IntroductionRecent advances in instrumentation allow for automation and small scale esterification of lipids, specifically by the Agilent 7696A Sample Prep Workbench (Figure 1). While the Workbench works well for oils and extracted lipids, transferring direct methods for use with the workbench is challenging. In this investigation, an automated method using the 7696A Sample Prep Workbench was developed based on a method by Golay, et al.1 Three different infant formulas were used for method investigation; results were compared to conventional direct methods for accuracy. Optimization of the automated method included evaluation of initial sample weights, aliquot size used for reaction, and solvent volumes. Data shows that the automated method has many advantages; precise control of reaction times, analyst labor savings, and reduction in solvent usage by 90%.
ResultsInitial testing resulted in lower than expected results (Table 1). All fatty acids were quantitated and evaluated, however palmitic, oleic, and linoleic were chosen as representative saturated, monounsaturated, and polyunsaturated fatty acids. Further testing was performed, using various sample weights, internal standard in MTBE volumes, and sample aliquots (Table 2). Reducing the sample aliquot improved method performance, particularly for sample IF1 (Table 3). Increasing the volume of internal standard in MTBE to 400 µL yielded, in general, higher results than 300 µL (Table 4).
Based on the results of trial 3, a 150 mg aliquot and 400 µL of internal standard in MTBE was used to analyze three infant formulas (IF1, IF2, and IF3). All fatty acids were quantitated and evaluated, however linoleic, α linolenic, arachidonic, and docosahexaenoic acid were chosen for display due to their importance in infant nutrition. Comparable results were generated using both methods (Figures 2-5). Note: Results for IF2 appear low, indicating further need for method optimization.
Materials and MethodsInfant Formulan Powdered formula, approximately 30% lipid (IF1)n Powdered formula, approximately 15% lipid (IF2)n Ready to feed formula, approximately 3.5% lipid (IF3)
Automated Method Reagentsn Internal standard solution: C11:0 methyl undecanoate + C13:0 tridecanoin in
methyl tert-butyl ether (MTBE) (1.667 mg/mL each) n 5% sodium methoxide in methanoln 0.1 g/mL disodium hydrogen citrate plus 0.15 g/mL sodium chloride in water
Methodsn Covance method: Direct methylation using 0.5 M NaOH in methanol, and 14%
BF3 in methanoln Final Automated Workbench method:
Preparation prior to Workbench:
1. Adjust sample weights for powders to obtain approximately 5 mg lipid in the final aliquot, record weight. (~0.24 g for IF1, ~0.45 g for IF2)
2. Add 2 mL H2O, record total weight. Mix well.
3. Allow to stand for at least 15 minutes.
4. Using a pipette, aliquot approximately150 µL into a 2 mL injection vial and record weight of aliquot. Cap vial.
5. Aliquot liquid samples directly into the 2 mL vial. Cap vial.
7696A Sample Prep Workbench was programmed to do the following steps:
1. Add 400 µL internal standard in MTBE (methyl tert-butyl ether).
2. Mix at 2500 rpm for 20 seconds
3. Add 500 µL 5% sodium methoxide
4. Mix at 2000 rpm for 40 seconds
5. Wait 10 seconds
6. Mix at 2000 rpm for 20 seconds
7. Add 100 µL hexane
8. Add 600 µL aqueous NaCl/disodium citrate
9. Mix at 2000 rpm for 40 seconds
The reaction was timed so that 185 seconds elapsed between the addition of sodium methoxide and hexane. A total of 240 seconds elapsed until the addition of NaCl/Citrate solution.
All final solutions were analyzed by gas chromatography using the same conditions (column SP-2560, 100 m x 0.25 mm x 0.20 micron film thickness) and external calibration standards.
Comparison of an Automated Method to Conventional Methods for the Determination of Fatty Acids in Infant Formula Barb Mitchell, Sarah Kanable, Christopher Leuenberger, Erin Meinholz, Meagan Dallman, Erika Vacha, John Richard, Chad Volkmann and Wayne EllefsonCovance Laboratories Inc., Madison, Wisconsin
Future WorkResults for IF2 (15% lipid) remained lower than the target values, further testing is needed to improve overall recoveries. Additional testing will focus on determining the most efficient sample to reagent ratios. In addition, other types of formula will be investigated.
ConclusionsAutomation of direct transesterification of triacylglycerols in infant formulae is possible using the Agilent 7696A Sample Prep Workbench. This automation reduces solvent use, frees analyst time, reduces exposure to hazardous chemicals, and allows for consistent timing of a critical reaction. Since the method tolerates water, small sample aliquots needed for analysis on the Workbench are possible without compromising the advantage of a larger sample size.
AcknowledgementsThe authors would like to first thank Agilent Technologies for the use of a 7697A Sample Prep Workbench for this investigation. We would also like to thank Chad Scheuerell for helping with continuing our ongoing investigation and Brent Rozema for his overall support of our research.
References1. Golay, Pierre-Alain, Giuffrida, Franscesca, Dionisi, Fabiola, Destaillats, Frédéric (2009) Journal of AOAC
International Vol. 92, No. 5: 1301-1309.
2. Suter, Bea, Grob, Konrad, Pacciarelli, Bruno (1997) Z. Lebensm Unters Forsch A 204: 252-258.
Figure 1. Agilent 7696A Sample Prep Workbench.
Table 1. Trial 1 Results - Initial Testing
Target (mg/g) Results (mg/g) % Deviation
IF1
Palmitic 52.7 36.1 -31.5
Oleic 104.3 64.8 -37.9
Linoleic 53.1 35.4 -33.3
IF2
Palmitic 22.8 13.0 -43.0
Oleic 51.8 27.2 -47.5
Linoleic 41.5 23.6 -43.1
Table 2. Workbench Method Experiments
Trial # Sample size Volume of H20
addedAliquot size
Volume of IS solution
1To obtain
50 mg lipid2.0 mls 250 mg 300 µL
2To obtain
50 mg lipid1.0 mls
100 mg200 mg
300 µL
3To obtain 5
mg lipid in the aliquot
1.0 mls100 mg150 mg200 mg
300 µL400 µL
Table 3. Trial 2 Results - Reduced Sample Aliquots100 mg aliquot 200 mg aliquot
Target (mg/g) Result (mg/g) % Deviation Result (mg/g) % Deviation
IF1
Palmitic 52.7 50.7 -3.8 31.4 -40.4
Oleic 104.3 100.7 -3.5 58.8 -43.6
Linoleic 53.1 51.0 -4.0 31.2 -41.2
IF2
Palmitic 22.8 15.1 -33.8 9.14 -59.9
Oleic 51.8 33.9 -34.6 19.7 -62.0
Linoleic 41.5 27.6 -33.5 16.5 -60.2
Table 4. Trial 3 Results - Increased Internal Standard in MTBE Volume
IF1 300 µL ether 400 µL ether
100 mg Target (mg/g) Result (mg/g) % Deviation Result (mg/g) % Deviation
Palmitic 52.7 51.2 -2.8 51.7 -1.9
Oleic 104.3 101.2 -3.0 103.4 -0.9
Linoleic 53.1 52.0 -2.1 52.6 -0.9
150 mg
Palmitic 52.7 42.7 -19.0 53.9 2.3
Oleic 104.3 81.3 -22.1 108.0 3.5
Linoleic 53.1 43.0 -19.0 54.7 3.0
200 mg
Palmitic 52.7 33.6 -36.2 47.7 -9.5
Oleic 104.3 61.4 -41.1 93.7 -10.2
Linoleic 53.1 33.3 -37.3 48.4 -8.9
IF2 300 µL ether 400 µL ether
100 mg Target (mg/g) Result (mg/g) % Deviation Result (mg/g) % Deviation
Palmitic 22.8 13.8 -39.5 16.5 -27.6
Oleic 51.8 30.5 -41.1 36.9 -28.8
Linoleic 41.5 25.1 -39.5 30.0 -27.7
150 mg
Palmitic 22.8 15.9 -30.3 19.6 -14.0
Oleic 51.8 34.9 -32.6 44.3 -14.5
Linoleic 41.5 29.1 -29.9 35.9 -13.5
200 mg
Palmitic 22.8 15.4 -32.5 20.7 -9.2
Oleic 51.8 33.4 -35.5 47.0 -9.3
Linoleic 41.5 28.4 -31.6 38.1 -8.2
Table 5. Overall Precision
Sample Type
Automated Base Transesterification
Method% RSD
18:2 Linoleic Acid
IF1 1.69IF2 3.12IF3 2.00
18:3 αLinolenic Acid
IF1 1.55IF2 2.93IF3 4.59
20:4 Arachidonic Acid
IF1 3.39IF2 7.03IF3 3.55
22:6 Docosahexaenoic
Acid
IF1 3.85IF2 11.2IF3 4.59
Figure 2. Linoleic Acid mean results for IF1, IF2, and IF3 comparing methods.
Figure 4. Arachidonic Acid mean results for IF1, IF2, and IF3 comparing methods.
Figure 3. α Linolenic Acid mean results for IF1, IF2, and IF3 comparing methods.
Figure 6. Comparison of organic solvent use per sample.
Figure 5. Docosahexaenoic Acid mean results for IF1, IF2, and IF3
comparing methods.
The automated method reduced manual steps for the analyst, allowed for precise control of reaction times, and reduced the amount of reagents used. (Figure 6). Not only does the Workbench reduce the amount of solvent used, it also reduces the chemists’ exposure to hazardous chemicals.
Overall precision was evaluated based on results over 3 days of analysis producing a total of 15 data points, with the exception of the IF2 which is based on 3 days of data producing a total of 13 data points (Table 5).
Presented at the125th AOAC Annual Meeting & ExpositionNew Orleans, Louisiana18–21 September 2011
Comparison of an Automated Method to Conventional Methods for the Determination of Fatty Acids in Infant FormulaBarb Mitchell, Sarah Kanable, Christopher Leuenberger, Erin Meinholz, Meagan Dallman, Erika Vacha, John Richard, Chad Volkmann and Wayne EllefsonCovance Laboratories Inc., Madison, Wisconsin
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