Optimized cleanup method for the determination of short chain polychlorinated n-alkanes in sediments by high resolution gas chromatography/electron capture negative ion-low resolution mass spectrometry

The performances of three adsorbents, i.e. silica gel, neutral and basic alumina, in the separation of short chain polychlorinated n-alkanes (sPCAs) from potential interfering substances such as polychlorinated biphenyls (PCBs) and organochlorine pesticides were evaluated. To increase the cleanup efficiency, a two-step cleanup method using silica gel column and subsequent basic alumina column was developed. All the PCB and organochlorine pesticides could be removed by this cleanup method. The very satisfying cleanup efficiency of sPCAs has been achieved and the recovery in the cleanup method reached 92.7%. The method detection limit (MDL) for sPCAs in sediments was determined to be 14 ng g⁻¹. Relative standard deviation (R.S.D.) of 5.3% was obtained for the mass fraction of sPCAs by analyzing four replicates of a spiked sediment sample. High resolution gas chromatography/electron capture negative ion–low resolution mass spectrometry (HRGC/ECNI–LRMS) was used for sPCAs quantification by monitoring [M−HCl]⁻ ions. When applied to the sediment samples from the mouth of the Daliao River, the optimized cleanup method in conjunction with HRGC/ECNI–LRMS allowed for highly selective identifications for sPCAs. The sPCAs levels in sediment samples are reported to range from 53.6 ng g⁻¹ to 289.3 ng g⁻¹. C₁₀- and C₁₁-PCAs are the dominant residue in most of investigated sediment samples.     

Quantification of free and total salicylic acid in plants by solid-phase extraction and isocratic high-performance anion-exchange chromatography

Salicylic acid (SA) is an important signaling compound in plants and is involved in various defense responses. Here we report a new method for quantification of free and total soluble SA in Arabidopsis thaliana with 5-fluorosalicylic acid (5-FSA) as internal standard. The SA was isolated from leaf extracts by solid-phase extraction with phenyl-phase cartridges and selectively eluted as the cationic iron(III)-complex. Recoveries of SA and 5-FSA were equal and exceeded 90%. Free SA was subsequently released from the iron(III)-complex by addition of 2,2-bipyridyl and high-performance anion-exchange chromatography was performed on an NH₂ column. The SA appeared as last peak with a retention time of 15 min, baseline-separated from other substances. On-line detection was performed fluorimetrically for both SA and 5-FSA at an excitation wavelength of 300 nm and an emission wavelength of 410 nm, because both substances give similar fluorescence spectra. The detection limit for SA was 5 ng g^–1 FW for a sample size of 100 mg. Thus the main advantages of the method are highly selective sample preparation, increased sensitivity, reduced analysis time compared with reversed-phase HPLC, and use of a novel internal standard detectable under the same conditions as SA. The techniques described are applicable to other plant materials.     

Station?re Phasen

Summary A method for the preparation of squalene ligand chemically bonded to silica gel is described. The chromatographic properties of the material for column liquid chromatography were examined by use of polyaromatics and alkylbenzenes. Owing to the existence of a — -interaction between squalene ligand and solute, the retention mechanism and the elution order for some solutes on this column was found to differ from those obtained on a RP-18 column.

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Teil 16: Siehe [1]

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Herrn Prof. Dr. Antonius Kettrup zum 50. Geburtstag gewidmet     

Determination of azidothymidine and its degradation product thymine in pharmaceutical dosage forms by HPLC and HPTLC densitometry

Summary HPTLC densitometry and HPLC are considered for the determination of azidothymidine and its degradation product thymine in pharmaceutical dosage forms. In HPTLC the substances were separated on silica gel with fluorescence indicator in methanol-chloroform (1090) and methanol-chloroform (1585) systems. Absorbance measurement (detection of reflectance) of the separated substances was carried outin situ at 268 nm using four-level calibration (external standard, linear regression function) in the concentration range of 25–100 ng thymine/spot and using single-level calibration (external standard) at the concentration of 100 ng azidothymidine/spot. HPLC was carried out using RP-18 stationary phase and methanol+aqueous 0.03 mol/l KH₂PO₄ (18+82, v/v) as the mobile phase. The temperature was 50°C and the detection wavelength 266 nm. The detection limit of thymidine was 0.05%. The concentration range for azidothymidine was 0.5–1.5 mg/ml and for thymine 1–40 g/ml (for an injection volume of 10 l). The results were evaluated by linear regression analysis.     

Preconcentration of transition metal ions from ethanol solution onN-acyl-N′-benzoylthiourea — Modified silica gel

Silica gel was chemically modified with anN-acyl-N-benzoylthiourea group. This material behaved as a selective means of preconcentrating Cu(II), Zn(II) and Cd(II) from ethanol by the column technique. Ethanolic solutions having 6 mol of the metal ions were percolated through the column and retentions of 100% were achieved for all metals. Zinc and copper were eluted quantitatively from the column bed with 5mL of ethanolic O.1molL^–1 (for Zn) and 0.9 mol L^–1 (for Cu) citric acid. Cadmium was recovered totally with 60 mL of ethanolic 2.0 mol L^–1 citric acid. The modified silica was shown to be chemically stable after various adsorption-elution cycles (at least twenty).     

Radio gas chromatography on capillary columns using a multi-column system (column-switching)

Summary A gas chromatographic system with capillary columns (fused silica) for the analysis of radiolabelled compounds is described. The system presented is based on a dual column gas chromatograph equipped with column switching facllity and a variable splitter at the column outlet combined with a dead-volume free adapter for the radioactivity monitor for continous measurement of radioactivity in the column effluent. The first column works as a separation column and the second is roughly shortened and used as a feed to the mass detector. The adjustment of the split ratio is regulated by the inlet pressures for the carrier gas supplying both columns. For mass detection all conventional systems can be used. Detection of radioactivity by a gas proportional counter (system based on a combustion technique). Three flow modes can be adjusted: a) total column effluent to the mass detector or b) to the radioactivity monitor, and c) simultaneous flow (dependent on the chosen split ratio) to mass-and radioactivity detectors. The system was developed for use in clinical chemistry and tested with labelled and unlabelled steroids. The method for peak identification by means of relative retention times and methylene units was possible also for radioactive peaks when a heart cutting technique was used. The radio gas chromatographic system presented allows the development of radiochromatograms with the same peak characteristics as in conventional capillary gas chromatography.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Determination of pyrene and benzo(a)pyrene residues in water by derivative synchronous solid-phase spectrofluorimetry

Benzo(a)pyrene (BaP) and pyrene (Pyr) are two polycyclic aromatic hydrocarbons (PAHs) showing native fluorescence in solution. Both compounds have been determined in water at trace levels by solid-phase spectrofluorimetry, in which BaP and Pyr are fixed on Sephadex G-25 gel and the relative fluorescence intensity is measured after the system is packed in a 1-mm silica cell. First-derivative synchronous spectra obtained at =38 nm were used to determine BaP and Pyr in the presence of other potentially interferent PAHs. The spectral characteristics of the PAHs-gel system are described, the applicable concentration ranges being 0.4–2.5 ng/ml for BaP and 0.7–4.5 ng/ml for Pyr. The relative standard deviations were 1.1% and 1.4% for BaP and Pyr respectively. The detection limits were 0.04 ng/ml for BaP and 0.1 ng/ml for Pyr. The method was applied to the analysis of both compounds in water at trace levels and a recovery study on tap, natural and sea waters was carried out.     

Five different columns in the analysis of basic drugs in hydrophilic interaction liquid chromatography

Five different columns (two silica, two cyanopropyl and one diol) were investigated in hydrophilic interaction liquid chromatography (HILIC). For the assessment of columns behavior in HILIC mode, six basic drugs (lamotrigine, thioridazine, clozapine, chlorpheniramine, pheniramine and sulpiride) were chosen. The assessment of the influence of the concentration of organic modifier on analytes’ retention on each column was provided by fitting the retention data into theoretical models. Utilizing the statistical analysis, the selection of the model that provides better prediction of the retention behavior was enabled. Dual RP-HILIC mechanism was suggested on cyanopropyl and diol columns, therefore the transition points between the two mechanisms on these columns were calculated. Furthermore, in order to investigate the impact of three factors simultaneously on the retention behavior of the analyzed substances on Betasil Silica column, chemometrically-aided empirical models were built. The experiments were conducted according to the matrix of Box-Behnken design and on the basis of the retention data, six quadratic models were obtained and their adequacy was confirmed using ANOVA test. The obtained coefficients of quadratic models enabled the elucidation of both single factor and factor interactions influence. This was also graphically presented in 3D response surface plots.      

Determination of the enantiomeric purity of albuterol on sorbents modified by macrocyclic antibiotics

The separation of albuterol enantiomers on sorbents with macrocyclic glycopeptide antibiotics immobilized on the silica surface was investigated. Commercial columns—Nautilus-E (BioKhimMak, Russia) with eremomycin as a chiral selector and ChirobioticTAG (Astec, United States) with teicoplanin aglycone as a chiral selector—were used for enantioseparation. Levalbuterol is the (R)-enantiomer of albuterol. We managed to separate albuterol enantiomers on both columns in a polar organic mode, but selectivity was higher on the ChirobioticTAG column (R _s = 1.7). The maximum resolution of enantiomer peaks (1.7) was observed in methanol–acetonitrile–triethylamine–acetic acid (90: 10: 0.05: 0.05) as the mobile phase. The detection limit of the compound calculated by a signal–background ratio of 3: 1 was 0.00002 mg/mL, which corresponds to 0.1% of (S)-enantiomer with respect to the total amount. The results made it possible to determine the enantiomeric purity of the active pharmaceutical substances of levalbuterol.     

Continuous-flow separation and pre-concentration coupled on-line to solid-surface fluorescence spectroscopy for the simultaneous determination of<Emphasis Type="Italic"> o</Emphasis>-phenylphenol and thiabendazole

A novel and single flow-injection system combined with solid-surface fluorescence detection is proposed in this work for the resolution of a mixture of two widely used pesticides (o-phenylphenol and thiabendazole). The continuous-flow methodology is based on the implementation of on-line pre-concentration and separation of both analytes on the surface of C₁₈ silica gel beads placed just inside the flow cell, implemented with gel-phase fluorimetric multi-wavelength detection (using 305/358 and 250/345 nm as excitation/emission wavelengths for thiabendazole and o-phenylphenol, respectively). The separation of the pesticides was possible owing to the different retention/desorption kinetics of their interactions with the solid support in the zone where the stream impinges on the solid material. No previous separation of the analytes before they reach the flow cell is needed thereby simplifying substantially both the procedure and the manifold. By using a sample volume of 2,600 L, the system was calibrated in the range 0.5–16 and 5–120 ng mL^–1 with detection limits of 0.09 and 0.60 ng mL^–1 for thiabendazole and o-phenylphenol, respectively. The RSD values (n=10) were about 1% for both analytes. The proposed methodology was applied to environmental water samples and also to various commercial pesticide formulations containing both analytes. Recovery percentages were 97–103% and 98–102% for thiabendazole and o-phenylphenol, respectively.     

Separation and quantitation of nicergoline and related substances by high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry

Summary This study demonstrated the utility of high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry (HPLC/API-MS) in the investigation of 10-methoxy-1,6-dimethylergoline-8-methanol 5-bromonicotinic acid ester (Nicergoline) and its related substances. The analysis was performed by using an ODS column with ammonium acetate and methanol mixture as the mobile phase. Nicergoline and its related compounds could be characterized by HPLC/API-MS in terms of their molecular weight. The use of multiple ion detection techniques for the quantitation of these compounds was also investigated. The detection limits of nicergoline and its related substances were 5 to 10 ng each at a signal-to-noise ratio of 4. The method was also applied to the study of the decomposition products of nicergoline in simulated gastric and intestinal fluids.     

The Essential Oil of Pistacia vera L. at Various Temperatures of Direct Thermal Desorption Using Comprehensive Gas Chromatography Coupled with Time-of-Flight Mass Spectrometry

The essential oil obtained from the hulls of Pistacia vera fruits using direct thermal desorption was resolved using comprehensive gas chromatography (GC×GC) coupled with time-of-flight mass spectrometry (TOF-MS). A range of temperatures (100, 150, 200, 250 °C) for thermal desorption were applied to extract volatile organic compounds from around 10mg of raw sample. Although different species profiles were produced at each temperature (notably browning products above 200 °C), -pinene and (Z)--terpineol were found to be the major components at all temperatures studied. The number of compounds observed increased with increasing temperature. A temperature of 150 °C is recommended as optimum for the direct thermal desorption of volatiles from matrices such as fruit hulls. The GC×GC separation chromatographically resolved several hundred components which when coupled for detection with TOF-MS gave high probability identifications for around 100 compounds. The comprehensive separation also separated a number of components which remained unresolved on a single GC column, such as camphene with butyrolactone and dodecane with p-cymene.     

Miniaturized CE System Based on Amperometric Detection and Horizontal Sampling

A novel miniaturized capillary electrophoresis (CE)-amperometric detection system has been fabricated by integrating a self-positioning electrode system and a 10-cm piece of fused silica capillary on a Plexiglas base plate. The self-positioning electrode system was composed of a disc working electrode positioned opposite the outlet of the capillary, with an Ag/AgCl reference electrode, a platinum auxiliary electrode, and a platinum grounding electrode accommodated in a rectangular detection cell on the plate. The platinum wire, close to the inlet of capillary, served as a contact to the high-voltage power supply. Alternate placement of the capillary inlet in vials containing sample or buffer solutions permits volume-defined electrokinetic sample introduction in a horizontal arrangement. Performance of this unique system was demonstrated by separating sugars, amino acids, and two phenols used in the pharmaceutical industry. The factors influencing the analytical performance of the new microsystem for the separation of p-aminophenol and acetaminophen have been characterized and optimized. The two analytes could be separated on baseline within 160s in 50mM acetate buffer (pH 5.0). Responses for p-aminophenol and acetaminophen were linear over the range of 10 to 1000µM with detection limits (S/N=3) of 2.1 and 2.9µM.     

Chromatographic Applications on Monolithic Columns: Determination of Triamcinolone, Prednisolone and Dexamethasone in Pharmaceutical Tablet Formulations Using a Solid Phase Extraction and a Monolithic Column

A method is described for extraction, separation and quantification of 3 corticosteroids in tablets (triamcinolone, prednisolone and dexamethasone). The extraction was achieved using a solid phase extraction (SPE) on RP-18 cartridges. The steroids eluates were examined by HPLC. The chromatographic separation was carried out on Chromolith^® Performance RP-18e column, a new packing material consisting of monolithic rods of highly porous silica, using isocratic binary mobile phase of MeOH and H₂O in the ratio 1: 1. A diode array detector was used at 254 nm for detection. The method was validated for system suitability, linearity, precision, limits of detection, specificity, stability and robustness. The limits of detection were 6.25, 6.25 and 12.50 ng per 25 L injected volumes for them respectively. The recovery values of this method were between 89 and 101% and the reproducibility was within 6.86.     

Rapid,sensitive and simultaneous determination of fluorescence-labeled designated substances controlled by the Pharmaceutical Affairs Law in Japan by ultra-performance liquid chromatography coupled with electrospray-ionization time-of-flight mass spectrometry

A simultaneous determination method based on ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) was developed for 16 “designated substances” (Shitei-Yakubutsu) controlled by the Pharmaceutical Affairs Law in Japan. These substances were first labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole at 60 °C for 2 h in 0.1 M borax (pH 9.3). The resulting fluorophores were well separated by reversed-phase chromatography using an Acquity UPLC™ BEH C₁₈ column (1.7 μm, 100 mm × 2.1 mm i.d.) by isocratic elution with a mixture of water and acetonitrile–methanol (20:80) containing 0.1% formic acid. The separated derivatives were sensitively detected by both FL and TOF-MS. However, the determination of several designated substances by FL detection showed interference from endogenous substances in biological samples. Therefore, the determination in real samples was carried out by a combination of UPLC separation and ESI-TOF-MS detection. The structures of the designated substances were identified from the protonated-molecular ions [M+H]⁺ obtained from the TOF-MS measurement. The calibration curves obtained from the peak area ratios of the internal standard (I.S.), i.e., 3-phenyl-1-propylamine, and the designated substances versus the injection amounts showed good linearity. The limits of detection ( \textS/N = 3 ) \left( {{\text{S/N}} = 3} \right) and the limits of quantification ( \textS/N = 10 ) \left( {{\text{S/N}} = 10} \right) in 0.1 mL of human plasma and urine for the present method were 0.30–150 pmol and 1.0–500 pmol, respectively. Good accuracy and precision (according to intraday and interday assays) were also obtained with the present procedure. This method was applied to analyses of human plasma, urine and real products.      

Encapsulation of Viable Aerobic Microorganisms in Silica Gels

The aim of this study was to develop techniques to sterilise the surface of silica gels containing encapsulated cells and the liquid broth they were immersed in, so that the observed metabolic activities could be unambiguously assigned to fully encapsulated cells. Gel surfaces were sterilised by UV-irradiation daily. The surfaces of the gels and the overlaying medium remained sterile for 20 days following irradiation, as demonstrated by the lack of visible surface growth and viable cells in the medium. We report the encapsulation of a viable, metabolically active, aerobic fungus Penicillium chrysogenum, and the aerobic Gram-positive bacterium Streptomyces rimosus in gels derived from aqueous silica sols. Carbohydrate consumption (catabolism) and antibiotic biosynthesis (penicillin or oxytetracycline) (anabolism) were monitored in both cultures, demonstrating that the encapsulated cells remained viable within the gel matrix. This demonstrates that the silica gels are sufficiently porous to sustain metabolic activities of aerobic cells, which require the diffusion of oxygen and other substrates within the gels nanopore network.     

Evidence for the role of the monB genes in polyether ring formation during monensin biosynthesis

Ionophoric polyethers are produced by the exquisitely stereoselective oxidative cyclization of a linear polyketide, probably via a triepoxide intermediate. We report here that deletion of either or both of the monBI and monBII genes from the monensin biosynthetic gene cluster gave strains that produced, in place of monensins A and B, a mixture of C-3-demethylmonensins and a number of minor components, including C-9-epi-monensin A. All the minor components were efficiently converted into monensins by subsequent acid treatment. These data strongly suggest that epoxide ring opening and concomitant polyether ring formation are catalyzed by the MonB enzymes, rather than by the enzyme MonCII as previously thought. Consistent with this, homology modeling shows that the structure of MonB-type enzymes closely resembles the recently determined structure of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis.     

CPS LG 313 Phenylalkanol — adsorbates on silica as types of immobilized drugs 1. Chemiadsorption on activated silica surfaces

The chemiadsorption of phenylalkanols onto a porous silica support (d _pore 10 nm) viaSi-O-C bonds was studied. The silica support was activated prior to the alcohol adsorption by annealing or by the introduction ofSi-Cl groups. Different reaction conditions were applied, namely time, temperature, solvent and drug-support ratio. The yield of chemiadsorption was dependent on both the reaction conditions and the structure of the alcohols. Long alkyl chains, especially those with an alcohol OH-group positioned in the vicinity of bulky substituents, reduce the yield considerably. Using chlorinated silica, a reasonable yield of chemiadsorption can be obtained even at relatively low reaction temperatures.Dedicated to Professor Dr. Dr. h. c. Armin Weiss on the occassion of his 60th birthday.     

Preparation and separation of metallobacteriochlorophylla by reversed-phase high-performance liquid chromatography

Summary Semi-preparative high-performance liquid chromatography has been used for the preparation of copper(II) bacteriochlorophylla [Cu(II)-BChl-a] and zinc(II) bacteriochlorophylla [Zn(II)-BChl-a]. Both compounds are separated on a reversed-phase Inertsil ODS-2 column using a mobile phase of acetone-methanol (2575, v/v). The fractionated metallobacteriochlorophylls (M-Bchl-a) are identified by fast atom bombardment mass spectrometry. The spectroscopic parameters such as the wavelength of absorption maxima and the molar extinction coefficients are determined using pure M-Bchl-a obtained by preparative HPLC. The HPLC method proposed here has been demonstrated to be useful for the purification and determination of components of M-Bchl-a.     

Packing and performance of microbore columns for adsorption and partition chromatography

Summary Microbore columns of 1 mm i.d. have turned out to be very suitable for the achievement of efficient columns.The packing procedure for stainless steel 1 mm i.d. columns from 5 to 100 cm in length was studied. Stationary phases used were: pure silica gel, octyl, octadecyl and amino bonded silicas. The main parameters (slurry composition, packing system, choice of materials) are discussed.Short columns packed with 3 or 5m particles allow high speed separations. A separation in 18 seconds is described.Very high plate numbers can be obtained with long columns. With 7–8m particles, a 1 m column can produce 50,000 plates (h=3). Columns can be joined without loss of efficiency. 270 000 theoretical plates were obtained on a 6 m adsorption column with a test mixture. In reversed-phase chromatography, bile acid sodium salts can be separated on a 1 m column. In adsorption chromatography, details are given of the separation of a polystyrene oligomer sample, as well as a light and a heavy petroleum distillate samples on a 2 m column with refractive index detection in the last-case.