PART III: LABORATORY METHODS OF ANALYSIS
C. Sample Preparation for Tryptophan
1. Weigh about 0.2 g sample into a stoppered tube.
2. Add 15ml fresh 4.3 N LiOH.H2O.
3. Flush with nitrogen gas or vacuum sealed.
4. Heat at 120ºC in oven for 16 hours.
5. Transfer hydrolysate to a beaker, add water and 9ml of 6 N HCI. Make sure total volume is less than 100 ml. Adjust pH to 4.5 using dilute HCI.
6. Dilute to 100 ml with water in a volumetric flask.
7. Filter through filter paper and finally filter a small aliquot of filtrate through a syringe filter (0.2 µm cellulose acetate membrane).
8. Inject 20 µl.
Reference
Waters AccQ Tag Amino Acid Analysis
152 3.3.6.1 DETERMINATION OF FATTY ACIDS COMPOSITION
METHOD A: FATTY ACID COMPOSITION IN OIL SAMPLE USING GCMS Principles
Gas chromatography (GC) is a technique for carrying out the separation and measurement of mixtures of materials that can be volatilized. These materials may be gases, liquids or solids with appreciable vapor pressures at temperatures up to a few hundred degrees. The sample should be vaporized and carried to the leading end of the column in negligible time. The detector, commonly flame ionization, monitors the composition of the carrier-gas stream as it leaves the flame ionization, monitors the composition of the carrier-gas stream as it leaves the column. Many compounds of interest do not posses sufficient volatility to be passed through a GC column. Examples of these are sugars, amino acids, metal and large fatty acids.
However, it is often possible to react these compounds with reagents to produce new compounds or derivatives which may be analyzed by GC. One of the most common derivatization procedures is replacement of strongly hydrogen bonding of acids or sugars with methyl groups. The decrease in hydrogen bonding of methyl esters then allows sufficient volatility for analysis.
Chemicals/Reagents
1. Sodium methoxide solution (0.2 ml; 2 M NaOCH3) 2. Hexane
3. Standard fatty acid methyl ester (FAME) mixture Apparatus/Instruments
1. Shimadzu GC 2010 2. Dropper
3. 5 ml vials 4. Vortex Procedures GC Setup
GC Model : Shimadzu GC 2010
Column Type : Capillary Column (BPX-70;30 m length x 0.25 mm Internal diameter x 0.2 µm film thickness)
Mobile phase : Nitrogen
Carrier gas flow-rate : Split 10
Detector : Flame Ionisation Detector (FID)
Detector Temperature : 250 ºC
Oven Temperature :
Programme : Holding for 1 min at 100ºC
Heating from 100ºC to 180ºC (8ºC/min) Holding for 2 min at 180ºC
Heating from 180ºC to 230ºC (8ºC/min) Holding at 230ºC for 1 min
Injection Temperature : 230ºC
3.3.6 FATTY ACIDS
153 Preparation of FAME:
1. Weigh 0.05 g of oil in a 5-ml vial and dissolve it with 1 ml hexane 2. Add 0.2 ml of sodium methoxide solution
3. Stopper the vial and vigorously shake using vortex for 1 minute 4. Allow the mixture to separate into two layers (centrifuge if necessary)
5. Carefully pipette the upper layer (supernatant) (containing FAME) into another 2 ml vial
6. Prepare FAME from three independent oil samples Retention Time for Standard FAME Mixture
1. Inject 1 µl of the standard FAME mixture and acquire the chromatogram.
2. Inject 1 µl of the upper layer (supernatant) into the GC column and acquire the chromatogram.
3. Identify the retem\ntion time for each fatty acid in both chromatograms.
Calculations
Quantification of Fatty Acid Concentration
From the standard chromatogram, for each fatty acid, record its area %, Response Factor (RF) and Corrected Response Factor (CRF) using the following formulations.
Area % = Area of each fatty acid x 100 Total area of all fatty acids
RF = Amount (Wt %) of each fatty acid Area % of each fatty acid RF = RF of each fatty acid RF of each fatty acid
Using the CRF obtained, calculate the concentration of each fatty acid in test oil and finally report the concentration in Normalized Weight % by using the following formula.
Amount of each fatty acid = (Area x CRF) of each fatty acid In test oil (WT %)
Normalized Weight (N.WT) % = Wt % of each fatty acid x 100 Total WT % of all fatty acids References
Yusof, HM, Miles, EA and Calder PC. 2008. Influence of very long-chain n-3 fatty acids on plasma markers of inflammation in middle-aged men.
Prostaglandins, Leukotrienes and Essential Fatty Acids 78: 219–228.
G.C. Burdge, P. Wright, A.E. Jones, S.A. Wootton, A method for separation of phosphatidylcholine, triacylglycerol, non-esterified fatty acids and cholesterol esters from plasma by solid-phase extraction, Br. J. Nutr. 84 (2000) 781–787.
154 METHOD B: FATTY ACID COMPOSITION IN FOOD SAMPLES USING GCMS Principles
This method is applicable to solids. The usual sample quantity is about 10 g. Extraction is optimized if samples are previously dried and ground. The solvents used are petroleum ether 60 – 80 ºC or less preferably 40 – 60 ºC. Hexane may also be used. Complete extraction can usually be achieved in 2 hours.
Materials and Reagents:
1. H2SO4 – 2 % w/v in methanol 2. NaCl – 5% in distilled water 3. KHCO3 – 2% w/v in distilled water 4. Toluene – AR
5. Hexane – AR or better 6. Chloroform
7. External standard – RM3, RM5, trans mix, PUFA – Sigma-Aldrich 8. Internal standard – C17:0
9. F.A.M.E. RM-3 mixture, Oil Reference Standard, AOCS No. 3 cat No. O7256- 1AMP (Supelco)
10. Polyunsaturated Fatty Acid (PUFA) Mix No.1 (Animal Source), Cat No. 47033 (Supelco)
11. Supelco 37 component FAME mix, Cat No. 47885U Supelco
12. Cis/ Trans FAME Column Performance mix, Cat No. CB-000711(Supelco) Apparatus/Instruments
1. Gas Chromatography Perkin Elmer Autosystem 2. Balance: analytical sensitivity ± 0.1 mg
3. IKA-VIBRAZ-VXR shaker 4. Nitrogen gas blower
5. Micropipette
6. Test-tube with condenser attachment: 20 mL 7. Heating block
8. Repeater pipette or graduated pipette and pipette-aid 9. Pasteur pipette
10. Test tube: 12 mL (to be used in centrifuge) 11. Vial
12. Vortex mixer 13. Centrifuge Procedures
A. Extraction of food samples
1. Measure 1-5 g of homogenize food sample into a 12 ml test tube.
2. Add 1 ml 0.9% NaCl shake vigorously, and add 1 ml ethanol and 4 ml hexane.
3. Shake for 1 hour at 1000 rpm with IKA-VIBRAZ-VXR shaker.
4. Centrifuge at 2000 rpm for 15 minutes and transfer to 7 ml Trident vial.
155 5. Put 2 ml hexane into the 12 ml test tube.
6. Repeat the extraction.
7. Combine the supernatant and dry by blowing with nitrogen gas.