The assay was conducted at the Center of Drug Discovery Research and Development, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt. The analysis was carried out using a LC-MS/MS system (Nexera with LC-MS-8045, Shimadzu Corporation, Kyoto, Japan) composed of HPLC (Nexera LC-30AD) coupled to a triple quadrupole mass spectrometer (Nexera with LC-MS-8045, Shimadzu Corporation, Kyoto, Japan). The HPLC was equipped with an auto-sampler (SIL-30AC), a temperature-controlled column oven (CTO-20AC), and a photodiode array detector (LC-2030/2040) with detection wavelengths of 235, 254, and 280 nm with λ max absorption at 220–400 nm. The LC-MS/MS was equipped with an RP-C18 UPLC column (Shimpack; 2 × 150 mm) possessing a particle size of 2.7 µm. The gradient elution system was composed of Acetonitrile (ACN) and 0.1% (v/v) formic acid in H2O. The elution was performed according to the following sequence: 0–2 min by 10% ACN, 2–26 min by 10–80% ACN, and finally 26–33 min by 100% ACN, with a flow rate of 0.2 mL/min. A positive mode was operated during LC-MS/MS with electrospray ionization (ESI). LC-MS/MS data were collected and processed by LC mass software, version 3.05 (Shimadzu Corporation, Kyoto, Japan).
Nexera lc 30ad
Manufactured by Shimadzu
Sourced in Japan
The Nexera LC-30AD is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. It features a binary pump capable of delivering solvent flow rates up to 10 mL/min, with a maximum pressure of 66 MPa. The system is equipped with an auto-sampler, column oven, and a variety of detection options, including UV-Vis, diode array, and refractive index detectors.
Automatically generated - may contain errors
Lab products found in correlation
Oasis hlb column, by Waters Corporation (2 mentions)
Xbridge c18 column, by Waters Corporation (2 mentions)
Lcms 8040, by Shimadzu (2 mentions)
Labsolutions software, by Shimadzu (2 mentions)
Nexera with lc ms 8045, by Shimadzu (1 mentions)
12 13 dihome d4, by Cayman Chemical (1 mentions)
12 13 epome d4, by Cayman Chemical (1 mentions)
Sil 30ac, by Shimadzu (1 mentions)
Lcms 8060, by Shimadzu (1 mentions)
4 protocols using nexera lc 30ad
1
LC-MS/MS Quantification of Compounds
2
Lipid Metabolite Profiling by LC-MS/MS
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Fatty acid metabolites were purified from lipid fractions by solid‐phase extraction with Oasis HLB columns (Waters Corporation). Purification and liquid chromatography were performed as described previously.17 , 18 Briefly, FA metabolites were extracted from 4 × 107 cells with internal standards. FA metabolites were then separated using a high‐performance liquid chromatography system (Nexera LC‐30 AD, Shimadzu Corporation) equipped with an XBridge C18 column (particle size 3.5 μm, length 150 mm, inner diameter 1.0 mm; Waters) and analyzed on a triple quadrupole mass spectrometer (LCMS‐8040; Shimadzu). Mass spectrometry analyses were carried out in negative ion mode while FA metabolites were analyzed via multiple reaction monitoring, as previously reported.17 , 18 For quantification, calibration curves were prepared for each compound and deuterated internal standards were used to monitor recoveries. Data analysis was performed with LabSolutions software (Shimadzu Corporation).
3
Quantitative Analysis of Oxidized Fatty Acids
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Total lipid fractions were extracted from purified beef tallow samples by single-step deproteinization using methanol. The oxidized fatty acid fraction was purified from the lipid fractions by solid-phase extraction with Oasis HLB columns (Waters Corporation, MA, USA). Hydroxy fatty acids were separated using a high-performance liquid chromatography system (Nexera LC-30AD, Shimadzu Corporation, Kyoto, Japan) equipped with an XBridge C18 column (particle size 3.5 µm, length 150 mm, inner diameter 1.0 mm; Waters) and analyzed on a triple quadrupole mass spectrometer (LC-MS-8040; Shimadzu, Kyoto, Japan).
Mass spectrometric analysis of hydroxy fatty acids was performed in negative ion mode with an injection volume of 15 µL containing 0.5 mg of the oxidized fatty acid fraction and 1500 pg of the internal standard (12,13-diHOME-d4, 13S-HODE-d4, 13-KODE-d3, 12,13-EpOME-d4; Cayman chemicals, Ann Arbor, Michigan USA). The quantification of hydroxy fatty acids was identified and quantified by multiple-reaction monitoring as reported for the determination of other lipid metabolites [47 (link)]. For quantitation, calibration curves were prepared for each compound, and recoveries were monitored using deuterated internal standards. Data analysis was performed using LabSolutions software (Shimadzu, Kyoto, Japan).
Mass spectrometric analysis of hydroxy fatty acids was performed in negative ion mode with an injection volume of 15 µL containing 0.5 mg of the oxidized fatty acid fraction and 1500 pg of the internal standard (12,13-diHOME-d4, 13S-HODE-d4, 13-KODE-d3, 12,13-EpOME-d4; Cayman chemicals, Ann Arbor, Michigan USA). The quantification of hydroxy fatty acids was identified and quantified by multiple-reaction monitoring as reported for the determination of other lipid metabolites [47 (link)]. For quantitation, calibration curves were prepared for each compound, and recoveries were monitored using deuterated internal standards. Data analysis was performed using LabSolutions software (Shimadzu, Kyoto, Japan).
4
Oxylipin Assay with UHPLC-MS/MS
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Oxylipin assays were performed on a UHPLC–MS/MS system consisting of the following Shimadzu® modules (Shimadzu Corporation, Marne-la-Vallée, France): a binary pump consisting of coupling two isocratic pumps Nexera LC30AD, an automated sampler SIL-30AC, a column oven CTO-20AC, and a triple-quadrupole mass spectrometer LCMS-8060 operating in the negative ion mode. Chromatographic separation was achieved on a Kinetex® C18 column maintained at 50 °C and gradient-elution chromatography using the following mobile phases: water with 0.01% acetic acid (A) and methanol (B) at a flow rate of 0.600 mL/min. Gradient was as follows: 0.0–0.5 min, 10% (B); 0.5–2.0 min, 10 to 70% (B); 2.0–5.0 min, 70 to 75% (B); 5.0–5.1 min, 75 to 98% (B); 5.1–6.9 min, 98% (B); 6.9–7.0 min, 98% to 10% (B); and 7.0–8.0 min, 10% (B). The source interface parameters and common settings were as follows: interface voltage: −3 kV; nebulizing gas flow: 3 L/min; heating gas flow: 10 L/min; drying gas flow: 10 L/min; interface temperature: 400 °C; DL (desolvation line) temperature: 250 °C; heat block temperature: 500 °C; and collision gas pressure: 300 kPa. Detection and quantification were performed by scheduled-MRM (multiple reaction monitoring) using a pause time of 3 ms and individual dwell times to achieve sufficient points per peak. Isobaric compounds of 14,15-DHET, 14,15-EET, and 15-HETE were used as IS.
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