Chromatographic analyses of tocopherols and tocotrienols in palm oil

Analyses of tocols (tocopherols and tocotrienols) in palm oil have been extensively reported in the past. However, due to the scarcity of individual tocotrienol standards, calibrations have mostly been carried out using only α-tocopherol as standard. Moreover, even if the individual tocotrienols are being used, their reliability is often questioned, because tocotrienols are highly susceptible to oxidation and deterioration. This paper reports on the study of the deterioration rate of individual tocotrienol standards upon storage as well as different calibration methods for the tocols in palm oil.     

Capillary electrochromatographic analysis of barbiturates in serum

A capillary electrochromatographic method was developed for the separation of barbiturates. The separation was optimized in a 75 microm ID capillary, packed with 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), studying the effect of buffer pH, buffer concentration, and mobile phase composition. Using an applied voltage of 20 kV and the short-end injection method (9 cm capillary effective length), the mobile phase of 1.0 mM citrate buffer (pH 5.0) containing 40% methanol provided the baseline separation of barbital, phenobarbital, secobarbital, and thiopental (internal standard) in less than 4.5 min. The method was successfully applied to the analysis of barbiturates in human serum. Under the optimal conditions, good repeatability and linearity were obtained in the range of 2.90-43.29 microg/mL for barbital, phenobarbital, and secobarbital.     

Capillary electrochromatographic study of sapphyrin-organophosphoric acid derivatives interaction

Interaction of phosphate moiety possessing compounds with sapphyrin was studied using open-tubular electrochromatography in sapphyrin-coated capillaries. It was revealed that phenylthiohydantoin (PTH)-phosphoserine and PTH-phosphothreonine exert such a strong interaction that they can not be eluted from the sapphyrin-coated capillary even at prolonged run times (70 min). Nucleoside polyphosphates show generally strong interaction (but weaker than the above mentioned serine and threonine derivatives) no matter whether they possess one or two bases. Also the number of phosphate residues present in nucleoside polyphosphates tested plays a secondary role only. p-Aminobenzylphosphoric (p-ABPA) acid exhibited an unexpected behavior. It was retained more in the phosphate containing buffer than in borate-acetate. This appears to indicate that other than complexing of the phosphate moiety may be involved in the interaction. As no such effects were observed with the PTH-derivatives of serine and threonine it was concluded that additional interaction (if involved) depends on the nature of the organic part of the molecule.     

Capillary electrochromatographic analysis of aliphatic mono- and polycarboxylic acids

The parameters influencing the electrochromatographic separation of aliphatic organic acids in a capillary column with a wall-coated macrocyclic polyamine have been studied. Indirect detection using chromate, pyromellitate, trimellitate, o-phthalate, benzoate and acetate as background electrolytes has been tested. A complete separation of polyprotic acids could be achieved with pyromellitate buffer (7.5 mM, pH 6.5), and satisfactory results for the simultaneous separation of monoprotic acids and polyprotic acids were found using a capillary column of 70 cm (50 cm effective length)x75 microm inner diameter, electrokinetic injection (-10 kV, 10 s), benzoate buffer (6 mM, pH 4.6), separation voltage of -10 kV, and detection at 220 nm. For the separation of the geometric isomers fumarate and maleate, acetate buffer was found the best choice among the background electrolytes tested. The method so established has been applied to the determination of organic acids in soy sauce, brandy, lemon juice, spinach juice and cigarette. From the retention behavior, it was found that the separation mechanism on the bonded phase was influenced by the macrocyclic effect, electrostatic attraction, hydrogen bonding, van der Waals forces, and anion exchange, in addition to the differences in electrophoretic mobility.     

Determination of tocopherols and tocotrienols in cereals by pressurized liquid extraction-liquid chromatography-mass spectrometry

A rapid analytical method including pressurized liquid extraction (PLE) and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) has been developed for the determination of tocopherols and tocotrienols in cereals. The pressurized liquid extraction parameters were optimized in order to maximize the extraction efficiency. The use of methanol as extraction solvent at a temperature of 50 °C and a pressure of 110 bar, using one cycle of extraction with a static time of 5 min, provided the best results. A good LC separation was achieved using a C₁₈ column and a solution of 6.0 mM ammonia in methanol/water (97:3, v/v) as the mobile phase at a flow rate of 0.2 mL min⁻¹. MS coupling with an ESI interface in the negative ion mode was used as the detection technique. In the present work, it is shown that the addition of a base to the mobile phase is required to enhance the ionization of tocopherols and tocotrienols in negative ion mode electrospray ionization. The applicability of the method to cereal samples was confirmed. The reproducibility of the procedure was good, with relative standard deviations in the 6-10% range. The recoveries of added tocopherols from cereal samples ranged from 91 to 109%.     

Normal-phase high-performance liquid chromatography of tocopherols and tocotrienols. Comparison of different chromatographic columns

Natural vitamin E is composed of eight different vitamers (alpha-, beta-, gamma- and delta-tocopherols and alpha-, beta-, gamma- and delta-tocotrienols). As these eight vitamers have different antioxidant and biological activities, it is necessary to have quantitative data on each substance separately. The aim of this study was to find universal HPLC columns for the separation of all eight components and to test if a few columns of the same material (different batches) will give reproducible results. Normal-phase HPLC separations of vitamin E compounds in a prepared mixture (containing oat extracts, palm oil and tocopherol standards) were tried on six silica, three amino and one diol columns. As shown by calculations of retention factors (k), separation factors (alpha), numbers of theoretical plates (N) and resolutions (Rs), the best separations were obtained on three silica columns and two amino columns using 4 or 5% dioxane in hexane as the mobile phase as well as on a diol column using 4% tert.-butyl methyl ether in hexane as the mobile phase.     

Capillary electrochromatographic separation of illicit drugs employing a cyano stationary phase

In this study, we present a capillary electrochromatographic method for separation of basic compounds of interest in forensic science (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, cocaine, codeine, heroin, morphine, and 6-monoacethylmorphine). Several analytical conditions were taken into account to completely separate in the same run the 10 drugs of abuse analyzed. Chromatographic retention, selectivity and efficiency were evaluated in dependence of the type of stationary phase (CN and RP-C₁₈ derivatized silica particles), mobile phase composition, buffer type and pH, sample injection. The optimum separation parameters were set up using a mixture of aqueous sodium phosphate buffer (pH 2.5)/acetonitrile (80/20, v/v) as the mobile phase, 10 kV and 20 °C as applied voltage and capillary temperature, respectively. Under these conditions all the studied analytes were baseline resolved within 20 min. The method performance was investigated in terms of precision, linearity, sensitivity and accuracy to demonstrate the applicability of the developed capillary electrochromatographic system to forensic analysis. Calibration curves provided a good linearity over a working range of 100–1200 ng/mL for all analytes. Limits of detection and quantification were in the range 5–12 ng/mL and 10–30 ng/mL, respectively. Then the method was applied to the analysis of a human urine sample spiked with a basic compounds’ mixture. Urine samples’ pre-treatment was carried out through a solid phase extraction (SPE) procedure on strong cation exchange (SCX) cartridges.     

Simultaneous determination of tocopherols and tocotrienols in hazelnuts by a normal phase liquid chromatographic method.

A normal-phase high-performance liquid chromatography (NP-HPLC) method for the determination of tocopherols and tocotrienols in hazelnuts is reported. Three extraction procedures (with and without saponification) were assayed; the best results were obtained with a simple solid-liquid extraction procedure. Chromatographic separation was achieved using an Inertsil 5 SI column using isocratic elution with hexane/1,4-dioxane (95.5:4.5, v/v) at a flow rate of 0.7 mL/min. The effluent was monitored by a series arrangement of a diode-array followed by a fluorescence detector. All compounds were separated in a short period of time (17 min). The method proved to be rapid, sensitive, reproducible and accurate, allowing the simultaneous determination of all vitamin E homologues.     

Liquid chromatographic determination of tocopherols and tocotrienols in vegetable oils, formulated preparations, and biscuits

A precise and selective liquid chromatographic procedure for determining tocopherol and tocotrienol isomers in vegetable oils, formulated preparations, and biscuits was developed and validated. The proposed method quantitates vitamin E in better conditions of recoverability and reproducibility than the standard saponification procedure. Tocopherols and tocotrienols were extracted in hexane from vegetable oils, passed through a silica Sep-pak, chromatographed on a mu-Bondapak C18 column with a mobile phase of methanol-water (95 + 5, v/v), identified at 292 nm, and detected with fluorescence procedure (excitation 296 nm, and emission 330 nm). The correlation coefficient on the calibration curve was 0.9995 over the range of 0.1 to 100 microg/mL. Overall recovery of vitamin E isomers was 93%; coefficients of variation for intra- and interday precision, < 2.25%. The results obtained from extraction methods 1 (with saponification) and 2 (without saponification) were compared by ANOVA test. Significant differences appeared between vitamin E isomers (p < or = 0.05).     

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.     

Capillary electrochromatographic separation of peptides using a macrocyclic polyamine for molecular recognition

A macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N(8)), in the bonded phase was employed as a molecular receptor for CEC separation of oligopeptides. Parameters affecting the performance of the separations were considered. Baseline separation for the mixture of angiotensin I, angiotensin II, [Sar(1), Thr(8)]-angiotensin II, beta-casomorphin bovine, beta-casomorphin human, oxytocin acetate, tocinoic acid, vasopressin, and FMRF amide could be achieved using phosphate buffer (30 mM, pH 7) as the mobile phase. Column efficiency with average theoretical plate numbers of 69 000 plates/m and RSDs of <1% (n = 6) was achieved. [Met(5)]-enkephalin and [Leu(5)]-enkephalin, which have identical pI values and similar masses could be completely separated using acetate buffer (30 mM) with pH gradient (pH 3 inlet side and pH 4 outlet side). The results suggest that the mechanism for the peptide separation was mediated by a combination of electrophoretic migration and chromatographic retention involving anion coordination and anion exchange. After long-term use, the deviation of the EOF of the column after more than 600 injections was still within 6.0% of that for a freshly prepared column.     

Capillary electrochromatographic separation of non-steroidal anti-inflammatory drugs with a histidine bonded phase

An open tubular wall-coated capillary column containing histidine functional groups was prepared and employed for the capillary electrochromatographic separation of non-steroidal anti-inflammatory drugs. The anion exchange along with the hydrogen bonding and hydrophobic properties of the surface coating allowed the separation of analytes with very similar ionic mobility. Selectivity and resolution were studied by changing the pH over the range from 3.5 to 5.0 and the concentration of the buffer from 10 to 25 mM, as well as variation of the organic modifier, such as methanol, ethanol and 1-propanol over the range 7.5 to 20%. The optimum experimental conditions for the separation of a drug mixture, which consisted of indoprofen, ketoprofen, suprofen, naproxen, flurbiprofen, fenoprofen and ibuprofen were using a mixture of acetate buffer (20 mM, pH 5.0)-ethanol (1:5, v/v) as background electrolyte and an applied voltage of -20 kV with UV detection at 220 nm. The separation of these drugs could be achieved with an average plate number of 1.0 x 10(5) m(-1).     

Capillary electrochromatographic separation of proteins on a column coated with titanium dioxide nanoparticles

A TiO2 nanoparticle (TiO2 NP)-coated open-tubular column for the capillary electrochromatographic separation of proteins is described. The surface chemistry of the TiO2 NPs on the inner wall of the fused silica was significantly affected by the running buffer. By varying of the phosphate buffer pH, only cathodic EOF was indicated. The results showed that TiO2 NPs are existed as a complexed form with the buffer ligand. Good separation of conalbumin (ConA), apo-transferrin (apoTf), ovalbumin (OVA), and BSA could be achieved with phosphate buffer (40 mM, pH 8.0) and an applied voltage of 15 kV. Five peaks of glycoisoforms of OVA were observed under these conditions. In comparison with the retention behavior of the analytes on the bare fused-silica column, the new column's high resolving power seems to be predominantly derived from the ligand exchange of the analytes with the phosphate adsorbed onto the TiO2 NPs. The method was also used to separate egg-white proteins. Both acidic and basic proteins in egg white were separated in a single run. The microheterogeneities of OVA could also be found in it. The separation efficiency for the main peak of OVA in egg white was around 10,000 plates/m.     

Capillary electrochromatographic separation of bovine milk proteins using a G-quartet DNA stationary phase

DNA oligonucleotides that form G-quartet structures were used as stationary phase reagents for separation of bovine milk proteins, including alpha-casein, beta-casein, kappa-casein, alpha-lactalbumin and beta-lactoglobulin. Both artificial protein mixtures and a skim milk sample were analyzed. The separations were performed using open-tubular capillary electrochromatography, in which the oligonucleotides were covalently attached to the inner surface of a fused-silica capillary. Better resolution was achieved using the G-quartet-coated capillaries than was achieved using either a bare capillary or a capillary coated with an oligonucleotide that does not form a G-quartet structure. A 4-plane G-quartet-forming stationary phase was able to resolve three peaks for alpha-casein and to detect thermal denaturation of the proteins in the milk sample. The results suggest that G-quartet stationary phases could be used to separate very similar protein structures, such as those arising from genetic variations or post-translational modifications.     

Capillary electrochromatographic separation of amino acid enantiomers with molecularly imprinted polymers as chiral recognition agents

The use of molecularly imprinted polymers polymerized in a capillary for the separation of amino acid enantiomers by electrochromatography is described. The substrate-selective polymers were prepared by using l-phenylalanine anilide as print molecule and methacrylic acid as the functional monomer. The treatment of the inside surface of the capillary, the composition of the polymer and the electrochromatographic running conditions were investigated. This preliminary report demonstrated a novel and simple method for capillary electrochromatographic separations of amino acid enantiomers using molecularly imprinted polymers. Received: 9 April 1996 / Revised: 8 August 1996 / Accepted: 8 August 1996     

Capillary electrochromatographic separation of metal ion species with on-line detection by inductively coupled plasma mass spectrometry

A bonded phase capillary column containing macrocyclic polyamine, [28]ane-N₆O₂ functional groups was used for the electrophoretic separation of arsenic, chromium and selenium species. A simple device interfacing this capillary electrochromatography (CEC) systems to an inductively coupled plasma mass spectrometer (ICPMS) is described. The dimension of the capillary column was 160 cm×100 μm i.d. To accommodate this electrophoretic separation, an auxiliary capillary was used with nitric acid (0.05 M) as makeup liquid. With the electrokinetic method at –20 kV, 20 s and a nebulizer gas flow rate of 1 l min⁻¹, the sample injected was analyzed with an applied potential of −20 kV. The background electrolyte buffer for the separation of CrO₄²⁻–Cr³⁺ was phosphate (20 mM, pH 6.5). That for HAsO₄²⁻–Ph₄As⁺ was pyromellitate (20 mM, pH 6.0) and for SeO₄²⁻–SeO₃²⁻ was acetate (20 mM, pH 6.0). The role of the buffer’s anion was also discussed. The separation efficiency of the bonded phase was compared with the bare fused silica. Concentration detection limits for these metal ions were in the low ppb range. In addition, the matrix effect of the established system with the bonded phase was found smaller than that with the bare fused silica.     

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.     

Development and optimization of ultra-high performance supercritical fluid chromatography mass spectrometry method for high-throughput determination of tocopherols and tocotrienols in human serum

The goal of this study was to develop an effective supercritical fluid chromatography method using single quadrupole MS for analysis of all isomeric forms of vitamin E. Finally, two fast and effective methods, the high resolution one and the high speed one, for the determination of 8 vitamin E isomers in human serum were developed.