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Applying Principles of High Speed Gas Chromatography to Reduce Analysis Times in the Speciation of Fats by GC Using EZGC Online Software

Jaap de Zeeuw, Rebecca Stevens*, Jennifer Rutherford, & Linx Waclaski.

Overview Determination of fat content in food is achieved through GC

analysis of the corresponding fatty acid methyl esters (FAMES)

Speciation of fats into saturated, monounsaturated, polyunsaturated, and trans categories is required for

nutritional labeling

A growing body of research has implicated elevated trans fat intake with cardiovascular disease, FDA recently determined that partially hydrogenated vegetable oils (PHVO’s) are no

longer generally recognized as safe (GRAS)

The separation of positional and geometric isomers (cis/trans) of unsaturated FAMES is challenging, standard

approaches involve 100m x 0.25mm ID capillary GC columns containing 90+% bis-cyanopropylpolysiloxane, analysis times

routinely exceed 1 hour

High Speed GC and Method Translation

Method translation is used to calculate new method parameters for different columns, carrier gases, and outlet

pressures. The elution temperature of each analyte is unchanged from the original method so that the

chromatographic profile is maintained

Software for performing method translation is freely available at http://www.restek.com/ezgc-mtfc

Separations and Calibration Analysis of PHVO’s in CandyFAME Peak # Alias

C4:0 1C6:0 2C8:0 3C10:0 4C11:0 5C12:0 6C13:0 7C14:0 8C14:1 (t9) 9C14:1 (c9) 10 MyristoleicC15:0 11C15:1 (t10) 12C15:1 (c10) 13C16:0 14C16:1 (t9) 15C16:1 (c9) 16 PalmitoleicC17:0 17C17:1 (t10) 18C17:1 (c10) 19C18:0 20C18:1 (t6) 21C18:1 (t9) 22 ElaidicC18:1 (t11) 23 VaccenicC18:1 (c6) 24C18:1 (c9) 25 OleicC18:1 (c11) 26C18:2(t9,t12) 27C19:1 (t7) 28C19:1 (t10) 29C18:2(c9,c12) 30 LinoleicC20:0 31C20:1T (t11) 32C18:3(c6,c9,c12) 33 γ-LinolenicC20:1 (c11) 34 EicosenoicC18:3(c9,c12,c15) 35 α-LinolenicC21:0 36C20:2 (c11,c14) 37C22:0 38C22:1T (t13) 39C20:3 (c8,c11,c14) 40 DGLAC22:1 (c13) 41 ErucicC20:3(c11,c14,c17) 42C23:0 43C20:4 (c5,c8,c11,c14) 44 ArachidonicC22:2 (c13,c16) 45C24:0 46C20:5 (c5,c8,c11,c14,c17) 47 EPAC24:1 (C15) 48 NervonicC22:6 (c4,c7,c10,c13,c16,c19) 49 DHAC18:1 (t9, OH-12) 50

A 50 component mix of FAMES in hexane at 0.90 mg/mL was used for evaluation of different high speed GC

configurations

FAMES spanned a range of 1-10 % w/w and are representative of major fatty acids found in meat, fish, dairy,

and vegetable oils

A shorter 70m x 0.18mm x 0.18mm bis-cyanopropylsiloxane column was employed as well as hydrogen carrier gas to

reduce analysis time while maintaining high peak capacity

Complex FAME separations are very sensitive to column loading, carrier gas flow, and oven temperature program

A commercial caramel candy listing partially hydrogenated soybean oil and cream as ingredients was chosen to

highlight a complex mixture of FAMES

A 2.16 g sample of candy was subjected to hydrolytic extraction and BF3-MeOH transmethylation according to AOAC method 996.06 followed by both high speed and

standard GC determination

The combination of dairy fat and PHVO gave a complex mixture of 18:1 and 18:2 trans isomers, several unknown isomers were observed but their area not peak summed.

Detailed separation of positional isomers is traded for speed on shorter, wider bore columns. Sample loading capacity was

acceptable for both 0.25mm ID and 0.18mm ID columns

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60Minutes

24 25 26 27 28 29 30 31 32 33

40 41 42 43 44 45 46 47 48 49 50 51 52 53

13 14 15 16 17 18

Overall Profile

100m x 0.25mm, He 60 min analysis

100m x 0.25mm, H2 36 min analysis

70m x 0.18mm, H2 19 min analysis

AOAC 996.06100m x 0.25mm, He

12 14Minutes

70m x 0.18mm, H2

GC Method Total Fat (mg)

Sat.(%)

MUFA(%)

PUFA(%)

Trans (%)

100m x 0.25mm, He 243.2 2.8 4.4 0.8 2.3

100m x 0.25mm, H2 236.2 2.8 4.2 0.8 2.2

70m x 0.18mm, H2 216.9 2.4 3.8 0.7 2.3

4x dilution, 50:1 split

4x dilution, 75:1 split

2 3 45

6

7

8

14

11

2022

27 31 38 46

22

25

21 26

2830 31

3437

39 40 43 4446

50

48

37

37

46

46

21

21

31

31

34

34

44

44

39

39

20

20

21-23

21-23

30

30

27

27

26

26

48

48

* Pharmasol Corporation, One Norfolk Avenue, South Easton, MA 02375