ISSN 1061 9348, Journal of Analytical Chemistry, 2011, Vol. 66, No. 7, pp. 572581. Pleiades Publishing, Ltd.

., 2011. Original Russian Text F.A. Chmilenko, N.P. Minaeva, L.P. Sidorova, 2011, published in Zhurnal Analiticheskoi Khimii, 2011, Vol. 66, No. 7, pp. 686695.

ARTICLES

Complex Chromatographic Determination of the Adulteration of Dairy Products: A New Approach

F. A. Chmilenko, N. P. Minaeva, and L. P. Sidorova
O. Gonchar National University, pr. Gagarina 72, Dnepropetrovsk, 49010 Ukraine
Received March 25, 2010; in final form, November 23, 2010

AbstractA new approach to complex chromatographic determination of the adulteration of dairy products was proposed, based on the determination of not only the total fatty acid composition but also the composi tion of the sterol fraction and the concentration of trans isomers of fatty acids. Procedures for the chromato graphic identification of low fat dairy products were developed, including the extraction of a watermilk alcohol emulsion with a hexaneether mixture followed by the chromatographic determination of the sterol fraction and trans isomers. Keywords: chromatography, dairy products, adulteration DOI: 10.1134/S1061934811070057

Contemporary technologies in oil and fat produc tion make the revealing of food adulterations difficult; thus, conventional methods for testing foodstuff are not suitable any more for the accurate and reliable determination of adulterated product and its composi tion [1, 2]. In recent years, there has been a consider able amount of adulterated products in the market; therefore, the problems of food chemistry, particularly, food quality control and certification, become of spe cial interest. Dairy products (butter, cream, sour cream, cheese, condensed and powdered milk, ice cream, and others) are the most often adulterated. Hydrogenated vegeta ble oils or their mixtures are often used for adultera tion; however, their use is restricted because of a high concentration of trans isomers of fatty acids (TFA) in them. Therefore, improve of product quality and the development of methods for control and standardiza tion are particularly important for dairy and oil and fat industries. The most reliable parameters of oil and fat prod ucts are the fatty acid and triglyceride composition [3 7] and the parameter of the sterol fraction, which are found by chromatographic methods [823]. Rough adulterations can easily be found by the total fatty acid composition (FAC). The revealing of fewer amounts of additions (up to 10%) is impeded by natural variations in a wide range of original products. Therefore, we propose a complex approach for the reliable identification of dairy products and the deter mination of small additives of vegetable oils (<10%), based on the results of determination of not only total FAC but also the composition of the sterol fraction and the concentration of trans isomers of fatty acids.

The determination of fatty acid composition is widely used only for revealing rough adulterations. A 1020% concentration of additives cannot be found by using solely this method because of natural varia tions of the fatty acid composition in a wide range. In recent years, many publications have been devoted to the analysis of data on the fatty acid composition, where much attention is paid to the statistical evalua tion of results [24, 25]. As shown in practice, these methods may not be completely suitable for the cur rent situation, because the results may depend on the apparatus used in analysis and the completeness of separation and determination of acids. There is no consensus on the boundary concentrations of these or those acids, because the criteria defining the natural ness of milk fat are not standardized and are still under discussion [2628], and there is no simple and opera tor friendly algorithm for interpreting the analytical results. The composition of the sterol fraction distinctly depends on what the fat nature is: animal or vegetable. If exclusively milk fat is used, only cholesterol should be found in the sterol fraction. Vegetable oils contain no cholesterol; however, there are other ste rols, namely, brassicasterol, campesterol, stigmaste rol, sitosterol, etc. The presence of these sterols in animal fats indicates the additives of vegetable oils. The determination of the adulteration of oil and fat products by the composition of the sterol fraction is the most reliable method that enables the determina tion of 2% additives and on of vegetable oils. In this case, peaks of phytosterols are recorded in the chro matogram; namely these components prove the fact of adulteration.

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COMPLEX CHROMATOGRAPHIC DETERMINATION OF THE ADULTERATION Chromatographic methods Gas chromatography (GC) Gasliquid chromatography (GLC) High performance liquid chromatography (HPLC) Fatty acids and their isomers Spectral methods

573

IR spectrometry Hybrid methods IR attenuated total internal reflection Mass spectrometry GLC + Ag TLC NMR spectrometry GLC + IR spectrometry
+

GC + IR spectrometry GC + mass spectrometry

Diagram 1. Methods for determination of fatty acids and their isomers.

To determine the concentration of trans isomers of fatty acids, contemporary methods are used such as chromatographymass spectrometry, IR spectrome try, capillary gasliquid chromatography (GLC), and high performance liquid chromatography (HPLC) [2938]. Silver ion HPLC is usually applied for sepa rating and determining of trans C18:1 isomers in par tially hydrogenated oils and milk fats [39, 40]. Hybrid methods are also in use: gas chromatography was com bined with thin layer chromatography [4145] or with mass spectrometric detection after methyl pre trans esterification of fatty acids [4648] (Diagram 1). New chromatographic, mass spectrometric, spec trometric methods enable the detection of trace amounts of trans fatty acids. However, the problems, related to the overlapping of cis and trans isomers, that is, to the incomplete separation of trans acid from its cis components, remain unsolved. There are no methods of determination universal for all samples of oil and fat products. The resolution of these and other problems of determination of TFA is being actively pursued. This work proposes a complex procedure for ana lyzing oil and fat products to establish its authenticity by three criteria: the total fatty acid composition, the composition of the sterol fraction, and the concentra tion of trans isomers of fatty acids. Moreover, the goal was to develop a procedure for the chromatographic identification of low fat (less than 50%) dairy prod ucts. For this purpose, a stage of accelerated sample preparation should be developed for recovering a fat layer from dairy products, followed by chromato graphic determination of the FAC, the composition of the sterol fraction, and TFA. The procedure should be rapid, with little time needed to separate the fat frac
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tion and the least possible consumption of organic sol vents for sample preparation. EXPERIMENTAL Determination of the fatty acid composition of oil and fat products with a high fat concentration (butter, spread, margarine, fat). The fatty acid composition was determined on an Agat gas liquid chromatograph with a flame ionization detector (FID; that is, the GCFID mode was used), using a glass capillary col umn (80 m in length, 0.35 mm in inner diameter) with a Carbowax 20M liquid stationary phase (layer thick ness, 0.2 m). The parameters of determination were given earlier in [44]. For qualitative identification of fatty acid methyl esters, we used a standard reference solution from SUPELCO, containing 37 fatty acid methyl esters (Catalogue no. 47885). For quantitative evaluation, the method of internal normalization was used. The procedure of sample preparation for the iden tification and determination of FAC is based on the alkaline hydrolysis of triglycerides followed by obtain ing the fatty acid methyl esters by the esterification reaction [44]. The results of identification and determination of the fatty acid composition of oil and butter (natural and adulterated) are presented in Table 1. The compo sition of the fatty acid methyl esters in butter was cal culated by the method of internal normalization. The mass fraction of each acid i was calculated by the equation
Xi = S i 100 , Si

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Table 1. Quantitative determination of the fatty acid composition of butter Natural butter Acids Peak height, mV Peak height, mV/sec Mass frac Mass frac Mass frac Peak Peak area, Peak Peak area, tion of fatty tion of fatty tion of fatty height, mV mV/sec height, mV mV/sec acids (wt %) acids (wt %) acids (wt %) Adulterated butter Vegetable oil

C4:0

5.336

24.778

6.11

2.234

12.456

3.07

C6:0

2.512

13.803

3.40

1.480

8.089

1.99

C8:0

1.256

6.733

1.66

0.888

4.685

1.16

0.876

4.606

1.14

C10:0

2.478

12.672

3.12

2.001

10.056

2.48

0.772

3.836

0.95

C10:1

0.223

1.070

0.26

0.204

0.984

0.24

C12:0

2.659

16.955

4.18

2.315

11.642

2.87

9.367

48.646

12.00

C14:0

6.536

46.460

11.46

5.626

44.176

10.89

2.730

18.754

4.62

C16:0

10.413

119.152

29.38

7.168

125.669

30.99

9.853

132.062

32.56

C18:0

2.388

48.853

12.05

1.858

54.047

13.33

0.680

15.330

3.78

C18:1

3.872

94.948

23.41

3.129

112.038

27.63

5.374

146.695

36.17

C18:2

0.465

11.836

2.92

0.515

12.550

3.09

1.370

34.397

8.48

C18:3

0.063

1.948

0.48

0.062

1.788

0.44

C20:0

0.146

4.022

0.99

0.161

4.968

1.22

C22:0

0.078

2.285

0.56

0.021

1.232

0.30

C22:1

0.021

1.155

0.28

0.033

1.210

0.30

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575

where Si was the peak area of a fatty acid methyl ester, iSi was the sum area of all identified peaks of fatty acid methyl esters in the chromatogram. Inconsistence of the fatty acid composition indicates the adulteration of oil and fat products and can be a result of both improper storage of oil or butter and violation of tech nological processes during production or packing (Table 1). The determination of the sterol composition of oil and fat products with a high fat concentration (more than 50%; that is, butter, spread, margarine, fat) was performed on a Shimadzu 14B chromatograph in the GCFID mode, using a capillary column (30 m in length, 0.25 mm in inner diameter) with a ZB 5 liquid stationary phase (layer thickness, 0.25 m). The tem perature of injector and detector was 300. The col umn thermostat was operated as follows: initial tem perature 200, maintaining for 2 min; temperature increase at 7C/min to the final value of 295. The carrier gas pressure at the injector inlet was 0.1 MPa; the flow split at the column inlet was 1 : 20 (19/20 of the flow was dumped into atmosphere at the injector outlet). Nitrogen was a carrier gas [44]. To identify the peaks in the chromatogram, we used the following reference samples from SUPELCO: cholesterol (catalogue no. 47127), sitosterol (cata logue no. S 1270), stigmasterol (catalogue no. 47132), campesterol (catalogue no. B 736), and brassicasterol (catalogue no. B 493). Sample preparation consisted of the alkaline hydrolysis of a sample of oil and fat products followed by the extraction of unsaponifiable additives with a nonpolar solvent and the separation of the sterol frac tion from unsaponifiable substances by thin layer chromatography (TLC). Capillary columns enable a better separation of the peaks of the mentioned sterols in comparison to packed columns. Determination of the sterol composition of low fat (less than 50%) dairy products (ice cream, milk, curd cheese). Since the test products contain less than 50% of fat, the procedure used for spreads and fat mixtures is not suitable here [5]. For further investigation, an additional stage of preconcentration of a certain amount of fat (no less than 2.5 g) should be intro duced. The procedure for recovering fat is described in [43]. The separated sterols were determined by gas chro matography similarly to that for the products with a high fat concentration. Determination of trans isomers of fatty acids in oil and fat products with a high fat concentration (more than 50%, that is, butter, spread, margarine, fat). The procedure of sample preparation is based on the alka line hydrolysis of triglycerides followed by obtaining the fatty acid methyl esters by the esterification reac tion.
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A portion of a premelted sample (50100 mg) is placed in a glass test tube and dissolved in 1 mL of tol uene. One milliliter of a 1 M sodium methylate solu tion in methanol is added into the toluene solution, and the mixture is heated to 7080 for 1520 min. The mixture is cooled to room temperature; 1 mL of distilled water and 1 mL of hexane are added; and the mixture is shaken for 10 sec. An upper organic layer is separated and placed in a hermetically sealed vessel (a vial with a Teflon stopper) with anhydrous sodium sulfate. A 1 mm3 portion of the resulting filtrate is injected into the chromatograph by a microsyringe. Trans isomers were identified using a Shimadzu GC 14B gas liquid chromatograph with a flame ion ization detector by the retention times of TFA under standard chromatographic condition. We used an SP 2560 capillary column (100 m in length, 0.25 mm in inner diameter) containing adsorbent bis cyanopropylpolysiloxane (layer thickness, 0.2 m). The temperature of the injector and the detector was 220; the column thermostat was set at 180; the isothermal mode was used. Hydrogen was a carrier gas (25 cm3/sec). The sample volume was 0.5 L; the flow split ratio was 100 : 1. The pressure of the carrier gas at the injector inlet was 0.2 MPa, the flow split at the col umn inlet was 1 : 20 (19/20 of the flow was dumped into atmosphere at the injector outlet). The chromatograms were processed using Multi khrom 1.5x software for Windows (Ampersend, Rus sia). A mixture of fatty acids C18:1, C18:2, and C18:3 in methylene chloride was used as a reference sample. Determination of trans isomers of fatty acids in low fat (less than 50%) products (ice cream, milk, curd cheese). According to GOSTs [8, 9], the procedure of sample preparation used for testing spreads and fat mixtures is not suitable for the products with the mass fraction of fat less than 50%. An additional stage of preconcentration of a certain amount of fat (no less than 0.5 g) is needed similar to procedure 3 described above. Then, the alkaline hydrolysis of the resulting fat fraction is performed, and the fatty acid methyl esters are obtained by the esterification reaction. The separated trans isomers are detected by gas chromatography, as for the products with a high fat concentration. Determination of fatty acid trans isomers in oil and fat products with preliminary TLC separation. The fatty acid methyl esters are prepared similarly to the procedure for determining FAC. For the separation of fractions, a Sorbfil plate (PTSKh AF V UV) is maintained at105 for 1 h. A reference solution of TFA (1 L) and sample solutions are applied as distinct spots 2 cm in length, which ensures a better separation. The fractions are separated by using a proposed mobile phase with an optimal ratio of hexanediethyl ether (90 : 10 vol ratio). After that, the plate is dried for 5 min on air and sprayed by
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Sample collection 4050 g

Melting, water bath 4050C

Filtration

Weighed portion 0.1 g Hexane 1.9 cm3 Dissolution A 2 M sodium methylate solution in methanol (0.1 mL)

Settling, filtration

Fatty acid methyl esters

Dropwise, 2 cm in length

Application to a TLC plate A mixture of diethyl ether and hexane (9 : 1 vol %) Rhodamine 6G

Chromatographic separation

Spraying

Evaluation in the UV light

Calculation of R f

Cutting off the area of FAT

GC

Diagram 2. Preliminary TLC separation in the determination of fatty acid trans isomers in oil and fat products.

Rhodamine 6G, and the results are evaluated in the UV light. The areas corresponding to trans and cis isomers of monounsaturated fatty acids are cut off by a scalpel and transferred in a flask. Five milliliters of ethyl acetate is added; the mixture is

refluxed for 15 min, filtered in a test tube (twice), evaporated in a rotor evaporator to dryness. The sample is dissolved in ethyl acetate (200 L), and a 1 L portion is injected into the chromatograph by a microsyringe (Diagram 2).
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577

Treatment of results. The relative mass of each component c, %, was calculated by the equation

Table 2. Examples illustrating the use of the proposed proce dure for different weighed portions of recovered fat, applied to the TLC plate No. Weighed portion, g Yield, %

x =

Ax 100 , At
mm2,

and t where A is the peak area of component x, is the sum area of all peaks except the solvent peak, mm2. Calculation of the mass fraction of fatty acid trans isomers. In the cases of oils and fats refined at high temperature and for other fat products free of milk fat, the mass fraction of FAT is calculated as a sum of rel ative masses of C18:1 trans, C18:2 trans, and C18:3 trans methyl esters of fatty acids. The maximum number of peaks that the trans isomers can yield are 1 (C18:1 trans), 2 (C18:2 trans), and 4 (C18:3 trans). For the fat products, containing milk fat and/or partially hydrogenated fats and oils, the mass fraction of FAT is calculated as a sum of the relative masses of all fatty acid methyl esters, containing double bonds in a trans configuration. RESULTS AND DISCUSSION Earlier it was shown that the adulteration of dairy products can be detected by FAC [44]; however, this method is suitable to reveal the concentration of vege table additions of more than 20%, because the fatty acid composition of milk fat is not constant. The vari ations in the composition of milk fat are related to sea sonal changes, peculiarities in feeding, breed of cows, and technological issues. The variability in the compo sition of vegetable oils is attributed to climate factors, cultivation of a crop, its type, ripeness of the seeds at harvest, and others. The concentration of lauric (C12:0), myristic (C14:0), palmitic (C16:0), stearic (C18:0), oleic (C18:1), and linolic (C18:2) acids in fats is the most studied. Therefore, namely these acids are considered as crite ria for identifying the naturalness of milk fat [12, 24, 25]. We found out that the difference in the concentra tion of these acids in natural and adulterated products did not exceed 10% (Table 1); therefore, the identifi cation by butyric acid (C4:0) is more preferable To reveal the adulteration of milk fat, the chro matographic method for determining the fatty acid composition can be used only under condition that butyric acid (C4:0) is quantified by using an internal standard. Only in the case of butter, the concentration of butyric acid (6.1 0.5%) falls in the concentration range (wt %) characteristic for milk fat (4.76.5%) [8]. In the adulterated product, the concentration of butyric acid is lower (3.1 0.3%); in vegetable oil, there is no this acid at all (Table 1). The findings for olive oil, butter, and adulterated butter are presented in Table 1.
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0.1

66

0.3

75

0.5

96

0.7

99

0.9

100

1.1

100

1.3

100

1.5

99

1.7

85

10

1.9

74

11

2.1

68

12

2.3

65

It was found later that the composition of the sterol fraction enabled the determination of vegetable oil additives in the concentration of 2% and more. In this case, the peaks of phytosterols are recorded in the chromatogram, which evidences the adulteration. In the determination of the composition of the ste rol fraction, a mobile phase was proposed for the TLC separation of components with optimal component ratios of hexaneethyl acetate (from 65 : 35 to 60 : 40 vol %) or chloroformethyl acetate (95 : 5 vol %).
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CHMILENKO et al. Cholesterol 2 20 Cholesterol 6 8 16 ()

20

Detector signal, mV

10

12

14

18 (b)

20

22

24

26

28 30 t, min

Detector signal, mV

Brassicasterol

Campesterol

Stigmasterol

Sitosterol 24 26

6 28 30 t, min

10

12

14

16

18

20

22

Fig. 1. Chromatogram of the sterol fraction of (a) sour cream and (b) sour cream adulterated by addition of vegetable oil: (1) cho lesterol, (2) brassicasterol, (3) campesterol, (4) stigmasterol, and (5) sitosterol; I, mV, is the detector signal.

In the use of the mobile phase of another ratio, sterols cannot be separated from methylsterols on the TLC plates; thus, the results will be unreliable. Moreover, the optimal amount of the sample applied to the TLC plate is experimentally determined. The quantity should not exceed 0.1 mg of unsaponifiable sub stances, which is equivalent to a 1 g sample of oil and fat products rather than a 5 g portion recommended in [8] (Table 2). The application of the samples as distinct spots also contributes into a better separation. A peak with the retention time characteristic for sitosterol, recorded in the chromatogram (height 2% of the entire scale), indicates the presence of this sterol in the test sample, that is, proves that there is vegetable oil in the product. The presence of the peaks

of other phytosterolscampesterol or stigmasterol also proves this conclusion. The chromatograms of the adulterated and natural sour cream are presented in Figs. 1a and 1b. These days, the determination of the fatty acid composition is needed to be accompanied with the study of the concentration of cis and trans isomers of fatty acids and their ratio (Table 3). In this work, we study these parameters for some oil and fat products (Table 4, Fig. 2). We found out that only complex chromatographic analysis of dairy products by three criteria simulta neously (FAC, composition of the sterol fraction, and concentration of TFA) allowed an unambiguous determination of adulteration.
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COMPLEX CHROMATOGRAPHIC DETERMINATION OF THE ADULTERATION Table 3. Fatty acids and their isomers the most often contained in foodstuff Conventional symbols of fatty acids C16:0 C16:1 C18:0 Name Systematic (IUPAC) nomenclature Hexadecanoic Hexadecanoic Octadecanoic Isomers Trivial nomenclature Palmitic Palmitoleic Stearic Oleic C18:1, 9 cis Elaidic C18:1, 9 trans C18:1 Octadecadienoic Oleic Isooleic acids: Petroselinic C18:1, 6 cis Vaccenic C18:1, 11 trans C18:2 C18:3 C20:0 C20:1 C20:2 C20:3 C20:4 C20:5 C22:0 C22:1 C22:2 C22:6 Octadecadienoic Octadecatrienoic Eicosanoic Eicosenoic Eicosadienoic Eicosatrienoic Eicosatetraenoic Eicosapentaenoic Docosanoic Docosenoic Docosadienoic Docosahexaenoic Linoleic Linolenic Arachic Gadoleic Gondoic Eicosadienoic Eicosatrienoic Arachidonic Eicosapentaenoic Behenic Erucic Docosadienoic Docosahexaenoic C22:1, 13 cis C22:2, 13,16 cis C22:6, 4,7,10,13,16,19 cis C20:1, 9 cis C20:1, 11 cis C20:2, 11,14 cis C20:3, 8,11,14 cis C20:3, 11,14,17 cis C20:4, 5,8,11,14 cis C20:5, 5,8,11,14,17 cis Linoleic C18:2, 6 (9 cis12 trans) Linolelaidinic C18:2, 6 (9 cis12 trans) C16:1, 9 cis

579

C18:3, 9,12,15 (trans cis trans, cis cis trans, cis trans cis, trans cis cis)

Table 4. Concentration of fatty acid trans isomers in (1) confectionery fat, (2) adulterated butter, (3) milk margarine, (4) Polish cookies, and (5) palm oil Sample no. 1 2 3 trans Isomers of oleic C18:1 and linoleic Retention time, min C18:2 acids C18:1 C18:1 C18:1 C18:2 C18:2 C18:2 C18:2 C18:2 C18:1 C18:1 C18:2 C18:2 46.24 48.34 46.40 49.71 50.43 50.85 50.43 50.85 38.67 46.47 49.69 50.38
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Height, mV 5.53 3.43 16.81 1.50 4.10 3.33 2.03 1.80 148.05 26.57 0.74 0.69
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Peak area, mV sec 99.10 18.84 215.94 13.41 53.08 37.12 22.27 19.42 1902.30 709.95 9.49 12.25

Concentration, % 10.43 5.83 6.81 0.42 1.67 1.17 0.44 0.38 44.46 16.59 0.22 0.29

4 5

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580 mV 26 23 18:1 18:1 trans 5.834 18:1 n9. 18:2n6 3.778

CHMILENKO et al.

21

22:6n3.......5.081

C22:2.....1.401

22

29

22:0.......1.183 52 t, min
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48

49

50

51

Fig. 2. Chromatogram for determining the fatty acid composition: a section containing trans isomers of fatty acids detected in adulterated butter.

In this work, we study the identification of the authenticity of butter using three criteria: analysis of the fatty acid composition including the concentra tion and the ratio of cis and trans isomers of fatty acids and the determination of the sterol fractions of the samples. The results are presented in Tables 1 and 4 and in Fig. 2. Such an approach including complex chromatographic determination of adulteration of dairy products enables the revealing adulterating vege table oils at a level of less than 1%. We consider that the chromatographic methods for determining the fatty acid composition, the concen tration of TFA, and the composition of the sterol frac tion can refine each other in the case of the rough adulteration of dairy products (>10% of vegetable oils) and should be compulsory for the products with a low concentration (<2%) of vegetable oils. REFERENCES
1. Dmitrichenko, M. and Pilipenko, T., Ekspertiza pi shchevykh zhirov, moloka i molochnykh produktov (Examination of Dietary Fat, Milk and Dairy Prod ucts), St. Petersburg: Piter, 2003, vol. 352. 2. Pavlova, I.V., Molochn. Prom st., 2006, no. 2, p. 54. 3. DSTU (State Standards of Ukraine) ISO 5508 2001: Vegetable Oils and Animal Fats. Gas Chromatographic Analysis of Methyl Esters of Fatty Acids.

4. GOST (State Standard) 30623 98: Vegetable Oils and Margarine. Detection of Falsification. 5. Rudakov, O.B., Masla Zhiry, 2003, no. 4, p. 8. 6. Yakovlev , V.S., Kulikovskaya, T .S., and Krapivkin, B.A., Maslodel, 2009, p. 1. 7. GOST (State Standard) R 51483 99: Vegetable Oils and Animal Fats. Gas Chromatographic Determination of Constituent Contents of Methyl Esters of Total Fatty Acid Content. 8. DSTU (State Standards of Ukraine) ISO 18609 2004: Animal and Vegetable Fats and Oils. Determination of Unsaponifiable Matter Using Hexane Extraction. 9. DSTU (State Standards of Ukraine) ISO 3594 2001: Milk Fat. Detection of Vegetable Fat by Gas Liquid Chro matography of Sterols (Reference Method). 10. DSTU (State Standards of Ukraine) ISO 6799 2002: Animal and Vegetable Fats and Oils. Determination of the Content of the Sterol Fraction. Gas Chromatographic Method. 11. DSTU (State Standards of Ukraine) ISO 3596 1 1998: Animal and Vegetable Fats and Oils. Determination of Unsaponifiable Matter Using Diethyl Ether Extraction. 12. GOST (State Standard) R 51471 99: Milk fat. Detection Method of Vegetable fat by Gas Liquid Chromatography of Sterols. 13. Stepycheva, N.V., Chesnokov, V.V., and Stepychev , S.G., Metody Otsenki Sootvetstviya, 2007, no. 6, p. 12. 14. Vyshemirskii, F.A., Molochn. Prom st, 2003, no. 9, p. 45.
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2011