OMB Meeting Book_9-11-14

solution 2, D ( p )( 2 ), and 25.0 mL of the calibration standard stock solution 3, D ( p )( 3 ). Then make up to the mark with n -hexane. Dilute accordingly to the type of injector used. Note : This solution keeps for about 6 months if stored in the dark at –20°C. To prevent contamination of the standard solution, immediately distribute the solution into different vials (ready to inject) and store at –20°C before use. Use each vial once then discard it. ( q ) Commercially available calibration standard FAME mixture solution .—Before use, allow the ampoule to come to room temperature (maximum 25°C) in the dark without heating. Cut the ampoule with a glass knife and using a Pasteur pipet, rapidly transfer the content of the ampoule into a 50 mL pre-tarred volumetric flask, weigh, and make up to the mark with n -hexane. Dilute accordingly to the type of injector used. Note : This solution keeps for about 6 months if stored in the dark at –20°C. To prevent contamination of the standard solution, immediately distribute the solution into different vials (ready to inject) and store at –20°C before use. Use each vial once and then discard it. E. Preparation of Test Sample Milk powder and infant formula .—Mix well to ensure that sample is homogeneous. ( 1 ) Class or group of fatty acids in 100 g fat.— Calculate the mass fraction of all fatty acids included in a group or in a class of fatty acids by simple addition of individual fatty acids results (expressed in g FA/100 g fat). ( 2 ) Performance of the transesterification .—Record the areas of the two internal standard peaks (methyl undecanoate and tritridecanoin) in the analyzed samples. The performance of transesterification, PT expressed in %, is calculated on the recovery of the tritridecanoin as a second internal standard as follows: ܲ ݐ = ݉ ௖ଵଵ × ܣ ௖ଵଷ × ܴ ௖ଵଷ × ܵ ௖ଵଷ (ܶ )ܩܣ ܣ ௖ଵଵ × ݉ ௖ଵଷ × 100 where m C11 is the mass, in milligrams, of C-11:0 internal standard added to the solution; A C13 is the peak area of C-13:0 internal standard in the chromatogram; R C13 is the response factor of C13:0 relative to C11:0, calculated according to G ( a )( 2 ); S C13 is the stoichiometric factor to convert C13:0 FAME into C13:0 TAG; A C11 is the peak area of C-11:0 internal standard in the chromatogram; and m C13 is the mass of liquid milk and liquid formula. Bring sample to room temperature and shake vigorously before use. F. Procedure As a check on method performance, carry out two single determinations in accordance with F ( a ) and ( b ). Note : An alternative procedure using fat-extraction tubes with siphon or wash-bottle fittings is given in Figures 2012.13A–C . ( a ) Resolution between C18:1 trans and cis .—Inject once the qualitative cis/trans FAME isomers standard mixture solution, D ( o ). ( b ) Calibrating solution for the determination of response factor .—Inject three times the calibrated solution, D ( q ). ( c ) Test portion .—Into a 25 mL centrifuge tube with a screw cap, weigh to the nearest 0.1 mg an equivalent quantity of sample in order to have ca 50 mg fat in the tube ( Example : For a sample containing 26 g fat/100 g product, the corresponding sample weight is approximately 190 mg).

© 2012 AOAC INTERNATIONAL The resolution test is acceptable when value calculated for R is equivalent or higher than 1.00 (±5%) (to determine with the ring test). Note : Example of the calculation is given in Figure 2012.13C . ( 2 ) Calculation of response factor.— Inject into the gas chromatograph 1.0 μL of the calibrating solution, F ( b ). Determine the area of peaks attributable to each FAME present in the calibration standard mixture, D ( q ), and calculate their respective response factors (Rf i ) relative to the internal standard (C11:0): ܴ݂ ௜ = ݉Ԣ ௜ ȉ ܣ Ԣ ை ݉Ԣ ை ȉ ܣ Ԣ ௜ Note 1 : For fatty acid analysis on fat extracted from foods, the same amount of fat is required. For milk powder or infant formula powder, add 2.0 mL water using a micropipet. Close the tube, and then dissolve gently using a vortex mixer. Wait for 15 min at room temperature. Note 2 : For liquid milk samples and fat extracted from foods, no pretreatment (water addition) is required. Pipet 5 mL of internal standard solution, D ( j ). Add with a pipet 5 mL of 5% (w/v) methanolic sodium methoxide solution, D ( d ). The transesterification time starts with the addition of the first drop. Close the tube hermetically and shake well for 10 s using a vortex mixer. After 180 s, open the tube and add 2 mL hexane. After 210 s add 10 mL disodium hydrogen citrate and sodium chloride aqueous solution, D ( f ). The transesterification time stops after the addition of the last drop. Shake gently using a vortex mixer. The transesterification time should not exceed 240 s. Note 3 : To respect the total reaction time (240 s), the transesterification reaction should be carried out with a maximum of six tubes at the same time. Rapid delivery system (dispenser) can be used to add reagents, but not for the addition of internal standard solution. Centrifuge the tube at 1750 rpm (or equivalent rpm to g = 375 ± 25) for 5 min. Into a 10 mL volumetric flask, pipet 200 μL of the supernatant and make up to the mark with n -hexane. Note 4 : The dilution factor is calculated for on-column and splitless injection only. When using split injection, adapt the dilution to obtain the desired peak responses accordingly to split ratio used (ensure sufficient and accurate detection level for small peaks especially). Stored in the dark at 4°C, the sample solution after dilution is stable for 2 days. G. Determination ( a ) Quantitative determination. ( 1 ) Resolution.— Inject into the gas chromatograph 1.0 μL of the calibrating solution, F ( a ). Determine peak width at half height and distance between the left of the chromatogram and the top of peak for C18:1 trans Δ13/14 and oleic acid methyl ester. The resolution criteria (R) is calculated as follows: ܴ = 1.18( ݐ ோଶ െ ݐ ோଵ )/ܹ ൫ ଵ ଶൗ ൯ଵ + ܹ ൫ ଵ ଶൗ ൯ଶ where t R1 = distance, in centimeters, between the left of the chromatogram and the top of peak 1 (C18:1 trans Δ13/14); t R2 = distance, in centimeters, between the left of the chromatogram and the top of peak 2 (oleic acid); W (½)1 = peak width, in centimeters, at half height of peak 1 (C18:1 trans Δ13/14); W (½)2 = peak width, in centimeters, at half height of peak 2 (oleic acid).