Near-infrared spectroscopy (NIRS) reflectance technology for the determination of tocopherols in alfalfa

The vitamin E (α- and (β+γ)-tocopherol) contents present in alfalfa (fresh or dehydrated) were analysed using near-infrared spectroscopy (NIRS) technology together with a remote reflectance fibre-optic probe. The range of vitamin E was 0.55–5.16 mg/100 g for α-tocopherol and 0.07–0.48 for (β+γ)-tocopherol. The regression method employed was modified partial least squares (MPLS). The equations developed using the fibre-optic probe for 69 samples of alfalfa (dehydrated and fresh) to determine the content of vitamin E in feeds had multiple correlation coefficients (RSQs) and prediction corrected standard errors (SEP (C)) of 0.946 and 0.321 mg/100 g for α-tocopherol and 0.956 and 0.022 mg/100 g for (β+γ)-tocopherol. The predicted values of vitamin E in feeds using NIRS technology applying the fibre-optic probe directly on the sample with neither previous treatment nor manipulation are comparable to those obtained using the chemical method, which included alkaline hydrolysis and hexane extraction of the vitamin from the unsaponifiable fraction before chromatographic determination.     

Determination of Natural Vitamin E from Italian Hazelnut Leaves

A new potential source of natural vitamin E from thirteen samples of Corylus avellana L. leaves was screened: the major Italian cultivar — Tonda romana (collected from Latium and Sardinia localities); ten local genotypes from Sardinia — Moro seme, Suconcale, Moro, Sarda grossa, Sarda grossa seme, Sarda schiacciata, Coccoredda, Sarda lunga, Sarda piccola and Sarda tardiva; wild genotype — Selvatico from Latium. The determination was performed after optimizing the high-efficiency pressurized liquid extraction (PLE) conditions of α-tocopherol from Italian hazelnut tree green leaves. Moro from Sardinia showed the highest content of α-tocopherol (237.4±0.3 μg/g d.w). Leaves of this genotype may be considered as a potential new source for natural α-tocopherol. __________ Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 538–540, November–December, 2005.     

Direct copper determination in whole milk, non-fat milk and whey milk by electrothermal atomic absorption spectrometry

A method for the direct determination of copper in samples of whole milk, non-fat milk and whey milk by electrothermal atomic spectrometry (ETAAS) was studied. The fat separation by centrifugation at 3200 rpm and the separation of casein mycelles to obtain the whey milk by ultracentrifugation at 31 000 g were investigated. In all cases Mg(NO₃)₂ was used as chemical modifier and Triton X-100 (0.2% w/v) as emulsifying agent. The optimum pyrolysis temperature was 1500° C. The detection limit was 0.4 μg/l of copper. The precision was studied for the whole milk and the coefficients of variation (CV) were 5.7, 4.0, 2.4 and 2.8% for 0, 5, 10 and 20 μg/l of copper added. The accuracy was determined by using the Reference Material Milk A-11 (IAEA) with a certified content of 378.4 ± 24 ng Cu/g; 359 ± 16 ng/g were found. The method was applied to ten cow milk samples, the levels of copper being determined for whole milk, non-fat milk and whey milk. A statistical study was applied and it was concluded that the majority of copper is in the non-fat milk. Received: 29 February 1996 / Revised: 19 April 1996 / Accepted: 28 April 1996     

Suitability of ultra-high performance liquid chromatography for the determination of fat-soluble nutritional status (vitamins A, E, D, and individual carotenoids)

Our aim was to assess the suitability of ultra-high performance liquid chromatography (UHPLC) for the simultaneous determination of biomarkers of vitamins A (retinol, retinyl esters), E (α- and γ-tocopherol), D (25-OH-vitamin D), and the major carotenoids in human serum to be used in clinical practice. UHPLC analysis was performed on HSS T3 column (2.1 × 100 mm; 1.8 μm) using gradient elution and UV–VIS detection. The system allows the simultaneous determination of retinol, retinyl palmitate, 25-OH-vitamin D, α- and γ-tocopherol, lutein plus zeaxanthin, α-carotene, β-carotene, α- and β-cryptoxanthin and lycopene. The method showed a good linearity over the physiological range with an adequate accuracy in samples from quality control programs. Suitability of the method in clinical practice was tested by analyzing samples (n = 286) from patients. In conclusion, UHPLC constitutes a reliable approach for nutrient/biomarker profiling allowing the rapid, simultaneous and low-cost determination of vitamins A, E, and D (including vitamers and ester forms) and the major carotenoids in clinical practice.     

Neutron activation analysis of Bawachi seeds

Seeds of (the plant) P. Corylifolia Linn (Bawachi) known for their therepeutic value in indigenous medicine have been analysed for Cu, As, Sb and Se by thermal neutron activation analysis involving substoichiometric extraction and precipitation technique. The amounts of Cu, As, Sb and Se per gram of seeds dried at 80°C are 16.0±1.1 μg, 0.90±0.4 μg, 12.1±0.6 μg and 4.0±0.2 μg respectively.     

Separation and preconcentration of Se(IV)/Se(VI) speciation on algae and determination by graphite furnace atomic absorption spectrometry in sediment and water

A novel method for the separation and preconcentration of Se(IV)/ Se(VI) with algae and determination by graphite furnace atomic absorption spectrometry (GFAAS) has been developed. The Se(VI) is extracted with algae from the solution containing Se(IV)/Se(VI) at pH 5.0, and the remaining Se(IV) is then preconcentrated pH 1.0. The detection limits (3σ, n = 11) of 0.16 μg L^–1 for Se(IV) and 0.14 μg L^–1 for Se(VI) are obtained using 40 mL of solution. At the 2.0 μg L^–1 level the relative standard deviation is 2.6% for Se(IV) and 2.3% for Se(VI). The method has been applied to the determination of Se(IV)/Se(VI) in sediment and water samples. Analytical recoveries of Se(IV) and Se(VI) added to samples are ?97 ± 5% and 102 ± 6% (95% confidence), respectively. Received: 10 February 1999 / Revised: 21 June 1999 / /Accepted: 22 June 1999     

Determination of Selenium and Tin in Human Brain by Graphite Furnace Atomic Absorption Spectrometry

 A method is reported for measuring Se and Sn in human brain tissue. The patients from whom the samples were taken had no diseases in their central nervous system. Microwave energy was applied to digest the brain samples. The digested samples were analyzed without dilution by transversely heated graphite atomizer for atomic absorption spectrometry with longitudinal Zeeman background correction. The dependence of integrated absorbance on various chemical modifiers has been examined. The most appropriate technique proved to be 5 μl sample injection using 20 μg prereduced palladium-nitrate for Se determination, and 20 μl sample injection applying 10 μg palladium-nitrate + 3 μg magnesium-nitrate for the measurements of Sn. The optimal temperature program was found to be 1200 °C pyrolysis and 2100 °C atomisation temperature for Se and 1500 °C pyrolysis and 2300 °C atomisation temperature for Sn. Accuracy of the applied techniques was tested by the analysis of standard reference materials. The precision was ±5% for Se and ±10% for Sn. The range of recovery values was 85–95% for Se and 95–105% for Sn. The mean Se concentrations in the investigated brain parts ranged from 200 to 700 ng/g, while the Sn concentrations were between 20 and 300 ng/g dry weight. Received October 3, 2000. Revision February 1, 2001.     

An Exopolysaccharide from Cultivated <Emphasis Type="Italic">Cordyceps sinensis</Emphasis> and its Effects on Cytokine Expressions of Immunocytes

The exopolysaccharide (EPS) is a polysaccharide from cultivated Cordyceps sinensis, which possesses immunomodulatory and antitumor effects, was purified by DEAE-32 cellulose and Sephadex G-200 gel. The preliminary characters of EPS were analyzed by IR and GC, and the molecular weight was estimated by gel filtration. The effect of EPS on proliferation ability of lymphocytes from ICR mice was assayed by MTT method. The mRNA and protein expression levels of several cytokines in spleen and thymus cells were detected by RT-PCR and ELISA. The results showed that EPS consists of mannose, glucose, and galactose in a ratio of 23:1:2.6. Its molecular weight is about 1.04 × 10⁵. EPS elevated proliferation ability of spleen lymphocytes only at 100 μg/ml after 48 h treatment. Tumor necrosis factor alpha (TNF-α), interferon-α (IFN-γ), and interleukin-2 (IL-2) mRNA levels in splenocytes and thymocytes were increased after EPS treatment for 2, 4, 8, or 20 h. EPS also significantly elevated splenic TNF-α and IFN-γ protein expressions at 100 μg/ml and increased thymic TNF-α and IFN-γ protein levels at 50 and 100 μg/ml. These data indicated that EPS may stimulate cytokine expressions of immunocytes.     

Prompt gamma rays from triton-induced reactions on oxygen and their use for analysis

Triton-induced reactions on oxygen were studied with a view to using the prompt γ-rays for analytical purposes. Five γ-rays were found to be potentially useful, of which three had a high intensity, the n(1, 0), n(2, 0) and p(1, 0) γ-rays, the other two being the n(3, 0) and the unresolved pair α(1, 0)–α(2, 0). The γ-rays are labelled according to the conventions(a,b), wheres is the light prompt product and the γ-photon is emitted by de-excitation from levela tob in the heavy product nucleus. The method had a relative precision of 2 to 3.5% for surface oxygen concentrations from 50 to 3 μg/cm². The sensitivity with 1 900 keV tritons was 0.13 μg/cm² on surfaces of steel or copper which did not yield interfering γ-rays, but 0.45 μg/cm² on aluminium where interfering γ-rays were emitted.     

UPTAKE AND BIOCONVERSION OF α-TOCOPHERYL ACETATE TO α-TOCOPHEROL IN SKIN OF HAIRLESS MICE

Abstract— The photoprotective effect of topically applied α-tocopheryl acetate (vitamin E acetate), a stable derivative of α-tocopherol (vitamin E), and its possible bioconversion to the active antioxidant species (α-tocopherol) was examined in skin tissue of female hairless mice (HRS/J) exposed to UV-B irradiation. Our results indicate that topically applied α-tocopheryl acetate is absorbed into and retained by skin tissue. Furthermore, skin tissue from UV-B-irradiated animals that received daily topical α-tocopheryl acetate treatments contained significantly higher levels (P < 0.001) of α-tocopheryl acetate than non-UV-B-irradiated mice that received identical daily topical α-tocopheryl acetate treatments. Finally, free α-tocopherol levels in skin also were significantly increased (P < 0.00 1) by topical applications of α-tocopheryl acetate and skin levels of free α-tocopherol were significantly greater (P < 0.001) in UV-B-irradiated animals that received daily topical α-tocopheryl acetate treatments than in non-UV-Birradiated animals. These results suggest that UV-B irradiation enhances both the absorption of α-tocopheryl acetate and its bioconversion to free α-tocopherol.     

Determination of chloramphenicol in milk by a fluorescence polarization immunoassay

A procedure for the determination of the drug chloramphenicol using a fluorescence polarization immunoassay (FPIA) was proposed. The optimum pairs of antibodies and antigens labeled with fluorescein were chosen, and the analytical characteristics of the procedure were determined. A rapid procedure for milk sample preparation with the use of a saturated solution of ammonium sulfate was optimized. The total time of sample preparation and determination of chloramphenicol in milk was no longer than 10 min. The detection limits of chloramphenicol in water and milk were 10 ng/mL and 20 μg/kg, respectively. The procedure developed for the determination of chloramphenicol was tested in the analysis of model and real milk samples. It was found that some milk samples contained chloramphenicol in concentrations of 38–41 μg/kg, which are several times higher than the maximum permissible concentration (MPC) (10 μg/kg).     

PRODUCTS AND RELATIVE REACTION RATES OF THE OXIDATION OF TOCOPHEROLS WITH SINGLET MOLECULAR OXYGEN

Abstract— The relative reactivity of singlet molecular oxygen, 0₂(¹Δ_g), α-,β-,Γ-and δ with -tocopherol (vitamin E) was investigated using microwave discharge generation as a uniquely clean source of singlet oxygen and using a hydrocarbon solvent to approximate the membrane environment. The relative efficiencies of the tocopherols for O₂(¹Δg) were found to decrease in the order: D-α-tocopherol > D-β-tocopheroI > D–Γ-tocopherol > D-δ-tocopherol. The reaction products in all cases were found to be mixtures of quinone and quinone epoxides apparently resulting from decomposition of the primary product, the hydroperoxydienone.     

Availability of essential trace elements in medicinal herbs used for diabetes mellitus and their possible correlations

Four plant parts (leaves, roots, fruits and seeds) of twenty samples of sixteen antidiabetic herbs including three commercially marketed capsules have been analyzed for 6 minor (Na, K, Ca, Cl, Mg, and P) and 21 trace (As, Ba, Br, Ce, Co, Cr, Cs, Cu, Eu, Fe, Hg, La, Mn, Rb, Sb, Sc, Se, Sm, Th, V and Zn) elements by instrumental neutron activation analysis (INAA). Further, Ni, Cd and Pb contents were determined by AAS. Elemental data were validated by simultaneously analyzing reference material (RM), MPH-2 Mixed Polish Herbs. Several elements such as Cr and V (1–2 μg/g), Rb (10–40 μg/g), Cs (80–300 ng/g), Se (∼100 ng/g) and Zn (25–60 μg/g) play an important role in diabetes mellitus. Interelemental linear correlations have been observed for Cu vs. Zn (r = 0.89) and Rb vs. Cs (r = 0.87). K/P ratio varies in a narrow range with a mean value of 6.2 ± 1.4. Toxic elements As and Hg were found in <1 μg/g whereas Cd and Pb were in ∼5 μg/g and <10 μg/g, respectively.     

Enhancement of Human γ-Interferon Production in Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Batch Cultivation

Development of inexpensive and simple culture media and appropriate induction conditions are always favorable for industry. In this research, chemical composition and stoichiometric data for γ-interferon production and recombinant Escherichia coli growth were used in order to achieve a simple medium and favorable induction conditions. To achieve this goal, the effects of medium composition and induction conditions on the production of γ-interferon were investigated in batch culture of E. coli BL21 (DE3) [pET3a-ifnγ]. These conditions were considered as suitable conditions for the production of γ-interferon: 2.5× M9 medium, supplemented with a mixture of amino acids (milligram per liter), including glutamic acid 215, aspartic acid 250, lysine 160, and phenylalanine 90, and induction at late-log phase (OD₆₀₀ = 4.5). Under these conditions, dry cell weight of 6 ± 0.2 g/l and γ-interferon concentration of 2.15 ± 0.1 g/l were obtained. Later, without changing the concentration ratio of amino acids and glucose, the effect of increase in the primary glucose concentration on productivity of γ-interferon was investigated. It was found that 25 g/l glucose will result in maximum attainable biomass and recombinant human γ-interferon. At improved conditions, a dry cell weight of 14 ± 0.2 g/l, concentration and overall productivity of γ-interferon 4.2 ± 0.1 g/l and 420 ± 10 mg/l h, respectively, were obtained.     

"Dilute-and-shoot" triple parallel mass spectrometry method for analysis of vitamin D and triacylglycerols in dietary supplements

A method is demonstrated for analysis of vitamin D fortified dietary supplements that eliminates virtually all chemical pretreatment prior to analysis, which is referred to as a “dilute-and-shoot” method. Three mass spectrometers, in parallel, plus a UV detector, an evaporative light-scattering detector (ELSD), and a corona charged aerosol detector (CAD) were used to allow a comparison of six detectors simultaneously. Ultraviolet data were analyzed using internal standard, external standard, and response factor approaches. The contents of gelcaps that contained 2,000 IU (50 μg) vitamin D₃ in rice bran oil, diluted to 100 mL, were analyzed without the need for lengthy saponification and extraction. Vitamin D₃ was analyzed using UV detection, extracted ion chromatograms, selected ion monitoring (SIM) atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and two transitions of multiple reaction monitoring (MRM) APCI-MS. The internal standard, external standard, and response factor methods gave values of 0.5870 ± 0.0045, 0.5893 ± 0.0041, and 0.5889 ± 0.0045 μg/mL, respectively, by UV detection. The values obtained by MS were 0.6117 ± 0.0140, 0.6018 ± 0.0244, and 0.5848 ± 0.0146 μg/mL by SIM and two transitions of MRM, respectively. The triacylglycerols in the oils were analyzed using full-scan APCI-MS, electrospray ionization (ESI) MS, up to MS⁴, an ELSD, and a CAD. The method proved to be very sensitive for vitamin D₃, as well as triacylglycerols (TAGs), allowing identification of intact TAGs containing fatty acids up to 28 carbons in length. LC-ESI-MS of glycerin polymers is also demonstrated.     

Microanalysis of “biometallic” compounds using nuclear and atomic activation

A novel method for the characterization of metal containing biological compounds has been developed which utilizes both nuclear activation [e.g.,¹²C(³He, α)¹¹C] and atomic activations (X-ray emission) induced by 8 MeV³He bombardment. Detection limits for carbon (≈1 μg), oxygen (≈1μg), and metals (1 to 10 ng for elements between P and Zn) were obtained under routine experimental conditions. The metal stoichiometry of six different compounds (alkaline phosphatase, amylase, carbonic anhydrase, diamine oxidase, my oglobin, vitamin B₁₂) were determined with typical relative precisions of ±25% for a 40 μg sample. A comparison of these ratios with carbon and metal measurements obtained by other methods showed a relative accuracy of 1 to 20%.     

Determination of 2-isopropyl thioxanthone and 2-ethylhexyl-4-dimethylaminobenzoate in milk: comparison of gas and liquid chromatography with mass spectrometry

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) and gas chromatography–mass spectrometry (GC-MS) have been compared for the analysis of 2-isopropyl thioxanthone (ITX) and 2-ethylhexyl-4-dimethylaminobenzoate (EHDAB). Pressurized liquid extraction (PLE) was applied for the extraction of ITX and EHDAB from milk and milk-based beverages. Samples were homogenized with sea sand and anhydrous sodium sulfate, and were extracted with ethyl acetate at 100 °C and 10.3 × 10⁶ Pa in one cycle of 10 min at 90% flush. Both, GC-MS and LC-MS/MS were suitable to determine these photoinitiators in the PLE extracts, providing appropriate identification and quantification. The recoveries obtained ranged from 70 to 99% for ITX and from 70 to 95% for EHDAB. These recoveries were equal as those obtained by a conventional liquid–liquid partitioning with acetonitrile and tert-butyl methyl ether–hexane. The quantification limits using GC-MS, based on a signal-to-noise ratio of 10, were 0.5 μg/L for ITX and 1 μg/L for EHDAB. The repeatability of the method, as indicated by the relative standard deviations, was within the range 0.9–16.1%. The same parameters calculated using LC-MS/MS result in quantification limits of 0.1 μg/L for ITX and 0.02 μg/L for EHDAB and repeatability within the range 5.2–19.4%. These results pointed out that both techniques are appropriate to determine these compounds in food samples. The method was applied to milk and milk-based beverages from different supermarkets. The ITX and EHDAB contents ranged from 2.5 to 325 μg/L and from 8 to 126 μg/L, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Thermal properties and phase transitions in blends of Nylon-6 with silk fibroin

The thermal and structural properties of binary blends of Nylon-6 (N6) and a chemically related biopolymer, Bombyx mori silk fibroin (SF), are reported in this work. Homopolymers and blends, in composition ratios of N6/SF ranging from 95/05 to 70/30, were investigated by thermogravimetric (TG) analysis, differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy and wide angle X-ray scattering (WAXS). Silk fibroin typically degrades at temperatures just above 210°C, which occurs within the melting endotherm of N6. In TG studies, the measured mass remaining was slightly greater than expected, indicating the blends had improved thermal stability. No beta sheet crystals of SF were detected by FTIR analysis of the Amide I region. Strong interaction between N6 and SF chains was observed, possibly as a result of formation of hydrogen bonds between N6 and SF chains. DSC analysis showed that the addition of SF to N6 caused a decrease in the crystallization temperature, the melting temperature of the lowest melting crystals and the crystallinity of N6. Furthermore, the α-crystallographic phase dominates and the γ-crystallographic phase was not observed in N6/SF blends, in contrast to the homopolymer N6, which contains both phases. We suggest that the addition of SF might result in changes of the chain extension of N6, which lead to the appearance of α-rather than γ-phase crystals.     

Analysis of vitamin E in food and phytopharmaceutical preparations by HPLC and HPLC-APCI-MS-MS

Summary The analysis of α, β, γ, δ-tocopherols, trienols, α-tocopheryl acetate and nicotinate (vitamin E) in complex matrices was carried out using a new liquid chromatographic (HPLC) method giving better separation efficiency, selectivity and sensitivity than that described in the literature. The use of normal-phase (NP)-HPLC on silica gel with issoctane-diisopropylether-1,4-dioxane as optimized mobilepphase yielded higher resolution than conventional reversed-phase (RP)-HPLC using methanol mobile phase. Identification of peaks was by UV-absorbance at 295 nm, diode array, or fluorescence detection (λ _ex = 295 nm,λ _ex = 330 nm). The latter was found to be more selective and ten times more sensitive than UV-absorbance detection. A quadrupole, ion-trap mass spectrometer with an atmospheric-pressure ionization (APCl) interface was used to detect vitamin E constituents in the femtomole range. With collision-induced dissociation (CID) in the ion source, which gave characteristic fragmentation, the identity of the investigated compounds could be confirmed. Plots of peak area versus amount injected allowed quantitation of α, β, γ, δ-tocopherols and-trienols, α-tocopheryl acetate and nicotinate in real samples such as peanut, almond, spinach, spelt grain bran, latex and tablets. The method described offers fast identification and quantitation of vitamin E constituents of complex biological origin. Dedicated to Professor Dr. Heinz Engelhardt on the occasion of his 65^th birthday.     

Determination of fluorine by the spectrometry of prompt gamma-rays

The use, for analysis, of prompt gamma-rays excited by 5 MeV alpha-particles from the reactions¹⁹F(α,α′γ)¹⁹F,¹⁹F(α, nγ)²²Na and¹⁹F(α, pγ)²²NE, was studied. The precision of the analyses depended on the gamma-ray energy used for the measurement. Relative standard deviations were ±1.8, ±0.9 and ±1.3% using the 110-, 197- or 1275 keV gamma-rays. The method was tested with N. I. M. standard materials of calcium fluoride and fluorspar, and was used as a rapid method for the determination of fluorine in cements.