Name: cellular lipid extraction for targeted stable isotope dilution liquid chromatography-mass spectrometry analysis
Uploaded: 2011-11-17T12:00:00
Description: Watch this Scientific Journal Video about Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis at JoVE.com

Most current bioanalytical methods available for the measurement of bioactive lipids are not as extensive as they do not include chiral normal phase chromatography or SID. Chiral normal phase LC is critical for the separation of enantiomers and for being able to distinguish between enzyme- or ROS-mediated metabolism. The use of SID ensures that human error or complications that arise during extraction or analysis are taken into account during quantification. These added components along with ECAPCI-MRM make this the most sensitive, selective method available for the analysis of bioactive lipids.

1. Standard and Internal Standard Mixes
<ol>
	<li>Before lipid extraction, one must prepare a standard mix (SM) and an internal standard mix (ISM). For the SM, aliquot the equivalent volume of 1 &mu;g of each standard (25 total) listed in the reagent table below, using a calibrated syringe, into a 10 mL volumetric flask. Dry the standards under nitrogen and reconstitute in 10 mL of acetonitrile (ACN). The final concentration of the standard mix will be 100 pg/&mu;L. Divide into 1 mL aliquots and store at -80&deg;C unti...

<ol>
	<li>Needleman, P., Turk, J., Jakschik, B. A., Morrison, A. R., Lefkowith, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arachidonic+acid+metabolism.">Arachidonic acid metabolism.</a> Annu. Rev. Biochem. 55, 69-102 (1986).</li><li>Nikolaev, V., Reddanna, P., Whelan, J., Hildenbrandt, G., Reddy, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereochemical+nature+of+the+products+of+linoleic+acid+oxidation+catalyzed+by+lipoxygenases+from+potato+and+soybean.">Stereochemical nature of the products of linoleic acid oxidation catalyzed by lipoxygenases from potato and soybean.</a> Biochem. Biophys. Res. Commun. 170, 491-496 (1990).</li><li>Panigrahy, D., Kaipainen, A., Greene, E. R., Huang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P450-derived+eicosanoids:+the+neglected+pathway+in+cancer.">Cytochrome P450-derived eicosanoids: the neglected pathway in cancer.</a> Cancer. Metastasis. Rev. 29, 723-735 (2010).</li><li>Basu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+eicosanoids:+role+of+prostaglandin+F(2alpha)+and+F-isoprostanes+in+inflammation+and+oxidative+stress+related+pathology.">Bioactive eicosanoids: role of prostaglandin F(2alpha) and F-isoprostanes in inflammation and oxidative stress related pathology.</a> Mol. Cells. 30, 383-391 (2010).</li><li>Sanchez-Mejia, R. O., Mucke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+A2+and+arachidonic+acid+in+Alzheimer's+disease.">Phospholipase A2 and arachidonic acid in Alzheimer's disease.</a> Biochim. Biophys. Acta. 1801, 784-790 (2010).</li><li>Hersberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+the+lipoxygenase+derived+lipid+mediators+in+atherosclerosis:+leukotrienes,+lipoxins+and+resolvins.">Potential role of the lipoxygenase derived lipid mediators in atherosclerosis: leukotrienes, lipoxins and resolvins.</a> Clin. Chem. Lab Med. 48, 1063-1073 (2010).</li><li>Allayee, H., Roth, N., Hodis, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyunsaturated+fatty+acids+and+cardiovascular+disease:+implications+for+nutrigenetics.">Polyunsaturated fatty acids and cardiovascular disease: implications for nutrigenetics.</a> J. Nutrigenet. Nutrigenomics. 2, 140-148 (2009).</li><li>Capdevila, J. H., Falck, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+molecular+properties+of+the+cytochrome+P450+arachidonic+acid+monooxygenases.">Biochemical and molecular properties of the cytochrome P450 arachidonic acid monooxygenases.</a> Prostaglandins Other Lipid Mediat. , 68-69 (2002).</li><li>Ciccimaro, E., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable-isotope+dilution+LC-MS+for+quantitative+biomarker+analysis.">Stable-isotope dilution LC-MS for quantitative biomarker analysis.</a> Bioanalysis. 2, 311-341 (2010).</li><li>Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron-capture+negative-ion+chemical+ionization+mass-spectrometry+of+lipid+mediators.">Electron-capture negative-ion chemical ionization mass-spectrometry of lipid mediators.</a> Methods in Enzymology. 187, 13-23 (1990).</li><li>Singh, G., Gutierrez, A., Xu, K., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+chromatography/electron+capture+atmospheric+pressure+chemical+ionization/mass+spectrometry:+analysis+of+pentafluorobenzyl+derivatives+of+biomolecules+and+drugs+in+the+attomole+range.">Liquid chromatography/electron capture atmospheric pressure chemical ionization/mass spectrometry: analysis of pentafluorobenzyl derivatives of biomolecules and drugs in the attomole range.</a> Analytical Chemistry. 72, 3007-3013 (2000).</li><li>Blair, I. A., Capriol, R. M., Gross, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Mass Spectrometry. , 283-307 (2005).</li><li>Lee, S. H., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+chiral+lipidomics+analysis+by+liquid+chromatography+electron+capture+atmospheric+pressure+chemical+ionization+mass+spectrometry+(LC-ECAPCI/MS).">Targeted chiral lipidomics analysis by liquid chromatography electron capture atmospheric pressure chemical ionization mass spectrometry (LC-ECAPCI/MS).</a> Methods in Enzymology. 433, 159-174 (2007).</li></ol>

The standards and internal standards used in this protocol provide a representation of a targeted lipidomics method. A Waters 2695 separation module and Thermo Fisher TSQ Quantum Ultra were used for the LC-MS analysis and the optimal parameter settings can be found in Tables 1 and 2. Additionally, this extraction protocol was designed for adherent cells, but can be modified for other cell types as well as other biological matrices including urine, blood, and tissue. Many lipid standards ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic-6,8,9,11,12,14,15-d7 acid</td>
			<td>Cayman Chemical</td>
			<td>334250</td>
			<td>[2H7]-5-oxoETE Internal Standard</td>
		</tr>
		<tr>
			<td>5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid</td>
			<td>Cayman Chemical</td>
			<td>334230</td>
			<td>[2H8]-5(S)-HETE Internal Standard</td>
		</tr>
		<tr>
			<td>12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid</td>
			<td>Cayman Chemical</td>
			<td>334570</td>
			<td>[2H8]-12(S)-HETE Internal Standard</td>
		</tr>
		<tr>
			<td>15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid</td>
			<td>Cayman Chemical</td>
			<td>334720</td>
			<td>[2H8]-15(S)-HETE Internal Standard</td>
		</tr>
		<tr>
			<td>20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic-16,16,17,17,18,18-d6 acid</td>
			<td>Cayman Chemical</td>
			<td>390030</td>
			<td>[2H6]-20-HETE Internal Standard</td>
		</tr>
		<tr>
			<td>9S-hydroxy-10E,12Z-octadecadienoic-9,10,12,13-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>338410</td>
			<td>[2H4]-9(S)-HODE Internal Standard</td>
		</tr>
		<tr>
			<td>13S-hydroxy-9Z,11E-octadecadienoic-9,10,12,13-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>338610</td>
			<td>[2H4]-13(S)-HODE Internal Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&alpha;,15S-trihydroxy-prosta-5Z,13E-dien-1-oic-17,17,18,18,19,19,20,20,20-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>316010</td>
			<td>[2H4]-PGF2&alpha; Internal Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&alpha;,15S-trihydroxy-(8&beta;)-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>316350</td>
			<td>[2H4]-8-iso-PGF2a Internal Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&beta;.,15S-trihydroxy-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>10008989</td>
			<td>[2H4]-11&beta;-PGF2 Internal Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic-17,17,18,18,19,19,20,20,20-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>312010</td>
			<td>[2H4]-PGD2 Internal Standard</td>
		</tr>
		<tr>
			<td>9-oxo-11&alpha;,15S-dihydroxy-prosta-5Z,13E-dien-1-oic-17,17,18,18,19,19,20,20,20-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>314010</td>
			<td>[2H4]-PGE2 Internal Standard</td>
		</tr>
		<tr>
			<td>5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic-6,7,14,15-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>320110</td>
			<td>[2H4]-LTB4 Internal Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11,15S-trihydroxy-thromba-5Z,13E-dien-1-oic-3,3,4,4-d4 acid</td>
			<td>Cayman Chemical</td>
			<td>319030</td>
			<td>[2H4]-TxB2 Internal Standard</td>
		</tr>
		<tr>
			<td>5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34250</td>
			<td>5-oxoETE Standard</td>
		</tr>
		<tr>
			<td>12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34580</td>
			<td>12-oxoETE Standard</td>
		</tr>
		<tr>
			<td>15-oxo-5Z,8Z,11Z,13E-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34730</td>
			<td>15-oxoETE Standard</td>
		</tr>
		<tr>
			<td>5R-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34225</td>
			<td>5(R)-HETE Standard</td>
		</tr>
		<tr>
			<td>5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34230</td>
			<td>5(S)-HETE Standard</td>
		</tr>
		<tr>
			<td>8R-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34350</td>
			<td>8(R)-HETE Standard</td>
		</tr>
		<tr>
			<td>8S-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34360</td>
			<td>8(S)-HETE Standard</td>
		</tr>
		<tr>
			<td>11R-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34505</td>
			<td>11(R)-HETE Standard</td>
		</tr>
		<tr>
			<td>11S-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34510</td>
			<td>11(S)-HETE Standard</td>
		</tr>
		<tr>
			<td>12R-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34560</td>
			<td>12(R)-HETE Standard</td>
		</tr>
		<tr>
			<td>12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34570</td>
			<td>12(S)-HETE Standard</td>
		</tr>
		<tr>
			<td>15R-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34710</td>
			<td>15(R)-HETE Standard</td>
		</tr>
		<tr>
			<td>15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>34720</td>
			<td>15(S)-HETE Standard</td>
		</tr>
		<tr>
			<td>20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>90030</td>
			<td>20-HETE Standard</td>
		</tr>
		<tr>
			<td>9R-hydroxy-10E,12Z-octadecadienoic acid</td>
			<td>Cayman Chemical</td>
			<td>38405</td>
			<td>9(R)-HODE Standard</td>
		</tr>
		<tr>
			<td>9S-hydroxy-10E,12Z-octadecadienoic acid</td>
			<td>Cayman Chemical</td>
			<td>38410</td>
			<td>9(S)-HODE Standard</td>
		</tr>
		<tr>
			<td>13R-hydroxy-9Z,11E-octadecadienoic acid</td>
			<td>Cayman Chemical</td>
			<td>38605</td>
			<td>13(R)-HODE Standard</td>
		</tr>
		<tr>
			<td>13S-hydroxy-9Z,11E-octadecadienoic acid</td>
			<td>Cayman Chemical</td>
			<td>38610</td>
			<td>13(S)-HODE Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&alpha;,15S-trihydroxy-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>16010</td>
			<td>PGF2&alpha; Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&alpha;,15S-trihydroxy-(8&beta;)-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>16350</td>
			<td>8-iso-PGF2&alpha; Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11&beta;,15S-trihydroxy-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>16520</td>
			<td>11&beta;-PGF2 Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>12010</td>
			<td>PGD2 Standard</td>
		</tr>
		<tr>
			<td>9-oxo-11&alpha;,15S-dihydroxy-(8&beta;)-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>14350</td>
			<td>8-iso-PGE2 Standard</td>
		</tr>
		<tr>
			<td>9-oxo-11&alpha;,15S-dihydroxy-prosta-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>14010</td>
			<td>PGE2 Standard</td>
		</tr>
		<tr>
			<td>5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid</td>
			<td>Cayman Chemical</td>
			<td>20110</td>
			<td>LTB4 Standard</td>
		</tr>
		<tr>
			<td>9&alpha;,11,15S-trihydroxythromba-5Z,13E-dien-1-oic acid</td>
			<td>Cayman Chemical</td>
			<td>19030</td>
			<td>TxB2 Standard</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Diethyl Ether</td>
			<td>Sigma</td>
			<td>346136</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Acros</td>
			<td>61030-1000</td>
			<td>anhydrous</td>
		</tr>
		<tr>
			<td>N,N-diisopropylethyl amine</td>
			<td>Sigma</td>
			<td>387649</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pentafluorylbenzyl bromide</td>
			<td>Sigma</td>
			<td>101052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma</td>
			<td>320331</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Fluka</td>
			<td>00650</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher</td>
			<td>A996-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A454-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher</td>
			<td>366927</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Fisher</td>
			<td>H303-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher</td>
			<td>A464-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Fisher</td>
			<td>W7-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pasteur Pipets</td>
			<td>Fisher</td>
			<td>13-678-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 mL Glass Centrifuge Tubes</td>
			<td>Kimble</td>
			<td>73785-10</td>
			<td>Screw cap</td>
		</tr>
		<tr>
			<td>Phenolic Screw Caps</td>
			<td>Kimble</td>
			<td>73802-13415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chiralcel ADH Column</td>
			<td>Chiral Technologies</td>
			<td>19325</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC vials</td>
			<td>Waters</td>
			<td>60000751CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC inserts</td>
			<td>Waters</td>
			<td>WAT094171</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cellular lipid extraction for targeted stable isotope dilution liquid chromatography-mass spectrometry analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

Watch this Scientific Journal Video about Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis at JoVE.com

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

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