Analytical Characterization of Fatty Acids Essay Example

Analytical Characterization of Fatty Acids Essay Example Analytical Characterization of Fatty Acids Essay Analytical Characterization of Fatty Acids Essay 2 Besides its familiarity for toxicity and poisoning,3 it has uses for a number of diseases like asthma, muscle spasm, whooping cough, skin ulcer, hemorrhoids, anti-rheumatic etc. Its oil based preparation is used for healing of all types of wounds in Ayurveda and Siddha practice of medicine since long ago. 4 Datura species produces a number of small seeds encapsulated in an apple like fruit capsule; hence the name â€Å"thornapple† is based on this fact. The most common and medicinally important tropane alkaloids such as hyoscyamine, atropine nd scopolamine have been isolated from Datura species. Clarification of the dual effect of this plant requires extensive research for the exploration of chemistry and pharmacology of the plant under investigation. The present work is a part of these investigations focusing on the determination of fatty acids in oil extracted from Datura seeds. Due to biological importance5,6 fatty acids have gained importance in food nutrition evaluation 7-10 and in the diagnosis of certain diseases and pharmacology. 1 Fatty acids with unsaturation, either monounsaturated or polyunsaturated, have been used in lowering the risks of heart disease, against inflammation and in enhancing the immunity or immune system. 12-17 A number of analytical techniques have been applied for the determination of fatty acids. These include: enzymatic, spectrophotometric, HPLC18-20 and gas chromatography (GC). 21-23 GC-MS is the method of choice for the analysis of fatty acids due to various reasons like speed, resolutions and sensitivity. 4,25 From the literature it appears that most of the research work performed is the determination of alkaloids which are main constituents of this genus. No remarkable work has been carried out for the determination of fatty acids in Datura alba seed oil which is needed in order to explore its pharmacological importance. 2. RESULTS AND DISCUSSION Table 1 summarizes the results obtained from the GCMS analysis showing the relative concentration of individual esterified fatty acids based on the external standard method and the standard deviation values among the three results in each case. Analyses were performed three times and the values of area and concentration in Table 1 are the average of three measurement results. Quantification of FAMEs was performed using three points calibration curve * Corresponding author. Tel: 0092-91-9216240-43; Fax: 0043-512-5072767; E-mail: [emailprotected] com Fatty Acids in Datura alba Seed Oil by GC-MS J. Chin. Chem. Soc. , Vol. 58, No. 2, 2011 237 Table 1. Quantification results of fatty acid methyl esters # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Name C6:0; Hexanoic acid, methyl ester C8:0; Caprylic acid, methyl ester C10:0; Capric acid, methyl ester C12:0; Lauric acid, methyl ester C14:0; Myristic acid, methyl ester C15:0; Pentadecanoic acid, methyl ester C16:0; Palmitic acid, methyl ester C16:1c; Palmitoleic acid, methyl ester C17:0; Margaric acid, methyl ester C18:0; Stearic acid, methyl ester C18:1c; Oleic acid, methyl ester C18:1n8T; Octadecanoic acid, methyl ester C18:2c; Linoleic acid, methyl ester C20:0; Arachidic acid, methyl ester C20:2; Eicosadienoic acid, methyl ester Retention time (min. ) 3. 060 4. 957 6. 796 8. 552 11. 000 12. 670 14. 692 15. 213 16. 985 19. 704 20. 266 20. 433 21. 912 27. 306 29. 756 AreaO 19654 11442 3454 2928 39973 2407 2674581 8191 13220 427823 816146 86541 1554375 30982 22200 Conc. (%)O 0. 16 0. 11 0. 02 0. 1 0. 18 0. 01 6. 59 0. 21 0. 09 1. 35 5. 41 0. 92 16. 22 0. 22 0. 65 Std. Dev. * 0. 002 0. 003 0 . 002 0. 003 0. 010 0. 003 0. 020 0. 002 0. 003 0. 010 0. 003 0. 004 0. 002 0. 004 0. 005 * Standard deviation values for the three measurement results; O Average of three measurement results. with R2 value less than 0. 99 (R2 0. 99) in each case. Fig. 1 shows the GC-MS chromatogram obtained from fatty acid standard mixture of 37 components while Fig. 2 is the GCMS chromatogram of Datura alba seed oil with properly labeled signals of analytes detected. Both the saturated and unsaturated fatty acids were found in the sample under investigations. Linoleic acid was found in highest concentration which is necessary for the maintenance of growth. It has been shown to be a potent inhibitor of cyclooxiginase-2 (COX-2) catalyzed prostaglandin biosynthesis. 29,30 Among the other fatty acids with concentrations more than 1% are: Palmitic acid (6. 59%), Oleic acid (5. 41%) and Stearic acid (1. 35%) were found. Amount of rest of the fatty acids were less than 1% (Table 1). From the results it is clear that Datura alba seeds, besides its toxicity, can also be used in various pharmaceutical products as it contains different bioactive compounds like fatty acids. The method applied is a reliable method of analyzing imultaneously many fatty acid components in a single run. 3. EXPERIMENTAL 3. 1. Chemicals and reagents Boron triflouride solution in methanol (10%) was purchased from Fluka Chemie (Buchs, Switzerland). Sodium hydroxide solution (methanolic; 0. 5 N) and sodium chloride (analytical grade) were obtained from Merck (Darmstadt, Germa ny) while methanol (HPLC grade), n-hexane (HPLC grade) were from Fischer Scientific (Leicestershire, UK). Helium gas (99. 9999%) from Pak gas (United Arab Emirates) was procured. Tridecanoic acid methyl ester and Fatty acid methyl esters (FAMEs) 37 components standard mix were obtained from AccuStandard (Newhaven, Connecticut USA). These 37 components are: methyl ester of hexanoic acid, caprylic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, myristoleic acid, pentadecanoic acid, pentdecenoic acid, palmitic acid, palmitoleic acid, margaric acid, heptadecenoic acid, stearic acid, oleic acid, elaidic acid, octadecenoic acid, linoleic acid, octadecadienoic acid, g-linolenic acid, linolenic acid, arachidic acid, eicosenoic acid, eicosadienoic acid, 8,11,14-eicosatrienoic acid, heneicosanoic acid, arachidonic acid, eicosatrienoic acid, eicosapentaenoic acid, behenic acid, eruccic acid, docosadienoic acid (C22:2), tricosanoic acid, tetracosanoic acid, docosahexaenoic acid and tetracosenoic acid. Deionized water was used through out the experimental work. 3. 2. Preparation of standard Internal standard was prepared by dissolving 13. 7 mg of tridecanoic acid methyl ester in 1 mL hexane. External standard was prepared by diluting 10 mg of 37 component FAMEs mix standard to 10 mL with di chloromethane. From this solution further working standard solutions were prepared. 238 J. Chin. Chem. Soc. , Vol. 58, No. 2, 2011 Qureshi et al. Fig. 1. GC-MS chromatogram of 37 components standard. Chromatographic conditions: inj. vol. : 1  µL, carrier gas: Helium, column: TRB-FFAP capillary column (length; 30 m, i. d. ; 0. 35 mm, thickness; 0. 50  µm, treated with polyethylene glycol), MS scanning: 85-380 m/z. 3. 3. Extraction of oil and preparation of FAMEs About 100 g powdered seed material was extracted with 250 mL n-hexane26 for six hours through soxhlet extraction apparatus. The extract was concentrated by recovering the solvent using rotary evaporator. Fatty acids are polar compounds and are not volatile. For gas chromatographic analysis it is necessary that the sample to be analyzed must be volatile. In order to make fatty acids present in the oil volatile, derivatizaion is performed prior to GC-MS analysis. Methylation is the most general method of converting non-vola tile fatty acids into volatile fatty acids methyl esters (FAMEs). 7 Methylation of fatty acids was performed with BF3-methanol as derivatizing reagent, which is the most accepted procedure for converting fatty acids into FAMEs. 23 Derivatization was performed according to the AOAC standard reference method. 28 To a known amount of sample (equivalent to 25 mg fat) was added 0. 1 mL internal standard (1. 37 mg) and 1. 5 mL of sodium hydroxide solution in methanol (0. 5 N), sealed and heated in boiling water bath for 5 minutes. The hydrolyzed sample was cooled and added 2. 5 mL of boron triflouride solution in methanol (10%). The solution was then sealed and heated in boiling water bath for 30 minutes and cooled. To the esterified solution was added 5 mL saturated sodium chloride solution and extracted twice with 1 mL hexane. The hexane extract was filtered through 0. 45 mm membrane filter and injected 1 ml to GCMS using auto injector system. 3. 4. Chromatographic separation of FAMEs A gas chromatograph from Shimadzu hyphenated to a mass spectrometer QP 2010 plus (Tokyo, Japan) equipped with an auto-sampler (AOC-20S) and auto-injector (AOC20i) was used. Helium was used as carrier gas. All chromatographic separations were performed on a capillary column (TRB-FFAP; Technokroma) having specifications: length; 30 m, i. d. ; 0. 35 mm, thickness; 0. 250  µm, treated Fatty Acids in Datura alba Seed Oil by GC-MS J. Chin. Chem. Soc. , Vol. 58, No. 2, 2011 239 Fig. 2. GC-MS chromatogram of Datura alba seed oil. Chromatographic conditions: as in Fig. 1. with polyethylene glycol. Other GC-MS conditions are: ion source temperature (EI); 250  °C, interface temperature; 240  °C, pressure; 100 KPa, solvent cut time; 1. 8 min. 1  µL of sample and standard were injected into the GC column. Injector was operated in a split mode with a split ratio 1:50. Injection temperature was 240  °C. The column temperature program started at 50  °C for 1 min and changed to 150  °C at the rate of 15  °C/min. The temperature was raised to 175  °C at the rate of 2. 5  °C/min and hold for 5 minutes. Then the temperature was increased to 220  °C at the rate of 2. 5  °C/min and kept constant for 3 minutes. Total elution time was 43 minutes. MS scanning was performed from m/z 85 to m/z 380. GC-MS solutions software provided by the supplier was used to control the system and to acquire the data. Identification of the compounds was carried out by comparing the mass spectra obtained with those of standard mass spectra from the NIST library (NIST 05). REFERENCES 1. Nadkarni, K. M. 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