Modifiers of mercaptopyruvate sulfurtransferase catalyzed conversion of cyanide to thiocyanate in vitro

The enzyme mercaptopyruvate sulfurtransferase appears to play an important role in the in vivo detoxification of cyanide. It does so by transferring sulfur to cyanide to produce thiocyanate, which is less toxic and may be excreted through the kidney. Several compounds were tested for their ability to affect the rate of enzyme catalyzed thiocyanate formation in vitro. The studies were carried out using both a partially purified bovine kidney extract and a highly purified enzyme preparation. Hypotaurine and methanesulfinic acid doubled sulfurtransferase activity in the partially purified extract at 30 mM, but inhibited the purified enzyme to 57% (hypotaurine) and 27% (methanesulfinic acid) of control activity at the same concentration. Pyruvate, phenylpyruvate, oxobutyrate, and oxoglutarate each inhibited the extract and purified forms of mercaptopyruvate sulfurtransferase. Phenylpyruvate was the most effective inhibitor, reducing activity to 0.2% of control values in the extract, and 11% of control values for purified MPST when added to the reaction at 30 mM. Other compounds tested (see Table 1) had a negligible effect on sulfurtransferase activity. A heat stable cofactor was found in boiled kidney extract which stimulated sulfurtransferase activity in the extract but inhibited sulfurtransferase activity in the purified enzyme, as was observed for hypotaurine and methanesulfinate. The boiled extract had no thiocyanate forming activity of its own. The cofactor operated in synergy with methanesulfinate, but independently of hypotaurine.     

Rapid quantitation of cyanide in whole blood by automated headspace gas chromatography

Cyanide (CN), a chemical asphyxiant, is a rapidly acting and powerful poison. We have developed a sensitive, rapid, simple, and fully automated method for measuring CN in whole blood. The assay is based on the use of gas chromatography (GC) with nitrogen-phosphorus detection and acetonitrile as an internal reference. Following the automated addition of phosphoric acid to the blood sample, the released hydrogen cyanide is analyzed using a fully automated headspace GC system. The assay, validated on human blood samples spiked with potassium cyanide and on clinical samples from fire victims who had smoke inhalation injury, can detect CN at a wide range of concentrations (30-6000 microg/l) in about 17 min (including incubation and GC run time, and <2 min for manual sample preparation). This automated, high-throughput, simple, and sensitive method is suitable for the rapid diagnosis of CN in clinical and forensic specimens.     

Simultaneous determination of lysophospholipids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl-cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A₂ and PAF-acetylhydrolase, on single phospholipids or their mixtures.     

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma and whole blood by high-performance liquid chromatography with ultraviolet and fluorescence detection

A method for the simultaneous determination of the three selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, paroxetine and their metabolites in whole blood and plasma was developed. Sample clean-up and separation were achieved using a solid-phase extraction method with C₈ non-endcapped columns followed by reversed-phase high-performance liquid chromatography with fluorescence and ultraviolet detection. The robustness of the solid-phase extraction method was tested for citalopram, fluoxetine, paroxetine, Cl-citalopram and the internal standard, protriptyline, using a fractional factorial design with nine factors at two levels. The fractional factorial design showed two significant effects for paroxetine in whole blood. The robustness testing for citalopram, fluoxetine, Cl-citalopram and the internal standard revealed no significant main effects in whole blood and plasma. The optimization and the robustness of the high-performance liquid chromatographic separation were investigated with regard to pH and relative amount of acetonitrile in the mobile phase by a central composite design circumscribed. No alteration in the elution order and no significant change in resolution for a deviation of ±1% acetonitrile and ±0.3 pH units from the specified conditions were observed. The method was validated for the concentration range 0.050–5.0 μmol/l with fluorescence detection and 0.12–5.0 μmol/l with ultraviolet detection. The limits of quantitation were 0.025 μmol/l for citalopram and paroxetine, 0.050 μmol/l for desmethyl citalopram, di-desmethyl citalopram and citalopram-N-oxide, 0.12 μmol/l for the paroxetine metabolites by fluorescence detection, and 0.10 μmol/l for fluoxetine and norfluoxetine by ultraviolet detection. Relative standard deviations for the within-day and between-day precision were in the ranges 1.4–10.6% and 3.1–20.3%, respectively. Recoveries were in the 63–114% range for citalopram, fluoxetine and paroxetine, and in the 38–95% range for the metabolites. The method has been used for the analysis of whole blood and plasma samples from SSRI-exposed patients and forensic cases.     

Simultaneous determination of serum cortisol and cortisone by reversed-phase liquid chromatography with ultraviolet detection

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cortisol and cortisone in a single extract of 1 ml of serum is described. The method employs meprednisone as the internal standard. The steroids were analysed isocratically by reversed-phase HPLC with an octadecylsilane-bonded (ODS) column using ultraviolet detection. The matrix effect was reduced by lowering the sample pH by adding glacial acetic acid to the sera. The samples were then filtered through regenerated cellulose membranes at 4°C and extracted with diethyl ether. The dried eluates were redissolved in the mobile phase and injected into the column. The detection limit of the assay for both steroids was 500 ng/l. Cortisol was determined in twenty serum samples by both HPLC and radioimmunoassay (RIA). The results were similar. Interference by other steroids and certain steroid analogue drugs was also studied. The HPLC method yielded no cross-reactivity between the different steroids as may occur with the RIA technique. The HPLC method was technically easy to perform and it allowed us to quantify both cortisol and cortisone in a single serum extract with high specificity.     

Simultaneous determination of tricarboxylic acid cycle metabolites by high-performance liquid chromatography with ultraviolet detection

Here we describe a simple high-performance liquid chromatography (HPLC) procedure for the simultaneous detection and quantitation in standard solutions of 13 important metabolites of cellular energy metabolism, including 9 tricarboxylic acid (TCA) cycle components and 4 additional metabolites. The metabolites are detected by their absorbance at 210 nm. The procedure does not require prior derivatization, and an analysis can be carried out at ambient temperature within 15 min. The significance of the current work is that the current HPLC procedure should motivate the development of simplified TCA cycle enzyme assays, isotopomer analysis, and determination of selected TCA metabolite levels in plasma/tissues.     

Simultaneous determination of eight beta-blockers by gradient high-performance liquid chromatography with combined ultraviolet and fluorescence detection in corneal permeability studies in vitro

A gradient HPLC method with combined ultraviolet and fluorescence detection was developed for the simultaneous determination of eight beta-blockers (alprenolol, atenolol, metoprolol, nadolol, pindolol, propranolol, sotalol and timolol) in corneal permeability studies in vitro. Fluorescence detection with excitation wavelength at 230 nm and emission at 302 nm was selective for six of the compounds, whereas UV detection at 205 nm was able to detect all the compounds. Calibration was performed with fluorescence detection for six compounds from 50 or 200 nM to 3 microM, and with UV detection for all the eight compounds from 100 or 200 nM to 30 microM. With optimized fluorescence detection, detection limits between 0.7 and 1.3 nM (0.035-0.065 pmol per 50 microl injection) were obtained for atenolol, metoprolol, nadolol and sotalol. A mixture of eight beta-blockers was used in cassette dosing permeability studies with a cultured corneal epithelium. The HPLC method revealed marked differences in the permeation between hydrophilic and lipophilic beta-blockers through the corneal epithelial cell culture model.     

Determination of urinary thiocyanate and nitrate using fast ion-interaction chromatography

A simple and rapid chromatographic method for determination of nitrite, nitrate and thiocyanate is reported, and applied to the analysis of non-, medium and heavy smokers' urine samples. Ion-interaction liquid chromatography was carried out on a short 30 mm x 4.6 mm C18 column (3 microm particle size) with a mobile phase of 10 mM tetrabutylammonium phosphate in 20% MeOH. The chromatography was performed at an elevated temperature of 45 degrees C, at a flow-rate of 1 ml/min. Detection was by direct UV absorption at 230 nm. Sample preparation involved centrifugation and dilution, followed by sample clean-up on a C18 solid-phase extraction cartridge. The developed method proved both precise (% RSD <2%) and sensitive (standard detection limits <0.1 mg/l), and yielded total run times of under 10 min when applied to urine analysis of smokers and non-smokers, with thiocyanate eluting in under 5 min.     

Simultaneous determination of ketamine and xylazine in canine plasma by liquid chromatography with ultraviolet absorbance detection

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of ketamine and xylazine in canine plasma is described. Plasma samples (500 microl) are cleaned up via liquid-liquid extraction. The analytes and the internal standard clonidine are separated on a cyano (CN) column using a mobile phase containing acetonitrile-0.005 M phosphate buffer adjusted to pH 5.5 (3:2) at a detection wavelength of 215 nm. The method was validated according to specificity, sensitivity, accuracy and reproducibility and was used to determine the plasma concentrations of both compounds in dogs after intramuscular injection.     

Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection

When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography–ultraviolet (HPLC–UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0–2000 pg/ml) and midazolam (range 0–400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (< 15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography–mass spectrometry (GC–MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC–UV system.     

Simultaneous determination of 11 designated hallucinogenic phenethylamines by ultra-fast liquid chromatography with fluorescence detection

To avoid the spreading of illegal drugs, a designated drug regulation system was introduced along with revision of the Pharmaceutical Affairs Law in Japan in 2006, and 32 substances including phenethylamine-type drugs were listed in April 2007. In this study, a new simultaneous determination method, based on ultra-fast liquid chromatography coupled with fluorescence detection (UFLC-FL), was developed for the 11 designated phenethylamine drugs. The phenethylamines were labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 degrees C for 2h in 0.1M borax (pH 9.3). The resulting 11 fluorophores were completely separated by reversed-phase chromatography using an ACQUITY UPLC BEH C(18) column (2.1 mm x 100 mm 1.7 microm) and fluorometrically detected at 550 nm (excitation at 450 nm). The calibration curves obtained from the peak areas versus the injection amounts of the phenethylamines showed a good linearity. The limits of detection (signal-to-noise ratio of 3: S/N=3) on the chromatogram were in the range from 10 fmol (PMMA) to 2.5pmol (MMDA-2). Good accuracy (%) and precision (CV) by intra-day assay and inter-day assay were also obtained using the present procedure. The method was applied to the qualitative and quantitative analyses of phenethylamine in real products obtained from the Japanese market. As the results, BDB (0.24 mg/mg), MMDA-2 (0.98 mg/mL) and 2C-I (0.016 mg/mg) were identified from the different products (powder, liquid and mushroom like). Because the procedure is simple, selective and sensitive, the present method seems to be useful for the qualitative and quantitative analyses of the designated phenethylamines in various samples including biological specimens.     

Determination of cyanide and thiocyanate in blood by gas chromatography and gas chromatography-mass spectrometry

We devised a sensitive and simple method for determining cyanide And its major metabolite, thiocyanate, in blood using an extractive alkylation technique. Pentafluorobenzyl bromide was used as the alkylating agent, and tetradecyldimethylbenzylammonium chloride was used as the phase-transfer catalyst. The derivatives obtained were analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with an electron-capture detection. The detection limits of cyanide and thiocyanate were 0.01 and 0.003 μmol/ml, respectively, while the gross recovery of both compounds was 80%. The calibration curve was linear over the concentration range from 0.02 to 1.0 μmol/ml for cyanide and from 0.01 to 1.0 μmol/ml for thiocyanate. The accuracy and precision of the method were evaluated, and the coefficients of variation were found to be within 10%. Using this method, the blood levels of two victims who had died from cyanide poisoning were determined.     

Simultaneous determination of alpha-lipoic acid and its reduced form by high-performance liquid chromatography with fluorescence detection

The simultaneous determination of alpha-lipoic acid (LA) and DHLA (reduced form of LA) was carried out by HPLC with fluorescence detection. DHLA in the sample was first labeled with ABD-F at room temperature for 10 min and then the LA was labeled with SBD-F at 50 degrees C for 1 h after conversion to DHLA using the reducing agent, TCEP. The resulting fluorophores, ABD-DHLA and SBD-DHLA, were separated by reversed-phase chromatography and detected at 510 nm (excitation at 380 nm). Both fluorophors were completely separated without any interference of endogenous thiols and disulfides in the sample and sensitively detected by fluorimetry. The proposed method was applied to the assay of the LA supplement and the determination in human plasma after the oral administration of LA tablets. The concentration (%) of LA in the tablet was reasonable to the stated amount. Furthermore, the result of a time course study in the plasma after the administration of LA did not differ from a previous report. Thus, the present method seems to be applicable to the simultaneous determination of LA and DHLA in various biological specimens.     

Simultaneous determination of five antipsychotic drugs in rat plasma by high performance liquid chromatography with ultraviolet detection

A significant percentage of psychiatric patients who are treated with antipsychotics are treated with more than one antipsychotic drug in the clinic. Thus, it is advantageous to use a rapid and reliable assay that is suitable for determination of multiple antipsychotic drugs in plasma in a single run. A simple and sensitive HPLC-UV method was developed and validated for simultaneous quantification of olanzapine, haloperidol, chlorpromazine, ziprasidone, risperidone and its active metabolite 9-hydroxyrisperidone in rat plasma using imipramine as an internal standard (I.S.). The analytes were extracted from rat plasma using a single step liquid-liquid acid solution back extraction technique with wash procedure, which provided the very clear baseline for blank plasma extraction. The compounds were separated on an Agilent Eclipse XDB C8 (150 mm x 4.6 mm i.d., 5 microm) column using a mobile phase of acetonitrile/30 mM ammonium acetate including 0.05% triethylamine (pH 5.86 adjusted with acetic acid) with gradient elution. All of the analytes were monitored using UV detection. The method was validated and the linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries, selectivity and stability were determined. The LLOQ was 2.0 ng/ml and correlation coefficient (R(2)) values for the linear range of 2.0-500.0 ng/ml were 0.998 or greater for all the analytes. The precision and accuracy for intra-day and inter-day were better than 7.44%. The recovery was above 74.8% for all of the analytes. This validated method has been successfully used to quantify the plasma concentration of the analytes for pharmacological and toxicological studies following chronic treatment with antipsychotic drugs in the rat.     

Simultaneous determination of nine fluoroquinolones in egg white and egg yolk by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method with fluorescence detection was developed for determination of nine fluoroquinolones (FQs) in egg white and yolk. Egg white samples were deproteinized with acidified ethanol (egg yolk samples with acetonitrile and acidified ethanol), followed by defatting with hexane once (white) or twice (yolk), and extracting FQs into acetonitrile. After acetonitrile was evaporated, the residue was dissolved in mobile phase, and FQs were detected in LC with a fluorescence detector. Recoveries for nine FQs from white and yolk were 74.7-85.6%, 79.1-91.2%, respectively, with excellent relative standard deviations. The limits of quantification were 5-20 ngg(-1).     

Simultaneous determination of byak-angelicin and oxypeucedanin hydrate in rat plasma by column-switching high-performance liquid chromatography with ultraviolet detection

A simple and sensitive column-switching HPLC method was developed for the simultaneous determination of two furocoumarin compounds, byak-angelicin and oxypeucedanin hydrate, which are the main components of hot water extract of Angelica dahurica root (AE), in rat plasma. Plasma sample was simply deproteinated with perchloric acid. After centrifugation, the supernatant was injected into a column-switching HPLC system consisting of a clean-up column (Symmetry Shield RP 8, 20×3.9 mm I.D.) and analytical column (Symmetry C₁₈, 75×4.6 mm I.D.) which were connected with a six-port switching valve. The flow-rate of the mobile phase (acetonitrile–water, 20:80) was maintained at 1 ml/min. Detection was carried out at wavelength 260 nm with a UV detector. The column temperature was maintained at 40°C. The calibration curves of byak-angelicin and oxypeucedanin hydrate were linear over the ranges 19.6 to 980 ng/ml (r²>0.997). The accuracy of these analytes was less than 4.4%. The intra- and inter-day relative standard deviations of byak-angelicin and oxypeucedanin hydrate were within 12.0% and 12.7%, respectively. The present method was applied for the analysis of plasma concentration from rats after administration of AE.     

Simultaneous determination of verapamil and norverapamil in biological samples by high-performance liquid chromatography using ultraviolet detection

In this paper we develop an high-performance liquid chromatographic method with ultraviolet detection for the determination of verapamil and its primary metabolite norverapamil in biological samples. Both compounds, as well as the internal standard, imipramine, were extracted from alkalinised blood, with n-hexane–isobutyl alcohol, back-extracted into 0.01 M phosphoric acid and determined using a reversed-phase column and ultraviolet monitoring at 210 nm. The average coefficient of variation obtained over the concentration range of 1–1000 ng/ml is about 3%. The detection limit is below 5 ng/ml for both compounds, and extraction recoveries close to 80%. The method was applied to a pharmacokinetic study of the drug and its active metabolite and used to analyse blood samples from verapamil treated rabbits.     

Simultaneous determination of oleuropein and hydroxytyrosol in rat plasma using liquid chromatography with fluorescence detection

Oleuropein, the main glycoside present in olives, and hydroxytyrosol, the principal degradation product of oleuropein present in olive oil, have been linked to reduction of coronary heart disease and certain cancers. In the present study a direct and sensitive reversed-phase high-performance liquid chromatographic assay was developed for simultaneous quantification of both oleuropein and hydroxytyrosol. The plasma protein was precipitated with acetonitrile, samples were then centrifuged and supernatants were dried, and reconstituted with water prior to injection. The chromatographic analysis was carried out using a phenyl column and an isocratic elution of acidified water and acetonitrile with fluorescence detection at 281 and 316 nm for excitation and emission, respectively. The calibration curve was linear and limits of quantification were 30 ng/ml and 3 microg/ml for hydroxytyrosol and oleuropein, respectively. The method has been successfully applied to monitor oleuropein and hydroxytyrosol plasma levels in the rat.     

Simultaneous determination of danofloxacin and N-desmethyldanofloxacin in cattle and chicken edible tissues by liquid chromatography with fluorescence detection

A rugged, simple, and selective method for the determination of danofloxacin and its primary metabolite, N-desmethyldanofloxacin, in cattle (liver, muscle, kidney, and fat) and chicken (liver and muscle) tissues was developed. The method is selective for danofloxacin and N-desmethyldanofloxacin over other veterinary important fluoroquinolones, such as enrofloxacin, ciprofloxacin, norfloxacin, and ofloxacin. Selectivity is achieved through a combination of extraction, chromatography, and fluorescence detection. The analytes were extracted from homogenized tissues using a methanolperchloric-phosphoric acid solution. After centrifugation, direct injection of extraction supernate was possible. The limit of quantitation was 20 pg on column. Separation was achieved on an Inertsil C₈ (5 μm, 100 Å) column with dimensions of 250×4.6 mm I.D. The mobile phase consisted of 0.05 M phosphate buffer (pH 3.5)-acetonitrile (88:12). A fluorescence detector was utilized with an excitation wavelenght of 280 nm and an emission wavelength of 440 nm. The assay was accurate and reproducible within the range of 10 to 500 ng/g for both danofloxacin and N-desmethyldanofloxacin. Intra-assay accuracy was between 98 and 101%, and precision was less than 4%. Inter-assay accuracy was between 99 and 102%, while precision was less than 2%. Recoveries for both analytes over the dynamic range were greater than 90% for all the tissues.     

Simultaneous determination of ZT-1 and its metabolite Huperzine A in plasma by high-performance liquid chromatography with ultraviolet detection

ZT-1 is a novel acetylcholinesterase (AChE) inhibitor. It is rapidly transformed to Huperzine A (Hup A) in vitro. A simple and rapid HPLC-UV method for the simultaneous determination of ZT-1 and its metabolite Hup A in plasma is described. The chromatographic separations were achieved on a C(18) ODS column (250 mm x 4.6 mm ID) using methanol-1 mmol/L ammonium acetate (70:30,v/v) as mobile phase. The flow rate was 0.7 mL/min, the detection wavelength was 313 nm and the column temperature was kept at 35 degrees C. Plasma samples were prepared as rapidly as possible and extracted immediately with 5 mL of chloroform:iso-propyl alcohol mixture (v/v, 9:1).The retention times of ZT-1 and Huperzine A (Hup A) were 18.7 and 14.4 min, respectively. The mean absolute recoveries of two analytes were >90%. Quantification limits were all 0.02 nmol/mL for ZT-1 and Hup A. This analytical method was reliable and convenient procedure that meets the criteria for the pharmacokinetic evaluation of ZT-1 on experimental animals.