Dynamic Analysis of Phorbol Esters in the Manufacturing Process of Fatty Acid Methyl Esters from <Emphasis Type="Italic">Jatropha curcas</Emphasis> Seed Oil

Jatropha curcas seed oil, which is unsuitable as an edible oil but has received attention as a novel vegetable fat and oil resource, contains tumor-promoting phorbol esters. Currently, six types of derivatives of 12-deoxy-16-hydroxyphorbol (DHPEs) in J. curcas oil have been identified, and their toxicological safety for humans is being discussed. However, it is reported that most DHPEs disappear during the transesterification process. We investigated the dynamics of phorbol esters in the manufacturing process of fatty acid methyl esters from J. curcas seed oil. With the assumption that the precursor ion was the fragment ion (m/z = 311) from the frame unit of phorbol esters and their derivatives, we developed an LC–MS method for detecting the product ion (m/z = 165), which was obtained by cleavage of the fragment ion. The derivatives generated from the structural changes of the phorbol esters existed in fractions of glycerine–water in the manufacturing process; however, phorbol esters and their derivatives were not detected in the fatty acid methyl esters that were produced via a high-vacuum distillation process. Investigation into the dynamics of phorbol esters confirmed that the contents of phorbol esters, including DHPEs, in the fatty acid methyl esters were under detection limits.     

Analysis of triacylglycerols in refined edible oils by isocratic HPLC‐ESI‐MS

A simple, fast and reproducible reversed‐phase high performance liquid chromatography (HPLC) method coupled to electrospray ionization mass spectrometry (ESI‐MS) for the analysis of triacylglycerols (TAGs) species in the commercial edible oils has been developed. The TAGs species were separated using isocratic 18% isopropanol in methanol and a Phenomenex C18 column. The ESI‐MS conditions were optimized using flow injection analysis of standard TAG. Fifteen, fourteen, and sixteen TAGs were separated and identified in corn oil, rapeseed oil, and sunflower oil, respectively. The presence of intense protonated molecular (M + H⁺), ammonium (M + ${\rm NH}_{4}^{ + } $ ), and sodium (M + Na⁺) adducts ions and their respective diacylglycerols ions in the ESI‐MS spectra showed correct identification of TAGs. Some minor potassium adducts (M + K⁺) were also found. In addition, the identity of the fatty acid, position of each fatty acid, and the location of the double bond in the fatty acid moiety were explained. It was found that this isocratic method is useful for fast screening and identification of triacylglycerols in lipids.     

Jatropha curcas,a promising crop for the generation of biodiesel and value‐added coproducts

The review highlights the specific features of the Jatropha curcas plant and its potential for the production of biofuel, protein concentrates as livestock feed and value‐added products that could enhance the economic viability of Jatropha seed oil‐based biodiesel production. The roles of the plant in carbon capture, enhancing socio‐economic conditions, food production in the tropical regions, and influencing micro‐climate, vegetation and soil quality are discussed. The paper also gives a comparative account of the toxic and non‐toxic genotypes of J. curcas from the point of view of their physical and chemical properties and their potential for biodiesel and livestock feed production. Future areas of research are also presented.     

Optimization of cordycepin extraction from cultured Cordyceps militaris by HPLC‐DAD coupled with uniform design

BACKGROUND: Uniform design was used to optimize the ultrasonic‐assisted extraction of cordycepin from cultured Cordyceps militaris. The peak area of cordycepin identified using high performance liquid chromatography (HPLC) at a detection wavelength of 260 nm was considered the detection index. Three factors, ethanol concentration, extraction time and volume ratio of solvent to sample were studied. Optimal quadric polynomial step by step regression was applied to process the experimental results. RESULTS: Results show that the main factors affecting cordycepin extraction yield were the volume ratio of solvent to sample, extraction time and ethanol concentration, in that order. Cordycepin extraction yield reached a peak with ethanol concentration 20.21%, extraction time 101.88 min, and volume ratio of solvent to sample 33.13 mL g^?1. An extraction model was developed based on the findings. CONCLUSION: A direct, reliable and accurate assay has been developed for the quantification of cordycepin in cultured Cordyceps militaris by a HPLC‐DAD method. The validation procedure confirmed that this method is appropriate for the quality control of cordycepin. Results show that the uniform experimental design approach is useful for finding polynomial functions describing the relationships between variables and responses and to find the best experimental conditions for the extraction of cordycepin in the experimental domain considered. Copyright © 2007 Society of Chemical Industry     

A simplified method for HPLC‐MS analysis of sterols in vegetable oil

We have developed a liquid‐chromatographic method using atmospheric pressure chemical ionization (APCI)‐mass spectrometry (MS) detection in positive mode. This method was used to separate and identify 15 sterols and 2 dihydroxy triterpenes in saponified oils, enabling the analysis of these compounds directly from saponified samples without recourse to thin‐layer chromatography; this fact thus significantly simplifies the process. The analyses were made using a Waters Atlantis 5 µm dC₁₈ 150×2.1 mm column with a gradient of acetonitrile/water (0.01% acetic acid) at a flow rate of 0.5 mL/min and a column temperature of 30 °C. The quantification of several of these compounds in soybean oil, palm oil, seed oil, sunflower oil, olive‐pomace oil and virgin olive oil was carried out using their commercial standards, and the results were compared satisfactorily with the official method.     

TAG Profiles of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. Seed Oil by Easy Ambient Sonic-Spray Ionization Mass Spectrometry

Easy ambient sonic-spray ionization mass spectrometry (EASI–MS) was used to follow the maturation of Jatropha curcas L. seeds via the monitoring of the triacylglycerides (TAG) profile of the oil. Results show that TAG composition is significantly modified during seed development but remains nearly unchanged during storage. The EASI–MS oil analysis performed herein is simple, requires just a tiny droplet of the oil and is performed without any pre-separation or chemical manipulation. The oil from Jatropha gossypifolia L. was also evaluated, and a very different TAG profile was obtained.     

Development and Validation of a Simple Reversed‐Phase HPLC‐UV Method for Determination of Malondialdehyde in Olive Oil

A simple, precise, accurate and selective method was developed and validated for determination of malondialdehyde (MDA) in olive oil. Separation was achieved on a reversed‐phase C₈ column using a mobile phase consisting of methanol/0.8 % phosphoric acid (10:90, v/v), at a flow rate of 1.0 ml/min and UV detection at 220 nm. This method was validated according to the requirements for new methods, which include accuracy, precision, selectivity, robustness, a limit of detection, limit of quantitation (LOQ), linearity and range. The current method demonstrated good linearity over the range of 0.5–1000 ppm of MDA with r² greater than 0.999. The recovery of MDA in olive oil ranged from 97.1 to 99.1 %. The method was selective where MDA was distinctly separated from other compounds of the oil with good resolution. The method was also precise where the RSD of the peak areas of replicate injections of MDA standard solution were less than 1 %. The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters and by changing the analytical operators proved that the method is robust and rugged. The low LOQ of MDA (0.5 ppm) using this method enables quantitation of MDA at low concentration.     

Removal of Phorbol Ester from Jatropha Seedcake Using Ozonation and Solar Irradiation

Jatropha curcas is a well-known source of non-edible vegetable oil that is being promoted as an energy source and high quality feedstock in biodiesel production, especially in developing countries. The potential that the resulting seedcake by-product from jatropha oil extraction (?70% by volume) could also be used as a component in animal feed raises the prospect that a commercially viable jatropha-based industry could be developed. To date, however, the use of jatropha seedcake in livestock feed formulation has been constrained by the presence of phorbol esters (PE), which are known promutagenic and toxic compounds, and by the inability to eliminate PE by cost-effective means. Using seedcake by-product collected from a commercial facility in West Africa that processes jatropha biodiesel, this study demonstrates cost-effective measures of eliminating PE from jatropha seedcake using a combination of solar irradiation and ozonation.     

Transesterification of the crude Jatropha curcas L. oil catalyzed by micro‐NaOH in supercritical and subcritical methanol

Transesterification of the crude Jatropha curcas L. oil catalyzed by micro‐NaOH in supercritical/subcritical methanol was studied. The effects of various reaction variables such as the catalyst content, reaction temperature, reaction pressure and the molar ratio of methanol to oil on the conversion of crude Jatropha curcas L. oil to biodiesel were investigated. The results showed that even micro‐NaOH could noticeably improve this reaction. When NaOH was added from 0.2 to 0.5 to 0.8 wt‐‰ of triacylglycerols, the transesterification rate increased sharply; when the catalyst content was further increased, the reaction rate was just poorly improved. It was observed that increasing the reaction temperature had a favorable influence on the methyl ester yield. For the molar ratio ranging from 18 to 36, the higher the molar ratio of methanol to oil was charged, the faster the transesterification rate seemed. At the fixed stirring rate of 400 rpm, when the catalyst content, reaction temperature, reaction pressure and the molar ratio of methanol to oil were developed at 0.8 wt‐‰ NaOH, 523 K, 7.0 MPa and 24 : 1, respectively, the methyl ester yield could reach 90.5% within 28 min. Further, the kinetics of this reaction was involved and the results showed that it was a pseudo‐first‐order reaction whose apparent activation energy was 84.1 kJ/mol, and the pre‐exponential factor was 2.21×10⁵.     

Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of <Emphasis Type="Italic">Jatropha curcas</Emphasis> Oil

Phorbol esters present in Jatropha curcas oil are toxic when consumed and are co-carcinogens. These could be a potential constraint in the widespread acceptance of Jatropha oil as a source of biodiesel. Phorbol esters were quantified in the fractions obtained at different stages of oil pre-treatment and biodiesel production. During degumming some phorbol esters were removed in the acid gums and wash water. This implies that the use of these acid gums in animal feed is not possible and care should be taken when disposing the wash water into the environment. Silica treatment did not decrease the phorbol esters, while stripping/deodorization at 260 °C at 3 mbar pressure with 1% steam injection completely degraded phorbol esters. Phorbol esters were not detected in stripped oil, fatty acid distillate, transesterified oil (biodiesel) and glycerine. The presence of possibly toxic phorbol ester degradation products in these fractions could not be ruled out.     

HPLC‐APCI analysis of triacylglycerols in milk fat from different sources

Triacylglycerols (TAG) from milk‐fat from different sources (cow, goat and human milks) were characterised using non‐aqueous reversed phase high‐performance liquid chromatography–atmospheric‐pressure chemical ionisation, coupled to MS/MS (RP HPLC‐APCI MS/MS). The fatty‐acid content of all samples was also established by methyl transesterification and GC‐MS analysis. Optimisation of the HPLC gradient, combined with APCI tandem MS, enables TAGs complex mixtures to be analysed without prior separation. More than 160 different glycerides were identified; between 50 and 70 compounds were identified in the chromatograms of each sample. This method also enabled the principal TAG regioisomers to be recognized. The study focused on the investigation of the structure of TAGs containing very‐long‐chain PUFA, namely all cis‐ 4,7,10,13,16,19‐ DHA (DHA, C_{22:6, n‐3}) and all cis‐5,8,11,14,17‐eicosapentaenoic acid (EPA, C_{20:5, n‐3}), both in human and in n‐3‐enriched cow's milks. Ten TAGs containing DHA were identified in human milk and only one in milk from cows fed an n‐3 enriched diet.     

Kinetic of epoxy resin formation by high‐performance liquid chromatography

This article is focused on the following of the cure of an epoxy resin by high‐performance liquid chromatography (HPLC) and the comparison of the data obtained with those obtained by differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) techniques usually employed for characterize curing processes. A reversed‐phase HPLC method with UV detection is developed to study the kinetic of the curing reaction of diglycidyl ether of bisphenol A (DGEBA) with 1,3‐cyclohexanebismethylamine (1,3‐BAC) at 60, 70, and 80°C, before and after gelation. The limits of quantification obtained permit the application of the proposed method until the last steps of the formation kinetic. HPLC and DSC analysis show a good correlation. The gel conversions obtained by HPLC and DMA agree well. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 497–504, 2003     

Solubilization Behavior of Phorbol Esters from Jatropha Oil in Surfactant Micellar Solutions

Phorbol esters (PEs) are important toxic compounds found in Jatropha curcas oil and pressed seeds. These compounds are tumor promoters; thus, their removal prior to further utilization of the pressed seed is important. This work aimed to investigate the solubilization behavior of PEs and Jatropha oil in nonionic [effect of the ethylene oxide number (EON), carbon‐chain length and temperature] and anionic (NaCl addition) surfactant systems. The results reveal that an increase in the EON of the nonionic surfactant molecules, rather than an increase in the carbon‐chain length, enhances PE solubilization. The hydrophile‐lipophile balance (HLB) value was correlated with PE solubilization for nonionic surfactant solutions. The solubilization of PEs decreased slightly with increasing temperature, in contrast to solubilization of the oil. Moreover, the mole fraction of PE solubilized in the micelle decreased with increasing electrolyte concentration in anionic surfactant solutions. The solubilization behavior of PEs in both nonionic and anionic solutions indicates that PE acts more like a polar compound than a nonpolar compound. In addition, the PEs in nonionic micelles are likely located in the palisade region (i.e., between the head group and the first few carbon atoms of the tail), whereas those in anionic micelles are likely near the outer core of the head group. This finding suggests that a nonionic surfactant with a higher EON has a greater potential to extract PE from Jatropha seeds. If an anionic surfactant is combined as co‐surfactant, a small amount of electrolyte should be added to increase PE solubilization.     

Potential of <Emphasis Type="Italic">Jatropha curcas</Emphasis> as a Biofuel,Animal Feed and Health Products

Jatropha curcas is a multipurpose plant with numerous attributes. It can potentially become one of the world’s key energy crops. Its seed weighs 0.53–0.86 g and the seed kernel contains 22–27% protein and 57–63% lipid indicating good nutritional value. The seeds can produce crude vegetable oil that can be transformed into high quality biodiesel. Several methods for oil extraction have been developed. In all processes, about 75% of the weight of the seed remains as a press cake containing mainly carbohydrates, protein and residual oil and is a potential source of livestock feed. The highly toxic nature of whole as well as dehulled seed meal due to the presence of high levels of shells, toxic phorbol esters and other antinutrients prevents its use in animal diet. The genetic variation among accessions from different regions of the world and rich diversity among Mexican genotypes in terms of phorbol ester content and distinct molecular profiles indicates the potential for improvement of germplasm of Jatropha through breeding programs. The extracts of Jatropha display potent cytotoxic, antitumor, anti-inflammatory and antimicrobial activities. The possibilities on the exploitation potential of this plant through various applications have been explored.     

Quality of Biodiesel Prepared from Phorbol Ester Extracted <Emphasis Type="Italic">Jatropha curcas</Emphasis> Oil

Jatropha curcas seeds are rich in oil (28–32%), which can be converted to high quality biodiesel. The oil is non-edible due to the presence of toxic compounds, namely, phorbol esters (PEs). PEs have a number of agricultural/medicinal/pharmaceutical applications and hence their recovery generates a value added co-product towards the biodiesel production chain. This study aims to assess the effects of PE extraction on quality of both the residual oil and the biodiesel production from it. Two Approaches (1, use of an Ultra-turrax; and 2, use of a magnetic stirrer) were used with an effective treatment time of 2 and 5 min, resulting in 80 and 78% extraction of PEs, respectively. The phosphorus content was reduced by 70.2 and 75.8%, free fatty acids by 55.3 and 55.6%, and the fatty acid composition did not change in the residual oils. The peroxide value increased from 2.69 (untreated oil) to 3.01 and 3.49 mequiv O₂/kg in the residual oils following Approach 1 and Approach 2, respectively. The biodiesel prepared from both residual oils met European (EN 14214:2008) and American biodiesel standard (ASTM D6751-09) specifications. Oxidative stability indices for both the biodiesels were well within the permitted limit. It is concluded that PEs could be isolated in active forms for various applications by either of the two methods with a high yield and the residual oil can be processed to produce high quality biodiesel.     

Content of trans,trans‐2,4‐decadienal in deep‐fried and pan‐fried potatoes

Trans,trans‐2,4‐decadienal is a by‐product of frying oil that is also transferred to fried food. This aldehyde has been found and quantified both in frying oils and fumes generated during frying. Furthermore, it has been reported that 2,4‐decadienal has cytotoxic and genotoxic effects and promotes LDL oxidation. In the present work trans,trans‐2,4‐decadienal was detected directly in fried potatoes (french‐fries). Moreover, the influence of frying conditions (deep‐frying, pan‐frying), the oil type (olive oil, sunflower oil, cottonseed oil, palm oil and a vegetable shortening) and the degree of thermal deterioration (eight successive frying sessions without replenishment) on the production of 2,4‐decadienal in oil and potatoes was studied. The isolation of the aldehyde was performed by methanol extraction, while the identification and quantification was performed by RP‐HPLC. The quantity of trans,trans‐2,4‐decadienal produced during successive pan‐frying demonstrated a peak at the third and fourth frying session. The highest concentration of trans,trans‐2,4‐decadienal was detected in potatoes fried in sunflower oil, and the lowest in olive oil. The quantity of trans,trans‐2,4‐decadienal in fried potatoes decreased during successive deep‐frying at the seventh frying session or remained stable, except for cottonseed oil. The quantity of trans,trans‐2,4‐decadienal in fried potatoes was considered to be dependent on the oil used, on the frying process and, to a lesser extent, on the oil deterioration. In all cases tested, the highest concentration of trans,trans‐2,4‐decadienal was detected during deep‐frying. The unsaturation degree of the frying oil was considered to promote the formation of trans,trans‐2,4‐decadienal. Considering the quantity of 2,4‐decadienal found in french‐fries and in the respective frying medium, direct quantification of 2,4‐decadienal is required in order to make an estimation of intake from french‐fries.     

Production and Quantitative Analysis of Trehalose Lipid Biosurfactants Using High‐Performance Liquid Chromatography

Trehalose lipids (THL) are glycolipid biosurfactants having a wide range of biomedical and environmental applications. Low yield, high purification cost, and the absence of a valid analytical method hinders their application. Hence, in the present study a simple, rapid, and reliable isocratic high‐performance liquid chromatography (LC) method was developed for the identification and quantification of trehalose lipid biosurfactants from Rhodococcus erythropolis. THL having a minimum surface tension of 24 mN m^?1 and a critical micellar concentration of 25 mg L^?1 were produced using hexadecane as a substrate. A standard was developed from the crude THL mixture using thin‐layer chromatography and column chromatography and its structure was confirmed using infrared spectroscopy, mass spectroscopy, and ¹H NMR. A high performance liquid chromatography (HPLC) method for quantitation was developed using a C18 column with water/acetonitrile (80:20) as the mobile phase at a 1 mL min^?1 flow rate and UV detection at 208 nm. This method was validated according to International Conference on Harmonization guidelines for linearity, precision, accuracy, robustness, LOD, and LOQ. This method was found to be linear over the range 10‐50 μg m L^?1 (r² =0.99801), precise, accurate, and robust. This method can detect minimum 3.2 μg mL^?1 and quantify minimum 9.2 μg mL^?1 of THL. Standards were developed from R. erythropolis, broth and purified standard trehalose 6,6′‐dimycolate from Mycobacterium bovis, having the same retention time of 2.0 min. The yield was calculated from the calibration curve and was found to be 25 g L^?1.     

Sensitive Colorimetric Detection of Explosive 2,6‐Bis(picrylamino)pyridine after Preconcentration by Dispersive Liquid‐Liquid Microextraction

An experimental investigation for sensitive spectrophotometric detection of explosive, 2,6‐bis(picrylamino)pyridine (BPAP) using dispersive liquid‐liquid microextraction was carried out. Based on this procedure, which is a dispersive‐solvent‐free technique, the extractant is dispersed in the aqueous sample solution using Aliquat 336 (acted as disperser agent and carrier to extraction solvent) and monitored with microvolume UV/Vis spectrophotometer. The effect of different variables such as pH, concentration of sodium hydroxide, type and volume of extraction solvent, concentration of Aliquat 336 solution and coexisting substances were systematically investigated and optimized. Interference tests showed that the developed method has a good selectivity and could be used conveniently for determination of explosive analyte. The proposed method is capable of determining BPAP over a range of 2.0–150.0 ng mL⁻¹ with a limit of detection 1.0 ng mL⁻¹. Relative standard deviations (RSD) for 20.0 and 80.0 ng mL⁻¹ of BPAP were 3.3 and 1.2 % (n=10), respectively. This colorimetric method was applied to determine BPAP in different water and soil samples.     

Effect of absorbent in solid‐phase extraction on quantification of phospholipids in palm‐pressed fiber

Palm‐pressed fiber (PPF) is a by‐product of palm oil milling and it has been found to contain a high percentage of phospholipids (PL). The aim of this work was to evaluate the best solid‐phase extraction (SPE) method to purify PL from PPF. The purified PL were analyzed using high‐performance liquid chromatography (HPLC) with an evaporative light‐scattering detector (ELSD). The oil was extracted from PPF using the Folch method and classes of PL were purified using SPE. Different solvent phases and normal‐phase SPE cartridges [silica (Si), aminopropyl‐bonded silica (NH₂) and diol‐bonded silica (2OH)] at the same ratio of oil to sorbent mass were used to study the separation of PL from the extracted oil. The recovery of each standard PL was performed in a model oil system with the same amount of standard PL being added, and the repeatability of the SPE method was investigated. The isolation of PL by SPE diol cartridge and subsequent analysis by HPLC/ELSD have shown to be an accurate and reproducible analytical method for the determination of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and lysophosphatidylcholine in PPF. This method provided a high yield and rapid separation of PL in PPF with less contamination from other lipid groups.     

Wax ester fraction of edible oils: Analysis by on‐line LC‐GC‐MS and GC×GC‐FID

The wax ester fraction of various plant oils was isolated by normal‐phase HPLC (NPLC) on‐line coupled to GC via the on‐column interface and applying concurrent eluent evaporation. The esters were analyzed by on‐line NPLC‐GC‐MS and by comprehensive two‐dimensional GC with flame ionization detection (GC×GC‐FID) off‐line combined with NPLC‐GC. GC×GC‐FID enables to group the various classes of wax esters, in particular the phytol esters, geranylgeraniol esters and the straight‐chain esters of palmitic acids and the unsaturated C₁₈ acids. Optimization of the GC×GC columns and the conditions must take into account the limited thermostability of the diterpene esters. Chromatograms are shown for a range of oils, with particular focus on the various classes of wax esters in olive oil and the geranylgeraniol esters 22:0 and 24:0 in a variety of oils.