Simultaneous determination of quinine and four metabolites in plasma and urine by high-performance liquid chromatography

The determination of quinine, (3S)-3-hydroxyquinine, 2′-quininone and (10R)- and (10S)-10,11-dihydroxydihydroquinine in plasma and urine samples is described. This is the first time the R and S configurations have been correctly assigned to the two metabolites of 10,11-dihydroxyquinine. One hundred microliter-plasma samples were protein precipitated with 200 μl cold methanol. Urine samples were 10–100× diluted and then directly injected into the HPLC. A reversed-phase liquid chromatography system with fluorescence detection and a Zorbax Eclipse XDB phenyl column and gradient elution was used. The within and between assay coefficients of variation of the method for quinine and its metabolites in plasma and urine was less than 13%. The lower limit of quantitation was in the range of 0.024–0.081 μM.     

Identification of quinine metabolites in urine after oral dosing in humans

A gas chromatographic–mass spectrometric method was used to separate quinine and its metabolites present in urine after oral dosing of 300 mg quinine in humans. The technique allowed the separation of quinine and ten metabolites. Four of these metabolites were definitely identified as 3-hydroxyquinine, 2′-quinone, O-desmethylquinine and 10,11-dihydroxydihydroquinine, by comparing their methane chemical ionization mass spectra with those of authentic standards prepared by organic synthesis. Six other metabolites are described for the first time in human urine. From their electron impact and chemical ionization mass spectra, we propose these compounds to be 3-hydroxy-2′-quinone, O-desmethyl-2′-quinone, O-desmethyl-3-hydroxyquinine, O-desmethyl-3-hydroxy-2′-quinone, 10,11-dihydroxydihydro-2′-quinone and 10,11-dihydroxydihydro-O-desmethylquinine. These secondary metabolites probably arose from further biotransformation of the four primary metabolites.     

Reverse micelle-based chemiluminescence system for quinine sulphate.

A new chemiluminescence (CL) method is proposed for the determination of quinine sulphate, which is based on the dichloromethane solvent extraction of the ion-pair complex of tetrachloroaurate (III) with quinine sulphate, and luminol CL detection in a reversed micellar medium formed by the cation surfactant cetyltrimethylammonium bromide in a dichloromethane-cyclohexane (3:7, v/v)-water (0.35 mol/L Na2CO3 buffer solution, pH 11.5). The ion-pair complex of tetrachloroaurate (III) with quinine sulphate produced an analytical CL signal when it entered the reversed micellar water pool. In optimum conditions, CL intensities are proportional to the concentrations of the studied drug over the range 0.015-10 microg/mL, with a detection limit of 1.5 ng/mL. The relative standard deviation (RSD) is 1.38% for 2.5 microg/mL quinine sulphate (n=11). The method has been applied to the determination of the studied drug in biological fluids, with satisfactory results.     

Liquid chromatographic determination of amodiaquine in human plasma

A normal-phase high-performance liquid chromatographic method using dichloromethane- methanol-1M perchloric acid (100:10:0.9, v/v/v) at a flow rate of 1.0 ml min(-1) on a LiChrospher Si column with UV (254 nm) detection has been developed for the determination of amodiaquine and its metabolites desethyl amodiaquine and bisdesethyl amodiaquine in plasma. The limit of quantification was 5 ng ml(-1). Mean within-day and day-to-day coefficients of variation (CV) were 4.10 and 6.27% for amodiaquine, 3.43 and 4.80% for desethyl amodiaquine and 3.53 and 5.23% for bisdesethyl amodiaquine, respectively. Mean extraction recovery of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine from plasma were 82.48, 74.50 and 69.65%, respectively. Chloroquine and its metabolite desethyl chloroquine, quinine, sulfadoxine and primaquine do not interfere in the detection of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine in plasma.     

Preservation of tomcat (Felis catus) semen in variable temperatures

The aim of our study was to estimate the viability of cat epididymal sperm in short time storage at +4 degrees C and in long term storage at -196 degrees C and to assess the percentage of live sperm in fresh semen using eosin/nigrosin staining compared to the flow cytometry method. The testes with epididymides were obtained after routine castration procedure. The sperm for further research were collected after flushing the epididymides using extender consist of: Tris 2.4 g, citric acid 1.4 g, glucose 0.8 g, 0.06% (w/v) Na-benzylpenicillin, 0.1% (w/v) streptomycin sulphate and distilled water. Half of each sample was equilibrated with the dilution and loaded in 0.25 ml plastic straws. The straws were placed on a rack in liquid nitrogen vapour at -120 degrees C for 10 min, plunged in liquid nitrogen for 10 min, replaced to marked goblets and loaded into canes for long term storage in liquid nitrogen at -196 degrees C. Sixty percent of motile spermatozoa was accomplished after thawing. However, the percentage of the sperm with intact acrosomes was decreased and the share of cells with midpiece and tail defects was increased. The storage of sperm flushed from epididymides at +4 degrees C for a short time and the usage of sperm during 2-3 days after collection seems to be better than cryopreservation. In our study, normospermia was present in 72.7 +/- 8.8% of fresh semen. The most common defect was the presence of distal droplets, imperfect heads or abnormal acrosomal outline. The motility of fresh sperm flushed from epididymides achieved 77.9 +/- 6.8%. The viability of sperm amounting to 52.5 +/- 13.8% was achieved on third day of conservation in the liquid extender. The percentage of viable sperm in fresh epididymal spermatozoa was 84.9 +/- 7.8%. Compared to these results, the percentage of live cells using SYBR-14/propidium iodide staining was insignificantly lower (82.2 +/- 8%). The live, non-apoptotic cells were 79.0 +/- 7.8%. The share of live, early-apoptotic spermatozoa and late-apoptotic spermatozoa was, respectively, 2 +/- 1.4% and 1.5 +/- 0.9%. The viability of sperm estimated by eosin/nigrosin staining was confirmed by the flow cytometry method. There was no statistical differences between the staining. The usage of apoptosis detection kit revealed, that the percentage of early-apoptotic and late-apoptotic cells was insignificant.     

Determination of pyronaridine in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies

A method is described for the determination of pyronaridine in plasma using high-performance liquid chromatography with fluorescence detection. The method involves liquid-liquid extraction with phosphate buffer (pH 6.0, 0.05 M) and diethyl ether-hexane (70:30%, v/v) and chromatographic separation on a C₁₈ column (Nucleosil, 250 × 4.6 mm I.D., 5 μm particle size) with acetonitrile-0.05 M phosphate buffer pH 6.0 (60:40%, v/v) as the mobile phase (1 ml/min) and detection by fluorescence (λ_ex = 267 nm, λ_em = 443 nm). The detector response is linear up to 1000 ng and the overall recoveries pyronaridine and quinine were 90.0 and 60.3%, respectively. The assay procedure was adequately sensitive to measure 10 ng/ml pyronaridine in plasma samples with acceptable precision (< 15% C.V.). The method was found to be suitable for use in clinical pharmacological studies.     

High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene–butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C₁₈ column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 μM, 4% at 227 nM and 9% at the lower limit of quantitation, 4.5 nM. In urine, the coefficient of variation was 11% at the lower concentration, 227 nM and was 3% at 56.8 μM. The between-assay coefficient of variation was 4% at the low concentration (5.1 nM) in plasma, 2% at 276.8 nM and 3% at 1.97 μM. In urine, the between assay coefficient of variation was 4% at 204.6 nM, 3% at 5.12 μM and 2% at 56.8 μM.     

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma by temperature-programmed packed capillary liquid chromatography with on-column focusing of large injection volumes

A miniaturized temperature-programmed packed capillary liquid chromatographic method with on-column large volume injection and UV detection for the simultaneous determination of the three selective serotonin reuptake inhibitors citalopram, fluoxetine, paroxetine and their metabolites in plasma is presented. An established reversed-phase C8 solid-phase extraction method was employed, and the separation was carried out on a 3.5-microm Kromasil C18 0.32x300 mm column with temperature-programming from 35 (3 min) to 100 degrees C (10 min) at 1.3 degrees C/min. The mobile phase consisted of acetonitrile-45 mM ammonium formate (pH 4.00) (25:75, v/v). The non-eluting sample focusing solvent composition acetonitrile-45 mM ammonium formate (pH 4.00) (3:97, v/v) allowed injection of 10 microl or more of the plasma extracts. The method was validated for the concentration range 0.05-5.0 microM, and the calibration curves were linear with coefficients of correlation >0.993. The limits of quantification for the antidepressants and their metabolites ranged from 0.05 to 0.26 microM. The within and between assay precision of relative peak height were in the range 2-22 and 2-15% relative standard deviation, respectively. The within and between assay recoveries were in the 61-99 and 54-92% range for the antidepressants, respectively, and between 52-102 and 51-102% for the metabolites.     

Alternative method for determination of pyrimethamine in plasma by high-performance liquid chromatography

A rapid, selective, sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative determination of pyrimethamine (PYR) in plasma is described. The procedure involved the two-step extraction of PYR and the internal standard, quinine (QN) with acetonitrile and dichloromethane at basic pH. Chromatographic separation consisted of the mobile phase (methanol-water containing 0.005 M octanesulfonic acid, 50:50, v/v) running through the column (Techopak-10 C₁₈) at a flow-rate of 1.6 ml/min. Detection was at UV wavelength of 240 nm. The mean recoveries of PYR and QN at a concentration range of 50 and 500 ng/ml were 98.9 and 89%, and 94.7 and 96% for PYR and QN. The within-day coefficients of variation were 2.1–5.1% for PYR and 5.9% for QN. The day-to-day coefficients of variation were 2.1–4.1% for PYR and 5% for QN. The minimum detectable concentrations for PYR and QN in plasma were 3 and 10 ng/ml. The method was found to be suitable for use in clinical pharmacokinetic study.     

Liquid chromatographic determination of irbesartan in human plasma

A simple and sensitive method was developed for determination of irbesartan by liquid chromatography with fluorescence detection. Irbesartan and losartan (I.S.) in human plasma were extracted using diethyl ether:dichloromethane (7:3, v/v) followed by back extraction with 0.05 M sodium hydroxide. Neutralized samples were analyzed using 0.01 M potassium dihydrogen phosphate buffer (containing 0.07% triethylamine as peak modifier, pH was adjusted with orthophosphoric acid to pH 3.0) and acetonitrile (66:34, v/v). Chromatographic separation was achieved on an ODS-C-18 column (100 mm x 4.6 mm i.d., particle size 5 microm) using isocratic elution (at flow rate 1.25 ml/min). The peak was detected using a fluorescence detector set at Ex 259 nm and Em 385 nm, and the total time for a chromatographic separation was approximately 13 min. The validated quantitation ranges of this method were 15-4000 ng/ml with coefficients of variation between 0.75 and 12.53%. Mean recoveries were 73.3-77.1% with coefficients of variation of 3.7-6.3%. The between- and within-batch precision were 0.4-2.2% and 0.9-6.2%, respectively. The between- and within-batch relative errors (bias) were (-5.5) to 0.9% and (-0.6) to 6.9%, respectively. Stability of irbesartan in plasma was >89%, with no evidence of degradation during sample processing and 60 days storage in a deep freezer at -70 degrees C. This validated method is sensitive and simple with between-batch precision of <3% and can be used for pharmacokinetic studies.     

Chiral resolution of some antimalarial agents by sub- and supercritical fluid chromatography on an (S)-naphthylurea stationary phase

The behavior of mefloquine, halofantrine, enpiroline, quinine, quinidine, chloroquine and primaquine is studied by subcritical fluid chromatography on a (S)-naphthylurea column (250 mm × 4.6 mm ID) with a subcritical mobile phase composed of carbon dioxide, methanol and triethylamine (flow rate of 3 ml/min). Except for primaquine and chloroquine, each enantiomer was separated at a temperature between 40 and 60°C, and at a pressure below 15 MPa. A 98/2, v/v CO₂/methanol 0.1% triethylamine mixture allowed the separation of halofantrine enantiomers while the enantiomers of the more polar metabolite (N-desbutylhalofantrine) were separated with a 80–20 v/v mixture as used for mefloquine, enpiroline, quinine and quinidine. The influence of temperature, pressure and of the nature of the mobile phase is discussed. © 1993 Wiley-Liss, Inc.     

High-performance liquid chromatographic determination of diazepam in plasma of children with severe malaria

A sensitive, selective and reproducible reversed-phase HPLC method with ultraviolet detection was developed for the quantification of diazepam in small plasma samples from children with severe malaria. The method involves plasma deproteinization with acetonitrile, followed by liquid–liquid extraction with ethyl acetate–n-hexane. Diazepam was eluted at ambient temperatures from a reversed-phase C₁₈ column with an acidic (pH 3.5) aqueous mobile phase (10 mM KH₂PO₄–acetonitrile, 69:31, v/v). Calibration curves in spiked plasma were linear from 10 to 200 ng (r²≥0.99). The limit of detection was 5.0 ng/ml, and relative recoveries at 25 and 180 ng were >87%. Intra- and inter-assay relative standard deviations were <15%. There was no interference from drugs commonly administered to children with severe malaria (phenobarbitone, phenytoin, chloroquine, quinine, sulfadoxine, pyrimethamine, halofantrine, cycloguanil, chlorcycloguanil, acetaminophen and salicylate). This method has been used for monitoring plasma diazepam concentrations in children with seizures associated with severe malaria.     

Cryopreservation of shoot primordia cultures of melon using a slow prefreezing procedure

Tissue-cultured shoot primordia of melon (Cucumis melo L. cv. prince melon) were successfully cryopreserved in liquid nitrogen (LN) using a slow prefreezing method. The highest survival and recovery were obtained with the following procedure. Three week-old shoot primordia clumps were dissected into pieces of 2-3 mm of diameter and precultured in standard medium for 3 days. They were directly soaked in CSP1 cryoprotective solution (10%w/v sucrose, 10%w/v dimethylsulfoxide and 5%w/v glycerol) and incubated at room temperature for 30 min. Samples were ice-inoculated at -8 °C and cooled at a rate of between 0.3 and 1 °C min⁻¹ with a programmable freezer to -30 °C for prefreezing. They were then plunged into LN for storage. After rapid thawing in 40 °C water, the cryoprotective solution was slowly diluted 5 fold in a dropwise manner with 3% sucrose and the shoot primordia were transferred onto regeneration medium. Under optimal conditions, more than 80% of cryopreserved shoot primordia were viable and 50 to 80% regenerated shoots after one month of reculture. Cryopreserved shoot primordia could be used both for reproducing a shoot primordia culture and for regenerating plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cryopreservation of a water-bloom forming cyanobacterium, Microcystis aeruginosa f. aeruginosa

A method for the Cryopreservation of Microcystis aeruginosa f. aeruginosa is described. For the five strains tested, dimethyl sulfoxide (DMSO) (3% v/v) was the only effective cryoprotectant for freezing to, and thawing from -196°C and allowed the successful recovery (>50%) of all the strains. The viability of frozen material was independent of the period of storage in liquid nitrogen. The strain NIES-44 (National Institute for Environmental Studies) had a recovery level of greater than 90% at 3–10% (v/v) DMSO in both two step and rapid cooling methods. The other three strains, NIES-87, 88 and 89 had greater than 60% of viability after freeze/thawing in presence of both 3% and 5% DMSO concentrations. On the other hand, the strain NIES-90 showed approximately 50% of viability in only 3% DMSO solution after two step cooling to and thawing from -196°C. This strain was damaged by greater than 4% DMSO and by rapid cooling to -196°C. It was found that cold shock injury and the cytotoxicity of DMSO were different at a strain level.     

Stability studies of selected doping agents in urine: caffeine

The stability of caffeine in urine samples has been studied. A high-performance liquid chromatography (HPLC) method for the quantification of caffeine in urine samples was validated for that purpose. The method consists of a liquid-liquid extraction at alkaline pH with chloroform-2-propanol (9:1, v/v) with a salting out effect. 7-Ethyltheophylline was used as internal standard (ISTD). Analyses were performed with an Ultrasphere ODS C18 column using water/acetonitrile (90:10, v/v) as a mobile phase at a flow rate of 1 ml/min. Ultraviolet absorption at 280 nm was monitored. Extraction recoveries for caffeine and 7-ethyltheophylline were 81.4+/-6.0 and 87.3+/-5.7%, respectively. The calibration curves were demonstrated to be linear in the working range of 6-30 microg/ml (r2>0.990). The limit of detection and the limit of quantitation were estimated as 0.7 and 2.0 microg/ml, respectively. Precisions in the range of 1.5-9.2 and 4.1-5.8% were obtained in intra- and inter-assay studies, respectively, using control samples containing 10, 14 and 26 microg/ml of caffeine. Accuracies ranging from 2.9 to 7.4% for intra-assay experiments, and from 3.9 to 5.4% in inter-assay studies were obtained. Stability of caffeine in urine samples was evaluated after long- and short-term storage at different temperature conditions. The batches of spiked urine were submitted to sterilization by filtration. No adsorption of the analyte on filters was observed. Before starting stability studies, batches of reference materials were tested for homogeneity. For long-term stability testing, caffeine concentration in freeze-dried urine stored at 4 degrees C and in liquid urine samples stored at 4, -20, -40 and -80 degrees C was determined at several time intervals for 18 months. For short-term stability testing, caffeine concentration was evaluated in liquid urine stored at 37 degrees C for 7 days. The effect of repeated freezing (at -20 degrees C) and thawing was also studied for up to three cycles. The stability of caffeine was also evaluated in non-sterile samples stored at -20 degrees C for 18 months. No significant loss of the compound was observed at any of the investigated conditions.     

Long-Term Preservation of Fungus Cultures with Liquid Nitrogen Refrigeration

A preservation technique was tested on 162 strains of culturally fastidious fungi sensitive to lyophilization, representing five classes. The results indicated that liquid nitrogen storage of frozen specimens may be used as an alternative to lyophilization for long-term preservation of stock cultures of fungi. The fungus was frozen in 10% (v/v) glycerol-water menstruum in heat-sealed ampoules. The cooling from ambient temperatures to -35 C was controlled at a rate of approximately 1 C per minute. Further cooling to the storage temperature of -165 to -196 C was uncontrolled and took place at an accelerated rate. Frozen ampoules were thawed in a water bath at 38 to 40 C. Viable and unmutated cultures were developed from reactivated specimens after storage for as long as 5 years.     

Storage of pathogenic leptospires in liquid nitrogen

The virulence and viability of various serovars of Leptospira interrogans were successfully preserved by storage in liquid nitrogen. Dimethyl sulphoxide at a final concentration of 2.5% (v/v) was added as cryoprotectant to a culture of leptospires grown in Ellinghausen-McCullough-Johnson-Harris medium. Ampoules were cooled at a controlled rate of 1 degree-3 degrees C/min to -70 degrees C, then transferred to the liquid phase of a liquid nitrogen storage unit. Glycerol was discounted as a cryoprotectant as it was found to be approximately 10 times more toxic than dimethyl sulphoxide to four of five serovars used in this study. The viability of nine strains has so far been observed over a period of 8-22 months storage in liquid nitrogen and full viability of all strains has been preserved over this period. Virulence of strains of serovars pomona and hardjo was well preserved, as demonstrated by challenge tests in guinea pigs and domestic pigs.     

Liquid chromatographic-tandem mass spectrometric method for the quantitation of sildenafil in human plasma

A method to determine sildenafil in human plasma involving liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Sildenafil and the internal standard (I.S.), diazepam, are extracted from human plasma with ether-dichloromethane (3:2, v/v) at basic pH and analyzed by reversed-phase high-performance liquid chromatography (HPLC) using methanol-10mM ammonium acetate pH 7.0 (85:15, v/v) as the mobile phase. Detection by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode was linear over the concentration range 0.125-40.0 ng/ml. Intra- and inter-day precision of the assay at four concentrations within this range were 2.5-8.0%. The method was used to evaluate plasma concentration-time profiles in healthy volunteers given an oral dose of 20mg sildenafil as a combination tablet also containing apomorphine.     

Isolation and characterization of a mildly thermophilic nonsulfur purple bacterium containing bacteriochlorophyll b

A method for transformation of whole Bacillus amyloliquefaciens cells by electroporation was developed. The procedure is as efficient as the protoplast transformation method, resulting in up to 10(5) transformants/micrograms plasmid DNA, but requires less effort and time. Cells for electroporation were grown to late exponential phase in a rich medium supplemented with 0.25 M sucrose, washed with and resuspended in 0.25 M sucrose, 1 mM HEPES, 1 mM MgCl2, 10% (v/v) glycerol, pH 7.0, at 3-5 x 10(10) cells/ml for storage at -80 degrees C. The highest transformation frequency was obtained at 7.5 kV/cm with a 25 microF capacitor. The transformation efficiency increased linearly with DNA concentration at least over the range 10 ng-12.5 micrograms/ml. Transformations with ligated DNA and of industrial strains were also successful. In addition, B. subtilis cells treated as above could be transformed by electroporation, resulting in 10(4) transformants/micrograms DNA at 12.5 kV/cm.     

Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken

A high-performance liquid chromatography (HPLC) method with UV detection was developed for the simultaneous determination of arsanilic acid, roxarsone, nitarsone, and carbarsone in the feeds of swine and chicken. Feed samples were extracted with methanol/1% acetic acid (90:10, v/v) in an ultrasonic bath and the protein was precipitated with 2% Cu(2)SO(4). The samples were further purified by solid phase extraction (SPE) on SAX cartridges. Separation was performed on a Zorbax SB-Aq C18 HPLC column using an isocratic procedure with methanol and 1% acetic acid (3:97, v/v) at a flow-rate of 0.7 mL min(-1), and the UV detector was set at a wavelength of 260 nm. The recoveries of organoarsenic compounds spiked at levels of 2, 20 and 200 μg g(-1) ranged from 81.2% to 91.3%; the inter-day relative standard deviation values were less than 7.0%. The limits of quantification for four organoarsenic compounds were 1.0-2.0 μg g(-1). This simple and fast method could be applied to the determination of multi-residues of organic arsenic compounds in animal feeds.