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Rt-2560 Columns Ensure Accurate, Reliable AOAC 996.06 and AOCS Ce 1j-07 FAMEs Analysis

By Rebecca Stevens and Kristi Sellers

Restek’s Rt-2560 GC column was originally developed for detailed analysis of FAMEs in foods and food products. Its highly polar biscyanopropyl stationary phase and long column format make it an effective tool for the difficult task of separating and accurately quantifying complex mixtures of FAMEs. Recently, Restek optimized the manufacturing process and implemented a new, application-specific, QC-testing procedure for all new Rt-2560 columns (cat.# 13198). As shown here, Rt-2560 columns produced using the optimized process exhibit excellent performance for both AOAC 996.06 and AOCS Ce 1j-07 FAMEs analysis and meet all method requirements.

AOAC 996.06
AOAC official method 996.06 describes a standard procedure for the determination of total, saturated, and unsaturated fat in foods using capillary GC-FID. The procedure involves hydrolytic extraction, methylation, and capillary GC-FID analysis of the resulting fatty acid methyl esters (FAMEs). Triundecanoin, the C11:0 triglyceride, is used as an internal standard for quantitation, and response factors are included in method AOAC 996.06.

The method recommends a 100 m x 0.25 mm x 0.20 µm column with a highly polar biscyanopropyl stationary phase, such as the Rt-2560. The column must be able to separate the adjacent C18:3 and C20:1 FAME isomers as well as C22:1, C20:3, and C20:4 with a resolution of 1.0 or better. The Rt-2560 is specifically designed for FAMEs analysis and readily achieves these required critical separations as shown by peaks 22 to 24 and 29 to 31 in Figure 1.

Figure 1: Application-specific QC testing ensures that all Rt-2560 columns meet AOAC 996.06 method requirements.

PeakstR (min)Conc.
(µg/mL)
Structural Nomenclature
1.Methyl butyrate13.1640C4:0
2.Methyl caproate15.5340C6:0
3.Methyl octanoate19.4340C8:0
4.Methyl decanoate24.5040C10:0
5.Methyl undecanoate27.2020C11:0
6.Methyl dodecanoate29.8740C12:0
7.Methyl tridecanoate32.4720C13:0
8.Methyl myristate34.9740C14:0
9.Methyl myristoleate37.0120C14:1 (c9)
10.Methyl pentadecanoate37.3720C15:0
11.Methyl pentadecenoate39.3620C15:1 (c10)
12.Methyl palmitate39.6860C16:0
13.Methyl palmitoleate41.3020C16:1 (c9)
14.Methyl heptadecanoate41.8620C17:0
15.Methyl heptadecenoate43.4520C17:1 (c10)
16.Methyl stearate43.9740C18:0
17.Methyl octadecenoate44.9220C18:1 (t9)
18.Methyl oleate45.3440C18:1 (c9)
19.Methyl linolelaidate46.3920C18:2 (t9,t12)
20.Methyl linoleate47.3220C18:2 (c9,c12)
21.Methyl arachidate47.9240C20:0
22.Methyl linolenate48.7720C18:3 (c6,c9,c12)
23.Methyl eicosenoate49.2020C20:1 (c11)
24.Methyl linolenate 49.5420C18:3 (c9,c12,c15)
25.Methyl heneicosanoate49.7820C21:0
26.Methyl eicosadienoate51.0920C20:2 (c11,c14)
27.Methyl behenate51.6040C22:0
28.Methyl eicosatrienoate 52.5220C20:3 (c8,c11,c14)
29.Methyl erucate52.8820C22:1 (c13)
30.Methyl eicosatrienoate 53.2820C20:3 (c11,c14,c17)
31.Methyl tricosanoate53.4220C23:0
32.Methyl arachidonate53.6520C20:4 (c5,c8,c11,c14)
33.Methyl docosadienoate54.8520C22:2 (c13,c16)
34.Methyl lignocerate55.3240C24:0
35.Methyl eicosapentaenoate56.0920C20:5 (c5,c8,c11,c14,c17)
36.Methyl nervonate56.7420C24:1 (C15)
37.Methyl docosahexaenoate62.1720C22:6 (c4,c7,c10,c13,c16,c19)
Nutritional Labeling FAMEs on Rt-2560 by AOAC Method 996.06
GC_FF1261
ColumnRt-2560, 100 m, 0.25 mm ID, 0.20 µm (cat.# 13198)
SampleFood industry FAME mix (cat.# 35077)
Diluent:Hexane/dichloromethane
Conc.:1,000 µg/mL
Injection
Inj. Vol.:1 µL split (split ratio 20:1)
Liner:Premium 4 mm Precision liner w/wool (cat.# 23305.1)
Inj. Temp.:225 °C
Oven
Oven Temp.:100 °C (hold 4 min) to 240 °C at 3 °C/min (hold 15 min)
Carrier GasHe, constant flow
Flow Rate:1.0 mL/min
DetectorFID @ 285 °C
Make-up Gas Flow Rate:45 mL/min
Make-up Gas Type:N2
Hydrogen flow:30 mL/min
Air flow:300 mL/min
Data Rate:20 Hz
InstrumentAgilent 7890A GC

AOCS Ce 1j-07
AOCS official method Ce 1j-07 is very similar in scope and principle to AOAC 996.06 and other standard methods for GC-based determination of fat in foods. This method recommends triundecanoin, the C13:0 triglyceride, as an internal standard. Some suitable methods for extraction and methylation are referenced in the method, but no single approach is specified.

The same type of 100 m biscyanopropyl capillary GC column is recommended for AOCS Ce 1j-07, which requires baseline separation of C18:1 (cis-9) and C18:1 (cis-11). The column must also separate adjacent C18:3 and C20:1 isomers with a resolution of 1.0 or more. Figure 2 demonstrates that the Rt-2560 column meets these requirements and is suitable for AOCS Ce 1j-07, as well as other similar methods. In the AOCS method, a high initial GC oven temperature and long isothermal hold (180 °C for 32 minutes) decrease resolution for the more volatile saturated FAMEs, but may result in enhanced resolution of the C18:1 and C18:2 geometric and positional isomer clusters.

Figure 2: Application-specific QC testing ensures that all Rt-2560 columns meet AOCS Ce 1j-07 method requirements.

PeakstR (min)Conc.
(µg/mL)
Structural Nomenclature
1.Methyl caproate7.5240C6:0
2.Methyl octanoate7.8840C8:0
3.Methyl decanoate8.4840C10:0
4.Methyl undecanoate8.9320C11:0
5.Methyl dodecanoate9.5140C12:0
6.Methyl tridecanoate10.2720C13:0
7.Methyl myristate11.2740C14:0
8.Methyl myristoleate12.4820C14:1 (c9)
9.Methyl pentadecanoate12.5720C15:0
10.Methyl pentadecenoate14.1520C15:1 (C10)
11.Methyl palmitate14.2860C16:0
12.Methyl palmitoleate15.9820C16:1 (c9)
13.Methyl heptadecanoate16.5120C17:0
14.Methyl heptadecenoate18.6820C17:1 (c10)
15.Methyl stearate19.4340C18:0
16.Methyl octadecenoate21.0820C18:1 (t9)
17.Methyl oleate21.8540C18:1 (c9)
18.Methyl linolelaidate24.0920C18:2 (t9,t12)
19.Methyl linoleate26.1420C18:2 (c9,c12)
20.Methyl arachidate28.2540C20:0
21.Methyl linolenate29.9820C18:3 (c6,c9,c12)
22.Methyl eicosenoate32.0520C20:1 (c11)
23.Methyl linolenate32.4120C18:3 (c9,c12,c15)
24.Methyl heneicosanoate33.6620C21:0
25.Methyl eicosadienoate35.3320C20:2 (c11,c14)
26.Methyl behenate36.6440C22:0
27.Methyl eicosatrienoate37.4420C20:3 (c8,c11,c14)
28.Methyl erucate38.5120C22:1 (c13)
29.Methyl eicosatrienoate38.7220C20:3 (c11,c14,c17)
30.Methyl arachidonate39.1220C20:4 (c5,c8,c11,c14,c17)
31.Methyl tricosanoate39.7420C23:0
32.Methyl docosadienoate41.6420C22:2 (c13,c16)
33.Methyl eicosapentaenoate43.0720C20:5 (c5,c8,c11,c14,c17)
34.Methyl lignocerate43.1140C24:0
35.Methyl nervonate45.3320C24:1 (c15)
36.Methyl docosahexaenoate54.0220C22:6 (c4,c7,c10,c13,c16,c19)
Nutritional Labeling FAMEs on Rt-2560 by AOCS Method Ce-1j-07
GC_FF1262
ColumnRt-2560, 100 m, 0.25 mm ID, 0.20 µm (cat.# 13198)
SampleFood industry FAME mix (cat.# 35077)
Diluent:Hexane/dichloromethane
Conc.:1,000 µg/mL
Injection
Inj. Vol.:1 µL split (split ratio 20:1)
Liner:Premium 4 mm Precision liner w/wool (cat.# 23305.1)
Inj. Temp.:235 °C
Oven
Oven Temp.:180 °C (hold 32 min) to 215 °C at 20 °C/min (hold 31.25 min)
Carrier GasHe, constant flow
Flow Rate:2.0 mL/min
DetectorFID @ 325 °C
Make-up Gas Flow Rate:45 mL/min
Make-up Gas Type:N2
Hydrogen flow:30 mL/min
Air flow:300 mL/min
Data Rate:20 Hz
InstrumentAgilent 7890A GC
NotesC4:0 Methyl butyrate (623-42-7) elutes in the solvent front.

Conclusion
The improved manufacturing process and application-specific quality control test both ensure that Rt-2560 columns provide a consistently high level of performance in the detailed analysis of FAMEs. The Rt-2560 column meets method requirements for standard methods of fat speciation in food, including AOAC 996.06 and AOCS Ce-1j-07. In addition, Rt-2560 columns can be used for other methods that call for a 100 m high-percentage biscyanopropyl capillary GC column.

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AOCS Ce 1j-07