Agilent Determination of Fatty Acid Methyl Esters (FAMEs) in Salmon Oil Using Automated Sample Preparation Manual
Contents
1. Authors Norbert Helle and Monika Bzduch TeLA GmbH Bremerhaven Rebecca Veeneman Agilent Technologies Inc Determination of Fatty Acid Methyl Esters FAMEs in Salmon Oil Using Automated Sample Preparation Application Note Area Introduction The automated derivatization of fatty acids FAs was performed with the Agilent 7696A Sample Prep WorkBench Since free fatty acids show tailing in gas chro matography transformation of fatty acids into fatty acid methyl esters FAMEs is widely used Manual sample derivatization is time consuming and may lead to poor repeatibility Automated derivatization shows significant enhancement of repro ducibility and saves time Especially for highly unsaturated fatty acids slight varia tions in reaction temperature and time can negatively affect repeatability when using manual procedures Salmon oil is an excellent source of polyunsaturated omega 3 fatty acids The two main fatty acids eicosapentaenoic acid EPA and docosahexaenoic acid DHA have been identified as important health factors and are correlated with a normal function of the heart The concentration of EPA and DHA is the crucial quality factor for salmon oil capsules This application note demonstrates the use of the Agilent 7696A Sample Prep WorkBench for derivatization and subsequent determination of both EPA and DHA from salmon oil capsules j Agilent Technologies Materials and Methods For sample preparation 10 mg2. ations of polyunsaturated fatty acids especially the automation is significantly more reliable than manual procedures Reference 1 Animal and vegetable fats and oils Gas chromatogra phy of fatty acid methyl esters Part 3 Preparation of methyl esters using trimethylsulfonium hydroxide TMSH ISO 12966 3 2009 For More Information These data represent typical results For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2012 Printed in the USA January 23 2012 5990 9799EN Agilent Technologies
3. f 0 85 for 25 Mean 23 42 s m rs 22 5 20 Mean 18 85 x z a 17 5 15 0 Sample number Repeatability data of of automated FAME determination in salmon oil Figure 3 EPA and 1 22 for DHA were achieved No outliers were Figure 4 Structure of EPA methyl ester left and DHA methyl ester right observed over the 10 samples The total runtime for sample preparation on the Agilent 7696A Sample Prep WorkBench was only 20 minutes per sample whereas the time for the manual derivatization depends on the skills of the laboratory technician and can take up to 2 hours pA 2 400 4 5 C20 5 EPA gt 350 amp S lt 18 2 300 C18 1 gt 2 250 C16 1 S z C16 0 ay C18 3 S cant 22 1 150 4 c18 0 can Gals C22 6 oo gt C24 1 lt DHA C14 0 ea is 100 4 3 Bo g 5 A Yg lt oss l BIR IN Aia i 04 A LJ A A Ris in SLA aA T T T T T a 15 20 25 30 35 40 min Figure 2 _ GC FID chromatogram of a salmon oil sample prepared using Agilent WorkBench 7696A Table 2 Percent Distribution of Fatty Acids in Salmon Oil Sample C14 0 C16 0 C16 1 C18 0 C18 1 18 2 C20 0 C18 3 C20 1 C18 3 C22 1 C20 4 C20 5 C24 1 C22 6 0 71 4 68 7 95 3 54 12 95 13 86 0 36 2 61 1 71 3 33 3 35 0 87 23 79 0 36 19 93 Conclusion The automated sample derivatization is easy fast and reli able For samples with high relative concentr
4. keup flow N 45 mL min Agilent WorkBench Program Add 500 uL of TBME to sample Mix sample 3 000 rpm 3 cycles bidirectional 25 seconds Wait for 15 seconds Add 250 uL of sample to empty vial Add 125 uL of TMSH to empty vial Mix empty vial 3 000 rpm 3 cycles bidirectional Heat empty vial at 80 C for 5 minutes Flag empty vial as Result Flow diagram of FAME sample preparation with the Agilent 7696A Sample Prep WorkBench DI amp S s Results and Discussion Figure 2 shows the separation of FAMEs from salmon oil on an Agilent 7696A WorkBench The separation allows the unequivocal identification of all FAMEs The two compounds of main interest show retention times of 35 07 minutes EPA and 40 55 minutes DHA Besides EPA 23 7 and DHA 20 0 salmon oil further consists of unsaturated fatty acids oleic 12 linoic 11 and palmitoleic 8 acid The con tent of saturated fatty acids palmitic and stearic acid is low 4 and 5 respectively For the repeatability test 10 individual salmon oil samples were derivatized and analyzed to determinate the reproducibil ity of the automatic sample preparation and chromatography As shown in Figure 3 excellent repeatability was obtained The absolute areas of the EPA and DHA signals showed stan dard deviations of less than 1 EPA 0 51 DHA 0 78 Moreover variations of the EPA and DHA relative concentra tions were stable Relative standard deviations o
5. of salmon oil was weighed into a 2 mL autosampler vial The sample was diluted in 500 uL of tert butyl methyl ether TBME using the liquid dispensing module of the Agilent 7696A Sample Prep WorkBench and mixed for 90 seconds with the onboard vortex mixer A 250 uL aliquot of the prepared sample was tranferred to an empty vial and 125 uL of a Trimethylsulfoniumhydroxide TMSH derivatization solution MachereyNagel D ren was added and the mixture was again mixed using the vortex mixer of the WorkBench The mixture was heated for 5 min utes at 80 C in the single vial heater The flow diagram for the automated procedure on the Agilent 7696A Sample Prep WorkBench is in shown in Figure 1 The gas chromatographic conditions were chosen as shown in Table 1 Table 1 GC FID Conditions Peak identification C14 0 Myristic acid C16 0 Palmitic acid C16 1 Palmitoleic acid C18 0 Stearic acid C18 1 Oleic acid C18 2 Linoleic acid 20 0 Arachidic acid C18 3 y Linolenic acid C20 1 Gadoleic acid C18 3 Linolenic acid C22 1 Erucic acid C20 4 Arachidonic acid C20 5 Eicosapentaenoic acid C24 1 Nervonic acid C22 6 Docosahexaenoic acid GC Conditions Instrument Column Injection volume Injector Carrier gas Temperature program Flow Agilent 6890 Series GC HP 88 100 m x 250 pm 0 20 pm 2 uL Split Splitless Split 50 1 Hy 70 C 260 C 1 4 mL min 250 C FID H flow 40 mL min Air flow 450 mL min Ma
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