Detection and Confirmation of Ginsenosides in Horse Urine by GC–MS and LC–MS

Ginseng has been used by the Chinese as a traditional herbal medicine for thousands of years. In view of the growing popularity in the use of ginseng preparations as natural remedies and food supplements worldwide, there is an increasing concern for their abuse in both human and animal sports. Ginsenosides are considered the major constituents of ginseng responsible for its pharmacological properties. In this study, a method was developed for the detection and confirmation of a number of ginsenosides in horse urine. The intact ginsenosides were detected and confirmed at 5–100 ng mL^?1 by LC–MS², and two deglycosylation metabolites, namely protopanaxadiol and protopanaxatriol, could both be detected and confirmed at 2 ng mL^?1 by GC–MS² after trimethylsilylation. The above GC–MS and LC–MS methods were then applied to study the in vitro metabolism of ginsenosides Rg1 and Rb1 and the in vivo urinary metabolites after oral administration of Rg1 to horses. Results obtained reveal the very first evidence for the existence of the metabolites, Rg1 and protopanaxatriol, as glucuronides in urine.     

Iron–cobalt and iron–cobalt–nickel nanowires deposited by means of cyclic voltammetry and pulse-reverse electroplating

Metal nanowires composed of Fe–Co and Fe–Co–Ni alloys were successfully prepared by means of cyclic voltammetry (CV) and pulse-reverse (PR) electroplating techniques from acidic metal chloride solutions. The anodic dissolution process in the CVs or in the reverse electroplating period was found to be the key factor influencing the formation of metal nanowires. The addition of nickel into the Fe–Co alloy was found to extend the diameter of these nanowires. The morphology and crystalline information of these alloy deposits prepared by CV and PR deposition techniques were obtained from the field-emission scanning electron microscopic (FE-SEM) photographs and X-ray diffraction (XRD) patterns, respectively.     

Sensitive Determination of Felodipine in Human and Dog Plasma by Use of Liquid–Liquid Extraction and LC–ESI–MS–MS

Summary A sensitive and selective liquid chromatographic method coupled with electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) has been developed for quantification of felodipine in human and dog plasma. Compounds were separated on a 2.0 mm × 150 mm, 5.0 m particle, C₈ column with 1 m m ammonium acetate–acetonitrile, 20:80, pH 6.0, as mobile phase at a flow rate of 200 L min^–1. Nifedipine was used as internal standard. Plasma samples were extracted with diethyl ether, the centrifuged upper layer was evaporated, the residue was reconstituted with mobile phase, and the reconstituted samples were injected. The analytical column lasted for at least 1000 injections. By use of multiple reaction monitoring (MRM) mode in MS–MS felodipine and nifedipine were detected without severe interference from the human or dog plasma matrix. Felodipine produced a protonated precursor ion ([M + H]⁺) at m/z 384 and a corresponding product ion at m/z 338. And internal standard (nifedipine) produced a protonated precursor ion ([M + H]⁺) at m/z 347 and a corresponding product ion at m/z 315. Detection of felodipine in human and dog plasma was accurate and precise, with a limit of quantification of 0.05 ng mL^–1. The method has been successfully applied to preliminary pharmacokinetic study of felodipine in human and dog plasma.     

Modification of tricine–SDS–PAGE for online and offline analysis of phosphoproteins by ICP-MS

This study describes a modification to tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis to make it more effective for the separation of low molecular mass proteins and for coupling with inductively coupled plasma mass spectrometry (ICP-MS). The modified method employs low-percentage polyacrylamide gels (7–10%) (w/v) and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. Using phosphopeptides as a model system, and offline analysis, we obtained recoveries of 73% (w/v) in a 9% gel compared with 55% in a conventional 16% gel. Online coupling was achieved by modification of a standard commercially available gel electroelution apparatus and casting of the gel into a 7.3-cm-long tube. Online separation of a digest of β-casein was demonstrated with recovery of the mono- and tetraphosphopeptides, which were identified by comparison with peptide standards. A mass balance study with the standards yielded recoveries of 95% for tetraphosphopeptides and 48% for monophosphopeptides. The factors affecting the separations and recoveries are discussed in detail. The detection limits for 10-μL samples of the mono- and tetraphosphopeptides were 0.7 μM (7 pmol) and 0.2 μM (2 pmol) respectively.     

Synthesis of complex Si–O–N–H derivatives by molecular bombardment of silicon and silicon dioxide

Bombardment of a silicon target in a high vacuum with a molecular beam (mixture of high energy H₂+N₂, obtained by charge exchange) and a thermal beam of O₂ produces on the target a variety of compounds. The target is then bombarded by the same molecular beams which produce, extracted by an electrostatic field at an energy of about 10 keV, molecular ions due to the compounds thus formed. These ions are analysed (electromagnet) to give a primary spectrum of ions according to their mass, which are individually selected and dissociated in a collision cell (same H₂+N₂ mixture). Mass analysis of the dissociation fragments leads to the identification of silicon clusters (Si)_n and of Si–O–N–H derivatives, the fragmentations of which permit a definitive determination of their molecular complexity. Dissociation spectra have thus been obtained for some of the most intense peaks of the primary spectrum, on the one hand, and on the other hand for some peaks of lower intensity but of special interest to us (see below). The composition of the fragments is confirmed by the study of the satellite ions derived from the natural 28, 29 and 30 isotopes of silicon, and by the use of deuterium instead of hydrogen. None of the Si–O–N–H derivatives obtained was apparently known earlier. It is shown that some of these molecules (those ‘of special interest to us’) may be identical with sila-analogues of standard amino acids and of nucleic bases: the fact that their fragmentations are identical with those of the corresponding carbon analogues speaks in favour of a structural identity. However, one cannot yet distinguish between the various possible isomeric arrangements, as none of them has been independently prepared, which excludes a direct comparison with reference samples, and as these isomers might give identical fragments; we hope to be able to resolve this ambiguity later. Anyhow, the substances formed are the most complex molecular silicon derivatives so far produced: e.g. Si₂O₂NH₅, Si₃O₂NH₇, Si₄O₃NH₉, Si₄O₂N₂H₄, Si₄ON₃H₅, Si₅O₃N₂H₁₀, Si₅O₂NH₁₁, corresponding to sila-glycine, sila-alanine, sila-threonine, sila-uracile, sila-cytosine, sila-valine, sila-glutamine, – or isomers. Similar results have been obtained using a silicon dioxide target and high energy molecular beams of hydrogen and of nitrogen, without thermal oxygen, or with a carborundum target. © 2000 Académie des sciences / Éditions scientifiques et médicales Elsevier SASmolecular impact / silicon derivatives / amino-acid silicon analogues     

Thermodynamics and kinetics of hydrogen absorption–desorption of vanadium synthesized by aluminothermy

This paper studies the addition (0–40% w/w) of natural zeolite (NZ, 84% clinoptilolite) in blended cements made with Portland cement (PC) with low and medium C₃A content. The isothermal calorimetry was used to understand the effect of NZ on the early cement hydration. For low C₃A cement, the addition of NZ produces mainly a dilution effect and then the heat released curve is similar to plain cement with lower intensity. For medium C₃A cement, the curve shows the C₃S peak in advance and a high intensity of third peak attributed to C₃A hydration. The high cation fixed of NZ reduces the ions concentration (especially alkalis) in the mixing water stimulating the PC hydration. The flowability decreases when the NZ replacement level increases. Results of Fratini’s test show that NZ with both PCs used presents slow pozzolanic activity. At early age, XRD and FTIR analyses confirm that hydration products are the same as that of the corresponding PC and the CH is progressively reduced after 28 days and some AFm phases (hemi- and monocarboaluminate) appear depending on the NZ percentage and the PC used. For low replacement levels, the compressive strength is higher than the corresponding PC from 2 to 28 days. For high replacement levels, the early compressive strength is lower than that of corresponding plain PC and the pozzolanic reaction improves the later compressive strength of blended cements.     

Kinetics of coal and char oxycombustion studied by TG–FTIR

Coal and char oxycombustion is a complex process because of very high reaction rate of oxygen with coals and chars carbon. Very important process during oxycombustion is diffusion of O₂ to surface of coal and char grain. This process can be minimized using small samples and high flow of the gas, but it is also dependent on temperature. For this reason, it is impossible to eliminate diffusion processes which cause significant impact on calculated kinetic parameters. This paper describes the results of thermogravimetric studies of oxycombustion process with evolved gas analysis by FTIR. Ultimate and proximate analysis of coal and char were made. Thermogravimetric experiments of coal and its char oxycombustion were conducted using five heating rates, namely 2.5, 5, 10, 20 and 40 K min^?1, and gas mixture composed of 20 % O₂ in CO₂. Activation energies of coal and char oxycombustion were calculated by isoconversional methods: integral Vyazovkin and differential Friedman. Activation energies for three ranges of heating rates were calculated. This paper shows influence of heating rate on calculated activation energy. The reason of this phenomenon is due to change of the mechanism of coal and char oxycombustion from the chemical kinetic control regime to mixed chemical kinetic–diffusion control regime.     

Speciation of aluminium in tea infusions by use of SEC and FPLC with ICP–OES and ES–MS–MS detection

Speciation of Al in tea infusions was studied by size exclusion chromatography (SEC) and anion-exchange fast protein liquid chromatography (FPLC). Fractions were collected throughout the chromatographic separations and Al was determined “off line” by inductively coupled plasma optical emission spectroscopy (ICP–OES). Black, green, and red tea samples were investigated. The total concentration of Al in tea infusions was determined by ICP–OES and ranged between 0.5 and 4 mg dm⁻³. The pH of tea infusions ranged between 5.3 and 5.5. Data from SEC–ICP–OES analysis indicated that 10–35% of total Al in tea infusions was eluted at a retention volume corresponding to a molecular mass of approximately 3800 Da. The remaining Al was adsorbed on the column resin. The same tea infusions were also analysed by anion-exchange FPLC–ICP–OES. It was found experimentally that the same percentage of total Al as from the SEC column was eluted at a retention volume that corresponded to negatively charged Al-citrate. The remaining Al was adsorbed on the column resin. Identification of Al-binding ligands eluting under the chromatographic peak was performed by electrospray ionisation tandem mass spectrometry (ES–MS–MS) analysis. It was proven that ionic Al species in tea infusions (10–35% of the total Al) corresponded to negatively charged Al-citrate. The remaining species that was adsorbed on the SEC or FPLC columns was most probably bound to phenolic compounds. Speciation of Al in tea with milk or lemon was also studied. Results for tea with milk indicated that Al-citrate was not transformed and that approximately 60% of total Al was transformed into high-molecular-mass Al species. This fraction was subjected to sodium dodecyl sulfonate polyacryl gel electrophoresis (SDS–PAGE). The results indicated that Al was occluded by milk proteins (mostly caseins). When citric acid was added to tea infusions the percentage of negatively charged Al-citrate remained either the same or increased to 40% of total Al.     

Simultaneous Determination of Six Steroidal Alkaloids of Veratrum dahuricum by HPLC–ELSD and HPLC–MSn

A high performance liquid chromatography coupled with evaporative light scattering detection (HPLC–ELSD) and electrospray ionization multistage mass spectrometry (HPLC–ESI–MSⁿ), respectively, has been performed for the simultaneous determination of six steroidal alkaloids, including pseudojervine, veratrosine, jervine, veratramine, 3-veratroylzygadenine, 3-angeloylzygadenine, in Veratrum dahuricum collected in different seasons. The plants were soaked in methanol and extracted ultrasonically. The six steroidal alkaloids were obtained by silica gel column chromatography, eluting with gradient petroleum and acetyl acetate. The intra-day and inter-day precisions of the method were evaluated and were less than 1.4%. The content of steroidal alkaloids in the plant varied significantly from spring to autumn, confirming the necessity to control the quality of V. dahuricum during its preparation and application.     

Compatibility of sildenafil citrate and pharmaceutical excipients by thermal analysis and LC–UV

The evaluation of sildenafil citrate (SC), the best-selling drug for treatment of impotence, for compatibility with various excipients was investigated using thermal and isothermal stress testing. Differential scanning calorimetry (DSC), hot-stage microscopy (HSM) and liquid chromatography (LC) with ultraviolet detection were successfully employed to investigate the compatibility between SC and various excipients commonly used in solid form in the pharmaceutical industry. The studies were performed using 1:1 (m/m) drug/excipient physical mixtures and samples were stored under accelerated stability conditions (40 °C at 75% relative humidity). All excipients tested (such as colloidal silicon dioxide, croscarmellose sodium, lactose, mannitol and sucrose) showed potential incompatibilities by DSC and LC analysis after accelerated stability testing. However, some incompatibilities were not detected by the DSC method and were observed only when LC analysis was performed. HSM was able to differentiate active pharmaceutical ingredient degradation from solubilisation, supporting the interpretation of DSC in excipients where thermal events either overlapped or disappeared. The combination of both the analytical techniques (DSC and LC) and use of a stability chamber is extremely helpful in detecting incompatibilities and providing more robust and accurate approaches for pre-formulation studies.     

Determination of Shikimic Acid in Fruits of Illicium Species and Various Other Plant Samples by LC–UV and LC–ESI–MS

A simple and specific analytical method for the quantitative determination of shikimic acid from the methanol extract of the fruits of Illicium species and from various plant samples was developed. The LC–UV separation was achieved by reversed-phase chromatography on a C18 column with potassium dihydrogen phosphate and methanol as the mobile phase. In the LC–MS method, the separation was achieved by a C12 column using water and acetonitrile, both containing 0.1% acetic acid as the mobile phase. The methods were successfully used to study the percentage compositions of shikimic acid present in nine species of Illicium and various other plant samples. The detector response was linear with concentrations of shikimic acid in the range from 1.0–500.0 μg mL^?1 by LC–UV and 100–1000 ng mL^?1 by LC–MS. Mass spectrometry coupled with electrospray ionization interface is described for the identification of shikimic acid in various plant samples. This method involved the use of the [M-H]^? ions of shikimic acid at m/z 173.0455 (calculated mass) in the negative ion mode with extractive ion monitoring.     

Simultaneous enhancement of phosphorescence and chirality by host–guest recognition of molecular tweezers

A novel type of host–guest recognition systems have been developed on the basis of a Au(III) molecular tweezer receptor and chiral Pt(II) guests. The complementary host–guest motifs display high non-covalent binding affinity (K_a: ～10⁴ L/mol) due to the participation of two-fold intermolecular π–π stacking interactions. Both phosphorescence and chirality signals of the Pt(II) guests strengthen in the resulting host–guest complexes, because of the cooperative rigidifying and shielding effects rendered by the tweezer receptor. Their intensities can be reversibly switched toward pH changes, by taking advantage of the electronic repulsion effect between the protonated form of tweezer receptor and the positive-charged guests in acidic environments. Overall, the current study demonstrates the feasibility to enhance and modulate phosphorescence and chirality signals simultaneously via molecular tweezer-based host–guest recognition.     

Study of tungstate–protein interaction in human serum by LC–ICP-MS and MALDI-TOF

Oral administration of sodium tungstate is an effective treatment for type 1 and 2 diabetes in animal models; it does not incur significant side effects, and it may constitute an alternative to insulin. However, the mechanism by which tungstate exerts its observed metabolic effects in vivo is still not completely understood. In this work, serum-containing proteins which bind tungstate have been characterized. Size exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) with a Phenomenex Bio-Sep-S 2000 column and 20 mM HEPES and 150 mM NaCl at pH 7.4 as the mobile phase was chosen as the most appropriate methodology to screen for tungsten–protein complexes. When human serum was incubated with tungstate, three analytical peaks were observed, one related to tungstate–albumin binding, one to free tungstate, and one to an unknown protein binding (MW higher than 300 kDa). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of the tungsten-containing fractions collected from SEC–ICP-MS chromatograms, after desalting and preconcentration processes, confirmed the association of tungstate with albumin and the other unknown protein. Figure SEC-ICP-MS // MALDI-TOF     

Determination of Pentachloronitrobenzene and Its Metabolites in Ginseng by Matrix Solid-Phase Dispersion and GC–MS–MS

A rapid method was developed for the determination of pentachloronitrobenzene (PCNB) and its metabolites pentachloroaniline, pentachlorothioanisole residues in ginseng. Extraction and clean-up were carried out in a single step and analysis was accomplished by gas chromatography–mass spectrometry with multiple reaction monitoring. The main parameters affecting extraction yield and selectivity, such as type and amount of dispersant material, clean-up co-sorbent and extraction solvent were evaluated. The best results were obtained using 1 g ginseng, 2 g florisil as dispersant sorbent, 0.5 g neutral alumina as clean-up co-sorbent, and subsequent extraction with 10 mL acetone–n-hexane (5:5, v/v) with assisted sonication and repeated with another 5 mL of the same solvent mixture. The method was validated by analysis of ginseng samples fortified at different concentration levels (0.01–0.10 mg kg^?1). Average recoveries (n = 5) ranged from 85 to 95% with relative standard deviation between 2.5 and 11.2%. Spiked blank samples were used as standards to counteract the matrix effect observed in the chromatographic determination. The detection limits ranged from 0.2 to 0.9 µg kg^?1 in ginseng. The method was applied to the analysis of PCNB and its metabolite residues in commercial ginseng samples.     

Multiresidue Analysis of Pesticides in Vegetables and Citrus Fruits by LC–MS–MS

In this work, an analytical multiresidue method using liquid chromatography tandem-mass spectrometry (LC–MS–MS) with triple quadrupole in selected reaction monitoring (SRM) mode for the simultaneous determination of 54 pesticides in vegetables (pepper and tomato) and citrus fruits (orange and lemon) has been developed. The procedure involves initial single phase extraction of sample with acetonitrile by agitation, followed by liquid–liquid partition aided by “salting out” process using NaCl. The average recovery by the LC–MS–MS method obtained for these compounds varied from 65.5 to 114.5% with a relative standard deviation between 2.3 and 8.3%. The method presents good linearity over the range assayed 10–500 μg L^?1 (except famoxadone 50–1,000 μg L^?1) and the detection limits for the pesticides studied varied from 0.03 to 14.9 μg kg^?1. The proposed method was used to determine pesticide levels in vegetables and citrus fruit samples from different experimental orchards and greenhouses from the Region of Murcia.     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

Simultaneous Determination of Tamsulosin and Dutasteride in Human Plasma by LC–MS–MS

A sensitive and selective liquid chromatographic tandem mass spectrometric (LC–MS–MS) method was developed for simultaneous identification and quantification of tamsulosin and dutasteride in human plasma, which was well applied to clinical study. The method was based on liquid–liquid extraction, followed by an LC procedure with a Gemini C-18, 50 mm × 2.0 mm (3 μm) column and using methanol:ammonium formate (97:3, v/v) as the mobile phase. Protonated ions formed by a turbo ionspray in positive mode were used to detect analytes and internal standard. MS–MS detection was by monitoring the fragmentation of 409.1 → 228.1 (m/z) for tamsulosin, 529.3 → 461.3 (m/z) for dutasteride and 373.2 → 305.3 (m/z) for finasteride (IS) on a triple quadrupole mass spectrometer. The lower limit of quantification for both tamsulosin and dutasteride was 1 ng mL^?1. The proposed method enables the unambiguous identification and quantification of tamsulosin and dutasteride for clinical drug monitoring.     

Microstructure and hydrogen production activity of Pt–TiO2 prepared by precipitation–photodeposition

A series of Pt–TiO₂ photocatalysts were prepared by a facile precipitation–photoreduction method under different pH conditions, using H₂PtCl₆ as platinum precursor. The microstructure and chemical state of Pt loaded on the surface of TiO₂ were analyzed by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). It was revealed that the size and distribution of Pt nanoparticles on TiO₂ surface is closely related to the initial pH of H₂PtCl₆ solution. The optimal pH value for forming highly dispersed Pt nanoparticles is 12. The photocatalytic activities of the prepared samples were investigated in terms of hydrogen production. The results indicated that the Pt–TiO₂ sample prepared by precipitation–photodeposition method shows much higher activity than that prepared by traditional photodeposition method.     

Determination of arsenic species and arsenosugars in marine samples by HPLC–ICP–MS

Arsenic-speciation analysis in marine samples was performed by high-pressure liquid chromatography (HPLC) with ICP–MS detection. Separation of eight arsenic species—As^III, MMA, DMA, As^V, AB, TMAO, AC and TeMAs⁺—was achieved on a C₁₈ column with isocratic elution (pH 3.0), under which conditions As^III and MMA co-eluted. The entire separation was accomplished in 15 min. The HPLC–ICP–MS detection limits for the eight arsenic species were in the range 0.03–0.23 μg L⁻¹ based on 3σ for the blank response (n=5). The precision was calculated to be 2.4–8.0% (RSD) for the eight species. The method was successfully applied to several marine samples, e.g. oysters, fish, shrimps, and marine algae. Low-power microwave digestion was employed for extraction of arsenic from seafood products; ultrasonic extraction was employed for the extraction of arsenic from seaweeds. Separation of arsenosugars was achieved on an anion-exchange column. Concentrations of arsenosugars 2, 3, and 4 in marine algae were in the range 0.18–9.59 μg g⁻¹. This paper was presented at the European Winter Conference 2005