Characterization of Leucaena ( Leucaena leucephala ) oil by direct analysis in real time ( DART ) ion source and gas chromatography

M. Alam, N.M. Alandis, E. Sharmin, N. Ahmad, B.F. Alrayes and D. Ali Research Center-College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia Department of Pharmaceutical Chemistry, College of Pharmacy, Umm Al-Qura University, P.O. Box 715, Makkah Al-Mukarramah 21955, Saudi Arabia Central Laboratory, College of Science, Riyadh-11451, King Saud University, Kingdom of Saudi Arabia Department of Zoology, College of Science, Riyadh-11451, King Saud University, Kingdom of Saudi Arabia Corresponding author: malamiitd@gmail.com


INTRODUCTION
The precise word 'leucocephala' derived from 'leu', meaning white, and 'cephala', meaning head, refers to flowers.Leucaena leucocephala is essentially a tropical species requiring warm temperatures (15-25 o C) for optimum growth (Mullen et al., 2003).It is found in regions of Australia, US, Philippines, Thailand, China, Pacific islands, Mexico and India.In India, it is commonly known as Subabul / Kababul.It belongs to the family Fabaceae (Leguminosae) and sub-family Mimosaceae.The legume size is 10-17cm long containing 15-25 seeds each (Muthukrishnan et al., 2013).L. leucocephala is a fast growing hedgerow, medium-sized shrub.It is used in alley cropping and can release enough nitrogen to satisfy the requirement of maize grown in the alley crops (Mulongy et al., 1998).Its pod extract is a cheap, nontoxic and environmentally friendly natural product, used as corrosion inhibitor in an acidic medium (Muthukrishnan et al., 2013).The need for vegetable oils characterization arises in many aspects such as product development, quality assurance, product shelf life, and detection of adulteration.The industrial value of vegetable oils depends on their fatty acid composition and the ease by which these can be modified (Bhatnagar et al., 2009).
The most common method for the analysis of vegetable oils is gas chromatography.This technique is based on the selection of stationary phases, mobile phases, detectors, column temperature program and an appropriate resolution of carbon chain.Several scientific instruments are used to develop rapid, reliable and cost effective techniques for the analysis of vegetable oils such as gas chromatography-mass spectroscopy (GC-MS), high performance liquid chromatography (HPLC), matrix assisted laser desorption/ionization mass spectrometry (MALDI) and others (Dugo et al., 2012;Mercy et al., 2016;Kubo et al., 2013).TOF-MS techniques are advanced, efficient, much faster, have high data acquisition rate and a small internal volume for mass analysis (Vaclavik et al., 2009).Other advantages include rapid screening for target analytes, identification of non-target analytes by accurate-mass determination of protonated or de-protonated molecules and their product-ion mass spectra, quantification capability or performance, and reproducibility.The most frequently employed MS-related technique for the direct analysis of samples is direct analysis-inreal-time (DART) coupled with quadrupole timeof-flight mass spectrometry (Q-TOFMS).
In TOF analysis, molecules are charged by applying voltage between orifice and optics lens of ion source; the mass profile produced by applied voltage can be varied with respect to the amount of charged fragmentation.Typically, at low potential difference, positively charged species pass into the flight tube while at higher potential difference, induced-collision takes place which provides more spectral and isomeric information through fragmentation.TOF software has the ability to collect various orifice voltages consecutively, giving a full profile of a sample (Mess et al., 2013;Gómez-Gonza et al., 2011).
The present study investigated the triacylglycerols (TAGs) of pure L. leucocephala oil [LUCO] and their FAMEs by TOF-MS, fatty acid composition by GC, structural analysis by FT IR, 1 H NMR, 13 C NMR and thermal stability by TGA-DSC.We also measured the cytotoxicity of LUCO.

Materials
We collected ripe legumes from the L. leucocephala plant (University Campus, King Saud University) and ginning seeds.Seeds were powdered, and the oil was extracted from powdered seeds through a soxhlet apparatus using petroleum ether as solvent.After extraction, petroleum ether was removed by a vacuum rotatory evaporator and yield was found as 5.67%.Petroleum ether, sulphuric acid (BDH, Poole, England), and methanol (Sigma-Aldrich, Steinheim, Germany) were used without any further purification.

Instrumentation and testing conditions
For the experiments, an Accu TOF LC plus (JMS-T 100LP) system consisting of a DART ion source (IonSense, DART), an Accu TOF LC plus a higher solution time-of-flight mass spectrometer (JEOL, Japan) and an auto injection valve were used.The operating conditions of the DART ion source were as follows: positive ion mode; discharge needle voltage: 3.0kV; perforated and grid electrode potentials: +100 and -100 V, respectively.Conditions of TOF-MS: cone voltage: 2000 V, monitored mass range: m/z 50-10000; acquisition rate: 1600 spectra min −1 ; resolving power: approx.6000 FWHM (full width at half maximum).The distance between the DART gun exit and mass spectrometer inlet was 7mm.The sampling glass rod was immersed for 1s into the sample containing approx.500µL of the respective sample and was transferred to the optimized position in front of the DART gun exit and desorbed for 5s, while the spectral data were recorded.For each sample/PEG standard, a minimum of three repeated measurements were carried out.At the end of each run, the mass spectra of polyethylene glycol (PEG, average relative molecular weight 600/1000, Sigma-Aldrich, USA) methanol solution (200µL/ mL −1 ) were acquired to perform mass drift compensation (Easter J L et al., 2014;Wang Wang Y et al., 2014).The influence of gas beam temperature on signal intensity, LUCO and FAMEs were analyzed at 200 o C. The final spectra were obtained after the background was subtracted.The operating conditions were as follows: Gas Chromatography, Clarus 500 GC with FID, Perkin Elmer, USA, Column: Supelco Wax, 30 m long, 0.32 mm id, phase thickness 0.25μm, injection mode: split; split ratio, 1:100, 1.0μL injection used, diluted (1/10 v/v, FAMEs/hexane), Column temperature 140-240 o C, 4 o C/min, an initial temperature hold at 140 o C for 5 min, FID temperature 260 o C, Helium carrier gas flow 1.0 mL/ min, Hydrogen 30 mL/min, Air 300 mL/min.The FAMEs mix standard was analyzed under the same operating conditions to determine the peak identity.The FAMEs were expressed as relative area percentage (Dodds et al., 2005;Fardin-Kia et al., 2013).
LUCO spectra were taken on a FT IR spectrometer, Spectrum 100, Perkin Elmer, USA. 1 H and 13 C NMR spectra were recorded on Jeol DPX400 MHz using deuterated chloroform as solvent and tetra methyl silane as an internal standard.The thermal analysis of LUCO was carried out by simultaneous thermogravimetric analysis-differential scanning calorimetry (TGA-DSC) (Model TGA/DSC1, Mettler Toledo AG, Analytical CH-8603, Schwerzenbach, Switzerland) in nitrogen atmosphere (99.997 purity).The metal analysis of acid digested LUCO oil was analyzed with Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS); ThermoScientific, Instrument, USA.

Preparation of FAMEs of LUCO
Initially LUCO was derivatized by the alkaline saponification of vegetable oil (to break down glycerides because of their low volatility in nature) and was further esterified in the presence of methanol and a catalyst.LUCO (500mg) was refluxed with a 5mL methanolic sodium hydroxide (0.5M) solution at 150±5 o C for 5 minutes on a hot plate.5 mL of a boron trifluoride methanol solution were added and the whole content was further heated at 150±5 o C for 5 minutes.The flask was removed from the hot plate; 5 mL of n-hexane and 10 mL of a saturated aqueous sodium chloride solution were added and the contents were agitated thoroughly.An aliquot of supernatant hexane layer was transferred into anhydrous sodium sulphate to remove moisture.The above 10 µL filtrate was analyzed by GC-FID (Lee et al., 1998;Reiter et al., 2001;Řezanka et al., 2001).

Digestion of LUCO for ICP-MS
A certain amount (250mg) of LUCO sample was put into a digestive vessel of 60 mL capacity, with 8 mL of 65% nitric acid and the mixture was shaken carefully with a clean polytetrafluroethylene (PTFE) bar.After 30 minutes, the vessel was closed.The sample and blank (without oil) vessels were placed in the Topwave Analytik Jena microwave for digestion and the program temperature was set at 140 o C, 50 bar pressure for 8 minutes (ramp).The vessels were then left to cool at room temperature.The digested aliquots were filtered with a whatman no.42 filter paper and the filtrate was diluted with 50 mL deionized water (Pehlivana et al., 2008;Hsu W H et al., 2013).ICP-MS calibration was carried out by external calibration using 10 ppm of multi element standard solutions (Aristar grade, BDH laboratory supplies, England) for the trace elements.The sample and blank were analyzed in triplicate.The results are summarized in Table 1.

TOF-MS spectra of LUCO
The representative TOF-MS spectra of LUCO are shown in Figure 1.The spectra were divided into three categories according to ion fragmentations.The elemental (C, H, O) compositions of the major peaks were calculated by a software based on the exact mass number of the elements.The mass differences of identified compounds differed from their theoretical mass numbers by less ).However, the differentiation of positional isomers of TAGs with two or three different fatty acids is not possible by TOF-MS, due to the absence of separation of the isobaric molecules prior to ionization.The relative intensities of molecular adducts, as well as molecular ions are present in TOF-MS spectra (Vaclavik et al., 2009;Hajslova et al., 2011;Bosque-Sendra et al., 2012;Xu et al., 2015).
In the TOF-MS spectra (Figure 1), the extensive fragmentation of TAG acquisition resulted in the occurrence of numerous types of ions under the experimental conditions.The most abundant were diacylglycerol fragment ions created by the loss of one fatty acid molecule from the glycerol backbone.
Ions at m/z 593 to 709 were identified as markers for diacylglycerols fragment ions: palmitolinolein (PL, m/z 593.493), oleyllinolein (OL, m/z 620.530), diolein (OO, m/z 623.530), palmitoylgadolein (PG, m/z 624.551), linolylarchidin (LnA, m/z  ).The main fragment ion (m/z 601.51) was the base peak in all spectra of LUCO; the least intensive was the monoglycerol ion of its corresponding fatty acid ion (Wang et al., 2014).It is, therefore, TOF-MS followed by an appropriate method for the differentiation of TAGs for the identification of compounds.These observations confirmed that the results obtained from TOF-MS data were good and it was the choice of instrument for the screening of vegetable oil.Therefore, it is necessary to resort to multivariate techniques, which are important and proven techniques for complex data analysis (Vaclavik et al., 2009).The fatty acids involved may all be the same or different number of acids in many possible combinations.It is precisely this variation which gives rise to the wide spectrum of vegetable oils.It is not possible here to write the exact fatty acid sequence of the triglycerides.TOF-MS provides us a new way for vegetable oil analysis with speed of analysis, simplicity of sample introduction, and high sensitivity (Lesiak A D et al., 2014).The TOF-MS technique has been applied for the first time for the profiling of LUCO and was done successfully.

FTIR(cm
FTIR shows -CH 2 , -CH 3 symmetrical and asymmetrical absorption bands at 2926.07cm -1 and at 2854.82cm -1 .The absorption bands for -CH= CH-str appear at 3008.54cm -1 and also the carbonyl ester band at 1746.51cm -1 .The presence of unsaturation is also supported by characteristic peaks in the 1 H NMR and 13 C NMR spectra at 5.330-5.362ppm and 127.966-130.298ppm,respectively.The rest of the characteristic peaks for typical functional groups in the LUCO backbone have been mentioned above.

HepG2 cells toxicity
Figure 7 shows the growth inhibition graph of LUCO at different doses (0, 50, 100, 300, 700 µL/mL).Taking the results of all concentrations into account, the cell death in HepG2 cells was found to be higher at (700 µL/mL) by the MTT assay.This result shows that the cytotoxicity of HepG2 cells was oil dose dependent.In this study, we quantified the cytotoxicity of oil in human hepatocarcinoma cells for 24 hour by employing the MTT test.Finally, IC 50 values were calculated for the oil according to the MTT test.

CONCLUSION
The TOF-MS technique was effectively applied to study the composition of the TAGs of LUCO.The applied operating conditions of the DART ion source were successfully investigated to obtain the best detection limit with high quality mass profile.The TOF-MS finger prints study could provide a useful technique for the rapid identification of vegetable oil, finding of adulteration, quality assurance, and the evaluation of similarities and improvement
DSC (Figure 5) thermogram of L. leucocephala oil shows two endotherms, first at -30.25 to -13.53 o C, centered at -24.51 o C, the second endotherm starting at -11.50 to 8.57 o C, centered at 3.33 o C. The first endotherm belongs to phase transition and the second to melting.The TGA-DTG (Figure 6) thermogram shows the onset of degradation at 350 o C, no weight loss was observed at this temperature; only 4% weight loss occurs at 380 o C due to moisture and volatile impurities.50% weight loss occurs at 460 o C. The TGA thermogram shows single step degradation.
At 525 o C, a complete decomposition of oil occurs.This decomposition is assigned to the degradation of the hydrocarbon chain of the oil.In the DTG curve, an endothermic transition between 375 o C and 525 o C assigned to the hydrocarbons was also observed.The maximum weight loss of the sample according to the DTG curve was observed at 375 o C to 525 o C.
of product quality.The fatty acid profile plays a key role to the physico-chemical properties for further advanced research.

Table 1 .
List of trace metals present in LUCO