Processing impact on tocopherols and triglycerides composition of soybean oil and its deodorizer distillate evaluated by high-performance liquid chromatography

In the present study, high-performance liquid chromatography (HPLC) was used for the separation of tocopherols and triglycerides of processed soybean oil and deodorizer distillate (DD). The results of normal and reversed-phase modes of HPLC revealed that concentrations of tocopherols and triglycerides content were decreased during the neutralization, bleaching, and deodorization processes. The loss of individual tocopherols ranged between 55.16% and 63.25%. During processing, triglycerides containing stearic-oleic-linoleic (SOL) moieties and palmitic-palmitic-linoleic (PPL) fragments showed greater reduction up to 38.14% and 37.69%, respectively. Among tocopherols and triglycerides; γ-tocopherol and oleic-oleic-oleic (OOO) were found to be in greater concentrations 5.53% and 19.78%, respectively in DD as compared to their counterparts. A maximum reduction of tocopherols was observed in the deodorization step. DD was found to be a rich source of bioactive components; therefore, it could be used for many industrial applications including pharmaceutical formulations, cosmetics, and food industries.     

Liquid chromatographic method for the analysis of tocopherols in malt sprouts with supercritical fluid extraction.

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the determination of tocopherols in malt sprouts. A supercritical fluid extraction (SFE) procedure was used to isolate tocopherols from the vegetal matrix before quantitative analysis. The analytes were separated on a Zorbax reversed-phase column using methanol-water as mobile phase and quantified by measuring its fluorescence at lambda(em)=328 nm after excitation of the analytes at lambda(exc)=303 nm. The limits of detection for alpha-, gamma- and delta-tocopherols were 0.04, 0.05, and 0.05 microg/ml, respectively. The calibration graphs of the method were linear from 0.1 to 1.5, 0.2 to 2.5, and 0.2 to 2.0 microg/ml, for alpha-, gamma- and delta-tocopherols, respectively. This SFE and HPLC procedure is simple, precise and accurate for the determination of tocopherols in malt sprouts.     

Quantitative determination of alpha-, beta-, gamma- and delta-tocopherols in human serum by high-performance liquid chromatography and gas chromatography-mass spectrometry as trimethylsilyl derivatives with a two-step sample preparation

Using a two-step sample preparation with Extrelut and silica gel extraction in Pasteur pipettes it is possible to quantify all tocopherols in human serum samples by means of normal-phase HPLC with fluorescence detection (lambda(ex) 295 nm, lambda(em) 330 nm) or by GC-MS of their trimethylsilyl (TMS) derivatives. The method has been used in pharmacoepidemiological studies concerning the exposition with vitamin E-containing drugs in Germany. The recovery for all tocopherols is 98% and the limit of detection is 50 pg for alpha-tocopherol in the HPLC and 40 pg for all TMS-tocopherols in the GC-MS method using the selected ion monitoring mode with a well-tuned GCQ system. Linearity of calibration is excellent for both methods over the full physiological relevant range. Due to the low sample amount needed, the method is suitable for epidemiological and paediatric research.     

A Fast and Efficient Ultrasound-Assisted Extraction of Tocopherols in Cow Milk Followed by HPLC Determination

A fast HPLC method with fluorescence detector (FD) was developed for the determination of three tocopherols (TOCs) in milk samples from Modicana cattle breed. The ultrasound-assisted procedure was optimized for the extraction of TOCs prior to HPLC/FD analysis, reducing sample preparation time and allowing a fast quantification of α-tocopherol, δ-tocopherol and γ tocopherol. The optimized ultrasonic extraction combines an efficient and simple saponification at room temperature and a rapid HPLC quantification of TOCs in milk. The precision of the full analytical procedure was satisfactory and the recoveries at three spiked levels were between 95.3% and 87.8%. The linear correlations were evaluated (R² > 0.99) and the relative standard deviation (RSD) values for intra-day and inter-day tests at three spiked levels were below 1% for the retention time and below 5.20% for the area at low level spiking. The proposed procedure, reducing the experimental complexity, allowed accurate extraction and detection of three TOCs in milk samples from Modicana cattle breed.     

Rapid analysis of the major classes of retinoids by step gradient reversed-phase high-performance liquid chromatography using retinal (O-ethyl) oxime derivatives

A rapid step-gradient reversed-phase high-performance liquid chromatography (HPLC) method is presented for analysis of the major classes of retinoids in tissues. Retinal was converted into a new derivative, retinal (O-ethyl) oxime, since the standard derivative, retinaloxime, co-elutes with retinol on reversed-phase HPLC. The most abundant naturally occurring retinyl esters, retinyl palmitate and retinyl stearate, were eluted within 12 min to complete the separation. Retinoids were extracted in the presence of an antioxidant, butylated hydroxytoluene, and a lipid carrier, cholesterol. Recoveries of 98-100% were obtained from tissue samples by internal addition for the retinoids tested (retinol, retinal and retinyl palmitate); and the absolute recovery of endogenous retinal from rat eyecups was confirmed by spectrophotometric measurements of rhodopsin. Extraction was carried out in an air atmosphere and under subdued incandescent light rather than requiring inert atmosphere and safe-light conditions used in most methods. Cis-trans isomers were not separated under the reversed-phase HPLC conditions employed. Quantitation was carried out using retinyl acetate as internal standard and the day to day precision was better than 3.5%. A sensitivity of about 1 ng is obtained for all retinoids using absorbance monitoring at 325 nm and a C18 5 micrometers column with 12% reversed-phase loading. The tocopherols can also be separated and detected simultaneously with similar sensitivity by this method using a fluorescence detector in series [G. J. Handelman, L. J. Machlin, K. Fitch, J. J. Weiter and E. A. Dratz, J. Nutr., 115 (1985) 807].     

Chemical characterisation of old cabbage (Brassica oleracea L. var. acephala) seed oil by liquid chromatography and different spectroscopic detection systems

We report an extensive chemical characterisation of fatty acids, triacylglycerols, tocopherols, carotenoids and polyphenols contained in the oil extracted from old cabbage (Brassica oleracea L. var. acephala) by cold-pressing of the seeds. Analyses were performed by GC-FID combined with mass spectrometry, HPLC with photodiode array, fluorescence and mass spectrometry detection. The 94% of the total fatty acids were unsaturated, rappresented by erucic acid (more than 50%) followed by linoleic, linolenic and oleic acids accounting for approximately 10% each. The most abundant triacylglycerols (>13%) were represented by erucic–gadolenic–linoleic, erucic–eruci–linoleic and erucic–erucic–oleic. Among tocopherols, γ-tocopherol accounted for over 70% of the total content. Thirteen carotenoids and 11 polyphenols were identified and measured. In particular, the total content in carotenoids was 10.9 ppm and all-E-lutein was the main component (7.7 ppm); among polyphenols, six hydroxycinnamic acids and five flavonoids, were identified by combining information from retention times, PDA and MS data.     

Simultaneous extraction of tocotrienols and tocopherols from cereals using pressurized liquid extraction prior to LC determination

Tocopherols and tocotrienols have been simultaneously determined in food samples using a rapid and simple analytical method including pressurized liquid extraction (PLE) and LC with electrochemical detection. Separation was carried out on a Phenomenex Synergi 4 μm Hydro‐RP 80A column, using a solution of 2.5 mM acetic acid/sodium acetate in methanol/water (99:1, v/v) as mobile phase at a flow rate of 1.0 mL/min. Column temperature was maintained at 30°C. Detection was performed by coulometric detection at 500 mV except for (β+γ)‐tocotrienol, in wheat and rye samples, which was at +350 mV. A palm oil containing a relatively large amount of γ‐tocotrienol and lower concentrations of α‐ and δ‐tocotrienols and α‐ and γ‐tocopherols was used to provide reference retention times for the tocotrienols. Analyte quantification was performed using the external standard method. The calibration equations of tocopherols were used to quantify both tocopherols and their corresponding tocotrienols. The extraction recoveries obtained using the optimized PLE conditions were in the 80–114% range, with RSDs lower than 15%. The method was successfully applied to the determination of tocotrienols and tocopherols in cereal (wheat, rye, barley, maize and oat) and palm oil samples.     

Development and validation of an HPLC method for the simultaneous determination of tocopherols, tocotrienols and carotenoids in cereals after solid-phase extraction

The increasing interest in antioxidant properties of cereal and cereal-based products has prompted the development of a simple and reliable HPLC method for the simultaneous determination of important phytochemicals like tocopherols (T), tocotrienols (T3) and carotenoids. Separation was carried out on a Nucleosil 100 C(18) column, 5 μm (250 mm × 4.6 mm) thermostated at 25 °C, using a linear gradient elution system starting with methanol and ending with a mixture of methanol-isopropanol-acetonitrile. All separated compounds including the internal standard (α-tocopherol acetate) were eluted within 16 min and detected by dual detection: fluorescence for tocopherols and tocotrienols at 290 nm excitation and 320 nm emission and UV-vis photodiode array detection for lutein and β-carotene at 450 nm. Detection limits ranged from 0.2 μg/g (β-carotene) to 1.60 μg/g (α-tocopherol). The intra- and inter-assay coefficients of variation were calculated by using cereals with different levels of lipophilic antioxidants. The extraction method involved sample saponification and clean-up by solid-phase extraction (SPE). The extraction recoveries obtained using OASIS HLB SPE cartridges and dichloromethane as eluent were in the range of 90.2-110.1%, with RSD lower than 10%. The method was successfully applied to cereals: durum wheat, bread wheat, rice, barley, oat, rye, corn and triticale.     

Microemulsion electrokinetic chromatography for the separation of retinol, cholecalciferol, delta-tocopherol and alpha-tocopherol

A microemulsion electrokinetic chromatographic method was used to separate fat-soluble vitamins. The separation of retinol, cholecalciferol, and delta- and alpha-tocopherol was performed using a microemulsion containing 0.75% (v/v) n-heptane, 30 mM bis(2-ethylhexyl)sodium sulfosuccinate (AOT), 5% (v/v) 1-butanol, 15% (v/v) 1-propanol and 15% (v/v) methanol in 20mM boric acid-sodium borate buffer. The effect of the different microemulsion constituents was studied, including the type and concentration of surfactant, buffer, oil and co-surfactants. The presence of methanol in the microemulsion was found to be necessary to achieve the separation of the tocopherols. Detection was carried out at 200, 265 and 325 nm for the tocopherols, cholecalciferol and retinol, respectively. Calibration curves and precision data were obtained for each analyte. Good linear relationships were found between the analytical signal and the analytes concentration in the 25-500 mg L(-1) for retinol and cholecalciferol, and 25-300 mg L(-1) for tocopherols ranges. The precision of the method afforded relative standard deviations in the 4.0-10% range.     

The simultaneous determination of capsaicinoids,tocopherols, and carotenoids in pungent pepper powder

From nutritional points of view, carotenoids, capsaicinoids, and tocopherols are valuable constituents in pungent peppers. A rapid and reliable high performance liquid chromatography (HPLC) method for the simultaneous determination of phytonutrients in spice red peppers and chili products was developed and validated. The method included simultaneous detection by fluorescence and diode-array detectors. The major capsaicinoids, two tocopherols and 43 carotenoid components, were simultaneously separated, detected, and identified in the appointed pepper powder (containing Capsicum annuum and Capsicum frutescens) for method validation. The separation was performed on a Nucleosil C18 reverse phase column and optimized gradient elution. Resolution ranged between 0.96 and 1.46 with the highest values corresponding to γ-tocopherol and α-tocopherol. The limits of detection and quantification of target compounds ranged between 18.77 and 148.08 ng mL^?1. Recoveries were between 89.83–100.26 and 79.72–88.86% when standard materials were spiked at low and high amounts, respectively. The most sufficient extraction of the different phytonutrients was achieved by mixture of methanol and acetone, although it was only slightly better than the mixture of methanol and acetonitrile. These results suggest that the developed method could be used for rapid, one-step determination of a wide range of phytonutrients in chili and pepper powders.     

Organic acid, tocopherol, and phenolic compositions of some Turkish grape cultivars

The organic acid, tocopherol, and phenolic compositions of three different grape cultivars, Emir, Kalecik karasi, and Narince were studied in order to evaluate their nutritive values and the contents of natural antioxidants. Organic acids, tocopherols, phenolic acids, flavonoids, and trans-resveratrol contents were analyzed by high-performance liquid chromatography (HPLC). In addition, pH, soluble solid, titratable acidity, and total phenolic contents were also determined. It was determined that the contents of organic acids, tocopherols, and phenolic compounds were changed according to the cultivars. Published in Khimiya Prirodnykh Soedinenii, No. 2, pp. 128–130, March–April, 2006.     

Effect of stationary phase structure on retention and selectivity tuning in the high-throughput separation of tocopherol isomers by HPLC

Four stationary phases containing different groups such as: C18, C30, alkylamide, and cholesterolic, were presented for simultaneous HPLC analysis of structural isomers of tocopherol. Especially, the influence of stationary phase structure and properties on tuning of the highly selective HPLC separation of beta- and gamma-tocopherol pair demonstrating, respectively, para- and ortho- arrangement of methyl substituents on the 6-chromanol ring, has been elucidated. It was pointed out that selectivity of each stationary phase has been a result of modulation in the mass transfer and set of unspecific interactions in the tertiary system comprising analyte <==> stationary phase <==> mobile phase. Differences in observed retention and specific selectivity of tocopherols together with the stationary phase structure investigations indicated that a spatial organization changing of chemically bonded ligands as predominantly a solvation consequence. Additional molecular modeling studies preliminary explained some of these complicated supramolecular phenomena which caused that cholesterolic stationary phase offered beneficial performance in screening of tocopherols by HPLC and biomimetic studies of not completely recognized interactions of tocopherol isomers and biological membranes.     

Chromatographic analysis of tocol-derived lipid antioxidants

This paper provides a comprehensive overview of existing chromatographic methods for the analysis of tocol-derived lipid antioxidants in various sample matrices. After a brief introductory discussion on biological and nutritional aspects of the vitamin E active compounds, the review focuses on various techniques for the isolation, purification, chromatographic separation, and detection of tocopherols and tocotrienols. Compiled published normal-phase (NP) and reversed-phase (RP) high-performance liquid chromatographic (HPLC) methods demonstrate general trends and analytical variability and versatility of HPLC methodology. The relative merits of the two HPLC methods are assessed. NP and RP elution characteristics are delineated to aid in the identification of antioxidant components. Technical novelty of certain analytical procedures for non-food samples warrants their inclusion in this review in light of the potential applicability in food assays.     

Rapid determination of vitamin E in vegetable oils by reversed-phase high-performance liquid chromatography

A quick and direct method for measuring tocopherols (alpha, beta+gamma and delta) in vegetable oils has been developed using RP-HPLC with UV detection. Previous extraction of tocopherols is not required. The oil is diluted in hexane and an aliquot is mixed with ethanol containing an internal standard (alpha-tocopherol acetate). The chromatographic system consists of an ODS-2 column with a methanol-water mobile phase. Tocopherols are detected at 292 nm in less than 5 min after injection. The method is precise (RSD=2.69%) and has a high mean recovery (98.14%).     

快速皂化-气相色谱-串联质谱法同时测定乳粉中胆固醇和维生素E 4种异构体

胆固醇和生育酚都是人体所必需的重要营养元素,是乳粉中的重要质量指标。现行国家食品安全标准对胆固醇和维生素E 4种异构体（α -生育酚、β -生育酚、γ -生育酚和δ -生育酚）的检测前处理复杂繁琐、耗时长,且不能同时测定,因此,开发简单、快速且可同时测定胆固醇和4种生育酚的检测方法具有重要的现实意义。该研究采用气相色谱-串联质谱（GC-MS/MS）建立了乳粉中胆固醇和4种生育酚的定性定量测定方法。样品经脂肪酶酶解,采用快速的碳酸钾-乙醇皂化法,同时对酶解时间、皂化温度、萃取溶剂的种类、萃取溶剂的体积、萃取时间等前处理进行优化,从而确定出最优的前处理方法。结果表明,样品在37℃下酶解4 h,然后室温（25℃）下振荡10 min皂化,最后使用5 mL正己烷萃取10 min,4000 r/min离心6 min,取上层正己烷过膜,经TG-5MS Sil色谱柱分离,GC-MS/MS多反应监测离子模式扫描分析,同时获得定性和定量结果。实验结果表明,胆固醇在0.5~50.0 mg/L、4种生育酚在0.25~25.0 mg/L范围内具有较好的线性关系,相关系数（r ² ）大于0.99,加标回收率为76.6%~93.1%,相对标准偏差为0.9%~3.3%,胆固醇定量限为10.0 μg/100 g,4种生育酚的定量限均为5.0 μg/100 g。随机抽取市面上出售的20种婴幼儿配方奶粉及4种低脂乳粉,对其胆固醇和4种生育酚含量进行了分析测试,结果显示,婴幼儿配方乳粉中胆固醇、4种生育酚的含量高于低脂乳粉,其中婴幼儿乳粉的4种生育酚中α -生育酚和β -生育酚含量较高。该方法简便、快速、灵敏、准确,可满足乳粉中胆固醇和4种生育酚生育酚的检测要求,为乳粉品质的快速检测奠定了理论基础。     

The establishment of tocopherol reference intervals for Hungarian adult population using a validated HPLC method

Evidence suggests that decreased α ‐tocopherol (the most biologically active substance in the vitamin E group) level can cause neurological symptoms, most likely ataxia. The aim of the current study was to first provide reference intervals for serum tocopherols in the adult Hungarian population with appropriate sample size, recruiting healthy control subjects and neurological patients suffering from conditions without symptoms of ataxia, myopathy or cognitive deficiency. A validated HPLC method applying a diode array detector and rac‐tocol as internal standard was utilized for that purpose. Furthermore, serum cholesterol levels were determined as well for data normalization. The calculated 2.5–97.5% reference intervals for α ‐, β /γ ‐ and δ ‐tocopherols were 24.62–54.67, 0.81–3.69 and 0.29–1.07 μm , respectively, whereas the tocopherol/cholesterol ratios were 5.11–11.27, 0.14–0.72 and 0.06–0.22 μmol/mmol, respectively. The establishment of these reference intervals may improve the diagnostic accuracy of tocopherol measurements in certain neurological conditions with decreased tocopherol levels. Moreover, the current study draws special attention to the possible pitfalls in the complex process of the determination of reference intervals as well, including the selection of study population, the application of internal standard and method validation and the calculation of tocopherol/cholesterol ratios.     

Cyclodextrin-modified microemulsion electrokinetic chromatography for separation of alpha-, gamma-, delta-tocopherol and alpha-tocopherol acetate

Different forms of tocopherols, together with tocotrienols, are collectively named as vitamin E, and each possesses different degree of medical, biological and physiochemical significance. The main difficulty of separating different forms of tocopherols lay in their highly structural similarities and hydrophobicities. Microemulsion electrokinetic chromatography (MEEKC), claimed to attain high peak efficiency with great solubilization power, has not previously been applied to the separation of tocopherols. The effects that various parameters, such as buffer system, type and concentration of cyclodextrins, temperature, and sample matrix, have on the separation of tocopherols by MEEKC have been investigated. By using a buffer mixture of 4% (w/w) sodium dodecyl sulfate (SDS), 6.6% (w/w) 1-butanol, 0.8% (w/w) n-octane, 20% (w/w) 2-propanol, 68.6% (w/w) phosphate (25mM, pH 2.5), and 25mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD), the separation of alpha-, gamma-, and delta-tocopherol, alpha-tocopherol acetate, as well as the antioxidant butylated hydroxytoluene (BHT) at -26kV, 25 degrees C was completed within 35min. The practical potential of the present approach has been further validated by the determination of tocopherols in a vitamin E preparation, with the result of 132.63 (RSD 1.25%), 176.51 (RSD 0.29%), and 64.32mg (RSD 3.34%) per 500mg capsule for alpha-, gamma- and delta-tocopherol, respectively.     

Determination of tocopherols in vegetable oils by CEC using methacrylate ester-based monolithic columns

The separation and determination of tocopherols (Ts) in vegetable oils by CEC using methacrylate ester-based monolithic columns has been developed. The effects of pore size of the monolithic columns were studied, and the composition of mobile phase was optimized. The optimal pore size of the monolith was obtained with 12 wt% 1,4-butanediol in the polymerization mixture. Excellent resolution between tocopherols was achieved within 10 min analysis time with a 99:1 v/v MeOH-aqueous buffer containing 5 mM tris(hydroxymethyl)aminomethane at pH 8.0. The LODs were lower than 2.3 microg/mL, and interday and column-to-column reproducibilities at 25 microg/mL were better than 5.6%. Using a 93:7 v/v MeOH-aqueous buffer, both tocopherols and tocotrienols (T(3)s) of grapeseed and palm oils were resolved. Application to the detection of olive oil adulteration with low-cost edible oils was demonstrated.     

Method for simultaneous analysis of eight vitamin E isomers in various foods by high performance liquid chromatography and fluorescence detection

The objective of this study was to optimize a method to investigate the occurrence and to quantify the full isomeric composition of vitamin E (α-, β-, γ- and δ-tocopherols and tocotrienols) in 6 vegetables (raw and cooked), 3 herbs/spices, raw and cooked eggs, vegetable oils (canola, olive and soybean), flaxseed and sorghum (flour and seeds) and soy (flour) by HPLC with fluorescence detection. Different conditions of extraction and analysis were tested. The optimized method consisted of direct extraction with solvent (hexane:ethyl acetate, 85:15, v/v). For analysis normal phase column was used with mobile phase consisting of hexane:isopropanol:acetic acid (98.9:0.6:0.5) with isocratic elution and fluorescence detection. Excellent separation of all isomers was obtained along with adequate quantification in the foods analyzed. Recovery rates of standards ranged from 91.3 to 99.4%. The linearity range for each isomer varied from 2.5 to 137.5 ng/mL (R2 greater than 0.995 in all cases). Detection limits ranged from 21.0 to 48.0 ng/mL for tocopherols and from 56.0 to 67.0 ng/mL for tocotrienols, while quantification limits ranged from 105.0 to 240.0 ng/mL for tocopherols and from 280.0 to 335.0 ng/mL for tocotrienols. The optimized method was considered simple, fast and reliable, and also preserved vitamin E isomers when compared to validated methods involving saponification.     

A Rapid HPLC-UV Protocol Coupled to Chemometric Analysis for the Determination of the Major Phenolic Constituents and Tocopherol Content in Almonds and the Discrimination of the Geographical Origin

Reversed phase-high-pressure liquid chromatographic methodologies equipped with UV detector (RP-HPLC-UV) were developed for the determination of phenolic compounds and tocopherols in almonds. Nineteen samples of Texas almonds originating from USA and Greece were analyzed and 7 phenolic acids, 7 flavonoids, and tocopherols (−α, −β + γ) were determined. The analytical methodologies were validated and presented excellent linearity (r² > 0.99), high recoveries over the range between 83.1 (syringic acid) to 95.5% (ferulic acid) for within-day assay (n = 6), and between 90.2 (diosmin) to 103.4% (rosmarinic acid) for between-day assay (n = 3 × 3), for phenolic compounds, and between 95.1 and 100.4% for within-day assay (n = 6), and between 93.2–96.2% for between-day assay (n = 3 × 3) for tocopherols. The analytes were further quantified, and the results were analyzed by principal component analysis (PCA), and agglomerative hierarchical clustering (AHC) to investigate potential differences between the bioactive content of almonds and the geographical origin. A decision tree (DT) was developed for the prediction of the geographical origin of almonds proposing a characteristic marker with a concentration threshold, proving to be a promising and reliable tool for the guarantee of the authenticity of the almonds.