Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

Direct injection high-performance liquid chromatographic method with electrochemical detection for the determination of ethanol and methanol in plasma using an alcohol oxidase reactor

A highly sensitive reversed-phase high-performance liquid chromatographic assay for ethanol and methanol in plasma, using a post-column enzymic reactor with electrochemical detection, has been developed. The alcohols, separated on the column, were converted by immobilized alcohol oxidase into their respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, detected via oxidation at a platinum electrode. As the chromatographic column, two glass cartridges (150 mm × 3 mm I.D.) in series, packed with 10-μm HEMA-S 1000^® packing, were used. Alcohol oxidase from Candida boidinii was immobilized onto HEMA-BIO 1000 VS-L (10 μm), packed in a 30 mm × 3 mm I.D. glass cartridge. The reaction product, hydrogen peroxide, was detected with an amperometric detector with a platinum electrode, operated at +500 mV vs. an Ag/AgCl reference electrode. A 20-μl volume of ten-fold diluted plasma was injected without any pre-treatment. Under the described conditions, methanol and ethanol were well resolved from each other and from the “front” of the chromatogram. The limit of detection was ca. 2.5 nmol for ethanol and 0.6 nmol for methanol in plasma, at a signal-to-noise ratio of 3. Excellent linearity was observed for ethanol, in the range 0.125–4 μg injected (r = 0.9999). In contrast, the response for methanol was markedly non-linear above 500 μg injected, presumably owing to progressive saturation of the reactor. The precision and accuracy of the assay were satisfactory, as was the reactor life (one month).     

Sulfalene concentrations in plasma and blood cells of Plasmodium falciparum malaria cases after treatment with metakelfin using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method using acetonitrile–methanol–1 M perchloric acid–water (25:9:0.8:95, v/v/v) at a flow-rate of 1.0 ml min⁻¹ on LiChrospher 100 RP 18 column (250×4 mm; 5 μm) with UV (254 nm) detection has been developed for the determination of sulfalene in plasma and blood cells after oral administration of the antimalarial drug metakelfin. Calibration curves were linear in the range 0.5–100 μg ml⁻¹. The limit of quantification was 50 ng ml⁻¹. Within-day and day-to-day coefficients of variation averaged 3.84 and 5.31%, respectively. Mean extraction recoveries of sulfalene from plasma and blood cells were 87.21 and 84.65%, respectively. Mean concentrations of sulfalene in plasma of P. falciparum cases on days 2, 7 and 15 were 44.58, 14.90 and 1.70 μg ml⁻¹, respectively; in blood cells concentrations of sulfalene were 7.77, 3.25 and 0.75 μg ml⁻¹, respectively, after oral treatment with two tablets (1000 mg) of metakelfin. Significant difference was recorded on day 2 for sulfalene concentration in blood cells of healthy and P. falciparum cases (t=9.49; P<0.001).     

Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin

A combination of rational design based on mimicking natural protein–carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, ¹H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 μmol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitatively eluted with 0.5 M α- -methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention times of saccharidic solutes showed that significant retardation was observed for α- -mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of α- -fucose. Interestingly, α- -fucose and α- -mannose share an identical configuration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatchard plots from partition equilibrium studies on the interaction of glucose oxidase and the p-nitrophenyl-glycosides of -mannose, -glucose, -fucose and -galactose with immobilised 18/18 establish that the affinity constants (K_AX) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3×10⁵ M⁻¹, 1.9×10⁴ M⁻¹ and 1.2×10⁴ M⁻¹ respectively. ¹H-NMR studies on the interaction of α- -methyl-mannoside with ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position. Molecular modelling suggests the formation of four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of α- -methyl-mannoside and the bridging and ring nitrogen atoms of the triazine scaffold, with aromatic stacking of a second ligand against the carbohydrate face. The greater specificity of ligand 18/18 for mannose and glucose than for galactose parallels that exhibited by concanavalin A.     

Determination of sodium artesunate in plasma using ion-pairing high-performance liquid chromatography

A chromatographic method is described for the determination of sodium artesunate in plasma. This includes cetyltrimethylammonium bromide as a cationic pairing ion in a reversed-phase system using an octadecylsilica 100×4.6 mm I.D. 3 μm analytical column with a mobile phase of acetonitrile/acetate buffer at pH7. Column switching incorporating a 5 μm octadecylsilica 100×4.6 mm I.D. precolumn is used in addition to off-line solid-phase extraction for pretreatment of plasma samples in order to eliminate interference from endogenous components. Detection is by post-column derivatisation with 1.0 M methanolic KOH followed by UV detection at 289 nm. Calibration is linear over the range 100–1600 ng ml⁻¹ and the limit of detection is estimated as 20 ng ml⁻¹. Illustrative results are shown of the artesunate plasma levels determined by the proposed method following the administration of artesunate as tablets and as suppositories to healthy volunteers.     

Fosfomycin determination in serum by capillary zone electrophoresis with indirect ultraviolet detection

Capillary zone electrophoresis with indirect ultraviolet detection was used for the determination of fosfomycin in serum. Running buffer consisted of a mixture of 200 mM sodium borate with 10 mM phenylphosphonic acid used as ultraviolet absorbing background electrolyte. Relationships between the pH of the buffer and the efficiency of the separation (migration times and selectivities) or the sensitivity of detection were investigated. The method was then validated over a 10–100 μg ml⁻¹ concentration range to be applied to further therapeutic drug monitoring. The choice of ethylphosphonic acid as internal standard is discussed. The specificity and the linearity of the technique are demonstrated. The inter-day precision was satisfactory with a relative standard deviation of less than 2%. Accuracy was calculated with a standard error near 0.5 and 18% for 100 and 10 μg ml⁻¹, respectively.     

Dinuclear complexes of transition metals containing carbonate ligands. VIII. Kinetics and mechanism of carbon dioxide uptake by the tri-μ-hydroxo-bis (1,5-diamino-3-aza-pentane)cobalt(III) ion in weakly basic aqueous carbonate solution

The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO₃²⁻35 mM and at an ionic strength of 0.1 M (LiClO₄). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M⁻¹ s⁻¹ with ΔH≠ = (13.2±0.7) kcal mol⁻¹ and ΔS≠ = (−15.1 ± 0.7) cal deg⁻¹ mol⁻¹. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes.     

Development and validation of a sensitive solid-phase extraction and high-performance liquid chromatography assay for the novel antitumour agent CT2584 in human plasma

A HPLC assay and solid-phase extraction technique from human plasma has been developed and validated for the novel anticancer agent CT2584, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, which has recently completed a phase I trial at the Christie Hospital, Manchester under the auspices of the CRC phase I/II committee. Following addition of CT2576, 1-(11-octylamino-10-hydroxylundecyl)-3,7-dimethylxanthine, as internal standard, a solid-phase extraction cartridge (100 mg cyanopropyl) was used to isolate the drug CT2584 from human plasma. Analysis was performed by reversed-phase chromatography. CT2576 was used as internal standard at a concentration of 4 μg ml⁻¹ for the quantification of CT2584 from plasma for the duration of this work. The lower limit of quantification for the drug CT2584 in buffer using this assay was found to be 0.0122 μM (0.008 μg ml⁻¹) and 0.048 μM (0.027 μg ml⁻¹) when extracted from human plasma.     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cm×4.6 mm I.D.) column, with mobile phase of acetonitrile–ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min⁻¹. Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05–10.0 μg ml⁻¹ in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 μg ml⁻¹ to 2.9% and 3.3% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 μg ml⁻¹ to 7.6% and 11.5% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4±1.77% and 76.1±7.26% for AQ4. The limit of detection was 50 ng ml⁻¹ with a 100 μl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

Ammonium excretion by a mutant of the nitrogen-fixing cyanobacterium Anabaena siamensis

Anabaena siamensis isolated from rice fields in Thailand is a fast growing cyanobacterium with a high nitrogen-fixing activity. Mutant strains resistant to the l-glutamate analogue, l-methionine sulfoximine (MSX) were isolated by ethyl methanesulfonate mutagenesis. A stable mutant named A. siamensis SS1, which released ammonium to the medium, was studied further. In batch cultures the rate of ammonium production peaked at the early log phase and gradually decreased until the 4th day of growth when the cultures reached a density of 90 μg chl ml⁻¹. To obtain constant release of ammonium by SS1, continuous culture experiments were performed at a cell density of 5 μg chl ml⁻¹ and the following results were obtained: (1) growth rate as the parent (μ:0·123 h⁻¹) in the presence and absence of 500 μm MSX; (2) 48% GS transferase activity when compared with the parent; (3) ammonium excretion at a rate of 8 μmol (mg chl)⁻¹ h⁻¹ as measured up to 20 generations (120 h); (4) depressed nitrogenase activity; and (5) 30% higher nitrogenase activity than that of the parent. SS1 immobilized in alginate beads (5 μg chl ml⁻¹) exhibited values of glutamine synthetase and nitrogenase activity similar to those of free cells. However, ammonium excretion at the rate of 11·61 μmol (mg chl)⁻¹ h⁻¹ was obtained only up to 20 h after loading in bioreactors, due to the fast growth of SS1 as also occurred in batch cultures.     

Phosphine in soils, sludges, biogases and atmospheric implications—a review

This is a review of previously published and unpublished results of research into the occurrence of phosphine (PH₃) in the environment in the form of matrix bound phosphine in soils, aquatic sediments and sludges (range ng kg⁻¹to μg kg⁻¹), free phosphine in formed biogases (range ng m⁻³ to μg m⁻³) and in the atmosphere (range pg m⁻³ to ng m⁻³).The reviewed data support the hypothesis of the existence of a small gaseous link in the phosphorus cycle, which could become important over the long term.Matrix-bound phosphine in soils can be interpreted as a stationary state concentration of phosphine between production and consumption. This phosphine turnover within the soil may be important even if the stationary state concentration (matrix-bound phosphine) is small. Under such circumstances, a slow migration process of phosphine in the interstitial gas sphere of soils is possible. Such a process would influence the balance of phosphorus in agricultural and wetland soil.The detection of easily oxidizable phosphine as a ubiquitous trace gas in the atmosphere can be interpreted as the residue of an important turnover of phosphine between widely distributed emission sources and sinks such as soils and sediments. The atmosphere can carry gaseous phosphorus to remote places.     

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller–Hinton broth for pharmacokinetic/pharmacodynamic studies

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C₈ 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.     

Specific high-performance liquid chromatography assay for determination of rifabutin plasma concentration following Extrelut column extraction

A specific, precise and accurate assay for determination of rifabutin in human plasma using Extrelut column extraction was developed and validated. Rifabutin concentrations were calculated with a standard curve ranging from 5 to 800 ng ml⁻¹, using a split-curve approach. Chromatographic peaks were separated by means of a 5 μm Symmetry Shield RP8 using a KH₂PO₄ (0.05 M) buffer–acetonitrile mobile phase. Detection wavelength was set at 275 nm. Chromatography was carried out at room temperature (20–25°C). The limit of quantification was 5 ng ml⁻¹. The recovery was over 71%. The intra-day precision of the assay was 5, 7, and 1% while the inter-day precision was 11.2, 8.1, and 5.8% at concentrations of 30, 150 and 500 ng ml⁻¹, respectively. The accuracy ranged from 99 to 108%. Forty of the drugs most commonly administered to HIV-positive patients were found not to interfere with the assay. The assay has been used in a comparative study of rifabutin pharmacokinetics in HIV-positive patients with or without wasting syndrome.     

In vitro cytogenetic effects of Andrographis paniculata (kalmegh) on arsenic

In vitro effects of arsenic in human peripheral lymphocytes (HPL) at three different doses – 3.6×10⁻⁴, 1.4×10⁻³ and 0.72×10⁻³ μM for 24 h before harvesting on sister chromatid exchanges (SCE), Cell cycle proliferative index/replicative index (CCPI/RI), %M₁, %M₂ and %M₃, population doubling time (PDT) and average generation time (AGT) were examined. Andrographis paniculata (commonly referred to as ‘kalmegh’) has been used for centuries in traditional Indian and Chinese herbal medicine as a safe, natural folk remedy for assorted health concerns. In the present study, kalmegh (0.01 μg/7 ml culture media) was used along with the highest dose of arsenic; the results showed that arsenic induced increase in these genotoxic endpoints were fairly diminished by kalmegh. In addition, mutagenic in vitro effect of ethyl methanesulphonate (EMS) was used as a positive control in this study. It is thus concluded from this study that Andrographis has a protective role in arsenic toxicity.     

Quantification of BNP7787 (dimesna) and its metabolite mesna in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A sensitive and accurate assay was developed and validated to determine BNP7787 (dimesna), a new protector against cisplatin-induced toxicities, and its metabolite mesna in plasma and urine of patients. Both analytes were measured as mesna in deproteinized plasma or in urine diluted with mobile phase using high-performance liquid chromatography with an electrochemical detector provided with a wall-jet gold electrode. The assays for BNP7787 and mesna in deproteinized plasma were linear over the range of 1.6–500 μM and 0.63–320 μM, respectively. In plasma, the mean recovery of BNP7787 over the whole concentration range was 100.6% and of mesna 94.6%. The lower limits of quantitation (LLQs) of BNP7787 and mesna in deproteinized plasma were 1.6 μM and 0.63 μM, respectively. For both compounds the within- and between-day accuracy and precision of the assay was better than 12%. The assays for BNP7787 and mesna in urine were linear over the range of 0.8–1200 μM and 0.63–250 μM, respectively. In urine, the mean recovery of BNP7787 over the whole concentration range was 94.1% and of mesna 93.1%. The LLQ of BNP7787 in urine was 0.8 μM and of mesna 1.6 μM. The within- and between-day accuracy and precision of the assay for BNP7787 and mesna was lower than 15%. The stability of mesna in urine increased with an increasing concentration of mesna, lower temperature and addition of EDTA (1 g/l) and hydrochloric acid (0.2 M). BNP7787 in urine was stable for at least 24 h at temperatures in the range of −20°C up to 37°C and independent of the concentration. The developed assays are currently applied for samples of patients with solid tumors participating in a phase I trial of BNP7787 in combination with cisplatin.     

Inversion of sucrose with β- -fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β- -fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min⁻¹ g⁻¹. The inversion of sucrose with immobilized β- -fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Immobilization of -glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and -glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized -glucose oxidase membrane was 0.34 units cm⁻² and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized -glucose oxidase membrane was 1.6 × 10⁻³ mol l⁻¹ and that of free enzyme was 4.8 × 10⁻² mol l⁻¹. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized -glucose oxidase membrane. The enzyme electrode responded linearly to -glucose over the concentration 0–1000 mg dl⁻¹ within 10 s. When the enzyme electrode was applied to the determination of -glucose in human serum, within day precision (CV) was 1.29% for -glucose concentration with a mean value of 106.8 mg dl⁻¹. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized -glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of -glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Suspension culture of gametophytes of transgenic kelp in a photobioreactor

Transgenic Laminaria japonica gametophytes producing a recombinant tissue-type plasminogen activator (rtPA) protein, which is an effective third-generation thrombolytic agent for acute myocardial infarction (AMI), were cultured in an illuminated bubble column bioreactor. A maximum final dry cell weight of 1120 mg l⁻¹ was obtained in batch culture with an initial dry cell weight of 126 mg l⁻¹ and with aeration rate of 1.2 l air min⁻¹ l⁻¹ culture, nitrate at 1.5 mM and phosphate at 0.17 mM. The yield of rtPA was 56 μg g⁻¹ dry cell wt. This is the first report regarding cultivation of a transgenic macroalga in a bioreactor.Revisions requested 27 January 2005 and 14 April 2005; Revisions received 6 April 2005 and 17 May 2005     

l-Malic acid formation by immobilized Saccharomyces cerevisiae amplified for fumarase

The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric acid to l-malic acid at a rate of 65 mM g⁻¹ h⁻¹ in free cell experiments, and approximately 87% of the fumaric acid was converted to l-malic acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871 μm mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-μm diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.