Simplified Method for the Simultaneous Gas Chromatographic Determination of Fatty Acid Composition and Cholesterol in Food

Lebensm.-Wiss. u.-Technol., 30, 202–209 (1997)

Simplified Method for the Simultaneous Gas Chromatographic Determination of Fatty Acid Composition and Cholesterol in Food Eva Paterson and Renato Amado* ` Swiss Federal Institute of Technology, Institute of Food Science, ETH-Zentrum, CH-8092 Zurich ¨ (Switzerland) (Received April 22, 1996; accepted June 7, 1996)

A rapid, reliable and accurate gas chromatographic method for the determination of fatty acid composition in food was developed and evaluated. The procedure consists of direct saponification of glycerides and hexane extraction of sterols from the nonsaponifiable matter, followed by acidification of the residual soap and methylation of the free fatty acids. The substantial advantage of this procedure is the possibility of simultaneous determination of the fatty acid composition and the cholesterol of phytosterol content in food. In addition, it eliminates the use of chlorogenic chemicals and minimizes solvent use. This method was compared to a slightly modified standard procedure, involving fat extraction prior to saponification and transmethylation of the fatty acids. For both methods the coefficients of variation for analysed fatty acids (C ≥ 8) ranged from 0.6 to 7.4%. For the majority of fatty acids no significant difference was observed between mean values at a 95% confidence level. ©1997 Academic Press Limited Keywords: fatty acids; cholesterol; gas chromatography

Introduction The main interest from a nutritional point of view with regard to fat fraction in food focuses on its fatty acid composition and cholesterol content. Both these food constituents have a very important influence on the prevalence of cardiovascular disease (1). Although the effects of the widely recommended low cholesterol, high ratio of polyunsaturated to saturated fatty acids, and low total fat content diet still remain controversial (2), it is useful to have at hand fast, simple, reliable and accurate methods for the estimation of these substances. According to recent publications (3,4), the preferred methods for cholesterol assay use a direct saponification of the food sample and extraction of the unsaponifiable matter with an organic solvent like hexane, rather than the initial lipid extraction usually performed using a chloroform–methanol mixture or diethylether as solvents, followed by saponification of the polar fraction. The advantages of this approach have been widely discussed (5). However, for quantitative determination of the fatty acid content in food, methods are still in use which are based on lipid extraction as a first step (6). Nevertheless, in biochemistry, attempts were undertaken to simplify the quantitative determination of fatty acids in plasma by omitting the lipid extraction step (7). Several one-step extraction/methylation methods were described based on the reaction with methanolic hydrochloric acid in *To whom correspondence should be addressed.

benzene or other solvents (8–11). Takeuchi et al. (12) recommended direct sample saponification in ethanolic alkali for fish diets and fish faeces if all fatty acids present are of interest. The procedure introduced in this paper consists of the direct saponification of food samples followed by the separation of saponifiable and unsaponifiable matters with hexane. These two fractions are used as starting material for the quantitative determination of both fatty acids and cholesterol in samples. This approach allows the most important fat components to be analysed in one procedure, thus saving time. Since no chlorogenic solvents are required in the extraction step, exposure to potentially hazardous vapours is reduced and disposal costs associated with these solvents are eliminated. In order to verify obtained results, the developed procedure was compared to the slightly modified standard method with initial fat extraction as described by Winter (13), followed by saponification and transesterification according to Metcalfe et al. (14). The modification consists of reducing the sample weight and the quantity of extractants by a factor of ten. This approach not only minimizes the use of hazardous solvents but also enables the addition of an internal standard at the beginning of the determination procedure. The compensating nature of the internal standardization method minimized the error source in all following steps. Due to the reduced amount of solvents, it is possible to accelerate the separation of the

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Food sample

chloroform–methanol–water mixture into two layers by centrifugation thus saving analysis time. The present paper describes the quantitative determination of the fatty acid composition of food samples. This method is not suitable for the determination of short chain fatty acids (C < 8). Quantification of cholesterol content in food has been extensively treated in another publication (15).

*enzymatic degradation of starch

lipid extraction

separation of lipids

Experimental Details

saponification

Chemicals All reagents were of analytical reagent grade unless specified otherwise. Pyrogallol, methyl tridecanoate, boron trifluoride ( ~ 1.3 mol/L) in methanol, n-hexane, ethanol, methanol, chloroform, anhydrous sodium sulphate, seasand (acid purified) and Clara-Diastase (No.10065) were obtained from Fluka Chemie AG (Buchs, CH). 5α-Cholestane, cholesterol and tridecanoic acid were supplied by Sigma Chemical Co. (Munich, D). GLC Standard Mixtures of fatty acid methyl esters (FAME) were purchased from Matreya Inc. (Pleasant Gap, PA, U.S.A.). These products are equal weight (10 or 12.5 mg) mixtures of FAME. Thermoresistent α-amylase preparation Termamyl 120L was supplied by Novo Nordisk A/S. (Bagsvaerd, DK).

Food sample

*enzymatic degradation of starch

saponification

separation

(hexane phase)

(water phase)

nonsaponifiable fraction

saponifiable fraction

cholesterol

recovery of fatty acids

phytosterols tocopherols

separation of fatty acids

esterification

fatty acid methyl esters

Fig. 1 Direct method for quantitative determination of fatty acids and sterols in foods. * = for starch-rich food samples only

esterification

fatty acid methyl esters

Fig. 2 Modified extraction method for determination of fatty acids in foods. * = for starch-rich food samples only

Solutions Calibration solution for FAME was prepared by transferring the GLC Standard Mixtures No. GLC-101, GLC-30, GLC-50, GLC-60 and GLC-100 into a 100 mL volumetric flask and diluting to the mark with hexane. The final concentration of the single FAME ranged from 10 to 22.5 mg/100 mL. The solution was stored at 4 °C. Internal standard solution for FAME determination was prepared by dissolving approx. 100 mg ( ± 0.1 mg) tridecanoic acid (C13:0) in 50 mL n-hexane. Internal standard solution for cholesterol determination was prepared by dissolving approx. 25 mg ( ± 0.1 mg) of 5α-cholestane in 50 mL n-hexane. Potassium hydroxide solution was made by carefully dissolving 50 g KOH pellets in 50 mL deionized water. Pyrogallol solution was prepared by dissolving 5 g pyrogallol in 500 mL ethanol, methanol or water, respectively, as required by the different procedures (see below).

Sample preparation Food samples for the analyses and all ingredients for the meal were obtained in a local shop (Migros, Ruschlikon, ¨ CH). The meal — pasta with meat sauce — was prepared as follows: 152 g minced beef was fried for 10 min in 10 g olive oil. The resulting weight was 110 g. The tomato sauce contained 30 g cut onions (fried for 3 min in 10 g butter) and 230 g of canned tomatoes. The sauce was boiled for 5 min. The final weight of the tomato sauce amounted to 245 g. Tomato sauce and fried meat were mixed with 250 g of cooked pasta (12 min in an excess of boiling water) and the meal was sprinkled with 10 g of Parmesan cheese. The finished test meal (615 g) was homogenized in a mixer, divided into small (approx. 10 g) portions and kept in the freezer at –18 °C until analysed.

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U.K.), heating block Reacti-Therm III™, heating/ stirring module (Pierce Chemical Co., Rockford, IL, U.S.A.) and evaporator Rotavapor RE 111 with Water Bath 461, (Buchi ¨ Laboratoriums-Technik AG, Flawil, CH).

5

10

15

20

C24:1 (15)

C22:0 C22:1 (13)

C20:3 (11, 14, 17)

C18:3 (9, 12, 15)

C20:2 (11,14)

C19:0

C21:0

C20:0 C20:1 (11)

Homogenized food samples of 1.5 to 2.5 g, accurately weighed, were placed in a 50 mL centrifuge tube. The sample weight of fat-rich food like butter or mayonnaise must be reduced to approx. 0.2 g (the limiting factor is the concentration of KOH). One millilitre each of both internal standard solutions (5α-cholestane and C13:0), 9 mL pyrogallol in ethanol (1 g/100 mL), 1 mL potassium hydroxide solution (50 g/100 g), a magnetic stirring bar, and approx. 1.5 g of seasand for a better fat dispersion were added and the sample vortexed for 20 s. The capped tube was placed in a water-bath at 60 °C and saponified for 1 h under continuous stirring. After cooling to room temperature, 5 mL of deionized water was added followed by 10 mL hexane and the sample vortexed at the highest setting for 2 min. The sample was then centrifuged for 3 min at 2200 3 g and the upper hexane layer transferred with a Pasteur pipette into a

C18:2 (9, 12)

C17:0

C16:1 (9)

C15:0

C14:0 0

Procedure of the direct method (Fig. 1)

C18:0 C18:1 (9)

C16:0

C12:0 C13:0 (I.S.)

C8:0 C10:0

Instrumentation and operating conditions A gas chromatograph (GC) Hewlett Packard Model 5890 Series II with a split/splitless capillary injection system and a flame ionization detector (FID) equipped with an autosampler HP 7673 (Hewlett Packard, Avondale, PA, U.S.A.) was used. Operating conditions were: helium carrier gas at 1.7 mL/min, head pressure 100 kPa, 1:40 split ratio, injection port temperature 240 °C, detector temperature 280 °C, initial oven temperature 160 °C held for 4 min, then increased up to 230 °C at a rate 4 °C/min and held for 3.5 min at this temperature. Injection volume was 2 µL. The GC separation was accomplished by using a fused silica capillary column Supelcowax™ 10, 30 m 3 0.32 mm i.d. and 0.25 µm film thickness (Supelco, Inc., Bellefonte, PA, U.S.A.). Peak areas were integrated and calculated by Hewlett Packard 3365 ChemStation software. Saponification and separation of layers were carried out in 50 mL screw capped centrifuge tubes Cortex® Brand (Corning Inc., Corning, NY, U.S.A.). The following apparatus were also used: water-bath Julabo U3 (Julabo Labortechnik GmbH, Seelbach, D), magnetic stirrer/hot plate Heidolph MR 2002 (Heidolph GmbH, D), shaker Vortex Genie 2™ (Scientific Industries Inc., Bohemia, NY, U.S.A.), centrifuge MSE Mistral 1000 (MSE Scientific Instruments, London,

25

Retention time (min)

Fig. 3 Gas chromatogram of the standard mixture of a fatty acid methyl esters. Column: Supelcowax™ 10, 30 m 3 0.32 mm i.d., film thickness 0.25 µm, carrier gas helium at 1.7 mL/min; oven temperature programme: 160 °C (4 min), to 230 °C (4 °C/min), 230 °C (3.5 min), injection 2 µL, split ratio 1:40

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C19:0 and BHA

Well homogenized food samples of 1.5 to 2.5 g, accurately weighed, were placed in a 50 mL centrifuge tube. Sample weight of fat-rich food like butter or mayonnaise must be reduced to approx. 0.5 g. One millilitre of internal standard solution for

C18:3 (9, 12, 15)

C18:2 (9, 12)

C18:1 (9)

Procedure of the modified extraction method (Fig. 2)

5

C17:0

C15:0 0

L). The tube was capped tightly and placed in a heating block for 4 min at 100 °C. Tubes were cooled in cold tap water to room temperature and 4 mL hexane added, followed by 2 mL water. The sample was vortexed vigorously for 2 min and centrifuged for 3 min at 2200 3 g. Approx. 1.5 mL of the hexane extract was transferred into the GC-sample vials and 2 µL injected into the gas chromatograph. In order to prevent incomplete extraction of fat and/ or cholesterol in starch-rich food samples like cooked pasta it is necessary to perform an enzymatic degradation of the gelatinized starch prior to saponification: 0.25 mL of thermoresistent α-amylase preparation Termamyl 120L was mixed with 4.75 mL pyrogallol in deionized water (1 g/100 mL), and added to the sample prior to saponification. The tube was capped and held for 15 min at 95 °C in a waterbath under continuous stirring. For extraction of the unsaponifiable matter addition of water was omitted.

C16:1 (9)

C12:0

C8:0

C10:0

C14:0

C18:0

C16:0

C13:0 (I.S.)

clean 50 mL centrifuge tube. Another 10 mL hexane was added to the water phase and the extraction and centrifugation step repeated. The combined hexane extracts can be directly used for cholesterol determination as described by Paterson et al. (15). The water phase was acidified with 3 mL hydrochloric acid (HCl) (25 g/100 g) to pH ≤ 1.5, and shaken briefly. Released free fatty acids were extracted with 10 mL hexane. The sample was vortexed at the highest setting for 2 min and centrifuged for 3 min at 2200 3 g. The upper hexane layer was transferred with a Pasteur pipette into a clean 50 mL centrifuge tube and another 10 mL hexane added to the water phase. The extraction and centrifugation steps were then repeated. The first and second hexane extracts were combined using the same pipette. In order to dry the extract, approx. 4 g of anhydrous sodium sulphate was added to the centrifuge tube and shaken vigorously for a short time. The extract was centrifuged for 3 min at 2200 3 g and 4 mL of the clear extract (containing 10 to 50 mg of fatty acids) transferred into a 15 mL culture tube fitted with a screw cap. The extract was concentrated in a rotatory evaporator under reduced pressure of 200 mbar for 3 min nearly to dryness. The residue was redissolved in 1 mL of hexane and then a magnetic rod placed in the tube. Fatty acids were converted into methyl esters by adding 5 mL boron trifluoride in methanol (1.3 mol/

10

15

20

25

Retention time (min)

Fig. 4 Representative gas chromatogram of fatty acid methyl esters in food sample (pasta with meat sauce). Chromatograph conditions as in Fig. 3

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Table 1 Fatty acid composition of minced beef, Parmesan cheese and mayonnaise in mg/100 g fresh sample Minced Beef Ext Fatty acid

Meana

CV

C 8:0 – C 10:0 – C 12:0 – C 14:0 163.1 2.2 C 15:0 27.1 1.9 C 16:0 1507.9 2.7 C 16:1(9) 214.7 3.3 C 17:0 73.3 4.3 C 18:0 939.2 2.6 C 18:1(9) 2398.5 2.9 C 18:2 (9,12) 214.2 1.0 C 18:3 (9,12,15) 54.8 2.1 C 19:0 – C 20:0 – C 20:1(11) – C 20:2 (11,14) – C 20:3 (11,14,17) 37.4 3.7 C 22:0 – C 22:1(13) – C 24:1(15) – Total fatty acids

Parmesan Cheese Dir

5630.2

Meana

Ext Meana

CV

Meana

CV

2.6 2.3 2.3 1.9 4.0 2.4 2.4 2.9 1.8

202.8 487.4 640.1 2261.4 268.5 5950.9 267.5 170.6 2308.2 3974.6 400.4 131.6 21.6 52.0 48.4

5.2 3.0 5.6 4.5 4.0 3.9 4.5 4.2 4.2 3.8 3.6 3.2 3.4 5.2 5.8

213.4 497.1 642.8 2235.2 263.1 5761.9 261.7 166.1 2214.6 3821.2 386.6 125.7 22.3 49.1 48.8

4.1 3.1 3.2 3.2 3.3 3.4 3.7 3.7 4.1 3.8 3.8 3.6 3.4 4.4 5.6

5.9 6.8

34.2 23.1

– – – – 39.0

Ext

CV – – –

161.0 26.4 1490.2 211.4 69.7 926.0 2350.8 220.9 55.6

Mayonnaise

Dir

– 3.5

32.2 24.6

– – – 5551.0

Meana

– –

– –

17242.8

16766.9

CV

Meana

– – –

CV – – –

59.4

2.4

62.0

–

4.9 –

5275.3 125.4

0.6 1.2

5300.7 125.1

0.7 0.8 1.0 1.1

3679.7 16214.2 44818.4 171.7

1.1 0.9

214.5 99.2

–

2.9 5.8 –

3736.9 16410.6 45397.1 169.6 –

3.0 2.8 2.2 4.9 –

223.0 100.0

– 3.3 3.1

Dir

– –

3.1 3.6 – –

581.7

1.6

551.4

–

2.4 –

156.3

1.5

72235.3

142.7

2.8

71379.6

a n= 4;

CV = coefficient of variation in %. – = content of fatty acid less than 10 mg/100 g fresh sample. Ext = modified extraction method; DIR = direct method.

FAME (C13:0) was added and mixed with 8 mL chloroform and 8 mL pyrogallol in methanol (1 g/100 mL). The sample was shaken vigorously for 2 min and another 8 mL chloroform added. The sample was

filtered with slight suction through a sintered glass funnel coated with approx. 2 g of Celite and rinsed with 4 mL mixture of chloroform/methanol 2:1. For separation into two phases, z mL water was added,

Table 2 Fatty acid composition of cooked pasta in mg/100 g fresh sample determined without and with enzymatic starch degradation Ext Enzyme (concentration and reaction conditions) Fatty acid C 8:0 C 10:0 C 12:0 C 14:0 C 15:0 C 16:0 C 16:1(9) C 17:0 C 18:0 C 18:1(9) C 18:2 (9,12) C 18:3 (9,12,15) C 20:0 C 20:1(11) C 20:2 (11,14) C 20:3 (11,14,17) C 22:0 C 22:1(13) C 24:1(15) Total fatty acids

Ext

none Meana

CV

Meana

– – – – – 89.3 6.9

CV

Meana

– – – – – 7.6 8.3

216.6 13.7

8.5 9.0 5.4 4.1

44.1 248.7 356.1 19.0

– 21.4 121.5 162.8 8.4

Ext

2.6 3.2

192.0 11.6

2.9 2.8 2.7 5.3

40.0 216.0 302.5 14.0

410.3

2.6 3.2

256.6 15.7

2.9 2.8 2.7 5.3

49.1 288.7 426.1 19.3

8.0

Dir

none

Termamyl 120 L (0.25 mL in 5 mL water, 95 °C, 15 min)

Meana

2.9 3.3

166.6 13.4

3.0 2.6 2.8 4.8

38.0 238.6 341.3 16.1

9.1

Meana

8.8 10.8

262.2 15.1

6.5 10.7 9.1 8.6

51.3 286.0 428.3 19.8

8.5

2.3 2.4 –

– – – 3.5

CV – – – – –

–

– – – 3.7

CV – – – – –

–

– – – 4.1

CV

Ext

– – – – –

–

– – – 7.4

CV

Meana

– – – – –

–

– – – – – – –

Dir

Clara-Diastase Termamyl 120 L Termamyl 120 L (0.15 g in 15 mL (0.25 mL in 5 mL (1.0 mL in 5 mL sodium acetate water, water, buffer, 40–45 °C, 1 h) 95 °C, 15 min) 95 °C, 30 min)

4.3 2.3 3.2 1.6 – – –

10.5

9.5

3.0

– – –

– – –

– – –

– – –

– – –

905.6

784.1

1064.6

822.5

1072.2

a n=4;

CV= coefficient of variation in %. –= content of fatty acid less than 7 mg/100 g fresh sample. Ext=modified extraction method; Dir=direct method.

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Table 3 Fatty acid composition of the prepared meal (pasta with meat sauce) in mg/100 g fresh sample determined without and with enzymatic starch degradation with Termamyl 120L With starch degradation Ext 1.0 mL in 5 mL water, 95 °C, 30 min

Without starch degradation Ext Fatty acid C 8:0 C 10:0 C 12:0 C 14:0 C 15:0 C 16:0 C 16:1(9) C 17:0 C 18:0 C 18:1(9) C 18:2 (9,12) C 18:3 (9,12,15) C 20:0 C 20:1(11) C 20:2 (11,14) C 20:3 (11,14,17) C 22:0 C 22:1(13) C 24:1(15) Total fatty acids

Dir

Meanb

CV

19.1 43.3 58.3 205.4 23.3 984.6 81.8 23.6 397.9 1842.3 324.1 36.9

5.5 5.2 3.6 3.7 3.6 4.4 4.4 4.2 5.9 5.3 6.6 5.2

Meanb 21.1 45.7 62.7 213.9 23.7 1020.8 85.3 24.4 396.1 1888.5 339.3 40.3

5.9 5.0 4.6 3.7 2.7 2.5 2.6 6.2 3.7 2.1 6.2 5.4

– – – 10.2

CV

Meana 20.5 43.9 61.2 217.6 24.8 1051.9 86.4 24.6 412.1 1909.7 310.3 38.6

– – – 4.3

10.7

CV 5.2 4.4 2.8 2.5 2.4 2.2 2.3 2.8 2.1 2.2 3.2 2.3

Dir 0.25 mL in 5 mL water, 95 °C, 15 min Meana 21.0 44.7 59.4 208.4 23.3 1005.8 83.0 25.6 393.2 1896.5 337.0 39.0

– – – 5.4

10.6

CV 4.7 5.7 3.0 3.1 3.7 3.7 3.2 4.6 2.2 3.4 3.3 4.6 – – –

7.4

12.2

2.1

– – –

– – –

– – –

– – –

4050.8

4172.5

4212.2

4149.1

a n= 4; bn = 8;

CV=coefficient of variation in %. – = content of fatty acid less than 10 mg/100 g fresh sample. Ext = modified extraction method; Dir=direct method.

where z = 6 – (P 3 W/100), P = sample weight in g and W = water content of sample in g/100 g. The sample was vortexed for 2 min and centrifuged for 3 min at 2200 3 g. The upper layer was removed with a Pasteur pipette and the remaining chloroform phase dried by adding approx. 2 g of anhydrous sodium sulphate. Four millilitre of the dried chloroform phase (containing 10 to 50 mg of fatty acids) was transferred into a 15 mL culture tube fitted with a screw cap and concentrated in a rotatory evaporator under reduced pressure of 300 mbar for 5 min nearly to dryness. The residue was redissolved in 1 mL hexane and a magnetic rod placed in the tube. The content was saponified by adding 4 mL of NaOH (0.5 mol/L) in dry methanol and placing the capped tube for 5 to 10 min into the heating/stirring block at 100 °C under continuous stirring. Conversion of the fatty acids into methyl esters and separation of methyl esters was made as described for the direct method, except that (because of the larger end volume) the content of the 15 mL culture tube must be transferred into a larger culture tube before separation. For starch-rich food samples it is necessary to apply an enzymatic degradation of gelatinized starch prior to fat extraction: 1 mL of thermoresistent α-amylase preparation Termamyl 120L was mixed with 4 mL pyrogallol in deionized water (1 g/100 mL) and this solution added to the sample. The tube was capped and held for 30 min at 95 °C in a water-bath under

continuous stirring. For separation into two phases z – 5 mL of water was added.

Optimization of the Termamyl treatment In order to optimize the lipid extraction for starch-rich food samples, incubation experiments with the α-amylase preparation Termamyl 120L were carried out at different enzyme concentrations and for different reaction times. Various amounts of Termamyl 120L (100, 200, 500 and 1000 µL) were added to the samples and incubated at 95 °C for 15, 30 and 60 min. The concentration of liberated D-glucose at the end of the incubation period was chosen as an indicator of optimal conditions for the Termamyl action. Determination was performed using an enzymatic assay according to the description given by the Food Analysis Boehringer Mannheim Test No. 716251.

Statistical analysis The measured values were evaluated by descriptive statistics (means and coefficients of variation). Differences between the mean contents of each fatty acid in all examined samples for both methods were analysed with the statistical program of Microsoft® Excel Version 5.0 (Microsoft Corporation, Redmond, WA, U.S.A.). The paired two-sample t-test for means at the 95% confidence level was used.

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Results and Discussion In order to compare the direct method (Fig. 1) with the modified extraction method (Fig. 2) for the determination of fatty acid content and distribution in foodstuff, a test meal (pasta with meat sauce), as well as some of its ingredients, and mayonnaise were analysed. The lower detection limit for the quantitative determination of fatty acids in the meal was found to be approx. 10 mg per 100 g fresh sample. The chromatogram of the calibration solution of the standard mixture of FAME (Fig. 3) and a representative chromatograph of the food sample (Fig. 4) showed excellent separation of all fatty acids of interest. The selection of fatty acids can be extended for example by C20:4 and C20:5 without any change of the operating conditions for GC (data not shown). The data for minced beef, Parmesan cheese, and mayonnaise are summarized in Table 1. The results indicated a high degree of precision for both methods as shown by low coefficients of variation (CV) for the single fatty acids, ranging from 0.6 to 6.8. It was evident that both methods led to equivalent results. Application of the paired two-sample t-test at the 95% confidence level revealed no significant differences between the two methods for most of the fatty acids. A possible explanation for the only fatty acid, Eicosatrienoic acid (C 20:3), showing a different outcome of the statistical analysis could be its low concentration in some food samples making accurate integration of peaks more difficult. However, substantial differences resulted for cooked pasta. The extraction method gave much lower yields of fatty acids when compared to the direct method (Table 2). It was obvious that part of the lipids was ‘entrapped’ by the gelatinized starch and was thus not extracted. Because of this problem, already described by the method for determination of cholesterol (16), it was supposed that even by the direct method not all lipids were liberated and analysed. The data from Table 2 confirmed this assumption. The necessity to degrade the gelatinized starch in order to release total lipids has already been recognized. The official method described in the Swiss Manual for Food Analysis (17) uses ClaraDiastase (an α-amylase preparation from Aspergillus oryzae) for starch degradation. In our experiments the effectiveness of the Clara-Diastase was compared to thermoresistent α-amylase Termamyl 120L. Clara-Diastase at the recommended concentration level improved lipid extraction from cooked pasta but the amount of determined fatty acids was lower as compared to the amount obtained by the use of Termamyl 120L (Table 2). Moreover, Clara-Diastase could not be used in the direct method prior to the saponification step because this enzyme needs buffered conditions for its action. Another important advantage of using Termamyl 120L in the extraction method is that only half the time (30 min instead of 60) is needed for sufficient starch degradation as compared to ClaraDiastase. The need for a preliminary degradation of starch for the test meal was also studied. The results are summarized in Table 3 and indicate that this step could

be omitted. This finding was expected because the percentage of fatty acids in pasta is low (Table 2) and the share of pasta in the whole meal makes approx. 42%. The diminution of the total fatty acid content in the meal caused by the insufficiently extracted pasta was 6.5% for the extraction method and 2.5% for the direct method only. It was therefore within the methods’ precision. Preliminary experiments proved the necessity to prevent the oxidation of unsaturated fatty acids in the course of sample preparation. Addition of 3-tert-butyl4-hydroxyanisole (BHA) solution (1 g/100 g) in ethanol (direct method) or methanol (modified extraction method) led to two problems. Figure 4 reveals that BHA coeluated with methyl ester of nonadecanoic acid (C19:OMe) under the selected operating conditions. This microbial fatty acid had been shown to be present in Parmesan cheese used in the meal (Table 1). Second, the insolubility of BHA in water prevented its use during the starch degradation step. Therefore pyrogallol at a concentration of 1 g/100 mL was used as antioxidant. This substance is soluble in water, ethanol and methanol but insoluble in apolar solvents like hexane or in chloroform. The protective effect of pyrogallol is therefore limited to the enzymatic starch degradation and to the saponification step. However, these two procedures would most likely cause oxidation because of the heating process involved. The accuracy of both methods was also evaluated by analysis of glycerine mono-oleate. Using the direct method an oleic acid recovery of 99.6% was obtained, whereas with the extraction method only 89.3% of the theoretical value for oleic acid was recovered.

Conclusions The proposed direct method represents an accurate, precise, simple and time saving method which enables quantitative determination of cholesterol and fatty acid contents in food on the same sample. It eliminates the use of hazardous chlorinated solvents. If the cholesterol content of a sample is not to be determined, the even simpler modified extraction method could be used for the fatty acid analysis. The main advantage of this modification is the possibility to introduce (like in the direct method) the internal standard to the sample at the very beginning of the analytical procedure which improves its precision. Another advantage of the procedure is a substantially reduced use of solvents.

References 1 WILSON, P. W., CASTELLI, W. P. AND KANNEL, W. B. Coronary risk prediction in adults (The Framingham Study). American Journal of Cardiology, 59, 91G–94G (1987) 2 TRUSWELL, A. S. Evolution of dietary recommendations, goals, and guidelines. American Journal of Clinical Nutrition, 45, 1060–1072 (1987)

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