Automated in-tube solid-phase microextraction–liquid chromatography–electrospray ionization mass spectrometry for the determination of ranitidine

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) was evaluated for the determination of ranitidine. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary column by repeated aspirate/dispense steps. In order to optimize the extraction of ranitidine, several in-tube SPME parameters such as capillary column stationary phase, extraction pH and number and volume of aspirate/dispense steps were investigated. The optimum extraction conditions for ranitidine from aqueous samples were 10 aspirate/dispense steps of 30 μl of sample in 25 mM Tris–HCl (pH 8.5) with an Omegawax 250 capillary column (60 cm×0.25 mm I.D., 0.25 μm film thickness). The ranitidine extracted on the capillary column was easily desorbed with methanol, and then transported to the Supelcosil LC-CN column with the mobile phase methanol–2-propanol–5 M ammonium acetate (50:50:1). The ranitidine eluted from the column was determined by ESI-MS in selected ion monitoring mode. In-tube SPME followed by LC–ESI-MS was performed automatically using the HP 1100 autosampler. Each analysis required 16 min, and carryover of ranitidine in this system was below 1%. The calibration curve of ranitidine in the range of 5–1000 ng/ml was linear with a correlation coefficient of 0.9997 (n=24), and a detection limit at a signal-to-noise ratio of three was ca. 1.4 ng/ml. The within-day and between-day variations in ranitidine analysis were 2.5 and 6.2% (n=5), respectively. This method was also applied for the analyses of tablet and urine samples.     

Method for the simultaneous determination of losartan and its major metabolite, EXP-3174, in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

A liquid chromatography–electrospray ionization tandem mass spectrometric method was developed for the simultaneous determination of losartan and its major active metabolite, EXP-3174, in human plasma. The two analytes and the internal standard (DuP-167) were extracted from plasma under acidic conditions by using solid-phase extraction cartridges containing a sorbent of copolymer, poly(divinylbenzene-co-N-vinylpyrrolidone). The analytes were separated by LC equipped with a reversed-phase C₁₈ column, and introduced into the mass spectrometer via the electrospray ion source with pneumatically-assisted nebulization. For LC–MS–MS samples, an isocratic mobile phase consisting of [0.1% triethylamine–0.1% acetic acid (pH 7.1)]–acetonitorile (65:35, v/v) was used, and the assay was monitored for the negative fragment ions of the analytes. The method demonstrated linearity from 1 to 1000 ng/ml for both losartan and EXP-3174. The limit of quantification for both compounds in plasma was 1 ng/ml. This assay method may be useful for the measurement of levels of the two compounds in clinical studies of losartan.     

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane–isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-μm particle size, C₁₈-bonded silica column and water–sodium acetate–heptanesulfonate–acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4±3.1% for TMB and 89.4±4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10–5000 ng/ml for TMB and 25–25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.     

Convenient method of threonine, methionine and their related amino compounds by high-performance liquid chromatography and its application to rumen fluid

A high-performance liquid chromatographic procedure for the quantitative determination of cysteine (Cys), homocysteine (Hcys), methionine sulfoxide (MSO), methionine sulfone (MSO₂), homoserine (Hser), glycine (Gly), threonine (Thr), 2-aminobutyric acid (2AB), methionine (Met), cystathionine (Cysta) and its application to rumen fluid are described. The samples containing Thr, Met and other related amino compounds were derivatized with 9-fluorenylmethyl chloroformate. The separation of compounds was accomplished with a methanol gradient in 25 mM sodium citrate buffer (obtaining pH 6.40 and 3.80 by addition of 25 mM citric acid). All derivatized compounds were separated on a Mightysil RP-18 GP (150×4.6 mm I.D., 5 μm particle size) column. All analytes were detected at 265 nm with UV detection. The limits of detection (μM) (S/N ratio, 3:1) and quantification (μM) (S/N ratio, 10:1) of Cys, Hcys, MSO, MSO₂, Hser, Gly, Thr, 2AB, Met and Cysta were 0.50 and 1.68; 1.76 and 5.85; 0.85 and 2.88; 0.92 and 3.09; 1.04 and 3.52; 0.76 and 2.52; 0.65 and 2.18; 0.39 and 1.36; 0.31 and 1.03; 0.17 and 0.58, respectively. The recoveries of all compounds in rumen fluid were 97.93–102.3% in the within-day study and 94.52–98.69% on different day (6 days) studies. The average contents (μM) of Cys, Gly, Thr, 2AB, Met and Cysta were 1.72, 45.6, 20.0, 4.3, 2.11 and 3.42 before morning feeding. The concentration of Thr, 2AB and Cysta in rumen fluid tended to increase with time after feeding whereas Met showed the opposite tendency.     

Determination of an arylether antiarrhythmic and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates using liquid chromatography–tandem mass spectrometry

A method was developed and validated for the quantification of (±)-trans-[2-morpholino-1-(1-naphthaleneethyloxy]cyclohexane monohydrochloride (RSD1070) and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography coupled with positive ion electrospray ionization and detection by tandem mass spectrometry. The assay was linear from 2.5 to 100 ng/ml and this range was used for validation. Inter- and intra-assay variability (n=6), extraction recovery, and stability in plasma were assessed. The estimated limit of quantitation was in the range 2.5–3 ng/ml for both analytes in rat plasma. The analytical method was used in a pharmacokinetic study of RSD1070 in rats after a single i.v. bolus of 12 mg/kg.     

Achiral and chiral high-performance liquid chromatographic methods for clinafloxacin, a fluoroquinolone antibacterial, in human plasma

Achiral and chiral HPLC methods were developed for clinafloxacin, a quinolone antimicrobial agent. For achiral assay, analytes were isolated from plasma by precipitating plasma proteins. Separation was achieved on a C₁₈ column using an isocratic eluent of ion pairing solution–acetonitrile (80:20, v/v) at 1.0 ml/min with UV detection at 340 nm. The ion pairing solution was 0.05 M citric acid, 1.15 mM tetrabutylammonium hydroxide and 0.1% ammonium perchlorate. Inter-assay accuracy was within 4.9% with an inter-assay precision of 3.7% over a quantitation range of 0.025 to 10.0 μg/ml. For chiral assay, analytes were isolated from plasma by solid-phase extraction. Separation was achieved on a Crownpak CR(+) column using an isocratic eluent of water–methanol (88:12, v/v) containing 0.1 mM decylamine at 1.0 ml/min with UV detection at 340 nm. Perchloric acid was added to adjust pH to 2. Inter-assay accuracy was within 3.5% with a inter-assay precision of 5.4% over a quantitation range of 0.040 to 2.5 μg/ml.     

Simultaneous determination of thioTEPA, TEPA and a novel, recently identified thioTEPA metabolite, monochloroTEPA, in urine using capillary gas chromatography

An assay for the simultaneous quantitative determination of thioTEPA, TEPA and the recently identified metabolite N,N′-diethylene-N″-2-chloroethylphosphoramide (monochloroTEPA) in human urine has been developed. MonochloroTEPA was synthesized by incubation of TEPA with sodium chloride at pH 8. Thus, with this assay monochloroTEPA is quantified as TEPA equivalents. Analysis of the three analytes in urine was performed using gas chromatography with selective nitrogen–phosphorous detection after extraction with a mixture of 1-propanol and chloroform from urine samples. Diphenylamine was used as internal standard. Recoveries ranged between 70 and 100% and both accuracy and precision were less than 15%. Linearity was accomplished in the range of 25–2500 ng/ml for monochloroTEPA and 25–5000 ng/ml for thioTEPA and TEPA. MonochloroTEPA proved to be stable in urine for at least 4 weeks at −80°C. ThioTEPA, TEPA and monochloroTEPA cummulative urinary excretion from two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples and that the excreted amount of monochloroTEPA exceeded that of thioTEPA on day 2 to 5 of urine collection.     

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.     

Metabolism of amiodarone: II. High-performance liquid chromatographic assay for mono-N-desethylamiodarone hydroxylation in liver microsomes

Amiodarone (AMI) is a potent antiarrhythmic drug. In vivo and in vitro, AMI is biotransformed to mono-N-desethylamiodarone (MDEA). Recently, it was observed that MDEA was further hydroxylated to n-3′-hydroxybutyl-MDEA (3′OH-MDEA). The performance of a HPLC–UV assay being developed for the quantification of the new compound was investigated. Liver microsomes isolated from rabbit, rat and human biotransformed MDEA to 3′OH-MDEA. Their estimates of Michaelis–Menten parameters were K_m=6.39, 25.2, 19.4 μM; V_max=560, 54, 17.3 pmol/mg protein/min), respectively. Thus, hydroxylase activity in mammals may be the origin of the species dependence observed in the AMI metabolism.     

Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion spray and turbo ion spray tandem mass spectrometric detection

Methods for the determination of a semi-synthetic cyclic hexapeptide (I, MK-0991) in human plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection using pneumatically assisted electrospray (ion spray, ISP) and turbo ion spray (TISP) interfaces were developed. Drug and internal standard (II, an isostere of I) were isolated from plasma by solid-phase extraction (SPE). The eluent from SPE was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The use of ISP, TISP and heated nebulizer (HN) interfaces as sample introduction systems were evaluated and showed that the heated nebulizer was not adequate for analysis due to thermal instability and/or adsorption of I and II to glass surfaces of the interface. Compounds I and II were chromatographed on a wide pore (300 Å), 150×4.6 mm C₈ analytical column, and the HPLC flow-rate of 1.2 ml/min was split 1:20 prior to introduction to the ISP or TISP interface of the mass spectrometric system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode (SRM). The precursor→product ion combinations of m/z 1093.7→1033.6 and 1094.7→1033.6 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10–1000 ng/ml using ISP, and 2.5–500 ng/ml of plasma using TISP with good precision and adequate accuracy. The effects of HPLC mobile-phase components on the ionization efficiency and sensitivity of detection in the positive ionization mode, the evaluation of the matrix effect, and limitations in sensitivity of detection of I due to the formation of multiply charged species are presented.     

Determination of trace amounts of ginkgolic acids in Ginkgo biloba L. leaf extracts and phytopharmaceuticals by liquid chromatography–electrospray mass spectrometry

Ginkgolic acids (GAs) are toxic phenolic compounds present in the fruits and leaves of Ginkgo biloba L. (Ginkgoacae). Their maximum level in phytopharmaceuticals containing ginkgo extracts has been recently restricted to 5 μg/g by the Commission E of the former Federal German Health Authority. In order to detect ginkgolic acids at these low levels, a sensitive and selective analytical method, based on liquid chromatography–electrospray mass spectrometry (LC–ES-MS) has been developed. The three main phenolic acids (1–3) of the chloroform fruit extract were isolated and used as standards for quantification. In the LC–ES-MS negative ion mode, calibration curves with good linearities (r=0.9973, n=6) were obtained in the range of 0.5–10 μg/g for compounds 1, 2 and between 0.1 and 7.5 μg/g (r=0.9949, n=6) for ginkgolic acid 3. The detection limits at a S/N ratio of 3 were 0.1 (3) and 0.25 μg/g (1, 2). Recoveries were around 101% at 5 μg/g for the substances detected in the leaf extracts. Good precision was achieved with relative standard deviations of less than 4% (n=6). The optimised method was applied to verify whether the amount of gingkolic acids was below 5 μg/g in a standardised leaf extract which is a constituent of a phytopreparation.     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

Evaluation of fused-core and monolithic versus porous silica-based C18 columns and porous graphitic carbon for ion-pairing liquid chromatography analysis of catecholamines and related compounds

This paper evaluates the performances of reversed-phase (RPLC) and ion-pairing chromatography (IPLC) coupled with UV detection for the analysis of a set of 12 catecholamines and related compounds. Different chromatographic columns (porous C18-silica, perfluorinated C18-silica, porous graphitic carbon, monolithic and fused-core silica-based C18 columns) were tested using semi-long perfluorinated carboxylic acids as volatile ion-pairing reagents. Much more promising results were obtained by IPLC than by RPLC and important improvements in analytes peak symmetry and separation resolution were observed when using the "fast chromatography" columns (monolithic and fused-core C18) under IPLC conditions. For UV detection, a satisfactory separation of the 12 selected analytes was achieved in less than 20 min by using a fused-core particles column (Halo C18) and a mobile phase composed of a 1.25 mM nonafluoropentanoic acid aqueous solution and methanol under gradient elution mode. The chromatographic method developed can be directly coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) in positive ionization mode and 10 solutes among those selected can be observed. The presence of the acidic ion-pairing reagent in the mobile phase makes this system incompatible with negative ionization mode and thus unable to detect the two acidic compounds that only responded in negative mode. In terms of MS detection, Monolithic C18 column proved to be the best one to reach the lowest detection limits (LODs) (from 0.5 ngmL(-1) to 10 ngmL(-1) depending on the neurotransmitter). The applicability of the optimized LC-MS/MS method to a "real world" sample was finally evaluated. The presence of the matrix leads to signal suppression for several solutes and thus to higher LODs.     

Synthesis and biological activity of 17α-alkynylamide derivatives of estradiol

Seven estradiol (E₂) derivatives with an alkynylamide side chain at the 17α position were synthesized starting from ethynylestradiol (EE₂). The main chemical step was the coupling reaction of the acetylide ion of EE₂ with carbon dioxide, glutaric anhydride or bromoalkyl ortho ester. The synthesis of these compounds is fast (3–6 steps according to the compound) and is easily achieved with good yield. Five compounds with different side chain lenghts were evaluated for uterotrophic and antiuterotrophic activity in the CD-1 mouse. None of the tested compounds shows estrogenic activity in this sensitive in vitro system. At low doses (1 and 3 μg), a 14–57% inhibition of E₂-induced uterine growth was observed while no additional inhibition was observed at the 10, 20 and 30 μg doses. In human breast carcinoma cells in culture, all compounds show estrogenic activity at high concentrations while only compound 39 (N-buty,N-methyl-8-[3′,17′β-dihydroxy estra-1′,3′,5′(10′)-trien-17′α-yl]-7-octynamide) possesses antiproliferative or antiestrogenic effects. No significant correlation could be demonstrated between alkynylamide side chain length and estrogenic or antiestrogenic activity. Among the compounds tested, the derivative of EE₂ possessing a five-methylene (CH₂) side chain (compound 39) possesses the best antiestrogenic activity (44 ± 7% in the CD-1 mouse uterus assay at the 3μg dose and 57 ± 4% at 0.1 nM in human ZR-75-1 cancer cells in culture).     

Determination of calcium dobesilate in human plasma using ion-pairing extraction and high-performance liquid chromatography

A rapid, simple reversed-phase high-performance liquid chromatographic method with ultraviolet absorbance detection has been developed for the determination of calcium dobesilate in human plasma. Sample processing is based on an ion-pairing extraction with tetra-n-butylammonium hydroxide as cationic pairing ion and dichloromethane. Separation of the investigated calcium dobesilate and 2,4-dihydroxybenzoic acid as internal standard was achieved on a Discovery RP-Amide C₁₆ analytical column with 50 mM, pH 2.5, potassium dihydrogenphosphate buffer–acetonitrile (75:25, v/v) mobile phase. The wavelength was set at 305 nm. The limit of quantitation is 100 ng/ml and the calibration curve is linear up to 50 μg/ml. Within-day and between-day precision expressed as the relative standard deviation is about 10% and the accuracy of the determination did not deviate from 100% by more than ±10%. The developed method was found to be suitable for application in human bioequivalence studies.     

Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography–tandem mass spectrometry

A column-switching liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 μl) were injected onto a C₁₈-alkyl-diol silica (ADS) column and the matrix compounds were washed to waste with a mixture of 20 mM ammonium acetate solution at pH 4.0–acetonitrile (97:3). The retained analyte fraction containing (E)- and (Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (internal standard) was backflushed to the analytical C₁₈ column, with a mixture of 20 mM ammonium acetate–acetonitrile (85:15) for the final separation at pH 7.0. The eluate was directed to the mass spectrometer after splitting (1:100). The mass spectrometer was operated in the negative ion mode and the deprotonated molecules [M−H]⁻ were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M−H]⁻ in MS–MS resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M−H−Glu]⁻, which were chosen as the product ions for single reaction monitoring. Quantitative studies showed a wide dynamic range (0.0025–100 μg/ml) with correlation coefficients better than 0.995. The method was repeatable within-day (relative standard deviation, RSD<7%) and between-day (RSD<14%) and the recovery (78–103%) was better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of (E)- and (Z)-isomers of entacapone glucuronide in plasma of rats used in absorption studies.     

Determination of naphthodianthrones in plant extracts from Hypericum perforatum L. by liquid chromatography–electrospray mass spectrometry

The determination of the naphthodianthrone constituents in extracts of dried blossoms of Hypericum perforatum L. by combined HPLC–electrospray mass spectrometry is described. Hypericin (1), pseudohypericin (2) and their precursor compounds produce intensive negative quasi-molecular ions by deprotonation provided a non-acidic eluent system is used in the HPLC separation. From the [M–H]⁻ ions formed in the electrospray ionization process characteristic daughter ion spectra can be obtained by collisional activation which have been studied by tandem mass spectrometry.     

Quantification of topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces by high-performance liquid chromatographic methods

Sensitive high-performance liquid chromatographic (HPLC) methods have been developed and validated for the simultaneous determination of the antitumor drug topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces. Both compounds are reversibly hydrolysed to their hydroxycarboxylate forms at physiologic pH. Separate HPLC systems have been developed for the determination of lactone and total (lactone plus hydroxycarboxylate forms) concentrations in plasma. The instability of the analytes in plasma requires immediate protein precipitation with ice-cold methanol. The lactone forms of the analytes were stable in the methanol extracts for at least 15 months when stored at −70°C. For the determination of the total levels, the plasma extracts were acidified with 25 mM phosphoric acid to convert the compounds into their lactone forms quantitatively. The sample pretreatment procedure for urine included dilution in methanol while the faecal samples were homogenized in distilled water and then extracted twice with an acetonitrile–ammonium acetate mixture. Separation was achieved on reversed-phase columns (Zorbax SB-C18) and detection was performed fluorimetrically at 380/527 nm. Within-run and between-run precisions were less than 10% and average accuracies were between 90 and 110%. The methods were used in a mass balance study in patients with malignant solid tumors to determine the disposition and routes of elimination of topotecan and N-desmethyltopotecan.     

Synthesis and properties of chitosan hydrogels modified with heterocycles

Preparation and properties of chitosan modified with heterocycles in absence or presence of gluteraldehyde as a cross linker is described. New modified chitosan–heterocyclic hydrogels were prepared from chitosan and heterocyclic compounds such as N,N′-biisomaleimide, N,N′-biisophthalimide, and N,N′-phthalimidomaleimide via a crosslinking reaction. The new hydrogels chemical structure was characterized by spectral analysis (IR), X-ray diffraction, thermal gravimetric analysis (TGA), solubility, and swellability in water and different organic solvents. Evaluation of the efficiency of the new hydrogels to uptake copper and cobalt ions from aqueous systems was carried out and promising results were obtained.     

Exudate flavones and flavanones in Origanum species and their interspecific variation

Surface flavonoids in nine species of Origanum, representing taxa from all three of the currently recognised subgeneric groups, were examined both by HPLC coupled to diode-array detection and APCI–MS. Many of the flavonoids present were characterised by O-substituent at C-6 (OH, OMe) and/or C-8 (OMe). In total, 25 flavones and flavanones are described in this study, of which 13 are new to the genus and 5,4′-dihydroxy-6,7,3′-trimethoxyflavanone is reported for the first time. Taxa in subgeneric Group A accumulated flavonoids with methoxyl groups at both C-6 and C-4′; however, taxa in subgeneric Group B did not accumulate 4′-methoxylated compounds, and taxa in Group C did not accumulate 6-methoxylated compounds.