LPC 16:0, LPC 18:0 and LPC 18:1 were quantified in BALF by UPLC-MS as described earlier36 (link). The samples were spiked with 140 ng/ml of LPC 12:0 (internal standard) and were extracted twice using liquid-liquid extraction (Methanol/Chloroform, 2:1). The mixtures were vortexed, incubated for 15 minutes on ice, followed by addition of 1 ml Chloroform and 1 ml water to separate the phases; vortexed the mixture before spinning at 1,750 × g at 4 °C for 10 minutes. The lower phases were combined; dried using inert gas at RT; dissolved in 100 μl methanol and 10 μl of it was injected into the ESI-MS ion source through auto- sampler of LC system. The system used was Waters APi Quattro micro triple quadrupole mass spectrometer equipped with a Waters UPLC system and a Waters equity C18 column. The detection limit and quantification limit were 9 pg and 30 pg respectively. The standard curves for the LPCs were formed over a range of 1.9–500 ng/ml with linear coefficient of determination (R2) = 0.995.
Api quattro micro triple quadrupole mass spectrometer
Manufactured by Waters Corporation
The APi Quattro micro triple quadrupole mass spectrometer is a laboratory instrument designed for high-performance mass analysis. It utilizes triple quadrupole technology to provide precise and sensitive detection of a wide range of analytes. The core function of this product is to perform qualitative and quantitative analysis of samples by separating and detecting ions based on their mass-to-charge ratio.
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4 protocols using api quattro micro triple quadrupole mass spectrometer
1
Quantification of LPC Lipids in BALF
2
Synthesis and Characterization of Ferrocenyl Dithiocarbamates
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All reagents and solvents were purchased from commercial suppliers. All reagents were purchased from Sigma-Aldrich or ABCR Chemicals and were used without further purification. Metal salts were kindly donated by AngloAmerican Platinum Limited. Methyl hydrazinecarbodithioate, 50 Schiff-base dithiocarbamates [ferrocenyl (1a), 34 acetylferrocenyl (1b) 35 Waters API Quattro Micro triple quadrupole mass spectrometer (samples were injected into a stream of 50% acetonitrile and 0.1% formic acid) and data were recorded using electrospray ionisation (ESI) mass spectrometry in the positive mode. Melting points were determined using the Büchi Melting Point apparatus B-540.
3
Quantification of Glycosphingolipids by ESI-LC-MS/MS
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Tissue samples (∼100 mg wet weight) were homogenized in water (0.6 mL) and chloroform:methanol (1∶2, v/v; 3 mL) using a PowerGen 35 (Fisher Scientific). Homogenates were shaken (15 min) and centrifuged (5 min at 1,000×g). Pellets were re-extracted with water (0.7 mL) and chloroform-methanol (1∶2, v/v; 3 mL). The combined extracts were centrifuged (10 min at 7,000×g). The supernatants were transferred to fresh tubes and the solvents evaporated under N2. Dried extracts were redissolved in chloroform-methanol-water (60∶30∶4.5, v/v/v; 15 mL) and desalted on Sephadex G-25 columns. Samples were then subjected to alkaline methanolysis and desalted. Glycosphingolipids from 4 mg equivalents of tissue samples were quantified by ESI-LC-MS/MS using a Waters Quattro Micro API triple quadrupole mass spectrometer (Milford, MA) interfaced with Acquity UPLC system as described [46] (link), [47] (link).
4
Quantification of Ursodeoxycholyl LPE in Mice
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Ursodeoxycholyl LPE dissolved in 0.5% carboxymethylcellulose (CMC) at 5 mg/mL was intraperitoneally injected to six mice to obtain a dosage of 30 mg/kg. Mice were killed at 0, 1, 4, 5, 8, and 24 h after i.p. injection. Blood and liver tissues were harvested, and liver homogenates at 1 g/mL phosphate-buffered saline were prepared. Twenty microliters of the internal standard 0.5 ng/μL d 4 -UDCA was added to a sample prior to lipid extraction with chloroform and methanol ( 16 ). Ursodeoxycholyl LPE in lipid extracts was quantified by liquid chromatography mass spectrometry (LC-MS/MS) on a Waters Quattro Micro API triple quadrupole mass spectrometer (Milford, Mass) interfaced with an Acquity UPLC system as previously described ( 16 ). Briefly, online chromatographic separation was achieved using a 100 × 2.0-mm-internal-diameter Luna 3 μm C18 column (Phenomenex, Torrance, Calif). Binary solvents were 95% water/methanol with 2 mM ammonium acetate (solvent A) and 95% methanol/water with 2 mM ammonium acetate (solvent B). For analysis of UDCA-LPE, the gradient was started with 90% solvent A for 1 min, changed to 100% solvent B in 0.5 min, held for 5 min, and finally switched back to initial condition within 0.1 min. Flow rate was 0.2 mL/min.
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