Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R_F values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213–216 nm, diode array UV detection, or fluorescence detection (λ_ex=330 nm; λ_em=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3′,4′,5′-trimethoxyflavone. The substances G1–G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC–electrospray ionization tandem mass spectrometry (ESI-MS–MS) of the flavones was carried out employing a 150×2 mm I.D. column packed with a 3 μm/100 Å octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water–acetonitrile (50:50). Comparative RP-HPLC–ESI-MS of the raw methanol extract and the isolated substances G1–G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC–ESI-MS–MS was used to identify substances G1–G6 in phytopharmaceutical drugs.     

Luminescent properties of carbon-rich starburst gold(I) acetylide complexes. Crystal structure of [TEE][Au(PCy3)]4 ([TEE]H4=tetraethynylethene)

Two carbon-rich starburst gold(I) acetylide complexes [TEE][Au(PCy₃)]₄ (3, [TEE]H₄=tetraethynylethene) and [TEB][Au(PCy₃)]₃ (6, [TEB]H₃=1,3,5-triethynylbenzene) were prepared and their UV–vis absorption, emission and excitation spectra have been recorded. In fluid CH₂Cl₂ solutions, 3 exhibits prompt ¹(ππ*) fluorescence with λ_0–0 and λ_max at 413 and 428 nm, respectively, while 6 displays ³(ππ*) phosphorescence with λ_0–0 and λ_max at 446 and 479 nm, respectively. The crystal structure of 3·CH₂Cl₂ has been determined.     

Determination of rhodamine 123 by sequential injection technique for pharmacokinetic studies in the rat placenta

The sequential injection (SIA) technique was applied in pharmacokinetic studies of the transporter-mediated passage of a model substrate, rhodamine 123 (Rho123), through the dually perfused rat term placenta. The method described was used for real-time monitoring of Rho123 concentrations in both the maternal and fetal compartments. Determination was processed by fluorescence detection (λ_ex=480 nm, λ_em580 nm); calibration curve was linear over the range 0.01–50 μmol l⁻¹ (r=0.99965), detection limit was 10 nmol l⁻¹ (3σ) and RSD2% (10 readings). Transport of Rho123 was scanned under various conditions (ATP-synthesis inhibition) and several inhibitors of P-glycoprotein transporter were tested (e.g. quinidine). The advantages of the modern SIA method—an automated analytical tool providing both fast and precise analysis—were successfully demonstrated for examination of transport profiles to investigate the effect of P-glycoprotein on the placental transfer of Rho123.     

Determination of pentoxifylline in pharmaceutical formulations using iodine as oxidizing agent

Simple, selective and sensitive spectrophotometric methods are described for the determination of pentoxifylline, based on the haloform reaction with a known and excess of standard iodine solution under alkaline conditions. The excess of iodine is determined at pH 3.0 with metol–isoniazid (λ_max=620 nm; method A) or wool fast blue BL (λ_max=540 nm, method B). All the variables have been optimised and the reaction mechanisms presented. Regression analysis of Beer's law plots showed good correlation in the concentration ranges 4.0–24.0 and 0.4–2.4 mg ml⁻¹ for methods A and B respectively. No interferences were observed from excipients and the validity of the methods was tested by analysing pharmaceutical formulations. Recoveries were 99.0–100.0%. The concentration measurements were reproducible within a relative standard deviation of 1.0%.     

Analysis of 5-hydroxy-7-methoxyflavones by normal-phase high-performance liquid chromatography

The separation and identification of flavones present in a chloroform extract of Baccharis trinervis leaves was investigated. The chromatographic system consisted of a amino-bonded column, gradient elution from hexane-chloroform (85:15) to chloroform-acetonitrile (40:60) and detection at 346 nm. Four flavones were found. From NMR and MS data they were identified as 5-hydroxy-7,4′-dimethoxyflavone (I), 5-hydroxy-7,3′,4′-trimethoxyflavone (II), 5,3′-dihydroxy-7,4′-dimethoxyflavone (III) and 5,4′-dihydroxy-7-methoxyflavone (IV). Flavone II and III have not been found in Baccharis trinervis before. The chromatographic system showed good selectivity for the separation of the flavones. The relation between t_R and the structure is discussed.     

Spectrofluorimetric determination of levofloxacin in tablets, human urine and serum

A spectrofluorimetric method to determine levofloxacin is proposed and applied to determine the substance in tablets and spiked human urine and serum. The fluorimetric method allow the determination of 20–3000 ng ml⁻¹ of levofloxacin in aqueous solution containing acetic acid–sodium acetate buffer (pH 4) with λ_exc=292 and λ_em=494 nm, respectively. Micelle enhanced fluorescence improves the sensibility and allows levofloxacin direct measurement in spiked Human serum (5 μg ml⁻¹) and urine (420 μg ml⁻¹), in 8 mM sodium dodecyl sulphate solutions at pH 5.     

Synthesis, structure, and fluorescence of two cadmium(II)-citrate coordination polymers with different coordination architectures

Two novel Cd(II)-citrate complexes were obtained with different metal/ligand ratios through hydrothermal method. Their structures were determined by single-crystal X-ray diffraction analysis. Although their topological structures are both 2-D layer network assemblies, both central Cd(II) ions and Hcit³⁻ ligands display completely different coordination modes. In polymeric complex 1, Hcit³⁻ serves as a μ₁₀-bridged and central Cd(II) ions adopt 6- and 8-coordinated configurations. In contrast, a μ₉-bridged and 6- and 7-coordinated environments between Cd(II) and Hcit³⁻ are established in the polymeric complex 2. Two Complexes remain stable up to approximately 300 °C. The complex 1 exhibits strong fluorescent emission band at 450 nm (λ=346 nm) as well as complex 2 exhibits strong fluorescent emission band at 430 (λ=346 nm).     

The analysis of serum effects on structure, size and toxicity of DDAB-DOPE and DC-Chol-DOPE lipoplexes contributes to explain their different transfection efficiency

The effect of serum on structural properties of dimethyl-dioctadecyl-ammonium bromide (DDAB)–1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposomes and DDAB–DOPE/DNA lipoplexes has been investigated by energy dispersive X-ray diffraction (EDXD) technique, at different cationic lipid/DNA weight ratios (ρ). The role of serum on the size of lipoplexes has also been studied by dynamic light scattering. Lipoplex transfection efficiency (TE) as a function of ρ, and lipoplex toxicity to C6 rat glioma cells have been evaluated in Dulbecco's Modified Eagle Medium (DMEM) with and without serum. A multi-parametric analysis concerning the role of size, structure and cytotoxicity on transfection efficiency contributes to explain the experimental observation that 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol)–DOPE/DNA transfect C6 cells better than DDAB–DOPE/DNA lipoplexes.     

Determination of histamine and some other amines by high-performance capillary electrophoresis with on-line mode in-capillary derivatization

We have developed a method for the determination of histamine (His), tyramine (Tyr) and cadaverine (Cad) using high-performance capillary electrophoresis with fluorescence detection and an on-line mode in-capillary derivatization with o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as derivatization reagent. HPCE separation of His, Tyr, Cad and Spermidine (Spd) was influenced by sodium dodecyl sulfate (SDS) and phosphate–borate buffer (pH 10) concentration. After optimization of the method, a 4-component amine solution containing His, Tyr, Cad and Spd could be separated and detected by using 2 mM OPA/NAC–20 mM SDS–20 mM phosphate–borate buffer (pH 10) as a run buffer at an applied voltage of 25 kV, with detection at 340 nm. Although a practical sensitivity level can be obtained by using fluorescence detection (λ_ex=340 nm, λ_em=450 nm) instead of ultraviolet–visible detection, Spd was not detected at all. The precision (relative standard deviation; n=15) of this method for within- and between-days is less than 2.9% (peak area) and 1.3% (migration time), respectively. Linearity for these analytes, except for Spd, was established over a concentration range of 0.02 to 1.00 μmol/ml and detection limits (S/N=3) range from 1 nmol/ml for His and Tyr to 5 nmol/ml for Cad. The determination of His and some amines in aging raw fish meat samples (room temperature, 48 h) was carried out using the described method with fluorescence detection.     

Determination of boron in water and pharmaceuticals by sequential-injection analysis and fluorimetric detection

This work reports the application of a sequential-injection analysis (SIA) method for the determination of boron. The method relies on the enhancement of the fluorescence (λ_ex=313 nm, λ_em=360 nm) of chromotropic acid (4,5-dihydroxynaphthalene-2,7-disulphonic acid-CA) as a result of its complexation with boric acid (BA). Individual zones of the sample, the CA solution in a suitable buffer and a NaOH solution were aspirated in the holding coil of the SIA apparatus. As the zones were propelled towards the detector, zone penetration in the sample–CA interfaces occurred resulting in the formation of the strongly fluorescent BA–CA complex. The native fluorescence of the CA was quenched by the alkaline environment established as a result of the mixing at the CA–NaOH interface. The chemical and instrumental parameters affecting the fluorescence intensity were investigated and the influence of potential interferents was investigated. After selecting the most suitable conditions, the calibration plot for boron was linear in the range of 8–350 μg l⁻¹ with a 3σ limit of detection of 3 μg l⁻¹ and a relative standard deviation of 2.7% at the 90 μg l⁻¹ boron level (n=8). Finally, the method was applied to the determination of boron in natural waters and pharmaceutical products with revoveries in the range of 96–106%.     

Catalytic flow injection determination of vanadium by oxidation of N-(3-sulfopropyl)-3,3',5,5'-tetramethylbenzidine using bromate

A catalytic flow-injection (FI) method was developed for the determination of 10⁻⁹ mol l⁻¹ levels of vanadium(IV, V). The method is based on the catalytic effect of vanadium(V) on oxidation of N-(3-sulfopropyl)-3,3′,5,5′-tetramethylbenzidine (TMBZ·PS) using bromate as oxidant to form a yellow dye (λ_max=460 nm). The use of 5-sulfosalicylic acid (SSA) as an activator enhanced the sensitivity of the method. The calibration graphs with a working range 0.05–8.0 ng ml⁻¹ were obtained for vanadium(V). Vanadium(IV) was also determined, being oxidized by bromate. The detection limit (signal/noise, S/N=3) was 0.01 ng ml⁻¹ (ca. 2×10⁻¹⁰ mol l⁻¹) vanadium. The relative standard deviations (R.S.D.) for 15 determinations of 0.5 ng ml⁻¹ vanadium, and for ten determinations of 0.1 and 1.0 ng ml⁻¹ vanadium were 0.41, 2.6 and 0.25%, respectively, with a sampling rate of 15 samples h⁻¹. The proposed method was successfully applied to the determination of vanadium in natural waters.     

Automated sequential injection fluorimetric determination and dissolution studies of Ergotamine Tartrate in pharmaceuticals

A fully automated flow system for drug-dissolution studies based on the sequential injection analysis (SIA) was described and used for monitoring dissolution profiles of Ergotamine Tartrate (ET) in pharmaceutical formulations. 50 μl of dissolution medium was taken for each measurement at a flow rate of 40 μl s⁻¹ and detected by fluorescence detector using λ_ex=236 nm (λ_em≥390 nm). The calibration curve was linear over the range 0.03–0.61 mg l⁻¹ of ET (sufficient for the dissolution tests). Equation of the calibration curve was calculated giving the following values: F=117.7 c+0.80 (n=6); r=0.9998. Detection limit was 0.01 mg l⁻¹ of ET. The R.S.D. is less than 0.54 and 0.86% (n=10) when determining 0.61 and 0.03 mg l⁻¹ of ET in standard solution, respectively. The dissolution test of Bellaspon tablets (0.3 mg of ET in 1 tablet) was programmed for 20 min, with a continuous sampling rate of 120 h⁻¹ under conditions required by BP 1993.     

Overpressured layer chromatography in comparison with thin-layer and high-performance liquid chromatography for the determination of coumarins with reference to the composition of the mobile phase

The retention behaviour of fifteen closely related coumarins in normal-phase overpressured layer chromatography (OPLC) was studied with the aim of comparing the retentions with those in normal-phase thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) when optimization of the mobile phase was carried out according to the PRISMA system. The mobile phase optimization was carried out on TLC plates in unsaturated chambers. The resulting mobile phases were transposed to off-line, non-equilibrated OPLC and further to HPLC. The retention in TLC was measured at 37 selectivity points and in OPLC and HPLC at 13 points. Capacity factors (k′) and separation factors () were calculated in order to study the retention behaviour in the different systems. Two- and three-dimensional evaluations of k′ against selectivity points showed similar retention behaviours for the coumarins in TLC, OPLC and HPLC. The values for TLC, OPLC and HPLC showed similar patterns in the three-dimensional evaluations. The retention behaviour at different solvent strengths was also examined. According to quadratic regression, k′ showed a dependence on the change in solvent strength. OPLC, which can be considered as a “planar column” technique, and TLC are closely related methods, whereas HPLC shows a different behaviour in the elution process with regard to solvent strength.     

Determination of adenosine disodium triphosphate using prulifloxacin-terbium(III) as a fluorescence probe by spectrofluorimetry

A new spectrofluorimetric method is developed for determination of adenosine disodium triphosphate (ATP). The interactions between prulifloxacin (PUFX)–Tb³⁺ complex and adenosine disodium triphosphate has been studied by using UV–vis absorption and fluorescence spectra. Using prulifloxacin–Tb³⁺ as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the prulifloxacin–Tb³⁺ complex at λ = 545 nm and the enhanced fluorescence intensity is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The dynamic range for the determination of ATP is 4.0 × 10⁻⁷ to 2.0 × 10⁻⁵ mol L⁻¹, and the detection limit (3 σ/k) is 1.7 × 10⁻⁸ mol L⁻¹. This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of ATP in real pharmaceutical samples. The mechanism of fluorescence enhancement of prulifloxacin–Tb³⁺ complex by ATP was also discussed.     

黄豆黄素与黄豆黄苷吸收光谱和荧光光谱的比较研究

研究了黄豆黄素和黄豆黄苷在不同pH条件下的吸收光谱和荧光光谱, 从分子结构的角度解释了二者呈现不同光谱特征的原因. 黄豆黄素分子基本无荧光. 在弱碱性时, 黄豆黄素分子发生7-OH质子的电离, 导致吸收光谱中320 nm的吸收峰红移至348 nm. 采用pH-光度法测得7-OH质子的电离常数pK_a1=7.08±0.04. 黄豆黄素一价阴离子呈现较强荧光, 最大激发和发射波长λ_ex/λ_em分别为334 nm/464 nm, 荧光量子产率为0.049. 在碱性溶液中, 黄豆黄素4'-OH质子电离, 导致吸收光谱中254 nm的吸收峰红移至260 nm, 电离常数pK_a2=9.96±0.01. 黄豆黄苷分子基本无荧光. 在碱性条件下, 黄豆黄苷分子的4'-OH质子发生电离, 导致吸收光谱中256 nm的吸收峰红移至 280 nm, 电离常数pK_a=9.81±0.03. 黄豆黄苷阴离子基本无荧光, 但在热碱性条件下发生γ-吡喃酮环裂解反应而产生较强荧光, λ_ex/λ_em为288 nm/388 nm, 裂解产物的荧光量子产率为0.056. 虽然, 黄豆黄苷与黄豆黄素是苷与苷元的关系, 但黄豆黄苷不能在热碱性条件下通过糖苷水解转变为黄豆黄素, 二者的荧光增强机理存在本质不同.     

Determination of gamma-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry

γ-Hydroxybutyric acid (GHB), a minor metabolite or precursor of γ-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid–liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 mM nicotinic acid (compound for indirect detection), 3 mM spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 μm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≥2 μg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2–24 and 5–60 μg/ml, respectively. Data obtained suggest that LOD and LOQ (both in μg/ml) can be estimated with the relationships 0.83 κ and 2.1 κ, respectively, where κ is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1→m/z 85.1 precursor–product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 μg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in μg/ml) that are ≥ 4 κ.     

Preparative isolation and dual column high-performance liquid chromatography of ginkgolic acids from Ginkgo biloba.

A chromatographic procedure for the preparative isolation of six different 6-alkylsalicylic acids (syn. ginkgolic acids) with as alkyl substituents C13:0, C15:0, C15:1, C17:1, C17:2 and, tentatively C17:3 from Ginkgo biloba leaves was developed. The procedure consisted of a combination of normal-phase, reversed-phase and argentation chromatography. The compounds were characterised by means of UV, ¹H-NMR and ¹³C-NMR spectroscopy, and mass spectrometry after silylation. A 15 cm C₁₈ RP-HPLC column connected in series with a 20 cm silver(I) loaded cation exchanger HPLC column in combination with the solvent methanol–water (93:7) acidified with 0.1% formic acid was capable of separating the ginkgolic acids C13:0, C15:1, C17:2, C15:0 and C17:1 within 21 min on an analytical scale. The separation is based on a combination of reversed-phase mechanisms and double bond complexation. Detection took place by UV at 311 nm. The separation is a good starting point for the development of a quantitative procedure for the five major ginkgolic acids in Ginkgo leaves and standardised extracts.     

An infrared study of CD3CN in isopropanol, dimethyl formamide, and dimethyl sulfoxide

The vibrational characteristics of deuterated acetonitrile dissolved in isopropanol, dimethyl formamide (DMF), and dimethyl sulfoxide (DMSO) have been studied. Observed vibrational bands show substantial frequency shifts, the amounts of which vary almost linearly with concentration. The absorption feature in the region of 2220–2280 cm⁻¹ was deconvoluted to the consisting absorption bands. The band at 2258 cm⁻¹ of pure CD₃CN, which is on the low frequency side of the monomer CN stretch (ν₂), is attributed to the CN stretch of the dimer (ν′₂). The shoulder found on the further low frequency side of the ν₂ band, particularly in dilute solution, is believed to be due to ν₅, and its frequency and intensity vary largely as a function of concentration along with those of other vibrational bands involved with the CD₃ group. The ν₅ band of pure CD₃CN is believed to be active and located at about 2251 cm⁻¹. Ab initio calculations have also been performed for the solute–solvent complexes, CD₃CN–DMF and CD₃CN–DMSO, at the MP2/6-31+G(2d,p) level assuming anti-parallel configurations. The calculated results show a good agreement with the observed results.     

Kinetic investigation of electronic energy transfer processes following the pulsed dye-laser generation of excited atomic barium, Ba[6s6p(P1)], in the presence of atomic strontium

The collisional behaviour of Ba[6s5d(³D_J)], 1.151 eV above the 6s²(¹S₀) electronic ground state, in the presence of atomic strontium, has been investigated in the ‘long-time domain' (ca. 100 μs–1 ms) following the pulsed dye-laser excitation of barium vapour at elevated temperature at λ = 553.5 nm (Ba[6s6p(¹P₁)] ← Ba[6s²(¹S₀)]. Ba(³D_J) is subsequently produced from the short-lived ¹P₁ state (τ_e = 8.37 ± 0.38 ns) by a number of radiative and collisional processes. It may then be monitored in the ‘long-time domain' by atomic spectroscopic marker methods involving either collisional activation of Ba(³D_J) by Ba(¹S₀) and He buffer gas to yield Ba[6s6p(³P_J)] with subsequent emission from the ³P₁ state (τ_e = 1.2 ± 0.1 μs): Ba[6s6p(³P₁)] → Ba[6s²(¹S₀)] + hv (λ = 791.1 nm). Alternatively, emission from Ba(¹P₁) may be monitored at long times following the generation of this short-lived state by energy pooling following self-annihilation of Ba(³D_J) + Ba(³D_J) from Ba[6s6p(¹P₁)] → Ba[6s²(¹S₀)] + hv (λ = 553.5 nm). The generation of Ba(³D_J) in the presence of atomic strontium yields emission in the long-time domain from Sr[5s5p(³P₁)] (τ_e = 19.6 μs): Sr[5s5p(³P₁)] → Sr[5s²(¹S₀)]  + hv (λ = 689.3 nm). Whilst the decay profiles at short times are complex in form, at long times all these atomic profiles show first-order kinetic removal with the decay coefficients for λ = 791.1 nm, 689.3 nm and 553.5 nm emissions in the ratio 1 : 2 : 2, consistent with overall third-order activation of the form: Ba(³D_J) + Ba(³D_J) + Sr(¹S₀) → Sr(³P_J) + 2Ba(¹S₀). The mechanism is modelled in detail, including measurement of integrated emission intensities, yielding kinetic data for fundamental collisional processes. The overall rate constant for the third-order collisional activation of Sr[5s5p(³P_J])from 2Ba[6s5d(³D_J)] + Sr[5s²(¹S₀)] takes the upper limit of 5.8 × 10⁻²⁷ cm⁶ atom⁻² s⁻¹ (T = 900 K). The rate constant for the two body collisional quenching of Ba[6s5d(³D_J)] by ground state atomic strontium, Sr[5s²(¹S₀)], is found to be (2.0 ± 0.1) × 10⁻¹² cm³ atom⁻¹ s⁻¹ (T = 900 K).     

(S)-(+)-1-Methyl-2-(6,7,-dimethoxy-2,3-naphthalimido)ethyl trifluoromethanesulfonate as a fluorescence chiral derivatizing reagent for carboxylic acid enantiomers in high-performance liquid chromatography

A new triflate-type fluorescence chiral derivatizing reagent, (S)-(+)-1-methyl-2-(6,7-dimethoxy-2,3-naphthalimido)ethyl trifluoromethanesulfonate, [S-(+)-MDNE-OTf], has been developed for the determination of the enantiomers of carboxylic acids. By introducing the two methoxy groups on the naphthalimido ring moiety, the red shift in the fluorescence spectrum and a high resolution in reversed-mode separation of the diastereomers of chiral carboxylic acids have been achieved. The detection limits (S/N=3) with ultraviolet and fluorescence detection are 8 fmol (λ_max=283 nm) and 4 fmol (λ_ex=283 nm, λ_em=467 nm), respectively.