweden), high-performance liquid chromatography-grade formic acid was provided by Thermo Fisher (USA), and distilled water was purchased from Watsons Food and Beverage Company (China). A Waters Acquity (Waters, USA) UPLC instrument and a Xevo G2 (Waters, USA) Q-TOF/MS system were used in this study.
7
Quantification of Ocular Drug Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ocular tissues were pretreated for quantification of each drug using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system. Each tissue was homogenized with zirconia balls in water using a beads homogenizer (Precellys® Evolution; Bertin Technologies, France). The homogenates were recovered and further pretreated by solid-phase extraction using an Oasis® HLB µElution plate (Waters Corporation, Milford, MA, USA). The UPLC-MS/MS system consisted of a UPLC instrument (Waters Corporation, Milford, MA, USA) and an API 4000 triple quadrupole mass spectrometer (AB SCIEX, Framingham, MA, USA). Phenytoin (FUJIFILM, Wako Pure Chemical Corporation, Osaka, Japan) was used as an internal standard for all analytes, and the analytes and the internal standard were detected in positive ion mode. Lower limits of quantification for azithromycin were 0.0220, 0.0825, and 0.0275 μg/g for the eyelid, conjunctiva, and cornea, respectively, and those for levofloxacin and ofloxacin were 0.0132, 0.0165, and 0.0132 μg/g for the eyelid, conjunctiva, and cornea, respectively. Assay performance was monitored by the quality control samples.
8
Metabolite Analysis by UPLC-ESI-MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The analysis for metabolites was also performed by Waters UPLC instrument through an ESI interface. Separations were carried out using an Acquity UPLC HSS T3 column (100 mm×2.1 mm，1.8 µm), the mobile phase was 0.1 % formic acid (A): Acetonitrile (B), the gradient elution condition was 0~3 min (99.8%→98%A), 3~5 min (98%→95%A), 5~8 min (95%→90%A), 8~12 min (90% →85%A), 12~17 min (85%→70%A), 17~22 min (70%→60%A), 22~23 min (60%→58%A), 23~25 min (58%A), 25~32 min (58%→45%A), and 32~37 min (45%→35%A), 0.4 mL•min -1 was the flow rate. Temperature for column and sample room was set at 40 ℃ and 8 ℃. The above conditions of mass spectrometry were used.
9
Quantitative Amino Acid Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At least 600 seedlings were pooled per sample and a total of four samples per genotype was analyzed. Approximately 50 mg to 100 mg fresh weight of each sample was weighed into an Eppendorf tube and frozen in liquid nitrogen. 10 μL of 2.5 mM 13 C-and 15 N-labeled amino acid internal standard was then added to each tube, followed by 600 μL extraction solution (3:5:12 water:chloroform:methanol) and 2 steel balls. These samples were then put onto a tissulizer to lyse cells and extract free amino acids. Samples were centrifuged to pellet tissue and the supernatant was transferred to a fresh Eppendorf tube. This extraction procedure was then repeated and combined with the first extraction to ensure thorough extraction of all amino acids. After the second extraction, 300 μL chloroform and 450 μL water were added to each tube, which were then mixed vigorously. Debris was separated by centrifugation, the supernatant was collected and dried with a Speedvac, and then the pellet was resuspended in 1.0 mL 80% methanol. The methanoldissolved samples were transferred to a vial for analysis by LC/MS/MS with a Velos ion trap, and separation was accomplished using a HILIC column combined with a Waters UPLC instrument.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!