Separation and determination of two sesquiterpene lactones in Radix inulae and Liuwei Anxian San by microemulsion electrokinetic chromatography

A novel microemulsion electrokinetic chromatography (MEEKC) method for separating and determining two sesquoterpene lactones, alantolactone (AL) and isoalantolactone (IAL), in Radix inulae and Liuwei Anxian San has been developed. The effects of several important factors such as internal organic phases, concentration of microemulsion, concentration of acetonitrile, injection time and running voltage were systematically investigated to determine the optimum conditions. The optimum microemulsion system was composed of n-hexane (0.32% w/w), SDS (1.24% w/w), 1-butanol (2.64% w/w), acetonitrile (10% w/w) and 10 mm sodium tetraborate buffer (85.80% w/w, pH 9.2). The applied voltage was 20 kV. The analytes were detected at 214 nm. Regression equations revealed linear relationships (correlation coefficients 0.9950 for AL and 0.9946 for IAL) between the peak area of each analyte and the concentration. The limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 0.45 microg/mL for AL and 0.56 microg/mL for IAL. The levels of the analytes were successfully determined with recoveries ranging from 98.2 to 104.3%. Furthermore, a simple and effective extraction method, with methanol in an ultrasonic water bath for 60 min, was used for sample preparing. Also, MEEKC was compared with micellar electrokinetic chromatography (MEKC) and shown better separation results.     

A rapid ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometric method for the qualitative and quantitative analysis of the constituents of the flower of Trollius ledibouri Reichb

A rapid ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometric (UPLC–ESI-MS/MS) method was developed for the qualitative and quantitative determination of the constituents of the flower of Trollius ledibouri Reichb. The analysis was performed on an AcQuity UPLC™ BEH C₁₈ column using gradient elution with a mobile phase of 0.1% acetic acid and acetonitrile over 20 min. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring (MRM) was used for the qualitative and quantitative analysis of the constituents, respectively. According to the mass spectrometric fragmentation mechanism and UPLC–ESI-MS/MS data, the chemical structures of 15 constituents of the flower of T. ledibouri Reichb. were identified on-line without time-consuming isolation and four of them, 2″-O-β-l-galactopyranosylorientin, 2″-O-β-arabinopyranosylorientin, orientin and vitexin, were quantified. The limits of quantification of these four flavonoids were 540, 321, 515 and 220 μg g⁻¹ plant material, respectively. Four commercial samples from different sources were analyzed. The UPLC–ESI-MS/MS method for analyzing the constituents can be used to evaluate the quality of the flower of T. ledibouri Reichb.     

Interaction of sodium cholate with dioctadecyldimethylammonium chloride vesicles in aqueous dispersion

Mixtures of dioctadecyldimethylammonium chloride (DODAC) cationic vesicle dispersions with aqueous micelle solutions of the anionic sodium cholate (NaC) were investigated by differential scanning calorimetry, DSC, turbidity and light scattering. Within the concentration range investigated (constant 1.0 mM DODAC and varying NaC concentration up to 4 mM), vesicle → micelle → aggregate transitions were observed. The turbidity of DODAC/NaC/water depends on time and NaC/DODAB molar concentration ratio R. At equilibrium, turbidity initially decreases smoothly with R to a low value (owing to the vesicle–micelle transition) when R = 0.5–0.8 and then increases steeply to a high value (owing to the micelle–aggregate transition) when R = 0.9–1.0. DSC thermograms exhibit a single and sharp endothermic peak at T_m ≈ 49 °C, characteristic of the melting temperature of neat DODAC vesicles in water. Upon addition of NaC, T_m initially decreases to vanish around R = 0.5, and the main transition peak broadens as R increases. For R > 1.0 two new (endo- and exothermic) peaks appear at lower temperatures indicating the formation of large aggregates since the dispersion is turbid. All samples are non-birefringent. Dynamic light scattering (DLS) data indicate that both DODAC and DODAC/NaC dispersions are highly polydisperse, and that the mean size of the aggregates tends to decrease as R increases.     

Nonaqueous electrochromatography on continuous bed columns of sol-gel bonded large-pore C18 material: separation of retinyl esters

A nonaqueous electrochromatographic reversed-phase separation method for retinyl esters using continuous bed columns has been developed. The packing material 7 μm Nucleosil 4000 Å C₁₈ was sol–gel bonded in 180 μm I.D. capillaries. The mobile phase used was 2.5 mM lithium acetate in N,N-dimethylformamide–acetonitrile–methanol (2+7+1, v/v). At 350 V/cm and 30°C, this mobile phase composition gave rise to an electroosmotic flow of 1 mm/s. No Joule heating nor bubble formation were observed even at 625 V/cm (17 μA). With a 36 cm L_eff column complete separation of the commercially available and synthesized standards (all-trans-retinyl acetate, palmitate, heptadecanoate, stearate, oleoate, and linoleoate) was obtained within 10 min. The within-day and between-day variations of retention times of all-trans-retinyl palmitate were <0.3% relative standard deviation (RSD) (n=3) and <2% RSD (n=6), respectively. The within-day and between-day variations of peak areas were both <2% (both n=3). The columns were used for more than 1 month without degradation. Liver extracts from arctic seal were analyzed.     

Microchannel-assisted thermal-lens spectrometry for microchip analysis

We present a new method for homocysteine quantitation in human plasma based on in-capillary reaction of homocysteine with 2,2′-dipyridyl disulfide. Homocysteine is in this so-called thiol-exchange reaction quantitatively transformed in mixed disulfide concomitantly with formation of an equimolar amount of 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined specifically at 343 nm. The concentration of homocysteine is thus estimated indirectly from the result of 2-thiopyridone determination. The linear detection range for concentration versus peak area for the assay was from 0.03–3 mM (correlation coefficient 0.994) with a detection limit of 6 μM and a limit of quantitation 20 μM. The inter-day reproducibility of the peak area and the migration time were 1.37% and 0.05%, respectively. The method is simple, relatively rapid and can be easily automated. Moreover the common capillary electrophoresis apparatus with a UV detector can be used to distinguish between normal and pathological hyperhomocysteinemia plasma samples.     

Application of extraction disks in dissolution tests of clenbuterol and levothyroxine tablets by capillary electrophoresis

Sample preparation procedures using octadecyl (C₁₈) extraction disks were developed to obtain accurate and reproducible results for determinations of clenbuterol (20 μg per dose) and levothyroxine (100 μg per dose) in dissolution media of solid oral dosage forms. Preconcentration of samples allowed final concentrations of 1.1 μg/ml of clenbuterol and 4.0 μg/ml of levothyroxine to be reached prior to CE analysis. The results obtained by CE were in good agreement with those of HPLC. The precision of the migration time, peak area, peak height and accuracy were determined in both intea-day (n = 6) and inter-day (n =18) assays. Linearity was demonstrated over the ranges 0.5–80.0 μg/ml of clenbuterol and 1.0–30.0 μg/ml of levothyroxine. The mean recoveries were higher than 94.0%, ranging from 50 to 125% levels with respect to dose potencies. The proposed methodology may be generally applied to determine drugs at ng/ml concentrations.     

Langmuir monolayer properties of the fluorinated-hydrogenated hybrid amphiphiles with dipalmitoylphosphatidylcholine (DPPC)

Surface pressure–area (π–A), surface potential–area (ΔV–A), and dipole moment–area (μ–A) isotherms were obtained for the Langmuir monolayer of two fluorinated-hydrogenated hybrid amphiphiles (sodium phenyl 1-[(4-perfluorohexyl)-phenyl]-1-hexylphosphate (F6PH5PPhNa) and (sodium phenyl 1-[(4-perfluorooctyl)-phenyl]-1-hexylphosphate (F8PH5PPhNa)), DPPC and their two-component systems at the air/water interface. Monolayers spread on 0.02 M Tris buffer solution (pH 7.4) with 0.13 M NaCl at 298.2 K were investigated by the Wilhelmy method, ionizing electrode method and fluorescence microscopy. Moreover, the miscibility of two components was examined by plotting the variation of the molecular area and the surface potential as a function of the molar fraction for the fluorinated-hydrogenated hybrid amphiphiles on the basis of the additivity rule. The miscibility of the monlayers was also examined by construction of two-dimensional phase diagrams. Furthermore, assuming the regular surface mixture, the Joos equation for analysis of the collapse pressure of two-component monolayers allowed calculation of the interaction parameter (ξ) and the interaction energy (−Δ) between the fluorinated-hydrogenated hybrid amphiphiles and DPPC. The observations by a fluorescence microscopy also supported our interpretation as for the miscibility in the monolayer state. Comparing the monolayer behavior between the two binary systems, no remarkable difference was found among various aspects. Among the two combinations, the mole fraction dependence in monlayer properties was commonly classified into two ranges: 0 ≤ X ≤ 0.3 and 0.3 < X ≤ 1. Dependence of the chain length of fluorinated part was reflected for the molecular packing and surface potential.     

Synthesis and characterization of V-MFI obtained in fluoride-containing medium

Crystallization fields for the formation of V-MFI were determined from gels of composition: xNa₂O–yVO₂–7NaF–ySO₃–zSiO₂–2TPABr–260H₂O at 190 °C with 3.6≤x≤14.4 and 2.1≤y≤7.1 for z=12.0 and with 0.3≤y≤4.2 and 4.0≤z≤12.0 for x=3.6; TPA=tetrapropylammonium ions. The crystallization curves were analysed together with the various intermediate phases using XRD, pH of mother liquors, thermal analysis and SEM. The final samples were analysed, in addition, by multinuclear NMR. It is concluded, that V can be introduced into the MFI framework as V(IV) ions, accompanied by the presence of two SiOH defect groups per V atom introduced. The ⁵¹V-NMR signal due to V(V) can only be detected when additional vanadium-containing siliceous phases are formed.     

Speciation analysis of arsenic and selenium compounds in environmental and biological samples by ion chromatography-inductively coupled plasma dynamic reaction cell mass spectrometer

An inductively coupled plasma mass spectrometer (ICP-MS) was used as an ion chromatographic (IC) detector for the speciation analysis of arsenic and selenium. The arsenic and selenium species studied included arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AsB), selenite [Se(IV)] and selenate [Se(VI)]. Gradient elution using (NH₄)₂CO₃ and methanol at pH 9 allowed the chromatographic separation of all species in less than 12 min. Effluents from the IC column were delivered to the nebulization system of ICP-DRC-MS for the determination of arsenic and selenium. The potentially interfering ³⁸Ar⁴⁰Ar⁺ and ⁴⁰Ar⁴⁰Ar⁺ at the selenium masses m/z 78 and 80 were reduced in intensity by approximately 3 orders of magnitude by using 0.6 mL min⁻¹ CH₄ as reactive cell gas in the DRC while an Rpq value of 0.3 was used. Meanwhile, arsenic was determined as the adduct ion ⁷⁵As¹²CHH⁺ at m/z 89, which is more sensitive than ⁷⁵As. The limits of detection for arsenic and selenium were in the range of 0.002–0.01 ng mL⁻¹ and 0.01–0.02 ng mL⁻¹, respectively, based on peak height. The relative standard deviation of the peak areas for five injections of 5 ng mL⁻¹ As and Se mixture was in the range of 2–4%. The concentrations of arsenic and selenium species have been determined in urine samples collected locally. The major As and Se species in urines were AsB, DMA and probably selenosugar at concentration of 20–40, 15–19 and 17–31 ng mL⁻¹, respectively. The recoveries were in the range of 94–105% for all the determinations. This method has also been applied to determine various arsenic compounds in two fish samples. In this study, a simple and rapid microwave-assisted extraction method was used for the extraction of arsenic compounds from fish. The arsenic species were quantitatively leached with an 80% v/v methanol solution in a focused microwave field during a period of 5 min.     

Surface charge and surface chemical characteristics of magnetites substituted with nickel, cobalt and chromium

Magnetite (MAG) and magnetites substituted with nickel, cobalt and chromium (NF, COF and CRF, respectively) have been prepared over the whole range of composition, Me_xFe_3−xO₄ (x=0–3, Me=Ni, Co or Cr) using the coprecipitation technique and have been characterized for their chemical composition, crystallinity and surface area. The types of sorption sites present in them were characterized by analysing the water sorption isotherms ultilizing the D'Arcy and Watt equation. Intrinsic dissociation constants, and point of zero charge, PZC, were evaluated by analysing the pH-titration curves in acid and alkaline media. A similarity in the trend was observed for the crystallinity, surface area, sorption sites and surface charge characteristics as a function of the composition of the substituted MAGs (i.e. x). The overall behaviour of the substituted MAGs could be grouped into two: mixed oxides having composition with x=0–1 and those in the composition region x=1–3. Those in the composition region x=0–1 were crystalline with a lower surface area, less hydrophilic in nature and have surface charge characteristics lower than that of MAG. Those mixed oxides in the composition region x=1–3 nearly had the opposite characteristics, with increasing value of x. The hydrophilicity of the substituted MAGs decreased in the sequence CRF>NFCOF, and the acidic nature decreased in the sequence NF>CRF>COF.     

Sequential injection analysis (SIA)-chemiluminescence determination of indomethacin using tris[(2,2'-bipyridyl)]ruthenium(III) as reagent and its application to semisolid pharmaceutical dosage forms

Automated sequential injection (SIA) method for chemiluminescence (CL) determination of nonsteroidal anti-inflammatory drug indomethacin (I) was devised. The CL radiation was emitted in the reaction of I (dissolved in aqueous 50% v/v ethanol) with intermediate reagent tris(2,2′-bipyridyl)ruthenium(III) (Ru(bipy)₃³⁺) in the presence of acetate. The Ru(bipy)₃³⁺ was generated on-line in the SIA system by the oxidation of 0.5 mM tris(2,2′-bipyridyl)ruthenium(II) (Ru(bipy)₃²⁺) with Ce(IV) ammonium sulphate in diluted sulphuric acid. The optimum sequence, concentrations, and aspirated volumes of reactant zones were: 15 mM Ce(IV) in 50 mM sulphuric acid 41 μL, 0.5 mM Ru(bipy)₃²⁺ 30 μL, 0.4 M Na acetate 16 μL and I sample 15 μL; the flow rates were 60 μL s⁻¹ for the aspiration into the holding coil and 100 μL s⁻¹ for detection. Calibration curve relating the intensity of CL (peak height of the transient CL signal) to concentration of I was curvilinear (second order polynomial) for 0.1–50 μM I (r = 0.9997; n = 9) with rectilinear section in the range 0.1–10 μM I (r = 0.9995; n = 5). The limit of detection (3σ) was 0.05 μM I. Repeatability of peak heights (R.S.D., n = 10) ranged between 2.4% (0.5 μM I) and 2.0% (7 μM I). Sample throughput was 180 h⁻¹. The method was applied to determination of 1 to 5% of I in semisolid dosage forms (gels and ointments). The results compared well with those of UV spectrophotometric method.     

Metal speciation by coupled in situ graphite furnace electrodeposition and electrothermal atomic absorption spectrometry

The technique of coupled in situ electrodeposition–electrothermal atomic absorption spectrometry (ED–ETAAS) is applied to the analytes Bi, Pb, Ni and Cu. Bi, Pb, Ni and Cu are deposited quantitatively from their EDTA complexes at E_cell=1.75, 2.0, 3.0 and 2.5 V, respectively (E_cell=E_anode−E_cathode+iR). By varying the cell potential, selective reduction of free metal ions could be achieved in the presence of the EDTA complexes. For Bi³⁺ and Pb²⁺ this utilised the voltage windows E_cell=0.6–1.0 and 1.8–2.0 V, respectively. For Ni, deposition at E_cell=1.7–2.0 V achieved substantial, but not complete, differentiation between Ni²⁺ (ca. 90–100% deposition) and Ni(EDTA)²⁻ (ca. 12–20% deposition). An adequate voltage window was not obtained for Cu. The ability of ED–ETAAS to differentiate between electrochemically labile and inert species was demonstrated by application of both ED–ETAAS and anodic stripping voltammetry to the time-dependent speciation of Pb in freshly mixed Pb²⁺–NaCl media. Application to natural water samples is complicated by adsorption of natural organic matter to the graphite cathode.     

Simultaneous determination of selenium and lead in whole blood samples by differential pulse polarography

The polarographic reduction of lead in the presence of selenite gives rise to an additional peak corresponding to the reduction of lead (Pb) on adsorbed selenium (Se) on mercury at −0.33 V. The selenium and lead content can be determined using this peak by the addition of a known amount of one of these ions first and then the second ion. The linear domain range of lead is 5.0×10⁻⁷–2.0×10⁻⁵ M and for selenium 5.0×10⁻⁷–1.0×10⁻⁵ M. Using this method 4.90×10⁻⁷ M Se(IV) and 1.47×10⁻⁶ M Pb(II) in a synthetic sample could be determined with a relative error of +2.0% and 1.8%, respectively (n=4). A recovery test after acid digestion for a synthetic sample was 97% for selenium and 96.5% for lead. The method was applied to 1 ml of digested blood, and 328±23 μg l⁻¹ Se(IV) and 850±62 μg l⁻¹ Pb(II) could be determined with a 90% (n=5) confidence interval.     

Structural characterisation of [Pt(NH3)4]2[W(CN)8][NO3]·2H2O donor–acceptor complex

The bimetallic [Pt(NH₃)₄]₂[W(CN)₈][NO₃]·2H₂O is characterised by single-crystal X-ray diffraction [S.G.P2₁/m(11), a=8.0418(7), b=19.122(2), c=9.0812(6) Å, Z=2]. All platinum centres have the square-plane D_4h geometry with average dimensions Pt(1)–N 2.042(2) and Pt(2)–N 2.037(10) Å. The octacyanotungstate anion has the square-antiprismatic D_4d configuration with average dimensions W(1)–C 2.164(13), C–N 1.140(12), W(1)–N 3.303(5) Å. The structure exhibits two different mutual orientations of Pt versus W units resulting in Pt(2)–W(1), W(1)^* separations of 4.77(2), 4.55(2)^* and Pt(1)–W(1) of 6.331(8) Å. A centrosymmetric structure reveals groups of two distinct columns: the first is formed by intercalated NO₃⁻ between parallel [Pt(1)(NH₃)₄]²⁺ planes and the second consists of [W(CN)₈]³⁻ interlayered by, parallel to square faces of W-antiprisms, [Pt(2)(NH₃)₄]²⁺. The structure is stabilised through a three-dimensional hydrogen bond network via nitrogen atoms of cyanide ligands, hydrogen atoms of NH₃ ligands, water molecules and oxygen atoms of NO₃⁻ counteranions. The vibrational pattern and the range of ν(CN) frequencies attributable to the electronic environment of W(V) and W(IV) are consistent with the ground state Pt(II)↔W(V) charge transfer.     

Liquid chromatographic–electrospray mass spectrometric study of the phthalides of Angelica sinensis and chemical changes of Z-ligustilide

High-performance liquid chromatography–electrospray ionization–mass spectrometry has been applied to analyze the chemical constituents of Danggui (the rhizome of Angelica sinensis) and to study chemical changes of Z-ligustilide. Twelve phthalides were unambiguously identified as senkyunolide I (3), senkyunolide H (4), sedanenolide (8), butylphthalide (9), E-ligustilide (13), Z-ligustilide (14), Z-butylidenephthalide (15), Z,Z′-6.8′,7.3′-diligustilide (16), angelicide (17), levistolide A (18), Z-ligustilide dimer E-232 (19) and Z,Z′-3.3′,8.8′-diligustilide (20) in Danggui extract. The existence of 12 other phthalides (2, 5–7, 11, 12, 22–27), ferulic acid (1) and coniferyl ferulate (10) in Danggui extract has also been demonstrated. Phthalides 3, 4, 16–18 and 20 were determined to be the products from chemical change of Z-ligustilide. This is the first report of the existence of 16 compounds (2–8, 10–12, 20, 22–25 and 27) in Danggui extract.     

Determination of arsenic in geological materials by x-ray fluorescence spectrometry after solvent extraction and deposition on a filter

Rock, soil, or sediment samples are decomposed with a mixture of nitric and sulphuric adds. After reduction from arsenic(V) with ammonium thiosulphate, arsenic(III) is extracted as the chlorocomplex into benzene from a sulphuric-hydrochloric acid medium. The benzene solution is transferred onto a filter-paper disc impregnated with a solution of sodium bicarbonate and potassium sodium tartrate, and the benzene allowed to evaporate. The arsenic present is determined by X-ray fluorescence. In a 0.5-g sample, 1–1000 ppm of arsenic can be determined. The close proximity of the lead L peak (2θ 48.73°), to the arsenic K peak (2θ 48.83°) does not cause any interference, because lead is not extracted under the experimental conditions. Arsenic values obtained are in agreement with those reported for various reference samples.     

Optimisation of sample treatment for arsenic speciation in alga samples by focussed sonication and ultrafiltration

A procedure for arsenic species fractionation in alga samples (Sargassum fulvellum, Chlorella vulgaris, Hizikia fusiformis and Laminaria digitata) by extraction is described. Several parameters were tested in order to evaluate the extraction efficiency of the process: extraction medium, nature and concentration (tris(hydroxymethyl)aminomethane, phosphoric acid, deionised water and water/methanol mixtures), extraction time and physical treatment (magnetic stirring, ultrasonic bath and ultrasonic focussed probe). The extraction yield of arsenic under the different conditions was evaluated by determining the total arsenic content in the extracts by ICP-AES. Arsenic compounds were extracted in 5 mL of water by focussed sonication for 30 s and subsequent centrifugation at 14,000 × g for 10 min. The process was repeated three times. Extraction studies show that soluble arsenic compounds account for about 65% of total arsenic.

An ultrafiltration process was used as a clean-up method for chromatographic analysis, and also allowed us to determine the extracted arsenic fraction with a molecular weight lower than 10 kDa, which accounts for about 100% for all samples analysed.

Speciation studies were carried out by HPLC–ICP-AES. Arsenic species were separated on a Hamilton PRP-X100 column with 17 mM phosphate buffer at pH 5.5 and 1.0 mL min⁻¹ flow rate. The chromatographic method allowed us to separate the species As(III), As(V), MMA and DMA in less than 13 min, with detection limits of about 20 ng of arsenic per species, for a sample injection volume of 100 μL. The chromatographic analysis allowed us to identify As(V) in Hizikia (46 ± 2 μg g⁻¹), Sargassum (38 ± 2 μg g⁻¹) and Chlorella (9 ± 1 μg g⁻¹) samples. The species DMA was also found in Chlorella alga (13 ± 1 μg g⁻¹). However, in Laminaria alga only an unknown arsenic species was detected, which eluted in the dead volume.     

Freezing of water bound in lichen thallus as observed by H NMR. II. Freezing protection mechanisms in a cosmopolitan lichen Cladonia mitis and in Antarctic lichen species at different hydration levels

Proton NMR spectra at 300 MHz for dehydrated and hydrated thallus of Cladonia mitis Sandst. [=C. arbuscula (Wallr.) Flot ssp. Mitis (Sandst.) Ruoss], Himantormia lugubris (Hue) Lamb and Usnea aurantiaco-atra (Jacq.) Bory were recorded. The temperature was decreased from room temperature down to −45 °C. Pulse length was set to π/2=8.3 μs, which allowed the observation of tightly bound and loosely bound/or free water fraction, whereas the signal from solid matrix of thallus was reduced. In hydrated thallus a narrow Lorentzian line coming from loosely bound/free water fraction was recorded. For the temperature range between −5 and −20 °C a discontinuous increase in line halfwidths, accompanied by a decrease in area under the peak, was observed. This was attributed to the cooperative freezing of bulk water present in lichen thallus. In dehydrated thallus the NMR line consists of two components: a narrow, Lorentzian one (coming from loosely bound/free water fraction) and a broad line (from water tightly bound in lichen thallus). The overall area under peak remains unchanged down to −5 °C, and then between −5 and −20 °C it continuously decreases due to non-cooperative water immobilisation. As the temperature is decreased, for temperatures above −5 °C, the contribution made by the broad line component increases at the expense of the narrow line component. The mechanism of loosely-to-tightly bound water transfer is, at least partially, responsible for the freeze-protection of thallus in the lichen species investigated. No significant differences between the freeze protecting loosely-to-tightly bound water transfer mechanism of Antarctic lichens and that of cosmopolitan lichens was noticed.     

Conformational and structural analysis of N-N′-bis(4-methoxybenzylidene)ethylenediamine

The Schiff base compound, N-N′-bis(4-methoxybenzylidene)ethylenediamine (C₁₈H₂₀N₂O₂) has been synthesized and its crystal structure has been investigated by X-ray analysis and PM3 method. The compound crystallizes in monoclinic space group P2₁/n with a=10.190(1), b=7.954(1), c=10.636(1) Å, β=111.68(1)°, V=801.1(1) ų, Z=2 and D_cal=1.229 Mgm⁻³. The title structure was solved by direct methods and refined to R=0.056 for 2414 reflections [I>3.0σ(I)] by full-matrix anisotropic least-squares methods. The energy profile of the compound was calculated by PM3 method as a function of θ[N1′–C9′–C9–N1]. The most stable molecular structure of the title compound is the anti conformation, which is different in energy by 5.0 and 1.0 kcal mol⁻¹ from the eclipsed conformation I and gauche conformations, (III and V), respectively.     

Analysis of glycolytic intermediates in Saccharomyces cerevisiae using anion exchange chromatography and electrospray ionization with tandem mass spectrometric detection

The purpose of this study is to selectively and quantitatively analyze several glycolytic intermediates in cells of Saccharomyces cerevisiae using high-performance anion exchange chromatography (HPAEC) coupled to electrospray ionization tandem mass spectrometry for the analysis. A sodium hydroxide gradient is used to separate the glycolytic compounds and after the column sodium hydroxide is reduced by proton exchange with a membrane device prior to introduction to the mass spectrometer. The detection limits for 10 μl samples are down to the 0.4–5 pmol range. This corresponds for the intracellular metabolites to a range of 2–20 nmol per gram biomass dry weight (DW). Standard addition did reveal some influence of the sample matrix on the measured concentrations. Separation and analysis is hardly affected by the high sulfate and phosphate concentrations (1 mM) in the fermentation medium and by the intracellular matrix. Validation of the glucose-6-phosphosphate LC–MS–MS analysis results with enzymatic analysis showed an excellent agreement between the two methods. The suitability of the method was clearly shown by analyzing a series of steady state S. cerevisiae samples from a carbon limited aerobic chemostat culture.