The authors would like to acknowledge the contribution of Meritxell Romeu-Nadal to the rat study.

1. Animal Procedures
<ol>
	<li>All animal work should be carried out in accordance with the Home Office Animals (Scientific Procedures) Act (1986).</li>
	<li>Mate Wistar rats aged 10 weeks old by monogamous breeding, and confirm pregnancy by the appearance of a vaginal plug. Record this as day 1 of gestation, and commence experimental diet. For virgin females, house each rat individually, and commence experimental diet.</li>
	<li>After feeding the experimental diets for 20 days euthanize rats by CO2 asphyxiation followed by cervical dislocation.</li>
	<li>Use dissection forceps and scissors to expose the abdominal cavity and excise the liver by cutting the ligaments, which connect the liver to the diaphragm, anterior wall of the abdomen, stomach and duodenum. Wash the liver in PBS and freeze in liquid nitrogen before storing at -80 &#176;C.</li>
</ol>
2. Preparation of a Total Lipid Extract9
<ol>
	<li>Add molecular sieves (to fill 1/10&#160;of solvent container) to all solvents to create &#8216;dry&#8217; solvents. Carry out all solvent work within a fume hood.</li>
	<li>Cut approximately 100 mg frozen liver and weigh. Place the tissue into a tube in an ice bucket and add 0.8 ml ice cold 0.9% NaCl. Homogenize the tissue.</li>
	<li>Add internal standards dissolved in 1 ml/mg of dry chloroform: methanol (2:1, v/v) containing&#160;butylated hydroxytoluene (BHT; 50 mg/l) as anti-oxidant. For 100 mg of rat liver add 100 &#956;g of CE standard (Cholesteryl heptadecanoate 17:0). Caution: Chloroform and BHT are hazardous.</li>
	<li>Add 5.0 ml dry chloroform: methanol (2:1, v/v) containing BHT (50 mg/L).</li>
	<li>Add 1.0 ml 1 M NaCl, mix thoroughly by vortexing until mixture looks uniform. Samples can be capped and stored at -20 &#176;C at this stage for up to a week.</li>
	<li>Centrifuge at 1,000 x g for 10 min, low brake at room temperature.</li>
	<li>Collect lower phase using glass Pasteur pipette, transfer to new screw cap glass tube and dry under nitrogen at 40 &#176;C. Samples can be capped and stored at -20 &#176;C at this stage for up to a week.</li>
</ol>
3. Separation of Lipid Classes by Solid Phase Extraction (SPE)10
<ol>
	<li>Connect the SPE tank to a vacuum pump and place aminopropyl silica SPE cartridge on the tank.</li>
	<li>Place new screw-cap glass tube labeled TAG and CE in the tank rack under the column to collect first fraction.</li>
	<li>Dissolve the total lipid extract in 1.0 ml dry chloroform and vortex.</li>
	<li>Apply sample to the column using a glass Pasteur pipette and allow to drip through into the screw-cap tube under gravity. When no further drips fall, remove the remaining liquid by vacuum.</li>
	<li>Elute the TAG and CE fraction under vacuum, wash the column with 2 x 1.0 ml washes of dry chloroform.</li>
	<li>When all liquid is removed, dry the TAG and CE fraction under nitrogen at 40 &#176;C. Samples can be capped and stored at -20 &#176;C at this stage for up to a week.</li>
	<li>Place new screw-cap glass tube labeled PC into the tank tray under the column.</li>
	<li>Elute the PC fraction under vacuum with the addition of 2 x 1.0 ml dry chloroform: methanol (60:40, v/v) until all liquid is removed from the column.</li>
	<li>Remove and dry PC fraction under nitrogen at 40 &#176;C. Samples can be capped and stored at -20 &#176;C at this stage for up to a week.</li>
	<li>Place a new screw-cap glass tube labeled PE into the tank tray and elute PE fraction with the addition of 1.0 ml dry methanol under vacuum.</li>
	<li>Remove and dry PE fraction under nitrogen at 40 &#176;C. Samples can be capped and stored at -20 &#176;C at this stage for up to a week.</li>
	<li>Place new screw-cap glass tube labeled NEFA into the tank tray and elute NEFA fraction under vacuum by the addition of 2 x 1.0 ml washes of dry chloroform: methanol: glacial acetic acid (100:2:2, v/v/v). Caution: Glacial acetic acid is hazardous.&#160;</li>
	<li>Remove collected NEFA fraction and dry under nitrogen at 40 &#176;C. Samples can be capped and stored at -20 &#176;C at this stage for up to a week.</li>
	<li>Place a new aminopropyl silica SPE cartridge on the SPE tank and place a screw-cap glass tube in the tank tray under the cartridge to collect waste.</li>
	<li>Wash the column with 3 washes of dry hexane&#160;under vacuum and then a final 1.0ml wash under gravity. Do not allow the cartridge to become dry (turn the cartridge column channels to a closed position when hexane level is close to the cartridge matrix). Caution: Hexane is hazardous.</li>
	<li>Replace waste tube with new screw-cap glass tube labeled CE.</li>
	<li>Dissolve the dried TAG and CE fraction (prepared in step 3.6) in 1.0 ml of dry hexane and vortex. Apply this to the column using a glass Pasteur pipette and allow to drip through under gravity.</li>
	<li>When no further drips fall, remove the remaining liquid under vacuum.</li>
	<li>Under vacuum, wash the column with 2 x 1.0 ml washes of dry hexane to elute CE and dry collected fraction under nitrogen at 40 &#176;C. Samples can be capped and stored at -20 &#176;C at this stage for up to a week.</li>
	<li>Place new screw-cap glass tube labeled TAG in the tank tray and elute TAG with the addition of 2 x 1.0 ml washes of dry hexane: methanol: ethyl acetate&#160;(100:5:5) under vacuum.</li>
	<li>Dry collected fraction under nitrogen at 40 &#176;C. Samples can be capped and stored at -20 &#176;C at this stage for up to a week. Caution: Ethyl acetate is hazardous.</li>
</ol>
4. Preparation of FAME from CE10
<ol>
	<li>Add 0.5 ml of dry toluene to the separated CE fraction (collected in step 3.19) and vortex.&#160;Caution: Toluene is hazardous.</li>
	<li>Prepare methylation reagent (dry methanol with 2% (v/v) H2SO4), of which 1.0 ml is required per sample. Dispense volume of dry methanol into glass or suitable plastic container with lid and add the required amount of H2SO4 dropwise then mix by inversion.&#160;Caution: Sulfuric acid is hazardous.</li>
	<li>Add 1.0 ml of the methylating reagent to the samples dissolved in dry toluene, cap the tubes securely, and mix gently.</li>
	<li>Heat the samples for 2 hr at 50 &#176;C.</li>
	<li>After 2 hr remove tubes from heat. Once cool add 1.0 ml neutralizing solution (0.25 M KHCO3&#160;0.5M K2CO3). Caution: Potassium bicarbonate and potassium carbonate are hazardous.</li>
	<li>Add 1.0 ml dry hexane and vortex.</li>
	<li>Centrifuge at 250 x g for 2 min, low brake at room temperature.</li>
	<li>Collect upper phase, which contains the FAME, and transfer into a new non screw-cap disposable glass tube.</li>
	<li>Dry the collected FAME under nitrogen at 40 &#176;C. Samples can be capped and stored at -20 &#176;C at this stage for up to a week.</li>
</ol>
5. Removal of Free Cholesterol Contamination from CE FAME14
(Free Cholesterol can contaminate the sample; see Figure 1 for example chromatograph traces with and without free cholesterol removal).
<ol>
	<li>Place a waste tube into SPE tank and place a silica gel SPE cartridge onto the tank.</li>
	<li>Wash column with 3 x 1 ml washes of dry hexane under vacuum and 1 x 1 ml under gravity.</li>
	<li>Remove waste washes and add new waste tube into tank.</li>
	<li>Dissolve CE FAME in 1 ml dry hexane, vortex.</li>
	<li>Apply to the column using a glass Pasteur pipette and allow to drip through under gravity.</li>
	<li>Wash column with 3 x 1 ml washes of hexane under vacuum.</li>
	<li>Remove waste washes and place new non screw cap tube into tank labeled CE FAME.</li>
	<li>Elute the CE FAME with 2 x 1 ml dry hexane: diethyl ether (95:5 v/v) washes.</li>
	<li>Dry under nitrogen at 40 &#176;C. Samples can be capped and stored at -20 &#176;C at this stage for up to a week. Caution: Diethyl ether is hazardous.</li>
</ol>
6. Transfer of FAME into GC Auto Sampler Vial
<ol>
	<li>Add 75 &#956;l dry hexane to sample, vortex, and transfer to a GC auto sample vial.</li>
	<li>Add a further 75 &#956;l dry hexane to sample, vortex and transfer to the same GC auto sample vial. Samples can be capped and stored at -20 &#176;C at this stage for up to a month.</li>
</ol>
7. Analysis Using the Gas Chromatograph14
<ol>
	<li>Analyze FAME on a gas chromatograph. Example set up: 30 m x 0.25 &#956;m x 0.25 mm BPX-70 fused silica capillary column with temperature protocol: 
	Initial temperature 115 &#176;C, hold 2 min, ramp 10 &#176;C/min to 200 &#176;C, hold 18.5 min, ramp 60 &#176;C/min to 245 &#176;C, hold 4 min. 
	Column: Helium gas, flow rate 1.0, pressure 14.6 and velocity 29. 
	Injector: Temperature = 300 &#176;C. 
	Detector: Hydrogen flow 40.0, air flow 184.0, make up gas Helium, flow 45.0, temperature = 300 &#176;C.</li>
	<li>Set split ratio as appropriate (e.g. 25:1 for CE FAME analysis).</li>
	<li>Determine the area under each peak using appropriate software and identify FAME by comparison with standards. See Figure 2 for example chromatograms.</li>
	<li>Use the area under the peak data to calculate the contribution of individual fatty acids as a percentage of total fatty acids.</li>
	<li>Calculate absolute concentrations of fatty acids by dividing the area of internal standard by the amount added. Divide the area of each fatty acid by this result to obtain absolute concentrations of each fatty acid within the amount of tissue used.</li>
</ol>

<ol>
	<li>Browning, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+eicosapentaenoic+and+docosahexaenoic+acids+into+lipid+pools+when+given+as+supplements+providing+doses+equivalent+to+typical+intakes+of+oily+fish.">Incorporation of eicosapentaenoic and docosahexaenoic acids into lipid pools when given as supplements providing doses equivalent to typical intakes of oily fish.</a> Am. J. Clin. Nutr. 96 (4), 748-758 (2012).</li><li>Cao, J., Schwichtenberg, K. A., Hanson, N. Q., Tsai, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+and+clearance+of+omega-3+fattyacids+in+erythrocyte+membranes+and+plasma+phospholipids.">Incorporation and clearance of omega-3 fattyacids in erythrocyte membranes and plasma phospholipids.</a> Clin. Chem. 52 (12), 2265-2272 (2006).</li><li>Lauritzen, L., Carlson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+fatty+acid+status+during+pregnancy+andlactation+and+relation+to+newborn+and+infant+status.">Maternal fatty acid status during pregnancy andlactation and relation to newborn and infant status.</a> Matern. Child Health. 7 (2), 41-58 (2011).</li><li>Kelsall, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+dysfunction+induced+in+offspring+by+maternal+dietary+fat+involves+altered+arterial+polyunsaturated+fatty+acid+biosynthesis.">Vascular dysfunction induced in offspring by maternal dietary fat involves altered arterial polyunsaturated fatty acid biosynthesis.</a> PLoS One. 7 (4), (2012).</li><li>Karpe, F., Dickmann, J. R., Frayn, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acids,+obesity,+and+insulin+resistance:+time+for+a+re-evaluation.">Fatty acids, obesity, and insulin resistance: time for a re-evaluation.</a> Diabetes. 60 (10), 2441-2449 (2011).</li><li>Mossoba, M. M., Moss, J., Kramer, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans+fat+labelling+and+levels+in+U.S.+foods:+assessment+of+gas+chromatographic+and+infrared+spectroscopic+techniques+for+regulatory+compliance.">Trans fat labelling and levels in U.S. foods: assessment of gas chromatographic and infrared spectroscopic techniques for regulatory compliance.</a> J. AOAC Int. 92 (5), 1284-1300 (2009).</li><li>Chee, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+content+of+marine+oil+capsules.">Fatty acid content of marine oil capsules.</a> Lipids. 25 (9), 523-528 (1990).</li><li>Folch, J., Lees, M., Sloane-Stanley, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipids+from+animal+tissues.">A simple method for the isolation and purification of total lipids from animal tissues.</a> J. Biol. Chem. 226 (1), 497-509 (1957).</li><li>Burdge, G. C., Wright, P., Jones, E. A., Wootton, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+separation+of+phosphatidylcholine,+triacylglycerol,+non-esterified+fatty+acids+and+cholesterol+esters+from+plasma+by+solid-phase+extraction.">A method for separation of phosphatidylcholine, triacylglycerol, non-esterified fatty acids and cholesterol esters from plasma by solid-phase extraction.</a> Br. J. Nutr. 84 (5), 781-787 (2000).</li><li>Sepp&#228;nen-Laakso, T., Laakso, I., Hiltunen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+fatty+acids+by+gas+chromatography,+and+its+relevance+to+research+on+health+and+nutrition.">Analysis of fatty acids by gas chromatography, and its relevance to research on health and nutrition.</a> Anal. Chim. Acta. 465 (1), 39-62 (2002).</li><li>Beesley, T. E., Buglio, B., Scott, R. P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantitative chromatographic analysis. , (2000).</li><li>Bartle, K. D., Myers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+gas+chromatography.">History of gas chromatography.</a> Trends Anal. Chem. 21 (9), 9-10 (2002).</li><li>Childs, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The effect of gender, pregnancy and diet upon rat tissue fatty acid composition and immune function. , 378 (2008).</li><li>Harris, S. W., Pottala, J. V., Ramachandran, S. V., Larson, M. G., Robins, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+erythrocyte+membrane+Trans+and+marine+fatty+acids+between+1999+and+2006+in+older+Americans.">Changes in erythrocyte membrane Trans and marine fatty acids between 1999 and 2006 in older Americans.</a> J. Nutr. 142 (7), 1297-1303 (2012).</li><li>Roberts, L. D., McCombie, G., Titman, C. M., Griffin, J. 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+matter+of+fat:+An+introduction+to+lipidomic+profiling+method.">A matter of fat: An introduction to lipidomic profiling method.</a> J. Chromatogr. B. 871 (2), 174-181 (2008).</li></ol>

The authors declare no competing financial interests.

Gas chromatography is an accurate technique to use for fatty acid analysis, and its high reproducibility deems this technique suitable for clinical analyses.&#160;Appropriate GC columns must be used to enable identification of fatty acids of interest, with available columns having variations in the polarity of the stationary phase, column length and internal diameter. The use of a fused silica capillary column in this method of analysis provides good thermal stability and high reproducibility of retention times due to it...

Gas chromatography (GC) is a well-established technique used to identify and quantify the incorporation of fatty acids into lipid pools and cell membranes1,2 during supplementation or physiological conditions such as obesity (and related diseases such as diabetes) or pregnancy3-5. It is also suitable for analyzing the types and quantities of fats in foods. This is useful when characterizing experimental diets, as well as ensuring that the food industry complies with regulations. For example, GC can be used to confirm the identity and quantity of fatty acids within a product such as a dietary supplement to ensure that labeling is correct and regulations are adhered to6,7. &#160;Analysis of fatty acids can provide valuable insights into lipid metabolism in health and disease, the impact of dietary change, and the effect of changes in physiological state8. Use of GC to study samples during pregnancy has provided important information on changes in fatty acid and complex lipid homeostasis3.

In advance of the chromatographic separation, lipids are typically extracted from the sample using the solubility of lipids in solvent mixtures of chloroform and methanol. Sodium chloride is added to facilitate the separation of the mixture into aqueous and organic lipid containing phases9,10. Complex lipid classes of interest can be separated from the total lipid extract by solid phase extraction (SPE). This separation technique elutes lipid classes based upon their polarity or binding affinity. Triacyglycerols (TAG) and cholesteryl esters (CE) are eluted first as a combined fraction, further classes, phosphatidylcholine (PC), Phosphatidylethanolamine (PE), and non-esterified fatty acids (NEFA) are eluted by increasing the polarity of the eluting solvent. The separation of TAG from CE exploits the binding of TAG only to a fresh SPE cartridge, allowing CE to be eluted. TAG can then be eluted by increasing the polarity of the eluting solvent9,10. This method allows multiple samples to be separated simultaneously with a higher yield than is achieved with thin layer chromatography, which means that relatively small&#160;sized samples (e.g. &#60;100 &#181;l plasma or serum, &#60;100 mg tissue) can be analyzed11,12.

GC is a well-established technique first described in the 1950s; it was suggested that the mobile phase in the then liquid-liquid systems could be replaced with vapor. It was initially used for petroleum analysis but rapidly expanded into other areas such as amino acid analysis and lipid biochemistry, which is still of major interest. Advances in GC equipment and technology such as the development of capillary columns from the previously used packed columns has led to our current techniques in which fatty acids are able to be separated more efficiently at lower temperatures resulting in GC being routinely used to identify and quantify fatty acids in a wide range of investigations13.

GC requires fatty acids to be derivatized in order that they may become sufficiently volatile to be eluted at reasonable temperatures without thermal decomposition. This usually involves the substitution of a functional group containing hydrogen to form esters, thioesters or amides for analysis. Methyl esters are commonly studied derivatives, which are produced by methylation. In this method the ester bonds in complex lipids are hydrolyzed to release free fatty acids, which are transmethylated to form fatty acid methyl esters (FAME).&#160;The resulting profile of FAME, determined by GC, is referred to as the fatty acid composition and may be easily compared between different experimental groups9,10. The technique allows both the proportions of individual fatty acids and their concentrations to be measured.

In addition to the use of GC for analyzing fatty acids in nutrition studies and within the food industry, the technique can be used across a wide range of analytical fields. For example, environmental analyses using GC include measuring water contamination by insecticides and soil analyses measuring chlorobenzene content. In toxicology, GC has also been used to identify illegal substances in urine and blood samples of individuals; such a sports performance enhancers12 and the ability to separate complex mixtures of hydrocarbons makes this technique popular in the petroleum industry for petrochemical analysis12.

Pregnancy is associated with significant changes to the fatty acid composition of maternal tissues, specifically in the content of omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFA)3.&#160; In the current study, we exemplify the use of GC in the measurement of fatty acids by describing its use in the analysis of the fatty acid composition of liver tissue taken from virgin and pregnant rats fed low and high fat diets with different oil sources. The experimental diets provided here were a low fat soybean oil based diet, a high-fat soybean oil-based diet (130.9 g total fat/kg total fat) or a high-fat linseed oil-based diet (130.9 g total fat/kg diet), provided for 20 days.&#160;The full nutrient and fatty acid composition of these diets have been described previously14. The soybean oil diets are rich in linoleic acid (18:2n-6) and contain some &#945;-linolenic acid (18:3n-3) while the linseed oil diet is rich in &#945;-linolenic acid. These high-fat diets represent different rations of linoleic to &#945;-linolenic acids (rations of 8:1 and 1:1, respectively). The method for isolation of individual lipid classes and analysis by GC is well established and validated, and has been published previously10 but without the detailed technical description found herein.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Methanol:</td>
			<td>Fisher Scientific</td>
			<td>M/4056/17</td>
			<td>&#39;CAUTION&#39; Fumes - HPLC Grade</td>
		</tr>
		<tr>
			<td>Chloroform:</td>
			<td>Fisher Scientific</td>
			<td>C/4966/17</td>
			<td>&#39;CAUTION&#39; Fumes - HPLC Grade</td>
		</tr>
		<tr>
			<td>BHT:</td>
			<td>Sigma- Aldrich</td>
			<td>W218405</td>
			<td>&#39;CAUTION&#39; Dust fumes - Anhydrous</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma- Aldrich</td>
			<td>S9888</td>
			<td>Anhydrous</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Fisher Scientific</td>
			<td>H/0406/17</td>
			<td>&#39;CAUTION&#39; Fumes - HPLC Grade</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma- Aldrich</td>
			<td>695084</td>
			<td>&#39;CAUTION&#39; Burns - 99.85%</td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma- Aldrich</td>
			<td>339741</td>
			<td>&#39;CAUTION&#39; Burns - 99.999%</td>
		</tr>
		<tr>
			<td>Potassium carbonate</td>
			<td>Sigma- Aldrich</td>
			<td>209619</td>
			<td>99% ACS Reagent grade</td>
		</tr>
		<tr>
			<td>Potassium bicarbonate</td>
			<td>Sigma- Aldrich</td>
			<td>237205</td>
			<td>99.7% ACS Reasgent grade</td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Fisher Scientific</td>
			<td>10204340</td>
			<td>&#39;CAUTION&#39; Fumes - 99+% GLC SpeciFied</td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Fisher Scientific</td>
			<td>T/2300/15</td>
			<td>&#39;CAUTION&#39; Fumes</td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma- Aldrich</td>
			<td>309966</td>
			<td>&#39;CAUTION&#39; Fumes</td>
		</tr>
		<tr>
			<td>Nitrogen (oxygen free) cylinder</td>
			<td>BOC</td>
			<td>44-w</td>
			<td>&#39;CAUTION&#39; Compressed gas - explosion risk</td>
		</tr>
		<tr>
			<td>Aminopropyl silica SPE cartridges</td>
			<td>Agilent</td>
			<td>12102014</td>
			<td>Cartridge - Bead mass 100mg</td>
		</tr>
		<tr>
			<td>Silica gel SPE cartidges</td>
			<td>Agilent</td>
			<td>14102010</td>
			<td>Cartridge - Bead mass 100mg</td>
		</tr>
		<tr>
			<td>Molecular seives</td>
			<td>Sigma- Aldrich</td>
			<td>334324</td>
			<td>Pellets, AW-300, 1.6mm</td>
		</tr>
		<tr>
			<td>Glass pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>FB50251</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass test tube</td>
			<td>Fisher Scientific</td>
			<td>FB59527</td>
			<td>Non-screw cap</td>
		</tr>
		<tr>
			<td>Screw thread glass test tubes</td>
			<td>Fisher Scientific</td>
			<td>FB59555</td>
			<td>13mm</td>
		</tr>
		<tr>
			<td>Caps for screw thread test tubes</td>
			<td>Fisher Scientific</td>
			<td>FB51353</td>
			<td>To fit 13mm tube</td>
		</tr>
		<tr>
			<td>Solida phase extraction (SPE) tank</td>
			<td>Agilent</td>
			<td></td>
			<td>VacElut 20 Manifold</td>
		</tr>
		<tr>
			<td>Luer stopcocks for SPE tank</td>
			<td>Agilent</td>
			<td>12131005</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Sigma- Aldrich</td>
			<td>2656-194GB-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>GC vials</td>
			<td>Kinesis</td>
			<td>STV 12-03TS</td>
			<td>Short thread 9mm, TPX 0.2ml fused glass insert</td>
		</tr>
		<tr>
			<td>GC vial lids</td>
			<td>Kinesis</td>
			<td>SCC09-0.2B</td>
			<td>Short thread 9mm blue</td>
		</tr>
		<tr>
			<td>GC inlet liners</td>
			<td>SGE Analytical science</td>
			<td>092002</td>
			<td>Split/splitless Focus liner ID 4mm, OD 6.3mm length 78.5mm</td>
		</tr>
		<tr>
			<td>GC septa</td>
			<td>SGE Analytical science</td>
			<td>041856</td>
			<td>11mm, MN material</td>
		</tr>
		<tr>
			<td>GC column</td>
			<td>SGE Analytical science</td>
			<td>054612</td>
			<td>Length 30m, ID 0.22mm, Film thickness 0.25&#181;m</td>
		</tr>
		<tr>
			<td>Gas chromatograph</td>
			<td></td>
			<td></td>
			<td>HP 6890 series</td>
		</tr>
	</tbody>
</table>

the use of gas chromatography to analyze compositional changes of fatty acids in rat liver tissue during pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

The success of this method is dependent on following the protocol precisely and on using clean solvents and reagents in order to reduce &#8216;noise&#8217; and contamination that can appear on a chromatogram. Contaminated samples are more challenging to analyze, lowering the accuracy of the area under the curve calculations. If the protocol is followed successfully a chromatogram with clear symmetrical, well defined peaks and with minimal background noise should be obtained as illustrated in Figure 3. If...

Watch this Scientific Journal Video about The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy at JoVE.com

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

the-use-gas-chromatography-to-analyze-compositional-changes-fatty

Research

JoVE Journal

Chemistry

29.9K Views.  University of Southampton. Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

Video: The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

Trace explosive vapors of TNT and RDX collected on sorbent-filled thermal desorption tubes were analyzed using a programmed temperature desorption system coupled to GC with an electron capture detector. The instrumental analysis is combined with direct liquid deposition method to reduce sample variability and account for instrumentation drift and losses.

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive ...

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin&#8217;s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Facile Preparation of 4-Substituted Quinazoline Derivatives

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted 2-aminobenzophenones and thiourea in the presence of dimethyl sulfoxide (DMSO). This is a unique complementary reaction system in which thiourea undergoes thermal decomposition to form carbodiimide and hydrogen sulfide, where the former reacts with 2-aminobenzophenone to form 4-phenylquinazolin-2(1H)-imine intermediate, whilst hydrogen sulfide reacts with DMSO to give methanethiol or other sulfur-containing molecule which then functions as a complementary reducing agent to reduce 4-phenylquinazolin-2(1H)-imine intermediate into 4-phenyl-1,2-dihydroquinazolin-2-amine. Subsequently, the elimination of ammonia from 4-phenyl-1,2-dihydroquinazolin-2-amine affords substituted quinazoline derivative. This reaction usually gives quinazoline derivative as a single product arising from 2-aminobenzophenone as monitored by GC/MS analysis, along with small amount of sulfur-containing molecules such as dimethyl disulfide, dimethyl trisulfide, etc. The reaction usually completes in 4-6 hr at 160 &#186;C in small scale but may last over 24 hr when carried out in large scale. The reaction product can be easily purified by means of washing off DMSO with water followed by column chromatography or thin layer chromatography.

A protocol for facile preparation of 4-substituted quinazoline derivatives from 2-aminobenzophenones, thiourea and dimethyl sulfoxide is presented.

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...

A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. In this method for two-step pyrolysis online coupled to gas chromatography mass spectrometry (py-GC-MS) volatile compounds are analyzed during a first desorption run. In the second run, the same dried sample is pyrolyzed for analysis of non-volatile compounds such as pigments and polymers. These can be identified by their specific decomposition patterns. Additionally, this method can be used to differentiate original from counterfeit inks. Easy screening methods for data evaluation using the average mass spectra and self-made pyrolysis libraries are applied to speed up substance identification. Using specialized evaluation software for pyrolysis GS-MS data, a fast and reliable comparison of the full chromatogram can be achieved. Since GC-MS is used as separation technique, the method is limited to volatile substances upon desorption and after pyrolysis of the sample. The method can be applied for quick substance screening in market control surveys since it requires no sample preparation steps.

This method for two-step pyrolysis online coupled to gas chromatography with mass spectrometric detection and data evaluation protocol can be used for multi-component analysis of tattoo inks and discrimination of counterfeit products.

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. ...

Nitrogen Compound Characterization in Fuels by Multidimensional Gas Chromatography

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is crucial. There are significant challenges to overcome when measuring trace compounds in a complex matrix such as fuels. Background interferences and matrix effects can create limitations to routine analytical instrumentation, such as GC-MS. In order to facilitate specific and quantitative measurements of trace nitrogen compounds in fuels, a nitrogen-specific detector is ideal. In this method, a nitrogen chemiluminescence detector (NCD) is used to detect nitrogen compounds in fuels. NCD utilizes a nitrogen-specific reaction that does not involve the hydrocarbon background. Two-dimensional (GCxGC) gas chromatography is a powerful characterization technique as it provides superior separation capabilities to one-dimensional gas chromatography methods. When GCxGC is paired with a NCD, the problematic nitrogen compounds found in fuels can be extensively characterized without background interference. The method presented in this manuscript details the process for measuring different nitrogen-containing compound classes in fuels with little sample preparation. Overall, this GCxGC-NCD method has been shown to be a valuable tool to enhance the understanding of the chemical composition of nitrogen-containing compounds in fuels and their impact on fuel stability. The % RSD for this method is &#60;5% for intraday and &#60;10% for interday analyses; the LOD is 1.7 ppm and the LOQ is 5.5 ppm.

Here, we present a method utilizing two-dimensional gas chromatography and nitrogen chemiluminescence detection (GCxGC-NCD) to extensively characterize the different classes of nitrogen-containing compounds in diesel and jet fuels.

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is ...