Serum protein determination by high-performance gel-permeation chromatography

A general high-performance gel-permeation chromatography (HPGPC) method was developed to determine protein in human serum with improved sensitivity and speed. The optimum UV wavelength for protein detection was found to be 210 nm, by comparing the protein values obtained by varying the UV wavelength of the HPLC detection system with the protein values obtained from spectrophotometric protein assays, i.e., the bicinchoninic acid (BCA) method and the biuret method. The analysis time was less than 1 min. Since this HPGPC serum protein assay method is simple and rapid, it is expected to be particularly well adapted for use in clinical laboratories.     

Quantitative determination of olanzapine in rat brain tissue by high-performance liquid chromatography with electrochemical detection

A sensitive assay was developed for the measurement of olanzapine in rat brain tissue using HPLC with electrochemical detection. The assay has a lower limit of quantitation of 0.5 ng/ml in tissue homogenate and utilizes a liquid–liquid extraction followed by reversed-phase HPLC for the quantitative analysis of olanzapine. The method provided a linear response for olanzapine over a concentration range of 0.5–100 ng/ml with a coefficient of determination (r²) greater than 0.9995. The extraction efficiencies of olanzapine and internal standard (LY170158) were greater than 82% in brain tissue. The intra-assay and inter-assay relative errors ranged from −5.38 to 17.60% and −3.25 to 10.53%, respectively. The intra-assay and inter-assay RSD values were in the range of 1.12 to 6.96% and 3.78 to 6.68%. Long-term stability studies showed that brain tissue homogenate samples spiked with olanzapine and internal standard are stable at −70°C for at least 110 days. However, a room temperature stability study showed that olanazapine was not stable in brain homogenate if the sample was exposed at 25°C longer than 2 h. This method has been used for the study of the disposition and pharmacokinetics of olanzapine in male Sprague–Dawley rats.     

Improved liquid chromatographic method for mitoxantrone quantification in mouse plasma and tissues to study the pharmacokinetics of a liposome entrapped mitoxantrone formulation

A simple, rapid HPLC method for quantification of mitoxantrone in mouse plasma and tissue homogenates in the presence of a liposome entrapped mitoxantrone formulation (LEM-ETU) is described. Sample preparation is achieved by protein precipitation of 100 microl plasma or 200 microl tissue homogenate with an equal volume of methanol containing 0.5 M hydrochloric acid:acetonitrile (90:10, v/v). Ametantrone is used as the internal standard (i.s.). Mitoxantrone and i.s. are separated on a C18 reversed phase HPLC column, and quantified by their absorbance at 655 nm. In plasma, the standard curve is linear from 5 to 1000 ng/ml, and the precision (%CV) and accuracy (percentage of nominal concentration) are within 10%. In mouse tissue (heart, kidney, liver, lung, and spleen) homogenates (5%, w/v), the standard curve is linear from 25 to 2000 ng/ml, with acceptable precision and accuracy. The method was used to successfully quantify mitoxantrone in mouse plasma and tissue samples to support a pharmacokinetic study of LEM-ETU in mice.     

Determination of a new proton pump inhibitor, YH1885, in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new proton pump inhibitor, YH1885 (I), in human plasma and urine, and rat blood and tissue homogenate using fenticonazole as an internal standard. The sample preparation was simple: a 2.5 volume of acetonitrile was added to the biological sample to deproteinize it. A 50-μl aliquot of the supernatant was injected onto a C₈ reversed-phase column. The mobile phase employed was methanol-0.005 M tetrabutylammonium dihydrogenphosphate (77:23, v/v), and it was run at a flow-rate of 1.0 ml/min. The column effluent was monitored using an ultraviolet detector at 270 nm. The retention times for I and the internal standard were 9.0 and 10.3 min, respectively. The detection limits for I in human plasma and urine, and in rat tissue homogenate (including blood) were 50, 100 and 100 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 8.84%) for human plasma and urine, and for rat tissue homogenate. No interferences from endogenous substances were found.     

The effect of gradual changes in temperature on the release of hormones from nerve endings isolated from bovine neural lobes.

Abstract— Homogenates of bovine neural lobe tissue were fractionated by differential centrifugation at 20°C or at 4°C and the distribution of activities of vasopressin and oxytocin among the fractions was compared. The ratio of total hormone to protein (mg) in the homogenate was similar at the two temperatures. At 20°C a much smaller proportion of the total hormone was recovered in the soluble fraction (100,000 g_av supernatant), than at 4°C with a corresponding increase in recovery in the nerve-ending fraction (800–3000 g sediment). Nerve endings isolated at 4°C did not, when incubated, release hormone in response to changes in temperature. Nerve endings isolated at 20°C released hormone when the temperature was reduced below 15°C. Gradual reduction in temperature led to hormone release unaccompanied by lactate dehydrogenase release. Incubation of nerve endings for 10 min at 10°C increased the release of vasopressin and of neurophysin without any increase in lactate dehydrogenase. These results demonstrate that release of vasopressin by cold stimulation occurs by way of exocytosis.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C₁₈ reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

The Recovery of Cytokinins during Extraction and Purification of Clubroot Tissue

Losses of one naturally occurring cytokinin (zeatin) and one synthetic cytoknin (kinetin) were determined during purification of turnips (Brassica compestris) infected by Plasmodiophora brassicae (clubroot). A known amount of zeatin and 8-¹⁴C-kinetin was added after homogenization of plant material in ethanol or water. The commonly used practice to purify the aqueous residues of the homogenate by partitioning with petroleum ether was omitted because of emulsion formation. Losses due to emulsion formation and occlusion of 8-¹⁴C-kinetin into non-water soluble plant material could be prevented by extractionof clubroot tissue with water instead of ethanol. To minimize enzyme activity the aqueous homogenate was kept at 100°C for 5 min. High molecular weight compounds were removed by dialysis against water and the diffusible fraction was partitioned with n-butanol at pH 8.2. It was shown that a rapid evaporation of n-butanol under reduced pressure at high temperature caused less breakdown of 8-¹⁴C-kinetin than prologned treatment at a low temperature. To minimize breakdown to zeatin riboside the butanol fraction was purified further on cation cellulose-phosphate exchanger instead of on strong acid Dowex H⁺. 8-¹⁴C-kinetin was separated from zeatin by column chromatography on Sephadex LH20, and yielded 86% of the amount originally added to a plant homogenate. The zeatin containing fractions were further purified on thin layer chromatography (TLC) silicagel plates and injected into a high pressure liquid chromatograph. A yield of 60% could be estimated from the amount (15 μg) orignally added to 50 g clubroot tissue.     

Determination of indomethacin residues in poultry by high-performance liquid chromatography

A HPLC method using a C₁₈ column and UV detection (254 nm) is described for the determination of indomethacin residues in chicken tissues (liver, muscle and fat). Drug extraction from tissue homogenate in phosphate buffer (pH 3.5) was performed with dichloromethane. Mobile phase was acetonitrile–acetic acid (0.5% in water) (50:50). Indomethacin detection limit was 20 ng/g for the studied tissues. After administration of an oral dose of indomethacin (2 mg/kg), only three of the eight poultry studied showed drug tissue levels, in those cases the levels were below 50 ng/g.     

Ion-pair high-performance liquid chromatographic assay method for the assessment of clarithromycin stability in aqueous solution and in gastric juice

A simple and selective ion-pair HPLC method has been developed for the analysis of clarithromycin in aqueous solutions and in gastric juice. A Hypersil ODS 5-μm (150 × 4.6 mm I.D.) column was used with a mobile phase consisting of acetonitrile-aqueous 0.05 M phosphate buffer (pH 4.6) containing 5 mM 1-octanesulphonic acid (50:50, v/v). The column temperature was 50°C and detection was by UV absorption (210 nm). The limits of detection of 50-μl samples were 0.4 μg/ml (aqueous) and 0.78 μg/ml (0.5 ml gastric juice) or better. The assay was linear in the range of 1.56 to 100 μg/ml with r² values greater than 0.99. The recovery from the gastric juice samples was 98.5±2.9%. The method was applied successfully to determine the stability of clarithromycin in 0.01 M HCl and gastric juice.     

PHOSPHOLIPASE ACTIVITY IN SQUID AND FROG AXONS

Evidence for the presence of phosphatide acylhydrolase activity (EC 3.1.1) in centrifuged homogenate supernatants and extracts of squid giant axons and centrifuged homogenate supernatants of frog sciatic nerve bundles is reported. The enzyme was assayed by measurement of the rate of deacylation of [U-¹⁴C]phosphatidyl choline. The deacylation activity in the nerve homogenate supernatants exhibits: a pH maximum at 7.2–7.4 (25°C); a calcium ion maximum at 12–13 mM-CaCl₂(aq); a K_m value of 3.4 × 10^?4 M (25°C); and a temperature maximum at 37°C. The activation energy over the range 8–37°C is 5.7 ± 0.2kcal-mol^?1.     

Simultaneous determination of nine antiretroviral compounds in human plasma using liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) method using UV detection was developed for the determination of nine antiretroviral compounds commonly found in plasma from patients receiving antiretroviral therapy. Analytes include indinavir, saquinavir, ritonavir, amprenavir, lopinavir, delavirdine, efavirenz, nelfinavir and its M8 metabolite. Analytes were isolated from plasma using tert.-butyl methyl ether and separation achieved via reversed-phase liquid chromatography on a C(8) column with a gradient mobile phase. Detection at 210 nm provided adequate sensitivity. Limit of quantification is 50 ng/ml and all analytes demonstrated linearity across 50-10000 ng/ml from a single 200-microliter plasma sample. Recovery from plasma was consistently high (>80%). This novel HPLC methodology allows us to simultaneously determine plasma concentrations of nine antiretrovirals, including lopinavir, in HIV-infected patients on a single HPLC system.     

Stability study of Prulifloxacin and Ulifloxacin in human plasma by HPLC–DAD

A new and specific HPLC–DAD method for the direct determination of Prulifloxacin and its active metabolite, Ulifloxacin, in human plasma has been developed. Plasma samples were analysed after a simple solid phase extraction (SPE) clean-up using a new HILIC stationary phase based high-performance liquid chromatography (HPLC) column and an ammonium acetate buffer (5?mM, pH 5.8)/acetonitrile (both with 1% Et₃N, v/v) mobile phase in isocratic elution mode, with Danofloxacin as the internal standard. Detection was performed using DAD from 200 to 500?nm and quantitative analyses were carried out at 278?nm. The LOQ of the method was 1?μg/mL of the cited analytes and the calibration curve showed a good linearity up to 25?μg/mL. For both analytes the precision (RSD%) and the trueness (bias%) of the method fulfil with International Guidelines. The method was applied for stability studies, at three QC concentration levels, in human plasma samples stored at different temperature of?+?25,?+?4 and ?20?°C in order to evaluate plasma stability profiles.     

Simple and sensitive high-performance liquid chromatographic method for the determination of an everninomycin,SCH 27899, in rat plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of SCH 27899, an everninomycin antibiotic, in rat plasma. The method involved plasma protein precipation with acetonitrile, followed by reversed-phase HPLC analysis using a polymeric column and a mobile phase containing acetonitrile and ammonium phosphate, pH 7.8. The linear relationship between detector response and concentration was demonstrated with a correlation coefficient of larger than 0.996 at concentrations ranging from 0.2 to 100 μg/ml. The results showed that the HPLC method was accurate (bias ≤6%) and precise (coefficient of variation, C.V.≤6%). The limit of quantitation was 0.2 μg/ml with a C.V. of 2.6% and bias of 5%. SCH 27899 was stable in rat plasma at −20°C for at least 40 days. The HPLC method has been utilized for the determination of SCH 27899 in plasma samples from rats following single intravenous administration (3 mg/kg).     

Determination of sulfadoxine in human blood plasma using packed-column supercritical fluid chromatography

A sensitive, rapid, selective and reproducible method has been developed to measure plasma levels of sulfadoxine, 4-Amino-N-(5, 6-dimethoxy-4-pyrimidinyl) benzensulfonamide; in healthy, human volunteers using packed-column supercritical fluid chromatography. Omeprazole, 5-methoxy-2-[[(4-methoxy-3, 5-di-methyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole; was used as the internal standard (i.s.) at 15.0 μg/ml. The drug and the i.s. were extracted from plasma using dichloromethane. Separation of sulfadoxine and i.s. was done on a Nucleosil (250×4.6 mm) 10 μm, RP-C₁₈ column with 7.4% (v/v) methanol-modified supercritical fluid carbon dioxide (2.5 ml/min) as the mobile phase. The column temperature was 40°C and the outlet pressure was set at 8.83 MPa. The detection was done using a UV–Vis detector set at 265 nm. The limit of quantification was 0.50 μg/ml using 1 ml plasma specimen. The mean extraction recovery of the drug from plasma was found to be 94.9%. The SFC method was directly compared to a published HPLC/UV method. With respect to speed and use of organic solvents SFC was found to be superior; while in all other aspects the results were similar to the published technique. The method has been successfully used to estimate the sulfadoxine levels in healthy human volunteers from 0 to 240 h following an oral dose of 500 mg of sulfadoxine in combination with 25 mg of pyrimethamine.     

Dynamic Behavior of Clobazam on High‐Performance Liquid Chromatography Chiral Stationary Phases

Clobazam, a 1,5‐benzodiazepin‐2,4‐dione, is a chiral molecule because its ground state conformation features a nonplanar seven‐membered ring lacking reflection symmetry elements. The two conformational enantiomers of clobazam interconvert at room temperature by a simple ring‐flipping process. Variable temperature HPLC on the Pirkle type (R)‐N‐(3,5‐dinitronenzoyl)phenylglycine and (R,R)‐Whelk‐O1 chiral stationary phases (CSPs) allowed us to separate for the first time the conformational enantiomers of clobazam and to observe peak coalescence‐decoalescence phenomena due to concomitant separation and interconversion processes occurring on the same time scale. Clobazam showed temperature dependent dynamic high‐performance liquid chromatography (HPLC) profiles with interconversion plateaus on the two CSPs indicative of on‐column enantiomer interconversion. (enantiomerization) in the column temperature range between T_col = 10°C and T_col = 30°C, whereas on‐column interconversion was absent at temperature close to or lower than T_col = 5°C. Computer simulation of exchange‐deformed HPLC profiles using a program based on the stochastic model yielded the apparent rate constants for the on‐column enantiomerization and the corresponding free energy activation barriers. At T_col = 20°C the averaged enantiomerization barriers, ΔG^?, for clobazam were found in the range 21.08–21.53 kcal mol^?1 on the two CSPs. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this article are consistent with the literature data measured by DNMR at higher temperatures and in different solvents. Chirality 28:17–21, 2016. © 2015 Wiley Periodicals, Inc.     

Spectrophotometric quantitation of anchorage-dependent cell numbers using the bicinchoninic acid protein assay reagent

A rapid and convenient method is described for the determination of the actual and relative number of adherent cells in tissue culture. The cell lines human melanoma C32, ATCC CRL 1585, mouse melanoma B16-F10, and pig epithelial LLC-PK1, suspended in Dulbecco's minimum essential medium containing no serum, were allowed to adhere to fibronectin adsorbed to wells of a 96-well microtiter plate. Nonadherent cells were removed by aspiration, wells were washed, and adherent cells were solubilized with 200 microliters of the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay reagent. Plates were heated to 60 degrees C for 30 min and absorbances read at 562 nm using a microtiter plate reader. A linear correlation was observed between the number of adherent cells in the range 2-8 X 10(5)/ml cells added and the protein content of the adherent cells as measured by the BCA protein reagent. The assay procedure gave absorbance values in the range of 0.100 to 1.30 making the method highly sensitive and reproducible. Blank wells containing only coupled protein and no cells gave little or no absorbance. Cell adhesion was fibronectin specific since little or no cell attachment was observed when microtiter plates were coupled with bovine serum albumin. Similar results were obtained with other cell types such as platelets. These results indicate that measurement of total cellular protein using the BCA protein reagent can be a rapid and sensitive assay for the detection and quantitation of adherent cells.     

Simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat plasma using HPLC with on-line solid-phase extraction

A HPLC method with on-line solid phase extraction (SPE) and DAD detection was developed for the simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat (SHR) plasma. Plasma samples (100 μL) were injected directly onto a CAPCELL MF C(8) SPE column. High-abundance proteins and most matrixes in plasma were removed by on-line SPE technology, while nitrendipine and hydrochlorothiazide trapped on the SPE column were effectively separated on a C(18) analytical column. The column temperature was maintained at 20°C. The optimal detection wavelength was 237 nm for NTDP and 271 nm for HCTZ. The total analytical run time was 34 min. The proposed method was linear over the range 5-500 ng mL(-1) for nitrendipine and 10-1000 ng mL(-1) for hydrochlorothiazide. The lower limit of detection (LLOD) was 0.5 and 0.6 ng mL(-1) for nitrendipine and hydrochlorothiazide, respectively. The sensitivity and precision of the method were within acceptable limits during validation period. The method was successfully used to investigate the pharmacokinetic characteristics of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rats.     

An improved method for tissue long-chain acyl-CoA extraction and analysis

We report an extensively modified method for the extraction, solid-phase purification, and HPLC analysis of long-chain acyl-CoAs from tissues. Tissue samples were homogenized in a glass homogenizer in KH2PO4 buffer (100 mM, pH 4.9) and again after the addition of 2-propanol. Acyl-CoAs were then extracted from the homogenate with acetonitrile (ACN). The acyl-CoAs in the extract were bound to an oligonucleotide purification column and eluted using 2-propanol. This eluent was concentrated and then loaded onto a C-18 column and eluted using a binary gradient system in which solvent A was KH2PO4 (75 mM, pH 4.9) and solvent B was ACN containing 600 mM glacial acetic acid. Initial flow rate was 0.5 or 0.25 ml/min depending upon the tissue used. The HPLC eluent was monitoring at 260 nm. Our modifications increased the recovery of the extraction procedure to 70-80%, depending upon tissue, with high reproducibility and significantly improved separation of the most common unsaturated and saturated acyl-CoAs. We also report, for the first time, the mass (nanomoles per gram wet weight) of the most common polyunsaturated acyl-CoAs in rat heart, kidney, and muscle tissues. The modifications and high recovery permit the use of tissue samples of less than 100 mg, making this method useful for the analysis of small tissue amounts associated with mice.     

Calculation of the molecular masses of two newly synthesized thermostable enzymes isolated from thermophilic microorganisms

Two thermostable enzymes synthesized by thermophilic microorganisms were isolated and purified. A thermostable ß-galactosidase was produced in a continuous fermentation process by Bacillus stearothermophilus TP 32 as an intracellular enzyme. After applying different concentration procedures the raw extract enzyme was prepurified on a Sephadex G-200 size exclusion column. The isolated ß-galactosidase fraction was then separated with HPLC on a TSK G 3000 SW size exclusion column to determine the molecular mass based on calibration curves of standard proteins. The other enzyme, a thermostable protease, was synthesized by Bacillus stearothermophilus TP 26 as an extracellular enzyme. After its concentration, the enzyme was purified on a classical size exclusion column (Sephacryl S-200) and on a HPLC size exclusion column (BIO-SIL TSK-250). The micropreparatively isolated fraction was separated again on this HPLC column to determine its molecular mass. The optimum temperature of both enzymes was approximately 75°C.     

Determination of saposin proteins (sphingolipid activator proteins) in human tissues

Saposins are small glycoproteins which are required for sphingolipid hydrolysis by lysosomal hydrolases. Each saposin (A, B, C, and D) stimulates a different enzymatic activity. A new simple HPLC method to determine the levels of saposins A, C, and D in tissue was developed. Tissues were homogenized in 20 vol of water, boiled, and centrifuged. The supernatant was lyophilized and redissolved in 5 ml of water. A 1.5-ml sample of the solution was applied to a reverse-phase HPLC column (C4 column) and eluted with an acetonitrile gradient. Most contaminants eluted from the column prior to the saposins, which were eluted later as a cluster of peaks. This cluster was collected and then analyzed by another HPLC system equipped with an AX-300 anion-exchange column using a NaCl gradient. Saposins D, A, and C eluted from the AX-300 column separately and in that order. Quantitation of the saposins was made by measuring the sizes of each peak. Standard curves made from pure saposins showed that quantification was linear over a range from 1 to 5 micrograms. Saposin B was measured by its stimulation activity on pure human liver GM1 ganglioside beta-galactosidase. Stimulation was linear up to 80 micrograms of saposin B. Application of this method to analysis of human tissues for their saposin content is presented.