OMB Meeting Book_9-11-14

techniques (Ag + ) followed by a capillary GLC analysis. A part of this phenomenon is explained by presence of some C18:1 cis isomers (Δ6-8) which elute in the C18:1 trans and consequently are indirectly added to the sum of C18:1 trans and compensate the fact that C18:1 trans Δ15 is not taken into account. C18:2 TFA .—Identify and group all TFA of linoleic (C18:2 n-6) acids ( see Figures 2012.13A and B ). For the total TFA of C18:2, include all the trans isomers present in milk fat sample as shown in Figures 2012.13A and B . C18:3 TFA .—Identify and group all TFAof linolenic (C18:3 n-3) acids ( see Figures 2012.13A and B ). Note : In presence of milk fat and/or fish oil in the sample, another isomer of C20:1 elutes just before C20:1 n-9. Depending on the column resolution, the retention time of this fatty acid may also correspond to a trans isomer of C18:3 n-3 (the c,t,c or t,c,c). When there is only one peak in the corresponding zone of C18:3 TFA, its correct identification corresponds to a C20:1 isomer. When two, three, or four peaks are encountered in the corresponding zone for C18:3 TFA, each peak area should be included in the total areas of C18:3 TFA ( see elution order and formation rules below). Interferences could be also observed between C18:3 TFA isomers (C18:3 c,c,t; c,t,c; or t,c,c) and C20:1 n-9. When C20:1 n-9 elute with C18:3 c,t,c, (the minor C18:3 TFA isomer), their contribution on the total C18:3 TFA is negligible. However, if C20:1 n-9 is interfered with C18:3 c,c,t or with C18:3 t,c,c the chromatography conditions should be slightly modified to obtain sufficient separation. Interference is also visible when wrong ratio between C18:3 n-3 c,c,t and C18:3 n-3 t,c,c is observed (normal ratio is always 5:4). The kinetics of C18:3 trans isomers formation in refined and deodorized oils have been analyzed using highly polar capillary column and described in the literature. They could be used as a

where m′ i

= mass fraction of FAME i

in the calibration standard

solution, D ( q ); A′ O

= peak area of C11:0 in the calibration standard = mass of C11:0 in the calibration

solution chromatogram; m′ O standard solution, D ( q ); and A′ i

= peak area of FAME i

in the

calibration standard solution chromatogram. The variation between three injections is optimal when coefficients of variation are less than 2. Note : The response factors calculated for C18:2 n-6 could be applied for C-18:2 CLA and that calculated for C18:3 n-3 cis could be applied for C18:3 trans isomers. ( 3 ) Determination of the test portion .—Inject 1 μL of the test portion, F ( b ), into the gas chromatograph applying the same conditions as used in the calibrating solution. ( b ) Fatty acid identification .—Identify the fatty acids in the sample solution chromatogram by comparing their retention times with those of the corresponding peaks in the calibration standard solution, D ( q ), and in the qualitative standard mixture containing TFAs and CLA, D ( o ). C18:1 TFA .—Identify and group all TFA of C18:1 (include also the peak area of trans C18:1 trans Δ16 eluted in the cis C18:1 region just after the oleic acid) according to Figures 2012.13A and B . Note : When milk fat is present, two trans isomers of C18:1 are eluted in the cis C18:1 region (the C18:1 trans Δ15 and C18:1 trans Δ16, respectively), but only one (C18:1 trans Δ16) is resolved with the 100 m length capillary column. The second isomer (C18:1 trans Δ15) is generally overlapped with the oleic acid peak (C18:1 cis Δ9) and its area is only quantifiable using a preliminary separation (TLC Ag + , HPLC Ag + ) followed by a capillary GLC analysis. According to recent findings, it has been demonstrated that there is not significant difference of total C18:1 trans amount when the area of C18:1 trans Δ15 (not resolved peak) is not included in the sum in comparison to the result obtained after preliminary separation

Figure 2012.13A. Example of GC chromatogram (enlarged view of C18:1 TFA, C18:2 TFA, and CLA) using split injection.

© 2012 AOAC INTERNATIONAL