An Automated Method for Accurate Determination of EPA and ... · n Fish Oil A (used for...
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Introduction In today’s world of consumer marketing, it is well established that marine oils positively contribute to cardiovascular health. These benefits rely heavily on EPA, DHA and ultimately, the Omega 3 fatty acid content that is present in products being marketed. Several analytical methods are currently used in industry, such as AOAC 991.39 1 , AOCS Ce 1i-07 2 , and the GOED voluntary monograph for EPA and DHA 3 . In this study, the above methods were scaled down for use with the Agilent 7696A Sample Prep Workbench (Figure 1). Concerns with the conventional methods include the need to methylate standards for each analytical run and to minimize the sample’s exposure to oxygen. This concern is addressed with the use of the Workbench, as precise methylation occurs in a closed system. Additionally, the automation reduces the scale of the process, reduces solvent costs, and minimizes labor. In this investigation, the automated method was compared to the conventional methods using three marine oil supplements. Data is presented showing accuracy, precision, repeatability and ruggedness. Methods and Materials Experimental Design To optimize the workbench, Fish Oil A was analyzed, varying the heating and timing steps. After optimization, to show method comparability, 3 different oils containing EPA and DHA were analyzed by four analytical methods. The fatty acid methyl esters were quantitated using the same GC parameters. With the exception of the GOED Monograph, the same standards were used to isolate variation to the preparation of the methyl esters. A total of fifteen data points were obtained over three days with five replicates per day, per oil, per method. Accuracy was determined by spike recoveries at 50, 100, and 200% of the nominal value in Fish Oil B. Conclusion The Agilent 7696A Sample Prep Workbench not only produced results comparable to three widely applied methods of analysis, it also significantly reduced analyst time and solvent use. Although the emphasis of this poster was on EPA and DHA, the proposed method shows much promise to be applied to many other oil matrices. Acknowledgements The authors would like to thank Agilent Technologies for lending Covance the 7696A Sample Prep Workbench for this research. We would also like to thank Brent Rozema for his overall support of our research. References 1. Association of Official Analytical Chemists, AOAC Official Method 991.39, Fatty Acids in Encapsulated Fish Oils and Fish Oil Methyl and Ethyl Esters, AOAC, Rockville, MD (2007). 2. Official Methods and Recommended Practices of the AOCS, Official method Ce 1i-07, The American Oil Chemists’ Society, Champaign, IL (2007). 3. Global Organization for EPA and DHA Omega-3s. (2006, July 1). GOED Voluntary Monograph (v. 3). Retrieved from GOED Website: http://www.goedomega3.com/portals/0/pdfs/GOEDMonograph.pdf. An Automated Method for Accurate Determination of EPA and DHA in Marine Oils Found in Today’s Supplement Market Sarah Kanable, Barb Mitchell, Christopher Leuenberger, Erin Meinholz, Meagan Dallman, Erika Vacha, John Richard, Chad Volkmann and Wayne Ellefson Covance Laboratories Inc., Madison, Wisconsin Figure 4. Krill Oil mean results for EPA and DHA comparing all four methods. Overall precision is based on the results from 3 analysts producing a total of 15 data points for each method. Percent relative standard deviations (% RSD) from the automated method are comparable with the other methods used for this investigation (Table 2). The automated method can be easily replicated between analysts and is ideal for a large scale operation (Table 3). Spike recoveries were performed on Fish Oil B using the automated method. Both EPA and DHA were spiked at 50, 100, and 200% of the inherent amount. Spike recoveries ranged from 95.2% to 101.8%. Mean recoveries for each level are displayed below (Table 4). Optimization Five replicates, from two different weighings of Fish Oil A, were analyzed by two different automated methods: n Reaction times equivalent to AOAC 991.39, temperature 70°C n Proposed automated method The results from these tests were compared to results obtained from Covance’s conventional method. The proposed method, with shorter reaction times, yielded slightly higher results that were comparable to the conventional methodology (Table 1). Test Materials (purchased commercially) n Fish Oil A (used for optimization only) n Fish Oil B n Algal Oil n Krill Oil Automated Method Procedure The sample was weighed into a 4 dram vial containing internal standard (C23:0 FAME) and diluted in toluene. The sample weights and volumes were adjusted to keep the EPA and DHA within the range of analytical standards. The 7696A Workbench was programmed to do the following steps: 1. Add 200 µL 0.5 M NaOH in methanol. 2. Mix at 2500rpm. 3. Add 250 µL 14 % BF3 in methanol. 4. Heat sample at 65°C for 5 min. 5. Add 250 µL heptane, mix at 2500rpm. 6. Add 250 µL H 2 0/NaCl, mix at 2500rpm. 7. Wait ~5 minutes to allow for separation. 8. Transfer top layer (~300 µL) to final GC vial for injection. Figure 1. Agilent 7696A Sample Prep Workbench Table 1. Optimization Data AOAC Reaction Times via Workbench Proposed Method via Workbench Validated Mean via Conventional Method Units EPA DHA 152.5 96.9 155.9 99.3 156.9 99.8 mg/g mg/g Results Based on the 15 replicates, comparable results were generated using all four methods. Results indicate no advantage to performing the transesterification step on the standards, as performed in the GOED monograph (Figures 2–4). Table 2. Overall Precision for All Four Methods Automated %RSD AOAC %RSD AOCS %RSD GOED %RSD EPA Fish Oil Algal Oil Krill Oil 1.95 1.85 1.65 1.26 2.16 2.26 1.37 1.46 4.84 1.68 0.983 2.84 DHA Fish Oil Algal Oil Krill Oil 2.98 1.60 2.19 0.900 1.83 2.12 1.36 2.17 4.70 1.59 1.04 2.60 Table 3. Ruggedness on Automated Method (n=5) Analyst 1 Analyst 2 Analyst 3 Mean (mg/g) %RSD Mean (mg/g) %RSD Mean (mg/g) %RSD EPA Fish Oil Algal Oil Krill Oil 183 19.8 140 2.01 1.21 0.796 184 20.2 141 2.42 1.06 2.64 180 19.4 142 0.451 0.540 0.961 DHA Fish Oil Algal Oil Krill Oil 80.5 367 59.1 1.73 1.26 0.755 83.4 377 60.1 2.60 1.13 2.61 78.8 366 57.9 0.705 0.342 0.521 Table 4. Accuracy by Spiked Recovery on Fish Oil B Spike Level EPA DHA 50% 100% 200% 97.3 100.1 101.1 96.4 98.8 100.3 Table 5. Demonstration of Labor Savings Due to Automation AOAC 991.39 Automated Method 1. Add IS to vial – Evaporate solvent 2. Weigh Sample 3. Add NaOH in MeOH 4. Reflux 5. Cool 6. Add BF3 7. Reflux 8. Cool 9. Add isooctane 10. Shake 11. Add Saturated NaCl 12. Shake 13. Transfer Isooctane 14. Add Isooctane 15. Shake 16. Transfer Isooctane 17. Evaporate to 1 mL 18. Transfer to injection vial 19. Inject on GC 1. Add IS to vial – evaporate solvent 2. Weigh sample 3. Dilute in toluene 4. Aliquot to injection vial 5. Fill resource vials on 7696A 6. Enter 7696A sequence 7. Start 7696A 8. Move finished vials to GC 9. Inject on GC Figure 5. Comparison of organic solvent use per sample. Significantly less solvent is needed using the Workbench (Figure 5). The largest volume of solvent used (2–5 mls) is toluene, in which the sample is initially dissolved. This volume could be further reduced by changes to the standard curve. An added benefit of using the workbench is a significant reduction in hands-on analyst time, as the number of steps in the method is vastly reduced (Table 5). Methods n AOAC 991.39 n AOCS Ce 1i-07 n GOED Voluntary Monograph for EPA and DHA n Proposed automated method Figure 3. Algal Oil mean results for EPA and DHA comparing all four methods. Figure 2. Fish Oil B mean results for EPA and DHA comparing all four methods.
Transcript of An Automated Method for Accurate Determination of EPA and ... · n Fish Oil A (used for...
IntroductionIn today’s world of consumer marketing, it is well established that marine oils positively contribute to cardiovascular health. These benefits rely heavily on EPA, DHA and ultimately, the Omega 3 fatty acid content that is present in products being marketed.
Several analytical methods are currently used in industry, such as AOAC 991.391, AOCS Ce 1i-072, and the GOED voluntary monograph for EPA and DHA3. In this study, the above methods were scaled down for use with the Agilent 7696A Sample Prep Workbench (Figure 1).
Concerns with the conventional methods include the need to methylate standards for each analytical run and to minimize the sample’s exposure to oxygen. This concern is addressed with the use of the Workbench, as precise methylation occurs in a closed system. Additionally, the automation reduces the scale of the process, reduces solvent costs, and minimizes labor. In this investigation, the automated method was compared to the conventional methods using three marine oil supplements. Data is presented showing accuracy, precision, repeatability and ruggedness.
Methods and MaterialsExperimental Design
To optimize the workbench, Fish Oil A was analyzed, varying the heating and timing steps. After optimization, to show method comparability, 3 different oils containing EPA and DHA were analyzed by four analytical methods. The fatty acid methyl esters were quantitated using the same GC parameters. With the exception of the GOED Monograph, the same standards were used to isolate variation to the preparation of the methyl esters. A total of fifteen data points were obtained over three days with five replicates per day, per oil, per method. Accuracy was determined by spike recoveries at 50, 100, and 200% of the nominal value in Fish Oil B.
ConclusionThe Agilent 7696A Sample Prep Workbench not only produced results comparable to three widely applied methods of analysis, it also significantly reduced analyst time and solvent use. Although the emphasis of this poster was on EPA and DHA, the proposed method shows much promise to be applied to many other oil matrices.
AcknowledgementsThe authors would like to thank Agilent Technologies for lending Covance the 7696A Sample Prep Workbench for this research. We would also like to thank Brent Rozema for his overall support of our research.
References1. Association of Official Analytical Chemists, AOAC Official Method
991.39, Fatty Acids in Encapsulated Fish Oils and Fish Oil Methyl and Ethyl Esters, AOAC, Rockville, MD (2007).
2. Official Methods and Recommended Practices of the AOCS, Official method Ce 1i-07, The American Oil Chemists’ Society, Champaign, IL (2007).
3. Global Organization for EPA and DHA Omega-3s. (2006, July 1). GOED Voluntary Monograph (v. 3). Retrieved from GOED Website: http://www.goedomega3.com/portals/0/pdfs/GOEDMonograph.pdf.
An Automated Method for Accurate Determination of EPA and DHA in Marine Oils Found in Today’s Supplement Market Sarah Kanable, Barb Mitchell, Christopher Leuenberger, Erin Meinholz, Meagan Dallman, Erika Vacha, John Richard, Chad Volkmann and Wayne EllefsonCovance Laboratories Inc., Madison, Wisconsin
Figure 4. Krill Oil mean results for EPA and DHA comparing all four methods.
Overall precision is based on the results from 3 analysts producing a total of 15 data points for each method. Percent relative standard deviations (% RSD) from the automated method are comparable with the other methods used for this investigation (Table 2).
The automated method can be easily replicated between analysts and is ideal for a large scale operation (Table 3).
Spike recoveries were performed on Fish Oil B using the automated method. Both EPA and DHA were spiked at 50, 100, and 200% of the inherent amount. Spike recoveries ranged from 95.2% to 101.8%. Mean recoveries for each level are displayed below (Table 4).
Optimization
Five replicates, from two different weighings of Fish Oil A, were analyzed by two different automated methods:
n Reaction times equivalent to AOAC 991.39, temperature 70°C
n Proposed automated method
The results from these tests were compared to results obtained from Covance’s conventional method. The proposed method, with shorter reaction times, yielded slightly higher results that were comparable to the conventional methodology (Table 1).
Test Materials (purchased commercially)n Fish Oil A (used for optimization only)n Fish Oil Bn Algal Oiln Krill Oil
Automated Method
Procedure
The sample was weighed into a 4 dram vial containing internal standard (C23:0 FAME) and diluted in toluene. The sample weights and volumes were adjusted to keep the EPA and DHA within the range of analytical standards. The 7696A Workbench was programmed to do the following steps:
1. Add 200 µL 0.5 M NaOH in methanol.
2. Mix at 2500rpm.
3. Add 250 µL 14 % BF3 in methanol.
4. Heat sample at 65°C for 5 min.
5. Add 250 µL heptane, mix at 2500rpm.
6. Add 250 µL H20/NaCl, mix at 2500rpm.
7. Wait ~5 minutes to allow for separation.
8. Transfer top layer (~300 µL) to final GC vial for injection.
Figure 1. Agilent 7696A Sample Prep Workbench
Table 1. Optimization DataAOAC Reaction Times
via WorkbenchProposed Method via
WorkbenchValidated Mean via
Conventional MethodUnits
EPADHA
152.596.9
155.999.3
156.999.8
mg/gmg/g
ResultsBased on the 15 replicates, comparable results were generated using all four methods. Results indicate no advantage to performing the transesterification step on the standards, as performed in the GOED monograph (Figures 2–4).
Table 2. Overall Precision for All Four MethodsAutomated
%RSDAOAC%RSD
AOCS%RSD
GOED%RSD
EPAFish OilAlgal OilKrill Oil
1.951.851.65
1.262.162.26
1.371.464.84
1.680.9832.84
DHAFish OilAlgal OilKrill Oil
2.981.602.19
0.9001.832.12
1.362.174.70
1.591.042.60
Table 3. Ruggedness on Automated Method (n=5)
Analyst 1 Analyst 2 Analyst 3
Mean (mg/g) %RSDMean (mg/g)
%RSDMean (mg/g)
%RSD
EPAFish OilAlgal OilKrill Oil
18319.8140
2.011.21
0.796
18420.2141
2.421.062.64
18019.4142
0.4510.5400.961
DHAFish OilAlgal OilKrill Oil
80.536759.1
1.731.26
0.755
83.437760.1
2.601.132.61
78.836657.9
0.7050.3420.521
Table 4. Accuracy by Spiked Recovery on Fish Oil B
Spike Level EPA DHA
50%100%200%
97.3100.1101.1
96.498.8
100.3
Table 5. Demonstration of Labor Savings Due to Automation
AOAC 991.39 Automated Method
1. Add IS to vial – Evaporate solvent2. Weigh Sample3. Add NaOH in MeOH4. Reflux5. Cool6. Add BF37. Reflux8. Cool9. Add isooctane10. Shake11. Add Saturated NaCl12. Shake13. Transfer Isooctane14. Add Isooctane15. Shake16. Transfer Isooctane17. Evaporate to 1 mL18. Transfer to injection vial19. Inject on GC
1. Add IS to vial – evaporate solvent2. Weigh sample3. Dilute in toluene4. Aliquot to injection vial5. Fill resource vials on 7696A6. Enter 7696A sequence7. Start 7696A8. Move finished vials to GC9. Inject on GC
Figure 5. Comparison of organic solvent use per sample.
Significantly less solvent is needed using the Workbench (Figure 5). The largest volume of solvent used (2–5 mls) is toluene, in which the sample is initially dissolved. This volume could be further reduced by changes to the standard curve.
An added benefit of using the workbench is a significant reduction in hands-on analyst time, as the number of steps in the method is vastly reduced (Table 5).
Methods n AOAC 991.39n AOCS Ce 1i-07n GOED Voluntary Monograph
for EPA and DHAn Proposed automated method
Figure 3. Algal Oil mean results for EPA and DHA comparing all four methods.
Figure 2. Fish Oil B mean results for EPA and DHA comparing all four methods.
Presented at the125th AOAC Annual Meeting & ExpositionNew Orleans, Louisiana18–21 September 2011
An Automated Method for Accurate Determination of EPA and DHA in Marine Oils Found in Today’s Supplement MarketSarah Kanable, Barb Mitchell, Christopher Leuenberger, Erin Meinholz, Meagan Dallman, Erika Vacha, John Richard, Chad Volkmann and Wayne EllefsonCovance Laboratories Inc., Madison, Wisconsin
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