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Asian Journal of Chemistry; Vol. 23, No. 11 (2011), 4768-4770

Determination of Fatty Acid Composition of Two Species by GC-MS

1 2 3 1 1,* E. HORJ , A. IORDACHE , A. TOMA , O. COZAR and M. CULEA

1Babes-Bolyai University, 1 M. Kogalniceanu St., Cluj-Napoca 400084, Romania 2National Research and Development Institute of Cryogenics and Isotopic Technologies - ICSI Rm. Valcea, 4 Uzinei St., Rm. Valcea 240050, Romania 3University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca-USAMV, 3-5 Manastur St., 400372 Cluj-Napoca, Romania

*Corresponding author: Tel: +40 264 405300; E-mail: [email protected]

(Received: 17 August 2010; Accepted: 11 July 2011) AJC-10148

A gas chromatography-mass spectrometric (GC/MS) technique was used to evaluate effects in quantitative fatty acids in fish plasma after Se addition in food, 0.03 mg/kg, in comparison with control. is a potent antioxidant protecting the body from damage due to oxidation by free radicals. (EPA) and (DHA) are the major fatty acids found in fish. These fatty acids are produced by unicellular and phytoplankton which are consumed and then accumulate in fish. The aim of this work was to establish the extraction procedure and quantitative method of the fatty acids from fish plasma. The fatty acids were derivatized to obtain methyl esters. The identification of fatty acids was obtained by comparison of fatty acids methyl esters mass spectra with the mass spectra of fatty acids methyl esters kits and of NIST library. Concentrations of majority of unsaturated fatty acids increased with Se supplementation.

Key Words: Fatty acids, GC-MS, Derivatization, Selenium.

INTRODUCTION as methyl ester derivatives1-3. Finally, the extracted analytes were detected by GC/MS in the electron impact (EI) mode4. The importance of the (n-3) poly unsaturated fatty acids The aim of the paper was to develop an analytical method 1 (PUFA) in human has led to considerable research for determination of fatty acids in biological samples. The effort in the last years. Subsequently, the efficacy of (n-3) PUFA method was validated by using fatty acid standard samples. in the prevention or attenuation of many of the inflammatory The method was applied to evaluate effects in quantitative fatty conditions that are prevalent in the developed world, including acids in fish plasma after Se addition in food, 0.03 mg/kg, in rheumatoid arthritis, atopic illness, inflammatory bowel disease, comparison with control. various neurological and cardiovascular diseases has been esta- Theoretical: 20 µg of C11:1 internal standard was added blished. Fish and particularly those with oily flesh, such as after extraction and derivatization to each sample. The fatty , and , represent a rich and almost acids were calculated according with the internal standard unique source of the (n-3) PUFA, especially eicosapentaenoic quantity and by using the response factors, for detector response acid [20:5(n-3), EPA] and docosahexaenoic acid [22:6(n-3), correction. The response factors were obtained by repetitive DHA]. Demand for is increasing, yet the tradi- injections into GC/MS of the standard mixture containing tional capture are declining worldwide such that the known quantity of each fatty acid. The fatty acids calculations potential shortfall in fish products must be met by in biological samples were performed following the formulae: production. Future expansion of aquaculture and in particular salmon production, can continue only if suitable and sustainable A i / A j F = alternatives to fish are developed and introduced2. (1) m i / m j A gas chromatography-mass spectrometry (GC/MS) tech- nique was used for qualitative and quantitative analysis of fatty Ai mi ()()µg = m j µg (2) acids in fish. The method involves extraction procedure, Fi ⋅A j derivatization and gas chromatography/mass spectrometric where mi is the quantity corresponding to the compound i; mj analysis (GC/MS). Fatty acids from plasma were derivatized is the internal standard quantity added before sample preparation; Vol. 23, No. 11 (2011) Determination of Fatty Acid Composition of Two Carp Species by GC-MS 4769

22.27 Ai and Aj are the peak areas of the compounds i and, respectively 100 C16:0 j; Fi are the response factors for compound i and j, respectively 95 (the internal standard) calculated by using standards. 90 85 C18:2+C18:1 EXPERIMENTAL 80 75

General procedure: A trace DSQ Thermo Finnigan 70 24.36 quadrupole mass spectrometer coupled with a Trace GC was 65 60 e DHA used. The extracts were separated on a Rtx-5MS capillary c n a

d 55 n

column, 30 m × 0.25 mm, 0.25 µm film thickness, using a u b

A 50 e v temperature program from 50 ºC, 1 min, 8 ºC/min at 300 ºC, i 28.16

t 24.30

a 45 l e C18:0 the flow rate 1 mL/min, with helium 5.5 as carrier gas. R ETA 40 C16:1 Undecaenoic acid (C11:1) was used as internal standard. The 35 EPA following conditions were followed: transfer line temperature 30 24.63 25 22.01 250 ºC, injector temperature 250 ºC; ion source temperature 26.16 20 250 ºC; splitter: 10:1. Electron energy was 70 eV and the 26.56 emission current, 100 µA. 15 10 21.96 23.03 The fatty acids were extracted from the plasma samples 28.08 5 28.31 29.24 after amino acids extraction on a Dowex 50W-W8 exchange 21.80 22.85 23.22 25.37 27.50 30.09 30.37 0 22 23 24 25 26 27 28 29 30 31 resin. 2 mL water were also passed and collected. The extraction Time (min) was then performed by adding 0.5 mL chloroform:methanol Fig. 1. Separation chromatogram of the important fatty acids methyl esters (2:1) and mixing 30 s. Then was followed a centrifugation for identified in fish plasma

5 min. The supernatant was removed. The extract was dried 22.27 24.36 28.16

60 24.28 with a nitrogen flow. Derivatization procedure was applied 26.14 40 24.62 for esterification with methanol-acetyl chloride (4:1 v/v) for 22.01 26.54 20 23.01 28.07 28.29 22.84 23.47 24.14 25.06 25.40 25.73 27.15 20 min at 80 ºC. The method was validated. The validation 0 22.27 24.35 28.16

60 parameters precision and sensitivity were tested. GC/MS analyses 24.28 24.62 40 22.01 26.14 were performed for the determination of fatty acids in carp 26.54 20 28.07 28.29 22.74 23.01 23.4624.13 25.06 25.38 25.73 26.61 27.48 0 fish plasma. Two different varieties of (Cyprinus carpio) e 22.27 24.36 28.16 c n a

d 60 were studied: Galitian and Lausitz. The fodder consisted of: n 24.28 u 26.14

b 24.62 40 22.01 A 26.54 38 % proteins, 5 % , 3.5 % pulp and 9 % humidity. For the e v i 20 28.07 t 23.01 27.15 28.29 a 22.84 23.47 24.14 25.06 25.40 25.73 l

e 0 experimental batch the fodder included also 0.03 mg organic R 22.27 24.36 28.16 Se (Sel-plex, All-Tech, USA) per kg. Plasma samples were 60 24.30 24.62 26.14 40 22.01 26.56 collected from each carp, 8 controls and 10 experiments of 23.01 28.07 28.29 20 23.12 22.84 24.16 25.06 25.38 25.75 27.21 28.02 the two varieties. 0 22.25 24.35 24.62 60 24.28 28.15 RESULTS AND DISCUSSION 40 26.14 20 22.01 22.84 23.03 23.47 25.08 25.38 25.54 26.54 27.48 28.07 28.48 0 The developed method is selective and specific. The mass 22.25 24.35 28.16 60 24.28 24.62 spectra recorded on each chromatographic peak permit the 26.14 40 22.01 26.54 precise identification of the fatty acids, by using NIST library 20 22.82 28.07 28.29 22.76 23.03 23.47 25.08 25.38 25.73 27.21 27.48 0 of mass spectra. Also, the overlapping of the compounds is 22 23 24 25 26 27 28 Time (min) easily discovered. The method was validated by using fatty Fig. 2. Comparative chromatogram profiles of plasma fatty acids methyl acid standards. Precision gave lower value than 10 % for RSD esters from different carp fish extracts and sensitivity value was lower than 10 ng of fatty acid injected. All the samples followed the same extraction and derivatization (ranging from 5-8 %) and 18:1ω9 (ranging from 14-21 %) steps. formed a considerable percentage of the total fatty acids. The Fig. 1 presents the separation chromatogram of fatty acids dominant species of SFA were 16:0 (ranging from 15-30 %) methyl esters (FAME) in a plasma fish sample. The comparison and 18:0 (ranging from 6-9 %). EPA (20:5ω3) and DHA of the separated FAME from different carp fish extracts showed (22:6ω3) were the dominant PUFA and percentages were 30 very similar profiles, as could be observed in Fig. 2. Plasma % (Table-1). No significant differences were found for the concentrations of fatty acids were measured and compared in majority of fatty acids in the two varieties of carp. carp fish plasma, after Se addition in food, 0.03 mg/kg, in Figs. 3-5 presents the comparison fatty acids in experi- comparison with control. mental and control of the two varieties of carps. Significant The fatty acids identified in the plasma samples of the increase of DHA and unsaturated fatty acids (UFA) in both carp fish are shown in Table-1. The analysis of the fatty acid varieties of experimental plasma was observed in comparison composition revealed that polyunsaturated fatty acids (PUFA) with control (Figs. 4 and 5). were the most representative (ca. 53 %), followed by saturated Eicosapentaenoic acid and docosahexaenoic acid, found fatty acids (SFA) (ca. 24 %) and finally by monounsaturated only in fish and other sea foods, possess profound beneficial fatty acids (MUFA) (ca. 21 %). Among MUFA, monoenic 16:1 properties for human health. In this research, significantly 4770 Horj et al. Asian J. Chem.

TABLE-1 AEO(white,n=5),AMO(black,n=5)

FATTY ACIDS COMPOSITION IN CARP FISH PLASMA. 25.00 THE VALUES ARE EXPRESSED AS PER CENT OF TOTAL FATTY ACIDS (n = 10) 20.00 15.00 FAME TR Mean ± DS Hexadecenoic acid (C16:1) 22.01 6.84 ± 1.21 10.00 Hexadecanoic acid (C16:0) 22.27 18.61 ± 4.53 5.00

9,12-Octadecadienoic acid (C18:2) 24.28 8.05 ± 2.04 0.00 9-Octadecenoic acid (C18:1) 24.36 17.59 ± 2.20 ETA ETA Octadecenoic acid (C18:1) 24.41 3.85 ± 0.48 Octadecanoic acid (C18:0) 24.62 6.28 ± 0.62 (C22:6)(DHA) (C18:1)

5,8,11,14-Eicosatetraenoic acid 26.14 7.68 ± 1.20 (C18:2)

(C20:4) ETA 9-octadecenoic acid acid (C20:4)(ETA) acid (C20:4)(EPA) 9,12octadecadienoic acid

5,8,11,14,17-Eicosapentaenoic acid 26.22 3.76 ± 1.03 5,8,11,14eicosatetraenoic octadecenoicacid (C18:1) octadecenoic acid (C18:1) octadecanoicacid (C18:0) hexadecenoic acid(C16:1) hexadecanoic acid (C16:0) (C20:5) EPA 5,8,11,14eicosapentaenoic ETA 28.07 2.85 ± 1.36 Fig. 4. Comparison of changes of fatty acids in experimental and control (C22:6)DHA* 28.16 14.85 ± 6.46 carp of two varieties (AEL: fatty acid levels in plasma of ETA 28.29 1.18 ± 0.58 experimental Lausitz carp and AEO: fatty acid levels in plasma of Σ SFA – 23.53 experimental Galitian carp; AML and AMO: fatty acid levels in plasma of control Lausitz and Galitian carp) Σ MUFA – 21.42 Σ PUFA – 53.17 Σ USFA/ ΣSFA – 3.2 AEL(1),AML(2)

DHA /EPA – 4.3 6.00 *4,7,10,13,16,19-docosahexaenoic acid 5.00 80

70 4.00

60 UFA/SFA 3.00 DHA/EPA 50 SFA UFA 2.00 40 ETA+EPA

%arie DHA 30 MUFA 1.00

20 0.00 10 1 2

0 1 2 3 4 5 6 7 8 910 AEG(1),AMG(2) FAME fish (experiment)

Fig. 3. Comparison among SFA, UFA, ETA +EPA, DHA and MUFA in 6.00 experimental fish

5.00 (p < 0.01) higher docosahexaenoic acid content was deter- mined in the plasma samples of experimental carps studied 4.00 versus control. UFA/SFA 3.00 DHA/EPA Conclusion 2.00 The method developed showed good precision in the analysis of fatty acids from plasma samples. The high values 1.00 of poly unsaturated fatty acid in fish plasma prove the high nutritious value of fish. Docosahexaenoic acid increase in 0.00 1 2 plasma samples of experimental fish could be related with the Fig. 5. Comparison of UFA/SFA and DHA/EPA ratios in experimental and antioxidant properties of selenium in the body. control fish of two varieties

AEL(white,n=5)AML(grey,n=5) ACKNOWLEDGEMENTS 25.00

20.00 This work is supported by the Romanian Research Foun- 15.00 dation, Project Number ID-501/2008. 10.00

5.00 REFERENCES

0.00 1. N. Shapira, P. Weill, O. Sharon, R. Loewenbach and O.J. Berzak, Agric.

ETA ETA Food Chem., 57, 2249 (2009). 2. A. Freije, J. Oleo Sci., 58, 379 (2009). (C22:6)(DHA) (C18:1) 3. S. Pinela, B.R. Quintella, P. Raposo de Almeida and M.J. Lança, (C18:2) Scientia Marina, 73, 785 (2009). 9-octadecenoicacid acid (C20:4)(ETA) acid (C20:4)(EPA) 4. M. Kolodziejczyk, Rocz Panstw Zakl Hig., 58, 287 (2009). 9,12octadecadienoicacid 5,8,11,14eicosatetraenoic octadecenoic acid (C18:1) octadecenoic acid (C18:1) octadecanoicacid (C18:0) hexadecenoicacid (C16:1) hexadecanoic acid(C16:0) 5,8,11,14eicosapentaenoic