Determination of the Content and Degree of Esterification of Uronic Acids in Plant Tissues and Products of Their Processing

A method for quantitative determination of uronic acids and the degree of esterification of their carboxyl groups in plant tissues and products of their processing is described. The method involves determination of the difference between the concentrations of Cu²⁺in solution before and after interaction of copper with the substance in question. Copper was determined spectrophotometrically in the form of a copper–ammonium complex.     

A simple method for estimation of phospholipids in biological objects

A simple method to estimate phospholipids is elaborated. This method is based on determination of optical density of phospholipid-molybdate complexes in chloroform. The method is used for the quantitative determination of phospholipids in biomembranes, liposomes and blood serum without their preliminary extraction, as well as in chloroform, methanol and chloroform-methanol solutions. It is also modified for the phospholipids determination in chromatographic fractions on silufol plates.     

A simple and rapid method of determining the titer of herpes simplex virus antibodies in a study by immunoenzyme analysis of a single serum dilution

The result obtained in the study of the possibility of using the method for the determination of the titer of antibodies to herpes simplex virus by EIA techniques in a single dilution of the serum under test are presented. This method is based on the determination of the optical density of the serum titer (rcut) in different groups of sera with the use of the assay system, permitting the evaluation of the positive results obtained in the determination of their final dilution. The results obtained with the use of this method showed that error was 50% for high-titer sera, 60% for medium-titer sera and 30% for low-titer sera.     

Molecular identification of species and ploidy of Carassius fishes in Lake Biwa,using mtDNA and microsatellite multiplex PCRs

We established an efficient and reliable method to identify the species and ploidy of Carassius fishes in Lake Biwa (C. cuvieri, C. buergeri, and the latter’s polyploid) using a combination of multiplex mitochondrial DNA haplotype-specific PCR and ploidy determination based on nuclear DNA microsatellite PCR. The high accuracy of ploidy determination was confirmed by flow cytometry. This method can also identify triploid clonal lines and provide information about their genetic diversity and structure. The method is applicable to ecological and evolutionary studies of Carassius fishes in Lake Biwa throughout their entire life history.     

Measurements of polyamines and their acetylated derivatives in cell extracts and physiological fluids by use of an amino acid analyzer

A fast and sensitive method for the determination of free polyamines and their acetylated derivatives is presented. The separation is carried out on a Durrum DC-6A cation-exchange resin with an automated amino acid analyzer. The determination is based on a stepwise elution with a sodium chloride—sodium citrate buffer system. Detection is done by fluorescence of the o-phthaldialdehyde—polyamine conjugates. The sensitivity is in the picomole range. No prior purification step is needed. The method has been applied to cell extracts and urine samples.     

Spectrofluorimetric determination of certain biologically active phenothiazines in commercial dosage forms and human plasma

A validated simple and sensitive spectrofluorimetric method was developed for the determination of chlorpromazine hydrochloride, promethazine hydrochloride, trifluperazine hydrochloride, thioridazine hydrochloride, perazine maleate and oxomemazine. The method was based on condensation of malonic acid/acetic anhydride (MAA) under the catalytic effect of the tertiary amine moiety of the studied phenothiazines to provide a deep yellow to brown colour with green florescence. Relative fluorescence intensity of the products was measured at λ_exc 398 nm and λ_em 432 nm. Different variables affecting the reaction were studied and optimized. The method was successfully applied for the determination of the studied drugs in commercial dosage forms. The lower detection limits allowed the application of this method for the determination of the compounds in plasma as an example of a biological fluid. In addition, the method was considered specific for the determination of tertiary amines in the presence of primary and secondary amines; as a result, it was deemed suitable for the determination of the cited drugs in the presence of their degradation products resulting from N‐dealkylation or oxidation of the corresponding sulphoxides or sulphones. Copyright © 2012 John Wiley & Sons, Ltd.     

应用荧光实时定量PCR方法检测重组慢病毒滴度及其感染效率

慢病毒载体已经广泛应用于动物模型中基因治疗的研究和转基因动物的制备．而准确地测定重组慢病毒的滴度和感染效率是其关键步骤．通过荧光实时定量PCR的方法定量分析重组慢病毒的颗粒数以及病毒的活性滴度,并以GFP报告基因的方法作为对照来验证定量PCR方法的准确性．研究结果显示,应用荧光实时定量PCR法与GFP报告基因法测定得到的病毒活性滴度成正相关,而且前者可以更加准确地测定病毒滴度和病毒感染效率．     

Validated spectrofluorimetric method for determination of two phosphodiesterase inhibitors tadalafil and vardenafil in pharmaceutical preparations and spiked human plasma

A valid, sensitive and rapid spectrofluorimetric method has been developed and validated for determination of both tadalafil (TAD) and vardenafil (VAR) either in their pure form, in their tablet dosage forms or spiked in human plasma. This method is based on measurement of the native fluorescence of both drugs in acetonitrile at λ_em 330 and 470 nm after excitation at 280 and 275 nm for tadalafil and vardenafil, respectively. Linear relationships were obtained over the concentration range 4–40 and 10–250 ng/mL with a minimum detection of 1 and 3 ng/mL for tadalafil and vardenafil, respectively. Various experimental parameters affecting the fluorescence intensity were carefully studied and optimized. The developed method was applied successfully for the determination of tadalafil and vardenafil in bulk drugs and tablet dosage forms. Moreover, the high sensitivity of the proposed method permitted their determination in spiked human plasma. The developed method was validated in terms of specificity, linearity, lower limit of quantification (LOQ), lower limit of detection (LOD), precision and accuracy. The mean recoveries of the analytes in pharmaceutical preparations were in agreement with those obtained from the comparison methods, as revealed by statistical analysis of the obtained results using Student's t‐test and the variance ratio F‐test. Copyright © 2015 John Wiley & Sons, Ltd.     

Preconcentration of pharmaceuticals residues in sediment samples using microwave assisted micellar extraction coupled with solid phase extraction and their determination by HPLC-UV

An analytical method combining microwave assisted micellar extraction (MAME) and solid phase extraction (SPE) has been developed to extract and preconcentrate a selected group of eight pharmaceutical compounds in sediment samples prior to their determination using liquid chromatography with an UV-DAD detector. A non-ionic surfactant, Polyoxyethylene 10 lauryl ether (POLE) was used for the MAME extraction and the different parameters for the optimization process were studied. Then, SPE was used to clean-up and preconcentrate the target analytes in the extract, prior to their determination using HPLC-UV. The method was applied to the determination of the selected pharmaceuticals compounds in several kinds of sediment samples with different characteristics. Relative recoveries for spiked sediment samples were over 70% and relative standard deviations (RSDs) were under 11% for all recoveries tested. Detection limits between 4 and 167ngg(-1) were obtained. The method was validated using Soxhlet extraction procedure.     

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-l-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.     

A sensitive, fast, durable, highly selective method for the determination of amino acids and biogenic amines in the cerebrospinal fluid and other body fluids and tissues

A sensitive, fast, durable HPLC method with high resolution for the determination of amino acids and some biogenic amines is described. It allows the simultaneous determination of more than 40 substances in the cerebrospinal fluid or other body fluids or tissues. The method allows to measure both, the free and the conjugated amino acids. It detects 5 X 10(-13) g of most amino acids and measures the relevant substances in their physiological concentrations with less than 0.1 ml cerebrospinal fluid. The precision is 1-2%.     

Protein content and osmotic behavior of isolated fat cells

Fat cells isolated by a modification of the method of Rodbell were washed in protein-free buffers, which allowed determination of their protein content. Osmotic behavior and response to norepinephrine and insulin were unimpaired.     

Sensitive fluorimetric method for the determination of putrescine,spermidine and spermine by high-performance liquid chromatography and its application to human blood

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles.The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 ± 3.1, and spermine 8.4 ± 2.8 nmoles/ml packed erythrocytes. Putrescine is not present in normal human erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml.The polyamine content of the erythrocytes from patients with malignant neoplasm was significantly elevated.     

Spectrophotometric determination of acid mucopolysaccharides

The present report describes a novel spectrophotometric method for the quantitative determination of acid mucopolysaccharides based on the interaction of these macromolecules with the zirconyl ion. The method is simple, accurate, and involves the determination of the acid mucopolysaccharide molecules rather than their hydrolytic components (as in the case of existing methods of analysis). Substances, which are normally present in the acid mucopolysaccharide preparations (such as protein, glycoprotein, and nucleic acid), do not interfere with the determination.     

Use of spin probe in the determination of oxygen consumption in human neutrophils

A quantitative method of the oxygen consumption rate measurement by human blood neutrophils upon their activation is described. This method is based on the spin exchange determination between 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxyl spin probe and oxygen molecules from the ESR spectra. Method allows the determination of about 1 micromolar concentrations of oxygen.     

Immobilized-metal-ion affinity chromatography as a tool for the qualitative study of pepsinogen phosphorylation.

Affinity chromatography on immobilized Fe(3+) ions--immobilized-metal-ion affinity chromatography (IMAC) method--was used for the determination of pepsin and pepsinogen phosphorylation. IMAC is a very powerful method for detailed studies of proteins. Dephosphorylation of the pepsinogens and pepsins has no effect on their proteolytic ability. For this reason, the determination of proteolytic activity was used for the detection of pepsinogen (pepsin) presence in the collected fractions as a very suitable and specific method. Pepsins and their zymogens probably have the same amounts of phosphate ions in their molecule. The exact definition of conditions is very important for the prepurification of the proteinases and for their analysis.     

The determination of phospholipase D activity in emulsion systems

Although phospholipase D (PLD) is often used in emulsion systems consisting of buffer and a nonpolar organic solvent, most activity assays have been designed to work in purely aqueous milieu. Here a method is described for the determination of PLD activity in emulsion systems. The assay is based on the transphosphatidylation of phosphatidylcholine with 1-butanol in dichloromethane/buffer with the subsequent densitometric quantification of the products after their separation by HPTLC and staining with a CuSO4/H3PO4 reagent. The method is particularly appropriate for the determination of enzymes such as PLD from Streptomyces sp. that prefer the exchange of the head group in glycerophospholipids to their hydrolysis. Since the application of an organic solvent in the PLD assay allows the determination of the enzyme in analytes insoluble in aqueous media, the method can also be used to determine PLD activity in the presence of high concentrations of phospholipids.     

Characteristics of the precipitation reaction at the stage of antigen-antibody complex aggregate formation studied by a spectroturbidimetric method]

The possibility of using the spectroturbidimetric method, one of the variants of the light scattering method, for the determination of the characteristics of the second phase of the antigen-antibody reaction was shown examplified by the interaction between rabbit antiserum and bovine serum albumin. The parameters of the aggregates formed in the process (their average size, their concentration by weight and number) were calculated for the whole period of the reaction and with its components taken in different proportions. The precipitation curve plotted by the above method correlated well with the result of an independent determination at an extinction coefficient of E278. A conclusion was drawn, based on special evaluation, that hydrophobic interactions have a predominant influence on the formation of antigen-antibody aggregates. A quantitative approach was proposed for studying the mechanism of the antigen-antibody aggregate formation and for the analytical determination of various classes of immunoglobulins.     

Spectrofluorimetric determination of certain adrenergic agonist drugs in their pure forms and pharmaceutical formulations: Content uniformity test application

A new, simple, sensitive and rapid spectrofluorimetric method has been developed for determination of certain adrenergic agonists such as isoxsuprine hydrochloride, ritodrine hydrochloride and etilefrine hydrochloride in their pure forms and pharmaceutical dosage forms. The method depends on micellar enhancement of the native fluorescence of investigated drugs by using 2% w /v sodium dodecyl sulfate (SDS) as an anionic surfactant. The enhanced fluorescence intensity of investigated drugs was measured at 305 nm after excitation at 278 nm. The interaction of studied drugs with SDS was studied, and the enhanced fluorescence intensity was exploited to develop an assay method for the determination of investigated drugs. The relative fluorescence intensity–concentration plots were rectilinear over the range 0.15–3.00 μg ml^?1, with low quantification limits of 0.132, 0.123 and 0.118 μg mL^?1 for isoxsuprine, ritodrine and etilefrine, respectively. The proposed method was successfully applied for determination of studied drugs in their pharmaceutical formulations. Moreover, the high sensitivity of the proposed method allows performing the content uniformity testing of the studied drugs in their tablets by using the official United States Pharmacopeia (USP) guidelines. Statistical comparisons of the results with those of the reported methods revealed excellent agreement and indicated no significant difference in accuracy and precision.     

Method of isolating alveolar macrophages and their functional capacity

A number of modifications are suggested concerning the method for isolation of alveolar macrophages and the technique for their further handling. To study phagocyte function of alveolar macrophages in suspension, the modification of the method is described, which had been offered by the authors before for the determination of the index of complete phagocytosis by peripheral blood leukocytes.