Determination of ketoconazole in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and specific high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) has been developed and validated for the determination of ketoconazole in human plasma. The method used diethyl ether to extract the ketoconazole and the internal standard (I.S.) R51012 from alkalinized plasma sample. The LC separation was on a C(18) column (50 x 3 mm, 5 microm) using acetonitrile-water-formic acid (75:25:1, v/v/v) mobile phase. The retention times were approximately 1.8 min for both ketoconazole and the I.S. The MS-MS detection was by monitoring 531.2-->82.1 (m/z) for ketoconazole, and 733.5-->460.2 (m/z) for the I.S. The dynamic range was from 20.0 to 10000 ng/ml based on 0.1 ml plasma, with linear correlation coefficient of > or =0.9985. The run time was 2.5 min/injection. The recoveries of ketoconazole and the I.S. were 102 and 106%, respectively. The precision and accuracy of the control samples were with the relative standard deviations (RSDs) of < or =4.4% (n=6) and the relative errors (REs) from -0.6 to 1.4% for intra-day assay, and < or =8.6% RSD (n=18) and -1.4 to 0.9% RE for inter-day assay. The partial volume tests demonstrated good dilution integrity. Three freeze-thaw cycles, keeping plasma samples at ambient for 24 h, storing extracted samples at ambient for 24 h, and storing frozen plasma samples at approximately -20 degrees C for up to 2 months did not show substantial effects.     

Quantification of antidepressants and antipsychotics in human serum by precipitation and ultra high pressure liquid chromatography-tandem mass spectrometry

The present article describes the quantification of mirtazapine, O-desmethylvenlafaxine, quetiapine, venlafaxine, and ziprasidone (group 1), and amitriptyline, citalopram, clomipramine, clozapine, desmethylclomipramine, desipramine, imipramine, and nortriptyline (group 2) in human serum for therapeutic drug monitoring. The method was developed to replace old techniques which applied solid phase extraction and ultra-violet detection. The old methods had reached their limit of capacity regarding the number of samples and co-medicated drugs interfering with the detection. Serum samples were precipitated with zinc sulphate and methanol containing a stable isotope labelled analog for each analyte. Quantitative analysis was performed by ultra high pressure liquid chromatography combined with a tandem mass spectrometer using a Zorbax SB-C8 column (2.0 × 50 mm; 1.8 μm) with a mobile phase consisting of 0.1% formic acid in water and methanol, respectively. The total run time of the chromatography was 4 min. Precision and trueness varied from 2.6% to 14.9% and 87.6% to 103.5%, respectively. At the lower limit of quantification, precision was up to 17.9% and trueness varied from 89.5% to 111.5%. A five point standard curve covering the clinically relevant ranges with a power function fit was applied for calibration. Ion suppression from matrix effects and internal standards were thoroughly investigated and are discussed. Process efficiency rates varied from 42% to 99%. The method has shortened the response time, reduced interference from other drugs, avoided acetonitrile usage, and reduced the amount of serum needed for analysis 50-fold.     

Spectrophotometric study of ketoconazole binding with citrate capped silver nanoparticles and its monitoring in human plasma samples

Ketoconazole or Nizoral is an antifungal medication used to treat various type of fungal infections. It has been reported that this antifungal agent is able to induce a variety of heart function side effects, such as long‐QT syndrome, and ventricular arrhythmias. Hence, prescribing, identifying and controlling the side effects of medications such as ketoconazole is essential for human safety. In this study, a distinct and fast colorimetric probe based on citrate capped silver nanoparticles (Cit‐AgNPs) was introduced for determination of trace amounts of ketoconazole. Cit‐AgNPs were synthesized trough a novel method and applied for the quantification of ketoconazole in human plasma samples. Ultraviolet‐visible spectroscopy, transmission electron microcopy, dynamic light scattering, and energy dispersive X‐ray spectroscopy analysis, have been utilized for characterization of Cit‐AgNPs. It was revealed that in the presence of ketoconazole, the absorption intensity of Cit‐AgNPs was decreased by increasing the ketoconazole concentration. Moreover, this reaction is accompanied by a color change from shiny yellow to brown/red in acidic pH. Under acidic pH and optimum condition, the calibration graph of the assay was linear in the range of 0.1 to 0.8 μM. According to the obtained results Cit‐AgNPs can be used as a novel probe for the sensitive and specific detection and determination of similar drugs in clinical samples. Also, this method open a new way to biomedical analyses of pharmaceutical samples which is necessary in TDM.     

Determination of amitriptyline-n-oxide,amitriptyline and nortriptyline in serum and plasma by high-performance liquid chromatography

A method for the determination of amitriptyline-N-oxide, amitriptyline and nortriptyline in serum and plasma has been developed. After extraction from serum or plasma the drugs were analysed by high-performance liquid chromatography.The detection limit was 10 ng/ml (2 ml serum or plasma actually used). The coefficient of variation for all three compounds was below 10%.Amitriptyline-N-oxide was found in rat plasma after an oral dose (10 mg/kg) of amitriptyline-N-oxide.     

A microassay for the determination of iodide and its application to the measurement of the iodination of proteins and the catalytic activities of iodo compounds

A microassay system based on the effect of the catalytic Sandell-Kolthoff reaction of iodide on the oxidation of arsenic(III) by cerium(IV) was developed to measure iodine-containing compounds. This rapid assay uses small quantities of reagents, is suitable for use with a photometric microplate reader, can test many samples simultaneously, and eliminates problems associated with the use of radiolabeled compounds to measure iodination. It can detect picogram quantities of iodide. We report the use of this assay to measure the conjugation of an iodine-containing hapten (iodinated Bolton-Hunter reagent, IBHR) to ovalbumin and human serum albumin. It has proven to be excellent for studying the relative molar catalytic activity of the iodine-containing compounds IBHR, thyroxine, 4-iodophenol, and lithium 3,4-diiodosalicylate. The interference by azide on the assay was investigated.     

Determination of nortriptyline in human serum by fully automated solid-phase extraction and on-line high-performance liquid chromatography in the presence of antipsychotic drugs

A fully automated on-line method for determination of nortriptyline in human serum was developed using an ASPEC XL (Gilson) solid-phase extraction apparatus in combination with high-performance liquid chromatography. Solid phase extraction was performed on cyanopropyl cartridges. HPLC was carried out using a C₁₈ column with a mobile phase of acetonitrile–0.01 M triethylamine (34:66 v/v) buffer, pH 3.0. UV detection was at 242 nm. The Inter-day CV% was <5%. Comparison with liquid–liquid extraction of serum from patients treated with nortriptyline showed good agreement. Studies of analytical interference from coadministered psychoactive drugs revealed that only imipramine and a methotrimeprazine metabolite interfered.     

Synthesis and antifungal activities of phenylenedithioureas

A total of 20 new phenylenedithiourea derivatives was synthesized by reaction of phenylenediisothiocyanates with aromatic amines as aminobenzoic, aminosalicylic acid and their derivatives. Their chemical structures were confirmed by elemental analysis, IR spectrometry and 1H NMR. The compounds were screened for in vitro antifungal, antibacterial activities and some of them have strong antifungal activities comparable to the activity observed for ketoconazole.     

Electrochemical determination of arylsulfatase activity using high-performance liquid chromatography

A highly sensitive assay for arylsulfatase A was developed using high-performance liquid chromatography with electrochemical detection. The retention time of the enzymatic product, p-nitrocatechol, on a reverse phase column was approximately 2.0 min. The assay was able to detect 0.43 pmol p-nitrocatechol in a 20-microliter ethanol extract of the reaction mixture. The coefficients of variation for seven determinations of the intra- and interassays were 9 and 8%, respectively. The levels of arylsulfatase A activity in human saliva samples and leukocyte and platelet lysates determined by this assay were within 6 and 11%, respectively, of the levels determined by a spectrophotometric assay. The high-performance liquid chromatography assay may have utility in measuring arylsulfatase A activity in biological samples with low activity or specific activity or in samples with compounds which interfere with the spectrophotometric assay.     

Quantitation of intracellular polychlorobiphenyls during growth of Rhodotorula glutinis

A quantitative method to assay accumulated polychlorobiphenyls (PCB) in Rhodotorula glutinis is described which avoids any interference of extracellular adsorbed compounds. This method is based on a prior appropriate treatment of the cells before the extraction of derivatives by supercritical CO2 and GC analysis. The prior treatment consists in a double fixation of the samples with glutaraldehyde and osmium tetroxyde. After this fixation process the elimination of the extracellular PCBs can be achieved by washing the fixed yeast cells with organic solvents.     

Characterization of volatile constituents of Scaligeria tripartita and studies on the antifungal activity against phytopathogenic fungi

The chemical composition of the essential oils obtained from stems and leaves, fruits and roots of Scaligeria tripartita oil was analyzed by gas chromatography-mass spectrometry. A total of 38 compounds were identified ranging 89-94% of the oil samples. Geijerene was found as a main compound in the oils of the stems and leaves (37%) and fruits (55%), whereas epoxypseudoisoeugenol angelate (37%) was found as a main compound in the root oil. Oils were subsequently evaluated for their antimalarial, antimicrobial against human pathogenic bacteria or fungi and antifungal activities against plant pathogens. Antifungal activity of Scaligeria oils was observed against the strawberry anthracnose-causing fungal plant pathogens Colletotrichum acutatum, C. fragariae and C. gloeosporioides using the direct overlay bioautography assay. Chemotaxonomically important pure compounds indicated in the bioautography assay were subsequently evaluated in a 96-well microdilution broth assay. The performance of overpressured layer chromatography (OPLC) and TLC for the analysis of Scaligeria essential oils was also compared.     

An evaluation of fluorescence polarization and lifetime discriminated polarization for high throughput screening of serine/threonine kinases

We used two kinases, c-jun N terminal kinase (JNK-1) and protein kinase C (PKC), as model enzymes to evaluate the potential of fluorescence polarization (FP) for high-throughput screening and the susceptibility of these assays to compound interference. For JNK-1 the enzyme kinetics in the FP assay were consistent with those found in a [gamma-33P]ATP filter wash assay. Determined pIC(50)s for nonfluorescent JNK-1 inhibitors were also consistent with those found in the filter wash assay. In contrast, fluorescent compounds were found to interfere with the JNK-1 FP assay, appearing as false positives, defined by their lack of activity in the filter wash assay. We also developed a second assay using a different kinase, protein kinase C, which was used to test a 5000 compound diversity set. As for JNK-1, interference from fluorescent compounds caused a high false positive rate. The Molecular Devices Corporation 'FLARe' instrument is capable of discriminating between fluorophores on the basis of their fluorescence (excited state) lifetime, and may assist in reducing compound interference in fluorescent assays. In both model FP kinase assays described here some, although not complete, reduction in interference from fluorescent compounds was achieved by the use of FLARe.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition

A high-performance liquid chromatographic method for the measurement of bumetamide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C₁₈ column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C₁₈ Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol—water—glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5–2000 ng/ml.     

Determination of l-glutamate in various commercial soy sauce products using flow injection analysis with a modified electrode

A flow injection analysis (FIA) system with a modified electrode has been developed and optimized for determination of l-glutamate using l-glutamate oxidase (GLOD) (EC 1.4.3.11). GLOD was immobilized on controlled-pore glass using glutaraldehyde. The optimal potential applied on the working electrode was +700mV against a platinum (Pt) reference electrode. The optimal pH and flow rate of the carrier buffer were 7.4 and 1.5ml/min, respectively. A modified electrode was integrated into the FIA system in order to eliminate electroactive interference and it was used to determine l-glutamate in 39 samples of Thai commercial soy sauce products. The results obtained were compared with those obtained from enzymatic assay using glutamate dehydrogenase and those from a chromatographic assay using an amino acid analyser. Good correlations were observed amongst these methods. The results indicated that use of an FIA system with a modified electrode was able to eliminate electroactive interference and was applicable to the determination of l-glutamate in food samples. The modified FIA was faster and simpler than the more common methods of enzymatic and chromatographic analysis.     

Development of a Transcreener kinase assay for protein kinase A and demonstration of concordance of data with a filter-binding assay format

A Transcreener kinase fluorescence polarization (FP) assay has been developed for the serine/threonine kinase protein kinase A (PKA). The PKA Transcreener kinase assay is an homogenous, competitive antibody-based FP assay that uses Far Red Alexa Fluor 633-labeled adenosine 5' disphosphate (ADP) tracer and mouse monoclonal anti-ADP antibody. The Transcreener PKA assay was validated with both known PKA inhibitors and library compounds. The Transcreener PKA assay is resistant to low-wavelength (or common) fluorescent interference from small-molecule library compounds and generates IC50 results comparable with current radioactive filter-binding assay.     

Determination of the antifungal agent, ketoconazole, in human plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic (HPLC) assay for the quantitative determination of ketoconazole, an orally active antifungal agent, in human plasma is described. After extraction of the drug from plasma, the compound is separated by HPLC using a reversed-phase column and detected by UV light at 205 nm. Quantitation is accomplished by external standardization and the determination of peak areas is performed with the aid of an integrating computer. The average recovery of ketoconazole over a concentration range of 0.1–20.0 μg/ml was 88.2 ± 4.07% S.D. The maximum sensitivity of the assay is less than 0.1 μg/ml. The assay is suitable for use in pharmacokinetic studies following the administration of therapeutic doses of ketoconazole to humans.     

Simultaneous determination of amitriptyline,nortriptyline and four hydroxylated metabolites in serum by capillary gas-liquid chromatography with nitrogen-phosphorus-selective detection

A method for the simultaneous quantification of the antidepressant drug amitriptyline, its demethylated metabolite nortriptyline and four hydroxy metabolites (E-10-hydroxyamitriptyline, Z-10-hydroxyamitriptyline, E-10-hydroxynortriptyline, Z-10-hydroxynortriptyline) in human serum or plasma has been developed. The method is based on a three-step liquid-liquid extraction followed by gas-liquid chromatography (split-splitless injection, HP-5, 25 m×0.2 mm I.D., 0.33 μm capillary) with nitrogen phosphorus-selective detection (GLC-NPD). The limits of detection are 1.5 ng/ml for amitriptyline, nortriptyline, E-10-hydroxyamitriptyline and Z-10-hydroxyamitriptyline and 3 ng/ml for E-10-hydroxynortriptyline and Z-10-hydroxynortriptyline. The within-day and between-day precision is between 6 and 15% at three concentrations (low, moderate and high) for amitriptyline, nortriptyline and E-10-hydroxy metabolites. At low concentrations of 10 ng/ml, the precision of the assay of the Z-10-hydroxy metabolites has been found to be up to 19%. Accuracy is between 91 and 115% for all analytes. The performance of the assay of the hydroxy metabolites is mainly determined by the cleanness and the deactivation of the quartz insert of the injector port. Therefore, every day a freshly cleaned and deactivated insert was used.     

Measurement of protein by dye-binding assay in the presence of metrizamide

Quantitative determination of protein using the binding of Coomassie Brilliant Blue G-250 was investigated with respect to interference with the density gradient material metrizamide, and compared with the corresponding interference using the Lowry method. The background absorption obtained with metrizamide in the absence of protein was less than 10% of that obtained with the Lowry method. In the presence of 0–4% metrizamide, parallel standard curves were obtained with 0–67 μg of protein in the samples. The curves overlapped in the range 0–40 μg of protein when metrizamide was included in the blanks. With up to 2% final concentration of metrizamide in the assay, the curves overlapped at all protein concentrations tested (0–67 μg). Correction for metrizamide interference is thus a simple procedure and a precise estimation of the metrizamide concentration is less critical than when the Lowry assay is used. The method is well suited for quantitation of protein in samples collected from metrizamide grandients.     

Rapid and sensitive anthrone-sulfuric acid assay in microplate format to quantify carbohydrate in biopharmaceutical products: method development and validation.

The need for an accurate, fast and reliable analysis of carbohydrate test is crucial for numerous biological processes. In that sense, anthrone-sulfuric acid assay is one of the most efficient quantification techniques successfully applied to carbohydrate determination. In this paper, a sensitive and accurate anthrone-sulfuric acid microplate assay was developed and validated for the quantitative estimation of yeast carbohydrates in the production of hepatitis B virus surface antigen, and the main component of the recombinant vaccine HEBERBIOVAC HB. A response surface methodology was applied to design and optimize the assay in order to maximize the differences on the expected effect and to minimize the number of experiments. The proposed method was linear over the concentration range from 10 to 120 microg/mL for glucose, with values for the coefficient of determination >0.99. Intra- and inter-assay variation coefficient ranged between 0.45-4.79% and 2.48-8.94%, respectively. The Student t-test used in the interference study, revealed good parallelism among curves (T(obs)< or =T(0.05)), which indicates the lack of interference in the working range. Yields obtained in accuracy test for two concentration levels varied between 90 and 105%, confirming the assay's reliability. In conclusion, the validated method, which has successfully been used for the process control monitoring of several samples generated from the production of hepatitis B vaccine, allows the quality and purity of the final product.     

Selective high-performance liquid chromatographic assay for itraconazole and hydroxyitraconazole in plasma from human immunodeficiency virus-infected patients

A sensitive and selective reversed-phase liquid chromatographic assay for itraconazole and hydroxyitraconazole in human plasma has been developed and validated. Itraconazole and hydroxyitraconazole were extracted from the matrix using solid-phase extraction on a strong cation-exchange sorbent. All compounds were detected using fluorescence at 265 and 363 nm for excitation and emission, respectively. The assay has been validated over the range 10-1,000 ng/ml for both compounds, 10 ng/ml being the lower limit of quantification. Accuracies ranged from 104 to 113% for itraconazole and from 91 to 103% for hydroxyitraconazole. The intra-assay precisions were all below 9% for itraconazole and below 8% for hydroxyitraconazole. The selectivity has been evaluated with respect to all registered anti-human immunodeficiency virus (HIV) drugs and other potential co-medications and a few of their metabolites, commonly used by HIV-infected individuals. Both itraconazole and hydroxyitraconazole were stable under relevant conditions for HIV-inactivation and storage of samples. The applicability of the assay was demonstrated for samples collected from a treated HIV-infected patient.