Analytical Characterization of Fatty Acids Composition of Datura alba Seed Oil by Gas Chromatography Mass Spectrometry

Abstract

Methyl ester derivatives of fatty acids were analyzed for the determination of the constituents of
Datura seed oil. Gas chromatography coupled to mass spectrometer was used for these analyses. Results
delivered that there were saturated as well as unsaturated fatty acids in Datura alba seed oil. Total of 15
different fatty acid components were identified and quantified.Methyl linoleate was found in highest concentration
(16.22%) among the identified analytes of interest. In addition methyl esters of Palmitic acid
(6.59%), Oleic acid (5.41%) and Stearic acid (1.35%) were found. Concentrations of rest of the detected
fatty acids were less than 1%. From the literature it appears that no such work has been performed for the
determination of fatty acids in Datura alba seed oil.

Full text

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Analytical Characterization of Fatty Acids Composition of Datura alba
Seed Oil by Gas Chromatography Mass Spectrometry
Muhammad Nasimullah Qureshi,* Muhammad Siddique, Inayat-ur-Rahman and Farina Kanwal
Medicinal Botanic Centre, PCSIR Laboratories Complex Peshawar, Pakistan
Received July 9, 2010; Accepted November 23, 2010; Published Online December 14, 2010
Methyl ester derivatives of fatty acids were analyzed for the determination of the constituents of
Datura seed oil. Gas chromatography coupled to mass spectrometer was used for these analyses. Results
delivered that there were saturated as well as unsaturated fatty acids in Datura alba seed oil. Total of 15
differentfattyacid componentswereidentified and quantified. Methyllinoleatewasfound in highestcon-
centration (16.22%) among the identified analytes of interest. In addition methyl esters of Palmitic acid
(6.59%), Oleic acid (5.41%) and Stearic acid (1.35%) were found. Concentrations of rest of the detected
fatty acids were less than 1%. From the literature it appears that no such work has been performed for the
determination of fatty acids in Datura alba seed oil.
Keywords: Datura alba; Fatty acids; Methylation; GC-MS.
1. INTRODUCTION
Datura alba (Family; Solanaccae) grows in warmer
parts of the world particularly in south and south east Asia
including Pakistan, India and Sri Lanka. This annual herb
is bushy, smooth, fetid, 0.5 to 1.2 m in height also attaining
6 feet or more in rich soils. The leaves are 18 cm long and
the flowers are white in colour.1,2Besides its familiarity for
toxicityandpoisoning,3ithasusesforanumberofdiseases
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.4Datura spe-
cies produces a number of small seeds encapsulated in an
apple like fruit capsule; hence the name “thornapple” is
based on this fact. The most common and medicinally im-
portant tropane alkaloids such as hyoscyamine, atropine
and scopolamine have been isolated from Datura species.
Clarification of the dual effect of this plant requires exten-
sive research for the exploration of chemistry and pharma-
cologyoftheplantunderinvestigation.Thepresentworkis
a part of these investigations focusing on the determination
of fatty acids in oil extracted from Datura seeds.
Duetobiologicalimportance5,6fattyacidshavegained
importance in food nutrition evaluation7-10and in the diag-
nosis of certain diseases and pharmacology.11Fatty acids
with unsaturation, either monounsaturated or polyunsatu-
rated, have been used in lowering the risks of heart disease,
againstinflammationandinenhancingtheimmunityorim-
mune system.12-17
Anumber of analytical techniques have been applied
for the determination of fatty acids. These include: enzy-
matic, spectrophotometric, HPLC18-20and gas chromatog-
raphy (GC).21-23GC-MS is the method of choice for the
analysis of fatty acids due to various reasons like speed,
resolutions and sensitivity.24,25From the literature it ap-
pears that most of the research work performed is the deter-
mination of alkaloids which are main constituents of this
genus. No remarkable work has been carried out for the de-
termination of fatty acids in Datura alba seed oil which is
neededinordertoexploreitspharmacologicalimportance.
2. RESULTS AND DISCUSSION
Table1summarizestheresultsobtainedfromtheGC-
MS analysis showing the relative concentration of individ-
ual 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
Fatty Acids in Datura alba Seed Oil by GC-MS
J. Chin. Chem. Soc., Vol. 58, No. 1, 2011
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* Corresponding author. Tel: 0092-91-9216240-43; Fax: 0043-512-5072767; E-mail: mnasimuq@yahoo.com

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with R2value 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 GC-
MS 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 in-
vestigations. Linoleic acid was found in highest concentra-
tion which is necessary for the maintenance of growth. It
hasbeenshowntobeapotentinhibitorofcyclooxiginase-2
(COX-2) catalyzed prostaglandin biosynthesis.29,30Among
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, be-
sides its toxicity, can also be used in various pharmaceuti-
cal products as it contains different bioactive compounds
like fatty acids. The method applied is a reliable method of
analyzing simultaneously 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). So-
dium hydroxide solution (methanolic; 0.5 N) and sodium
chloride (analytical grade) were obtained from Merck
(Darmstadt, Germany) while methanol (HPLC grade),
n-hexane (HPLC grade) were from Fischer Scientific
(Leicestershire, UK). Helium gas (99.9999%) from Pak
gas(UnitedArabEmirates)wasprocured.Tridecanoicacid
methylesterandFattyacidmethylesters(FAMEs)37com-
ponents 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, hep-
tadecenoic acid, stearic acid, oleic acid, elaidic acid, octa-
decenoic acid, linoleic acid, octadecadienoic acid, g-lino-
lenic acid, linolenic acid, arachidic acid, eicosenoic acid,
eicosadienoic acid, 8,11,14-eicosatrienoic acid, heneico-
sanoic acid, arachidonic acid, eicosatrienoic acid, eicosa-
pentaenoic acid, behenic acid, eruccic acid, docosadienoic
acid (C22:2), tricosanoic acid, tetracosanoic acid, docosa-
hexaenoic 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 dichloromethane.
From this solution further working standard solutions were
prepared.
2
J. Chin. Chem. Soc., Vol. 58, No. 1, 2011
Qureshi et al.
Table 1. Quantification results of fatty acid methyl esters
# Name
Retention time
(min.)
AreaØ
Conc.
(%)Ø
Std. Dev.*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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
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
19654
11442
3454
2928
39973
2407
2674581
8191
13220
427823
816146
86541
1554375
30982
22200
0.16
0.11
0.02
0.01
0.18
0.01
6.59
0.21
0.09
1.35
5.41
0.92
16.22
0.22
0.65
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;ØAverage of three measurement
results.

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3.3. Extraction of oil and preparation of FAMEs
About 100 g powdered seed material was extracted
with 250 mL n-hexane26for six hours through soxhlet ex-
traction apparatus. The extract was concentrated by recov-
ering 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 per-
formed prior to GC-MS analysis. Methylation is the most
general method of converting non-volatile fatty acids into
volatile fatty acids methyl esters (FAMEs).27Methylation
of fatty acids was performed with BF3-methanol as deri-
vatizing reagent, which is the most accepted procedure for
converting fatty acids into FAMEs.23Derivatization was
performed according to the AOAC standard reference
method.28To 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 mLsatu-
rated sodium chloride solution and extracted twice with 1
mL hexane. The hexane extract was filtered through 0.45
?m membrane filter and injected 1 ?l to GCMS using auto
injector system.
3.4. Chromatographic separation of FAMEs
AgaschromatographfromShimadzuhyphenatedtoa
mass spectrometer QP 2010 plus (Tokyo, Japan) equipped
with an auto-sampler (AOC-20S) and auto-injector (AOC-
20i) was used. Helium was used as carrier gas. All chro-
matographic 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. 1, 2011
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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; 0.250 µm, treated with polyethylene
glycol), MS scanning: 85-380 m/z.

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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.
Injectiontemperaturewas240°C.Thecolumntemperature
programstartedat50°Cfor1minandchangedto150°Cat
therateof15°C/min.Thetemperaturewasraisedto175°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
tom/z380.GC-MSsolutionssoftwareprovidedbythesup-
plier was used to control the systemand to acquire the data.
Identification of the compounds was carried out by com-
paring the mass spectra obtained with those of standard
mass spectra from the NIST library (NIST 05).
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