High-performance liquid chromatographic determination of tianeptine in plasma applied to pharmacokinetic studies

An improved analytical method for the quantitative measurement of tianeptine and its main metabolite MC₅ in human plasma was designed. Extraction involved ion-paired liquid–liquid extraction of the compounds from 1.0 ml of human plasma adjusted to pH 7.0. HPLC separation was performed using a Nucleosil C₁₈, 5 μm column (150×4.6 mm I.D.) and a mixture of acetonitrile and pH 3, 2.7 g l⁻¹ solution of sodium heptanesulfonate in distilled water (40:60, v/v) as mobile phase. UV detection was performed using a diode array detector in the 200–400 nm passband, and quantification of the analytes was made at 220 nm. For both tianeptine and MC₅ metabolite, the limit of quantitation was 5 μg l⁻¹ and the calibration curves were linear from 5 to 500 μg l⁻¹. Intra- and inter-assay precision and accuracy fulfilled the international requirements. The recovery of tianeptine and its metabolite from plasma was, respectively, 71.5 and 74.3% at 20 μg l⁻¹, 71.2 and 70.8% at 400 μg l⁻¹. The selectivity of the method was checked by verifying the absence of chromatographic interference from pure solutions of the most commonly associated therapeutic drugs. This method, validated according to the criteria established by the Journal of Chromatography B, was applied to the determination of tianeptine and MC₅-metabolite in human plasma in pharmacokinetic studies.     

Fully automated analytical method for codeine quantification in human plasma using on-line solid-phase extraction and high-performance liquid chromatography with ultraviolet detection

A simple, sensitive and fully automated analytical method for the analysis of codeine in human plasma is presented. Samples are added with oxycodone, used as internal standard (I.S.), and directly loaded in the autosampler tray. An on-line sample clean-up system based on solid-phase extraction (SPE) cartridges (Bond-Elut C₂, 20 mg) and valve switching (Prospekt) is used. Isocratic elution improved reproducibility and allowed the recirculation of the mobile phase. A Hypersil BDS C₁₈, 3 μm, 10×0.46 cm column was used and detection was done by UV monitoring at 212 nm. Retention times of norcodeine (codeine metabolite), codeine and oxycodone (I.S.) were 5.5, 6.4 and 9.1 min, respectively. Morphine was left to elute in the chromatographic front. Detection limit for codeine was 0.5 μg l⁻¹ and inter-assay precision (expressed as relative standard deviation) and accuracy (expressed as relative error) measured at 2 μg l⁻¹ were 5.03% and 1.82%. Calibration range was 2–140 μg l⁻¹.     

Effects of Growth Regulators on Direct Flowering of Isolated Ginseng Buds <Emphasis Type="Italic">in vitro</Emphasis>

An in vitro method for obtaining gingseng inflorescences directly from explants of gingseng (Panax ginseng) is reported. Isolated shoot-buds of somatic embryo-derived plantlets ginseng were used as explants and incubated in B5 medium supplemented with 1 mg l⁻¹ benzyladenine (BA) and 1 mg l⁻¹ gibberellic acid (GA₃). About 15% of the buds flowered directly without developing vegetative organs. Cytokinin was found to be the key factor for inducing these isolated buds to proliferate and flower, but both these processes also occurred when benzyladenine (BA) was replaced by thidiazuron (TDZ). The optimal concentration of TDZ for obtaining the best ratios of bud proliferation and total flowering was 0.1 mg l⁻¹, while the highest number of vegetative shoots was obtained in medium supplemented with 1 mg l⁻¹ GA₃ and 0.1 mg l⁻¹ TDZ. The explant elongated abnormally in the presence of 10 mg l⁻¹ GA₃. Although a low concentration (1 mg l⁻¹) of NAA increased the bud proliferation ratio in the medium supplemented with 0.1 mg l⁻¹ TDZ and 1 mg l⁻¹ GA₃, a high concentration (5 mg l⁻¹) of NAA reduced the bud proliferation ratio and inhibited the flowering.     

Selection of atrazine-resistant plants by <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

The effects of atrazine on cotyledon cultures of Capsicum annuum (L.) cv. G₄ were investigated with a view of establishing a system for in vitro selection of resistant mutants. At low levels of herbicide produced little growth inhibition, some chlorophyll loss occurred associated with the production of albino shoots. At 20 mg l⁻¹ atrazine bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg l⁻¹). Differential morphogenetic responses were observed when the levels of sucrose (0.5–5%) were altered. Shoot regeneration was maximum in 2 sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3%–5%). However, lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of explants and permitted the selection of herbicide-resistant plants. Complete atrazine-resistant plantlets were obtained after rooting of regenerated green shoots on rooting medium containing 10 mg l⁻¹atrazine, 1.0 mg l⁻¹IAA and 0.5% sucrose. Leaf-segment assay of differentiated plants revealed that all regenerants were resistant to the atrazine. Reciprocal crosses between atrazine-resistant and -sensitive plants showed a non-Mendelian transmission of resistance trait.     

Determination of phytic acid by gas chromatography–mass spectroscopy: application to biological samples

A GC–MS method is reported for the determination of phytic acid based on purification by anion-exchange chromatography, enzymatic hydrolysis of phytic acid to myo-inositol and derivation to trimethylsilyl derivative, with scyllo-inositol as an internal standard. Analytical features of the method are: limit of detection 9 μg l⁻¹ phytic acid, linear working range 18–500 μg l⁻¹ phytic acid, and coefficient of variation 1.9%. The method has been successfully applied to a variety of biological samples: various rat organs (kidney, liver, brain and bone), human plasma and urine and kidney stones. A comparative study of sample treatments, including deproteization, lipid extraction and the presence of a chelator, is also reported. Phytic acid amounts found in rat organs ranged from 1.07 g kg⁻¹ for bone to 32.0 g kg⁻¹ for brain. Phytic acid in human plasma was of the order of 0.14 mg l⁻¹. In kidney stones, phytic acid was found in calcium containing stones.     

Simultaneous high-performance liquid chromatographic determination of quinupristin, dalfopristin and their main metabolites in human plasma

Quinupristin–dalfopristin (30:70, w/w) is a new streptogramin, which has been developed for intravenous use. A specific and sensitive HPLC method was developed to measure simultaneously quinupristin (RP 57669) and dalfopristin (RP 54476) and their main metabolites in human plasma. The metabolites measured by this method were RP 69012 (glutathione-conjugated) and RPR 100391 (cysteine-conjugated) from quinupristin and RP 12536 (natural pristinamycin IIA), from dalfopristin. Solid-phase extraction with disposable cartridges was combined with reversed-phase HPLC and fluorimetric detection for RP 57669, RP 69012 and RPR 100391 and UV detection for RP 54476 and RP 12536. The method provided good recovery and low limits of quantitation (0.025 mg l⁻¹ for RP 57669, RP 54476 and RP 12536, and of 0.010 mg l⁻¹ for RP 69012 and RPR 100391). The validated range of concentrations of the method was: 0.025–5000 mg l⁻¹ for RP 57669, RP 54476 and RP 12536 and 0.010–0.750 mg l⁻¹ for RP 69012 and RPR 100391.     

Determination of zearalenone and its metabolites α- and β-zearalenol in beer samples by high-performance liquid chromatography–tandem mass spectrometry

A fast, robust and sensitive LC–MS–MS method for the determination of zearalenone (ZON) and its metabolites α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in beer samples is described. Sample preparation was performed by direct RP-18 solid-phase extraction of undiluted beer samples followed by selective determination of analytes by LC–MS–MS applying an atmospheric pressure chemical ionization (APCI) interface. Using the negative ion mode limits of determination of 0.03–0.06 μg l⁻¹ beer and limits of quantification of 0.07–0.15 μg l⁻¹ beer were achieved, which was distinctly more sensitive than in the positive ion mode. Twenty-three beer samples from different countries, produced from different grains and under different brewing conditions, were investigated by this method, but only in one sample could β-ZOL and ZON be detected. Independently of the type of beer, relative standard deviations between 2.1% and 3.3%, a linear working range of 0.15 μg l⁻¹ to 500 μg l⁻¹ beer and recovery rates around 100% could be achieved when zearalanone (ZAN) was used as internal standard.     

Quantification of perifosine, an alkylphosphocholine anti-tumour agent, in plasma by pneumatically assisted electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

An HPLC assay with tandem mass spectrometric detection in the positive-ion Turbo-Ion-Spray^™ (TISP) mode for the fast and sensitive determination of perifosine ((I), D-21266) in human plasma was developed, utilising the structural analogue, miltefosine ((II), D-18506), as internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250-μl plasma aliquots, at pH 6.5, allowed a reliable quantification of perifosine down to 4 ng/ml. Injection of 200 μl of plasma extracts onto a 100×3 mm normal-phase analytical column at a flow-rate of 0.5 ml/min provided retention-times of 2.4 and 2.1 min for perifosine (I) and the internal standard (II), respectively. The standard curves were linear from 4 to 2000 ng/ml using weighted linear regression analysis (1/Y²). The inter-assay and intra-assay accuracies for the calibration standards were within +0.9% and −0.2%, exhibiting precisions (C.V.) of ±6.5 and ±7.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.     

Shoot regeneration,re-flowering and post flowering survival in bamboo inflorescence culture

In vitro grown inflorescences of Bambusa edulis were used to investigate the process of vegetative shoot growth in detail. The findings revealed that auxins and ACC could be significant growth regulators in this process. Overall, auxins [NAA, indolebutyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)] induced inflorescences to grow vegetative shoots. However, the efficiency of shoot regeneration varied. A greater percentage (27.3–34.5) of inflorescences in the 5 mg l⁻¹ NAA, 10 mg l⁻¹ NAA, and 1 mg l⁻¹ 2,4-D treatments formed more vegetative shoots than those exposed to other treatments. IBA promoted shoot regeneration less effectively than NAA and 2,4-D. Fifty percent of regenerated vegetative shoots flowered after 2 months when the medium was supplemented with 5 mg l⁻¹ NAA. All shoots that received 1 mg l⁻¹ 1-amino-cyclopropane-1-carboxylic acid (ACC) flowered in 5 mg l⁻¹ NAA medium. Rooted plantlets were used to examine their survival following in vitro flowering. All plantlets with vegetative shoots, even those with inflorescences, survived and grew.     

Production of a self-activating CBM-factor X fusion protein in a stable transformed Sf9 insect cell line using high cell density perfusion culture

Factor Xa is a serine protease, whose high selectivity can be used to cleave protein tags from recombinant proteins. A fusion protein comprised of a self-activating form of factor X linked to a cellulose-binding module, saCBMFX, was produced in a stable transformed Sf9 insect cell line. The activity of the insect cell produced saCBMFX was higher than the equivalent mammalian cell produced material. A 1.5 l batch fermentation reached a maximum cell concentration of 1.6 × 10⁷ cells ml⁻¹ and a final saCBMFX concentration of 4 mg l⁻¹. The production of saCBMFX by this cell line was also analyzed in a 1.5 l perfusion system using an ultrasonic filter as a cell-retention device for flow rates up to 3.5 l day⁻¹. The cell-retention efficiency of an air backflush mode of acoustic filter operation was greater than 95% and eliminated the need to pump the relatively shear sensitive insect cells. In the perfusion system over 4 × 10⁷ Sf9 cells ml⁻¹ were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 × 10⁷ cells ml⁻¹ the saCBMFX production rate was doubled to 6 mg l⁻¹ day⁻¹. The saCBMFX volumetric productivity of the perfusion system was higher than the batch fermentations (0.6 mg l⁻¹ day⁻¹) by an order of magnitude.     

Inhibitory effect of 4-methylpyrazole on antipyrine clearance in rats.

Pyrazole and 4-methylpyrazole (4-MP) are potent, effective inhibitors of alcohol dehydrogenase. Pyrazole and its derivatives also have been shown to affect the cytochrome P-450 dependent monooxygenase system. This study was performed to investigate the effect of 4-MP on the disposition kinetics of antipyrine (AP). Groups of male Fisher 344 rats were given an ip injection of 4-MP (100 mg/kg) or 4-MP HCl (equivalent to 4-MP 100 mg/kg) or an equivalent volume of saline. AP (20 mg/kg) was injected intravenously via the jugular vein catheter 30 minutes later. Blood samples were collected upto 24 hours and assayed by HPLC. 4-MP pretreatment significantly decreased AP clearance from 0.490 +/- 0.032 to 0.095 +/- 0.014 (4-MP HCl) and 0.076 +/- 0.008 (4-MP) L/hr.kg (p less than 0.01). The volume of distribution of AP decreased from 0.82 +/- 0.07 to 0.65 +/- 0.06 (4-MP HCl) and 0.56 +/- 0.04 (4-MP) L/kg (p less than 0.05). Mean residence time increased from 1.68 +/- 0.09 to 6.91 +/- 0.58 (4-MP HCl) and 7.39 +/- 0.56 (4-MP) hr (p less than 0.01). These results demonstrate a significant inhibitory effect of 4-MP on the cytochrome P-450 isozyme(s) which is responsible for AP metabolism in intact animals.     

Determination of 5-S-cysteinyldopa in plasma and urine using a fully automated solid-phase extraction–high-performance liquid chromatographic method for an improvement of specificity and sensitivity of this prognostic marker of malignant melanoma

5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S- -cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l⁻¹ and 25 to 5000 μg l⁻¹ for plasma and urine samples, respectively, a limit of detection of 0.17 μg l⁻¹, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons.     

Synthesis of -Dopa by Escherichia intermedia cells immobilized in a carrageenan gel

Escherichia intermedia cells were immobilized by entrapment in a carrageenan gel and used for -DOPA synthesis from catechol, pyruvate, and ammonia. A preparation containing 75 mg of cell per gram of gel retained 60–65% of its original activity. The effect of substrate concentrations on the initial rate of -DOPA synthesis was very similar for free and immobilized cells, and substrate inhibition was observed for the three substrates. In batch reactors, up to 7.8 g l⁻¹ of -DOPA was obtained in 20 h (productivity 0.39 g l⁻¹ h⁻¹). Cells immobilized in a carrageenan gel showed higher -DOPA synthesis, in both initial rates conditions and batch reactors, than cells immobilized in a polyacrylamide gel.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of the heavy metal tolerant and hyperaccumulator <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> (Brassicaceae)

Plants were regenerated from root explants of Arabidopsis halleri (L.) O’Kane and Al-Shehbaz via a three-step procedure callus induction, induction of somatic embryos and shoot development. Callus was induced from root segments, leaflets and petiole segments after incubation for 2 weeks in Murashige and Skoog medium (MS) supplemented with 0.5 mg/l⁻¹ (2.26 μM) 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.05 mg/l⁻¹ (0.23 μM) kinetin. Only calli developed from root segments continued to grow when transferred to a regeneration medium containing 2.0 mg/l⁻¹ (9.8 μM) 6-γ-γ-(dimethylallylamino)-purine (2ip) and 0.05 mg/l⁻¹ (2.68 μM) α-naphthalenacetic acid (NAA) and eventually 40 of them developed embryogenic structures. On the same medium 38 of these calli regenerated shoots. Rooting was achieved for 50 of the shoots subcultured in MS medium without hormones. The regeneration ability of callus derived from root cuttings, observed in this study, makes this technique useful for genetic transformation experiments and in vitro culture studies.     

Rapid regeneration of <Emphasis Type="Italic">Phaseolus angustissimus</Emphasis> and <Emphasis Type="Italic">P. vulgaris</Emphasis> from very young zygotic embryos

A rapid regeneration protocol for proembryos of Phaseolus angustissimus as young as 1 day after pollination (DAP) involving pod culture for 1 week followed by embryo culture for 2 weeks and embryo germination for 1 or 2 weeks is provided. Optimization of the media was conducted with pods collected 3 DAP. The best pod culture medium was composed of basal medium [(Phillips and Collins 1979) salts with (Geerts et al. 2001) vitamins], 1000 mg l⁻¹ glutamine, 1000 mg l⁻¹ casein hydrolysate, 3% sucrose and 0.5% agar. Embryo culture medium consisted of basal medium with 500 mg l⁻¹ glutamine, 250 mg l⁻¹ casein hydrolysate, 1.9 μM ABA, 3% sucrose and 0.5% bacto-agar. Embryos developed into plantlets on germination medium containing basal medium with 0.25 μM BA, 3% sucrose and 0.7% bacto-agar. Fertile, normal plants were recovered from direct embryogenesis and from micrografted embryo-derived shoots. Embryos obtained from pods collected 3 DAP regenerated plantlets at a rate of 29.3%, while embryos from pods collected 2 DAP and 1 DAP regenerated at rates of 20.2 and 4%, respectively. A second accession of P. angustissimusregenerated at a rate of 26.2%. Using this 5-week protocol for P. vulgaris resulted in a plantlet regeneration rate of 12.5%.     

Efficient Plant Regeneration System From Leaf Discs of Zonal (Pelargonium x hortorum) and two scented (P. capitatum and P. graveolens) Geraniums

An efficient and rapid plant regeneration system was established for zonal and scented geraniums using leaf discs as explants. Several explants, medium and culture conditions were studied to optimize shoot induction. Leaf discs taken from 4–5 weeks old in vitro grown plants, whatever the genotype, were more effective for shoot regeneration than those taken from greenhouse grown plants. Darkness proved to be a stimulating factor for shoot regeneration and the combination between NAA and two cytokinins gave the best results. Direct shoot regeneration (100%) was obtained from leaf discs of P. capitatum on half-strength MS medium supplemented with 0.5 mg l⁻¹ NAA in combination with 1 mg l⁻¹ of BAP and zeatin in darkness (11.4 shoots per explant). In the same medium and culture conditions, all P. graveolens leaf discs also exhibited direct shoot regeneration (7.3 shoots per explant). For P. x hortorum, 100% of leaf discs underwent shoot regeneration on a MS medium supplemented with 0.2 mg l⁻¹ NAA in combination with 0.5 mg l⁻¹ of BAP and zeatin in darkness (8.8 shoots per explant) or under low light conditions with 0.2 mg l⁻¹ NAA and 1 mg l⁻¹ of BAP and zeatin (7.5 shoots per explant). For this species, the best results for shoot elongation were obtained on half-strength MS medium gelled with Phytagel 0.3% (v/v). Whatever the genotype, all shoots rooted readily when transferred to diluted MS medium (MS/2) containing 1 mg l⁻¹ IAA. Acclimatized plants grew normally and flowered in greenhouse conditions. Flow cytometry analysis made on leaves of acclimatized plants revealed that all the scented geranium plants are similar to mother plants while 71% of P. x hortorum plants which showed strong growth were tetraploid.     

Quantification of metronidazole in small-volume biological samples using narrow-bore high-performance liquid chromatography

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of metronidazole in small volumes of rat plasma, gastric aspirate and gastric tissue. The extraction procedure involves liquid–liquid extraction and a protein precipitation step. A microbore Hypersil ODS 3 μm (150×2.1 mm I.D.) column was used with a mobile phase consisting of acetonitrile–aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90). The column temperature was at 25°C and the detection was by UV absorbance at 317 nm. The limit of detection was 0.015 μg ml⁻¹ for gastric juice aspirate and plasma and 0.010 μg g⁻¹ for gastric tissue (equivalent to 0.75 ng on-column). The method was linear up to a concentration of 200 μg ml⁻¹ for plasma and gastric juice aspirate and up to 40 μg g⁻¹ for tissue, with inter- and intra-day relative standard deviations less than 14%. The measured recovery was at least 78% in all sample matrices. The method proved robust and reliable when applied to the measurement of metronidazole in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.     

Newly Detected Specific Hydrogenation of the Conjugated Double Bond of Unsaturated Alkaloid Lactones by Aspergillus sp.

A new isolate of Aspergillus sp. hydrogenated the γ,δ-double bond of securinine (143 mg l⁻¹) to give 14,15-dihydrosecurinine at over 98% (w/w) yield after 8 h. It also hydrogenated the C11(13) double bond of 3-hydroxy-1(10),3,11(13)-guaiatriene-12,6-olide-2-one (HGT) (200 mg l⁻¹) to give 3-hydroxy-1(10),3-guaiadiene-12,6-olide-2-one with over 98% (w/w) conversion after 24 h.     

Callus induction and high-frequency plant regeneration from hypocotyl and cotyledon explants of Arctium lappa L.

Summary This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l^&#x2212;1 2,4-dichlorophenoxyacetic acid and 0.5&#x2013;2.0 mg l^&#x2212;1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0&#x2013;2.0 mg l^&#x2212;1 &#x03B1;-naphthaleneacetic acid (NAA) and 0.5&#x2013;2.0 mg l^&#x2212;1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l^&#x2212;1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl^&#x2212;1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.     

Determination of quinolones in plasma samples by capillary electrophoresis using solid-phase extraction

The potential of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) have been investigated for the separation and quantitative determination of 10 quinolone antibiotics. The influence of different conditions, such as the buffer and pH of the electrolyte, the surfactant and the ion-pairing agents added to the electrolyte and the organic modifier were studied. A buffer consisting of 40 mM sodium tetraborate at pH 8.1 containing 10% (v/v) methanol was found to be a highly efficient electrophoretic system for separating lomefloxacin, enoxacin, norfloxacin, pipemidic acid, ofloxacin, piromidic acid, flumequine, oxolinic acid, cinoxacin and nalidixic acid. A solid-phase extraction method to remove the sample matrix (pig plasma samples) was developed on a C₁₈ cartridge using a mixture of methanol–water (70:30, v/v). The method is specific and reproducible and mean recoveries were in the range 94.0±4.2% and 123.3±4.1% for pig plasma samples over the range used. A linear relationship between concentration and peak area for each compound in pig plasma samples was obtained in the concentration range 5–20 mg l⁻¹ and detection limits were between 1.1 and 2.4 mg l⁻¹.