Lipid mediators in crude lung homogenates were extracted and analyzed by liquid chromatography-tandem mass spectrometry. LMs were extracted from lung tissue, in 10 μl/mg homogenization buffer (phosphate-buffered saline), with solid-phase extraction (SPE) using Strata-X (Phenomenex, Torrance, CA). The method was modified for Strata-X SPE columns from a previously described method (50 (link)). Data were quantified with Masshunter B0502, using external calibration for each compound and internal standard [PGD2-d4, PGE2-d4, PGF2-d4, and 5- and 12-HETE-d8; 1,000 pg of each (Cayman Chemicals, Ann Arbor, MI)] to correct for losses and matrix effects. Extracted and quantified LMs included: DHA-derived pro-resolving 17-hydroxydocosahexaenoic acid (17-HDHA) and protectin D1 (PD1); EPA-derived pro-resolving prostaglandin E3 (PGE3) and LM intermediates 2-, 5-, 9-, 11-, 15-, and 18-hydroxyeicosapentaenoic acid (HEPE); AA-derived pro-inflammatory intermediates 5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE); AA-derived prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), and prostaglandin F2α (PGFα2); and thromboxane B2 (TBXB2).
Masshunter b0502
Manufactured by Cayman Chemical
Sourced in United States
The Masshunter B0502 is a high-performance liquid chromatography (HPLC) system designed for analytical chemistry applications. It is capable of performing precise and accurate liquid chromatography analysis. The Masshunter B0502 features a modular design, allowing for customization to meet specific research or testing requirements.
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4 protocols using masshunter b0502
1
Quantifying Lipid Mediators in Lung Tissue
2
Lipidomic Analysis of Lung Homogenates
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LMs in crude lung homogenates were analyzed with liquid chromatography-tandem mass spectrometry. Seventeen-hydroxy-docosahexaenoic acid (17-HDHA); 5-, 11-, 12-, 15- and 18-hydroxy-eicosapentaenoic acid (HEPE); 5-, 8-, 9-, 11-, 12-, and 15-hydroxy-eicosatetraenoic acid (HETE); prostaglandin D1 (PGD1); PGE2; PGE3 and PGD2 concentrations were measured. LMs were extracted from ~50 mg lung tissue, in 10 µL/mg homogenization buffer, with solid-phase extraction using Strata-X (Phenomenex, Torrance, CA, USA). The method was modified for Strata-XSPE columns from a previously described method [25 (link)]. Data were quantified with Masshunter B0502, using external calibration for each compound and internal standards (PGD2-d4, PGE2-d4, PGF2-d4 and 5- and 12-HETE-d8; 1000 pg of each (Cayman Chemicals, Ann Arbor, MI, USA)) to correct for losses and matrix effects.
3
Quantifying Lipid Mediators in Lung Tissue
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Lipid mediators in crude lung homogenates were extracted and analysed by liquid chromatography-tandem mass spectrometry. Lipid mediators were extracted from lung tissue, in 10 µl/mg homogenization buffer (phosphate-buffered saline), with solid-phase extraction using Strata-X (Phenomenex, Torrance, CA). The method was modified for Strata-XSPE columns from a previously described method (33 (link)). Data were quantified with Masshunter B0502, using external calibration for each compound and internal standards [PGD2-d4, PGE2-d4, PGF2-d4 and 5- and 12-HETE-d8; 1000 pg of each (Cayman Chemicals, Ann Arbor, MI)] to correct for losses and matrix effects. Extracted and quantified LMs included: DHA-derived pro-resolving 17-hydroxydocosahexaenoic acid (17-HDHA) and protectin D1 (PD1); EPA-derived pro-resolving LM intermediates 2-, 5-, 9-, 11-, 15- and 18-hydroxyeicosapentaenoic acid (HEPE); AA-derived pro-inflammatory intermediates 5-, 8-, 9-, 11-, 12- and 15-hydroxyeicosatetraenoic acid (HETE); AA-derived prostaglandin D1 (PGD1), prostaglandin E2 (PGE2), prostaglandin F2α (PGFα2); and thromboxane B2 (TBXB2).
4
Quantification of Lipid Mediators in Lung
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Lipid mediators in crude lung homogenates were analysed with liquid chromatography-tandem mass spectrometry. 17-Hydroxydocosahexaenoic acid (17-HDHA); 5-, 11-, 12-, 15-and 18-hydroxyeicosapentaenoic acid (HEPE); 5-, 8-, 9-, 11-, 12-and 15-hydroxyeicosatetraenoic acid (HETE); prostaglandin (PG)D 1 ; PGE 2 ; PGE 3 and PGD 2 concentrations were measured. Lipid mediators were extracted from ∼50 mg lung tissue, in 10 μl/mg homogenisation buffer, with solid-phase extraction using Strata-X (Phenomenex). The method was modified for Strata-XSPE columns from a previously described method (22) . Data were quantified with Masshunter B0502, using external calibration for each compound and internal standards (PGD 2 -d4, PGE 2 -d4, PGF 2 -d4 and 5-and 12-HETE-d8; 1000 pg of each (Cayman Chemicals)) to correct for losses and matrix effects.
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