Chemometrics-Assisted Optimization of Beta-/Gamma-Tocol Separation on a C<Subscript>30</Subscript> Stationary Phase in Reversed-Phase LC

This paper presents a chemometrics-assisted optimization study to improve the separation of tocopherol (-T) and tocotrienol (-TT) homologues on a C₃₀ stationary phase in reversed-phase HPLC. The HPLC settings were optimized using a central composite design and the response surface methodology. Flow rate, column temperature, and mobile phase composition were chosen as independent variables. Peak resolution (R_s), analysis time (t_R), and peak symmetries of the tocopherol isomers were chosen as response variables. Optimum performance in terms of R_s was obtained at a flow rate of 0.31 mL min^?1, a temperature of 8.70 °C, and % B content (methyl tert-butyl ether: methanol: water, 80:18:2, v/v/v) in the mobile phase of 38.12%. The analysis of variance and regression analysis gave adjusted R² values of 0.9841 for R_s, 0.9850 for t_R-(α-T), 0.9853 for t_R-(β-T), and 0.9204 for the peak symmetry of β-T. This confirms the good agreement of experimental data with predicted values. The close eluting peaks of β-/γ-tocol could be baseline separated at the optimized conditions at a minimized analysis time. Empirical second-order polynomial models were derived that gave statistically high significances (P?<?0.0001). Hence, the models can be successfully employed to predict the optimum separation conditions of co-eluting peaks of β-/γ-tocols. The optimized method was successfully applied to determine the individual tocol homologues in various cold pressed edible oils. Total contents ranged from 15 to almost 2600 mg tocol kg^?1 oil.     

Determination of lipoic acid, Trolox methyl ether and tocopherols in human plasma by liquid-chromatography and ion-trap tandem mass spectrometry

A method for the simultaneous determination of lipoic acid and/or Trolox methyl ether, along with α‐, γ‐ and δ‐tocopherol was developed using liquid chromatography–tandem mass spectrometry with negative electrospray ionization (HPLC‐ESI‐MS/MS) in an ion‐trap mass spectrometer. Detection and quantification were accomplished by a multiple reaction monitoring method, using specific transitions from precursor ion to product ion for each analyte. Chromatographic separation was achieved in a 12 min run using a C₁₈‐bonded phase and methanol–aqueous ammonium acetate elution gradient. Linear correlations of the chromatographic peak area (r.u. × s^?1) to the injected amount (ng) gave the slope values (r.u. × s^?1 × ng^?1) 2.34 × 10⁴ for α‐tocopherol, 5.05 × 10⁴ for γ‐tocopherol, 1.27 × 10⁵ for δ‐tocopherol, 8.86 × 10⁵ for lipoic acid and 1.23 × 10⁵ for Trolox methyl ether. The lower limit of quantification ranged between 0.02 and 1.22 ng for Trolox methyl ether and lipoic acid. MS³ experiments of γ‐ and δ‐tocopherol suggest ion‐radical reactions and dependence of the tocopherol fragmentation pattern on the phenolic ring methylation degree. The method is shown to be applicable to measurement of these metabolites in human serum after extraction. Copyright © 2012 John Wiley & Sons, Ltd.     

Efficient separation of tocopherol homologues in vegetable oil by ionic‐liquid‐based countercurrent chromatography using a non‐aqueous biphasic system

Tocopherol homologues are important fat‐soluble bioactive compounds with high nutritional value. However, it is of great challenge to separate these homologues because of their high structural similarities. In this work, ionic‐liquid‐based countercurrent chromatography was used for the separation and purification of tocopherol homologues. Conventional countercurrent chromatography and ionic‐liquid‐based countercurrent chromatography solvent systems were evaluated in respect of partition coefficient, separation factor, and stationary phase retention factor to separate these targets. Kind of ionic liquids, amount of ionic liquid, and sample amount were systematically optimized. A novel countercurrent chromatography non‐aqueous biphasic system composed of n‐hexane‐methanol‐1‐butyl‐3‐methylimidazolium chloride was established. The baseline separation of tocopherol mixtures was obtained in one cycle process. The ionic liquid played a key role in the countercurrent chromatography separation, which resulted in difference of partition behavior of individual tocopherol in the whole system through different hydrogen‐bonding affinity. Finally, n‐hexane‐methanol‐1‐butyl‐3‐methylimidazolium chloride (5:5:3, v/v) water‐free biphasic system was successfully applied to separate tocopherol homologues from vegetable oil that was not achieved beforehand. This method can be widely employed to separate many similar molecules such as tocotrienols, tocomonoenols, and marine‐derived tocopherol in food samples.     

Effect of mobile phase on resolution of the isomers and homologues of tocopherols on a triacontyl stationary phase

Reversed-phase liquid chromatographic (RPLC) separation of isomers and homologues of similar polarity is challenging. Tocopherol isomers and homologues are one such example. α, β, γ, and δ-tocopherols have been successfully separated by RPLC on triacontyl (C₃₀) stationary phase. System suitability was tested by using four mobile phases, and observed chromatographic separations of β and γ-tocopherols were compared. Comparison indicated that methanol–tert-butyl methyl ether (TBME) 95:5 (v/v) at a flow rate of 0.75 mL min^?1 was the best mobile phase. Detection systems were also evaluated on the basis of limit of quantification; it was concluded that fluorescence detection was best. The method was validated by analysis of two homologues and two isomers of tocopherol in sesame, maize, and soybean samples. MS coupled with an ESI interface in negative-ion mode [M ? H]^? was used for identification of individual components. It was concluded that addition of TBME to methanol was required to enhance the separation of β and γ-tocopherols, although methanol alone provided similar results. The applicability of the method to cereal, pulse, and oilseed samples was confirmed. The reproducibility of the procedure was good, with relative standard deviations in the range 1.7–3.9 %. Recovery of tocopherols added to sesame samples ranged from 91 to 99 %.

Crystal morphology of the γ (triclinic) phase of isotactic polypropylene and its relation to the α phase

γ-phase crystals of isotactic polypropylene (iPP) obtained from low-molecular-weight extracts of pyrolyzed polymers are examined by electron microscopy and electron diffraction. γ-phase crystals differ from α-phase crystals in three important respects: (i) they are elongated along the b^* rather than the a^* axis, (ii) the chain axis is inclined at 50° to the lamellar surface (indexed as 101) rather than normal to it, and (iii) they show screw dislocations, while α crystals do not. γ crystals are nucleated on the lateral (010) faces of a α crystals; the b_α and b axes are parallel. Virtually no nucleation of the α phase takes place on the γ phase, which is therefore not involved in the repetitive lamellar branching leading to iPP quadrites. Crystallization of the γ phase appears to be favored by or linked to the absence of chain folds and may be involved in the macroscopic curvature of iPP branches.     

Molecular relaxation in the blends of polystyrene/poly(2,6-dimethyl-1,4-phenylene oxide) at low temperature

Molecular relaxation behavior in terms of the α, β, and γ transitions of miscible PS/PPO blends has been studied by means of DMTA and preliminary work has been carried out using DSC. From DSC and DMTA (by tan δ), the observed α relaxation (T_α or T_g) of PS, PPO, and the blends, which are intermediate between the constituents, are in good agreement with earlier reports by others. In addition, the β transition (T_β) of PS at 0.03 Hz and 1 Hz is observed at −30 and 20°C, respectively, while the γ relaxation (T_γ) is not observed at either frequency. The T_β of PPO is 30°C at 0.03 Hz and is not observed at 1 Hz, while the T_γ is −85°C at 0.03 Hz and −70°C at 1 Hz. On the other hand, blend composition-independent β or γ relaxation observed in the blends may be a consequence of the absence of intra- or intermolecular interaction between the constituents at low temperature. Thus it is suggested that at low temperature, the β relaxation of PS be influenced solely by the local motion of the phenylene ring, and that the β or γ relaxation of PPO be predominated by the local cooperative motions of several monomer units or the rotational motion of the methyl group in PPO. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 1981–1986, 1998     

Characterization of the Phytochemical Profiles and Biological Activities of Ajuga chamaepitys subsp. chia var. chia and Ajuga bombycina by High-Performance Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry (HPLC–ESI–MSn)

This study investigates into the pharmacological potential of three solvent extracts (ethyl acetate, methanol, and water) of two Ajuga species (Ajuga chamaepitys subsp. chia var. chia and Ajuga bombycina) based on their antioxidant activity and enzyme inhibitory effects along with establishing the phytochemical profile. Spectrophotometric and high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MSⁿ) were used to determine the total and individual phytocompounds, respectively. Antioxidant potential was assessed using different assays such as DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal chelation. Enzyme inhibitory effects were studied against acetylcholinesterase, butyrylcholinesterase, tyrosinase, α-amylase, and α-glucosidase. The aqueous extract of both plants showed better ABTS scavenging, FRAP, and metal chelating activities. The methanol extracts displayed the highest tyrosinase inhibitory and antioxidant activity in the phosphomolybdenum assay while the ethyl acetate extracts of both plants showed better butyrylcholinesterase (BChE), α-amylase, and α-glucosidase inhibition. The total phenolic content was highest in the aqueous extract of A. chamaepitys while the methanolic extract of A. bombycina showed the highest flavonoid content. Identification by HPLC–ESI–MSⁿ revealed the presence of some individual compounds including phenolic acids, flavonoids, iridoid glycosides, phenylethanoid glycosides, and other compounds. To conclude, both A. chamaepitys and A. bombycina can be considered as rich sources of phytocompounds to manage chronic diseases.     

Radiation-induced polymerization at high dose rate. II. α-Methylstyrene in bulk

Bulk polymerization of α-methylstyrene was carried out in a wide dose rate range, 7.6–256 rad/sec by γ rays and 8.5 × 10³–2.1 × 10⁵ rad/sec by electron beams. At high dose rate by electron beams, cationic polymerization took place along with formation of oligomeric product of DP _n = ～4. At low dose rate by γ rays, radical polymerization was found to occur in water-saturated monomer. The cationic polymerization at high dose rate proceeds with essentially the same mechanism as was already known in γ-ray polymerization of dry monomers. Relatively low reaction rate of the cationic polymerization compared with that of styrene is explained with the fact that the propagation of α-methylstyrene is much more easily inhibited by a slight amount of water.     

Development of radiation indicators to distinguish between irradiated and non-irradiated herbal medicines using HPLC and GC-MS

The effects of high dose γ-irradiation on six herbal medicines were investigated using gas chromatography–mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC). Herbal medicines were irradiated at 0–50 kGy with ⁶⁰Co irradiator. HPLC was used to quantify changes of major components including glycyrrhizin, cinnamic acid, poncirin, hesperidin, berberine, and amygdalin in licorice, cinnamon bark, poncirin immature fruit, citrus unshiu peel, coptis rhizome, and apricot kernel. No significant differences were found between gamma-irradiated and non-irradiated samples with regard to the amounts of glycyrrhizin, berberine, and amygdalin. However, the contents of cinnamic acid, poncirin, and hesperidin were increased after irradiation. Volatile compounds were analyzed by GC/MS. The relative proportion of ketone in licorice was diminished after irradiation. The relative amount of hydrocarbons in irradiated cinnamon bark and apricot kernel was higher than that in non-irradiated samples. Therefore, ketone in licorice and hydrocarbons in cinnamon bark and apricot kernel can be considered radiolytic markers. Three unsaturated hydrocarbons, i.e., 1,7,10-hexadecatriene, 6,9-heptadecadiene, and 8-heptadecene, were detected only in apricot kernels irradiated at 25 and 50 kGy. These three hydrocarbons could be used as radiolytic markers to distinguish between irradiated (>25 kGy) and non-irradiated apricot kernels.     

Quantitative Determination of Underivatized α-Tocopherol in Cow Milk, Vitamin and Multivitamin Drugs by GC-FID

A simple and rapid method for direct determination of α-tocopherol (α-T, vitamin E) in pharmaceutical preparations (vitamin and multivitamin tablets) and cow milk obtained from different villages of Erzurum in Turkey was developed and validated by GC-FID. Separation of underivatized α-T in pure substance, milk samples, vitamin and multivitamin tablets was performed in about 8.4 min, using an HP-5 capillary column. The range of quantification for the GC-FID was 1–30 μg mL^?1. Within-day and between-day precision (RSD %) were less than 8.5%, and accuracy (relative error) was less than 11.0% (n = 6). LOQ and LOD values were found to be 0.35 and 0.30 μg mL^?1, respectively. The developed method was applied directly and easily to the analysis of α-T in vitamin and multivitamin preparations and cow milk. RSD values were found to be 6.59% (Grandpherol soft gelatine capsule: 200 I.U.), 0.59% (Megadyn film tablet: 10 mg) and 1.54% (Supradyn drage: 10 I.U.). The developed method was also applied to cow milk samples and mean values of α-T content was found 2.99 μg mL^?1 in cow milk samples. This developed and validated GC–FID method, in conjunction with other methods, could be successfully applied for routine laboratory because of its simplicity, rapidity, sensitivity, precision and accuracy.     

Stereoelectronic and steric effect in the Baeyer–Villiger rearrangement of steroidal α-chlorocyclobutanones

The regioselectivity of the Baeyer–Villiger oxidation of α-chlorocyclobutanone derivatives is markedly affected by substituents in position γ to the carbonyl group. The reaction of steroidal α-chlorocyclobutanones with m-chloroperoxybenzoic acid results in the formation of γ-chloro-γ-lactones and/or α-chloro-γ-lactones depending on the substitution pattern of the four-membered ring. In the reactions of γ-unsubstituted α-chlorocyclobutanones, the exclusive formation of γ-chloro-γ-lactone results from the migration of the chlorinated substituent (CHCl) versus the alkyl group (CH₂), contrary to the expected migratory aptitude. In explaining the unusual regioselectivity observed in these reactions, steric effects and dipole interactions in the formation of the reactive Criegee intermediates are considered and the stereoelectronic effect is postulated to be more important than the intrinsic migratory aptitude.     

Relaxation of shear and normal stresses in step-shear deformation of a polystyrene solution. Comparison with the predictions of the Doi–Edwards theory

The shear stress σ, two components of birefringence, and extinction angle were measured for a concentrated polystyrene solution in step-shear deformation of magnitude of shear 0.3 ≤ γ ≤ 4.0. The stress-optical coefficient did not depend on either γ or time. The first and the second normal-stress differences v₁ and v₂ were evaluated with the use of the stress-optical law. Over a certain range of long times, σ could be factored as σ = γh(γ)G(t) and the quantity h(γ) agreed with the prediction of the Doi–Edwards theory based on the de Gennes tube model of entangled polymer chains. At short times the effect of γ on σ/γ was smaller than at long times. The relaxation spectrum became approximately independent of γ at the short-time end of the rubbery plateau region. The ratios v₁/σ and v₂/v₁ were independent of time and were in quantitative agreement with those predicted by the Doi–Edwards theory: v₁/σ was equal to γ, v₂/v₁ was negative, and |v₂/v₁| decreased with increasing γ.     

Microwave-assisted synthesis of α-aminophosphonates with sterically demanding α-aryl substituents

Abstract

A series of N-benzhydryl protected α-aminophosphonates with α-phenyl, α-(1-naphtyl), α-(9-anthryl) or α-(1-pyrenyl) substituents was synthesized by the Kabachnik–Fields condensation of diphenylmethylamine (benzhydrylamine), the corresponding aryl aldehyde and a dialkyl phosphite under MW irradiation. X-ray studies performed at low temperatures for a few of these α-aminophosphonates confirmed the presence of unusually short intramolecular C_α–H^δ+ ··· ^δ+H–C_peri contacts.     

Dérivés C-Glycosyliques XXXI. Utilisation en chimie des sucres du synthon bromo-2-cyano-2-éthényle. Communication préliminaire

C-Glycosyl derivatives XXXI. Some uses in Carbohydrate Chemistry of the 2-bromo-2-cyano ethenyl synthon Sugars bearing the synthon 2-bromo-2-cyano-ethenyl, ? CαH?Cγ(Br)C?N, reacted with binucleophiles, gave rise to α, α, α, β or α, γ ring-forming reactions. C-glycosyl-dioxolanes, -aziridines, -dithianes, -pyrimidines, -furanes and -pyrazoles were so obtained. The stereochemical paths of some of these reactions are discussed.     

FTIR spectroscopic characterization of structural changes in polyamide‐6 fibers during annealing and drawing

First, we report the development of Fourier transform infrared (FTIR) spectroscopic methods to determine the α/γ‐crystalline phase ratio of polyamide‐6 fibers and, in combination with density measurements, the total crystallinity. Using density determinations of the crystallinity of pure α and pure γ samples, we found the absorption coefficient ratio for the 930 (α) and 973 cm⁻¹ (γ) bands to be 4.4, from which we could obtain the α/γ ratio for any polyamide‐6 sample. The application of this FTIR method to the quantitative analysis of phase changes during thermal treatment and the drawing of polyamide‐6 was then made. We confirmed that crystallization during thermal treatments involved increases in both phases and did not involve crystal‐to‐crystal transformation, whereas drawing involved both crystallization of the amorphous phase in the α form and γ→α transformation. Finally, we revisited the band assignments for the amorphous phase of polyamide‐6 and found that the band at 1170 cm⁻¹ was not an amorphous band but, because its absorbance was independent of crystallinity, could be used as an internal reference band. The band at 1124 cm⁻¹ was reliably attributed to the amorphous phase. © 2001 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 39: 536–547, 2001     

Structural differences between two crystal modifications of poly(γ-benzyl L-glutamate)

X-ray diffraction patterns were obtained for as-cast and oriented films of poly(γ-benzyl L -glutamate) and a comparison was made of the molecular packing of the α-helices in forms B and C. Form B snowed Bragg reflections on the layer lines as well as on the equator. The spacings were explained by a monoclinic unit cell comprising two chains, with a = 29.0₆ Å, b = 13 2₀ Å, c = 27.2₇ Å α = γ = 90°. and β = 96°. The chains contained in this unit cell and consequently alternating in the crystal have opposite chain directions. Form C showed continuous scattering on the layer lines and reflections on the equator. This form, therefore, is a nematiclike paracrystal in which the packing of α-helices is periodic in the direction lateral to the chain axis (a = 14.8–115.2 Å, b = 14.3–14.8 Å, c = 27 Å, and γ = 118°–120°), but the relative levels of the chains along the chain axes are displaced. The formation of form C may be attributed to random placement of two chains with mutually opposite chain directions.     

Chronopotentiometric analysis of tocopherols using a glassy carbon vessel as the working electrode

Using a specific electrochemical cell with a glassy carbon vessel as the working electrode, a significant enhancement of the chronopotentiometric determination of tocopherols was achieved. Due to the large area of the working electrode, the method sensitivity was increased approximately 50-fold compared to the common electrochemical cell with a glassy-carbon disc electrode. Limits of quantitation of approximately 0.35 mg/dm³ for α-, γ- and δ-tocopherol were obtained. The influence of the most important experimental factors was investigated and the method defined was applied for tocopherol determination in crude vegetable oils. The tocopherol content was determined by standard solutions prepared in a matrix of animal fat. An absolute method for γ- and δ-tocopherol quantitation is also described.     

LC/ESI‐MS/MS characterisation of lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain

Lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain showed an interesting anti‐adhesion activity against biofilm formation of human pathogenic bacterial strains. The chemical characterisation of the crude extract of V9T14 strain was first developed through electrospray ionisation mass spectrometry (ESI‐MS) and ESI‐MS/MS direct infusions: two sets of molecular ion species belonging to the fengycin and surfactin families were revealed and their structures defined, interpreting their product ion spectra. The LC/ESI‐MS analysis of the crude extract allowed to separate in different chromatogram ranges the homologues and the isoforms of the two lipopeptide families. The extract was then fractionated by silica gel chromatography in two main fractions, I and II. The purified biosurfactants were analysed through a new, rapid and suitable LC/ESI‐MS/MS method, which allowed characterising the composition and the structures of the produced lipopeptides. LC/ESI‐MS/MS analysis of fraction I showed the presence of C₁₃, C₁₄ and C₁₅ surfactin homologues, whose structures were confirmed by the product ion spectra of the sodiated molecules [M + Na]⁺ at m/z 1030, 1044 and 1058. LC/ESI‐MS/MS analysis of fraction II confirmed the presence of two main fengycin isoforms, with the protonated molecules [M + H]⁺ at m/z 1478 and 1506 corresponding to C₁₇ fengycin A and C₁₇ fengycin B, respectively. Other homologues (C₁₄ to C₁₆) were revealed and confirmed as belonging to fengycin A or B according to the retention times and the product ions generated, although with the same nominal mass. Finally, a relative percentage content of each homologue for both lipopeptides families in the whole extract was proposed. Copyright © 2010 John Wiley & Sons, Ltd.     

Spectral and electrochemical investigation of p-sulfonatocalix[4]arene-stabilized vitamin E aggregation

The host–guest interaction of α-tocopherol (vitamin E) with p-sulfonatocalix[4]arene (p-SC4) in solution state is studied using emission and cyclic voltammetric techniques. The lipid soluble α-tocopherol (α-T) forms a solid complex with p-SC4. FTIR and NMR spectral analysis of the solid complex reveals the tight packing of α-T inside the cavity of p-SC4. The structural deformation is confirmed by XRD analysis. SEM images differentiate the highly porous gel like structure of vitamin E aggregate and the solid structure of the host–guest complex prepared. NOESY spectra confirm the tight penetration of α-T within the hydrophobic cavity of p-SC4.     

Controlling the α/γ-Reactivity of Vinylogous Ketone Enolates in Organocatalytic Enantioselective Michael Reactions

The first regio-, diastereo-, and enantioselective direct Michael reaction of β,γ-unsaturated ketones with nitroolefins is enabled by Brønsted base/hydrogen-bonding bifunctional catalysis. A squaramide-substituted tertiary amine catalyzes the reaction of a broad range of β,γ-unsaturated ketones to proceed at the α-site exclusively, giving rise to adducts with two consecutive tertiary carbon stereocenters in diastereomeric ratios of up to >20:1 and enantioselectivities generally in the 90–98 % ee range.