New Method For Quantitative Determination Research Articles

Development of a spectrophotometric technique for the quantitative determination of ademol

According to statistics, craniocerebral injuries are often the cause of disability among the population. The choice of medicines for traumatic brain injuries is one of the most difficult problems in the complex treatment of such patients. Ademol (1-adamantylethyloxy-3-morpholino-2-propanol hydrochloride) is a promising medicinal substance that has a huge positive therapeutic effect of a protective act on the damaged brain and can be presented in medicinal preparations in different dosages. Therefore, the urgent task is the development of highly accurate, reliable, affordable, and fast methods of quantitative determination of ademol. The aim of the work is to study the optimal conditions for the “ademol – bromocresol green” reaction and to develop an express, sensitive and easy-to-implement method for the quantitative determination of ademol. Materials and methods. Bromocresol green (BCG) grade “chemically pure” and acetone grade “pure for analysis” were used as reagents and solvents. Analytical equipment: spectrophotometer Specord 200, electronic scales AVT-120-5DM, ultrasonic bath Elmasonic E 60H, class A measuring vessels. Results. A new method of quantitative determination of ademol by the spectrophotometric method was developed. The optimal conditions of the flow between the substance to be determined and the reagent have been studied, the concentration limits have been established, in which subordination to the basic law of light absorption is observed. A forecast of the complete uncertainty of the results of the specified method of quantitative determination was made to assess the correctness of the reproduction of the developed method in other laboratories. The proposed method is relevant according to the requirements of the State Pharmacopoeia of Ukraine. Conclusions. According to the experimental data, the method of quantitative determination of ademol can be correctly reproduced and suitable for use.

Validation Parameters as Applied to Methods for Quantification of Microorganisms in Medicinal Products

Validation/verification of microbiological methods is a prerequisite for quality control of non-sterile drugs. However, the use of existing procedures for validation/verification of analytical methods is challenging, since a number of factors, such as microorganism distribution in the sample, cell morphology, and metabolic activity of microorganisms contribute to the error in microbiological testing.The aim of the study was to assess the feasibility of using the microbiological method validation parameters for validation/verification of the agar plate method.Materials and methods: 18 non-sterile medicinal products were used in the study. Experiments included determination of antimicrobial activity. The quantification of viable bacteria, yeasts and moulds was performed using the modified pour plate method. The statistical processing of the obtained results was performed using Microsoft Excel 7.0 and Statistica 8.0.Results: the paper provides the results of quantitative determination of test microorganisms inoculated into non-sterile drugs. The results were obtained as part of validation/verification of the agar plate method of the State Pharmacopoeia of the Russian Federation, XIV ed.Conclusions: the validation/verification of the test method for isolation and quantification of microorganisms revealed no deviations of the study results from the established acceptance criteria. This proves the feasibility of using the following validation parameters: accuracy, precision, robustness, and limit of quantitation when validating new methods for quantitative determination of microorganisms or verification of previously validated methods.

New Method to Obtain Quantified Data on Color of Marine Sediments

A new method of quantitative determination of color using an original photodevice is presented for marine sediments, which are a specific geological object. The advantages of the photodevice over the main color-measured tool (colorimeter) are shown for the example of numerous sedimentary columns from the Pacific and Arctic oceans.

A novel arginine bioprobe based on up-conversion fluorescence resonance energy transfer

Arginine is an important amino acid in humankind bodies and is of essential clinical significance. This paper presents a novel bioprobe based on fluorescence resonance energy transfer (FRET), which can be used to detect arginine efficiently and economically. In this bioprobe system, positively charged up-conversion phosphor NaYF4 (NYF) acts as energy donor, and negatively charged gold nanoparticle (AuNP) acts as energy acceptor. The oppositely charged donor and acceptor come into close proximity through electrostatic attraction effect, which results in the occurrence of FRET between NYF and AuNP. The FRET process is thus in the “on” state, meanwhile the system is in the “off” state, and the emitting light of NYF quenched. When positively charged arginine is added into the system, the guanidyl of arginine binds to AuNP and leads to the negatively charged AuNP becomes positively charged one, and the AuNP separates from NYF because of the electrostatic repulsion. The FRET process is blocked and the system switches to the “on” state because the distance between NYF and AuNP becomes longer. In the “on” state, the intensity of the restored emitting light is proportional to the concentration of arginine. This approach brings a good linear relationship between the fluorescence intensity and the concentration of arginine in the concentration range of 14.42–115.04 μM. The limit of detection is as low as 2.9 μM. A new method for quantitative determination of arginine by just measuring the fluorescence intensity of the system is established.

Quantitation of Glutathione by Quinoline-5, 8-Dione-Based Tag Strategy Using MALDI Mass Spectrometry.

In the present work, we developed an UV-absorptive and highly reactive tag aromatic molecule, quinoline-5,8-dione (QLD), for robust quantitative analysis of GSH by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The QLD could react with GSH with high efficiency at room temperature, and the resultant QLD-GSH conjugate could be readily detected by MALDI MS without interferences. By using the QLD tag, the detection limit of GSH was lowered to 10fmolμL-1, which was four orders of magnitude higher than that detected without using the QLD tag. Furthermore, accurate quantitative measurements of GSH in solution were successfully demonstrated by using glutamic acid-cysteine-alanine (ECA) as an internal standard. By properly adjusting the ECA concentrations, the intensity ratio value of QLD-tagged GSH (QLD-GSH) to QLD-tagged ECA (QLD-ECA) displayed a good linearity with GSH concentrations in a broad range from 4 to 4000μM. Finally, the GSH level in HeLa cell lysates was also successfully detected, and the results are consistent with that obtained by a colorimetric assay. In summary, the proposed QLD-based tag method should be a rapid, cost-/time-effective, and sensitive new method for quantitative determination of GSH by MALDI MS. Graphical Abstract.

A new method for quantitative determination of renalase based on mass spectrometric determination of a proteotypic peptide labelled with stable isotopes.

Renalase is a recently discovered kidney secretory protein, which is considered as an important component involved in blood pressure regulation. Although altered levels of renalase have been detected in plasma and urine of patients with various kidney diseases, there is certain inconsistency of changes in the renalase levels reported by different laboratories. The latter is obviously associated with the use of the ELISA as the only available approach for quantitative analysis of renalase. Thus there is a clear need for the development of antibody-independent approaches for renalase quantification. We have developed a new method for quantitative determination of human renalase, which is based on mass spectrometric detection of a proteotypic peptide containing С-terminal 13 C15 N-labelled lysine. It corresponds to a tryptic peptide of human renalase, which has been previously detected in most mass spectrometric determinations of this protein. Using the labelled peptide H-EGDCNFVAPQGISSIIK-OH, corresponding to positions 100-116 of the human renalase sequence, as an internal standard and recombinant human renalase we have generated a calibration curve, which covered the concentration range 0.005-50ng/mL with a limit of quantitation of 5pg/mL. Using this calibration curve we were able to detect urinary renalase only after enrichment of initial urinary samples by ammonium sulfate precipitation (but not in untreated urine). Results of our study indicate that quantitative determination of renalase based on mass spectrometric detection of a proteotypic peptide labelled with stable isotopes gives significantly lower values of this protein in human urine than those reported in the literature and based on the ELISA.

Quantitative determination of interfacial copolymers from co-extruded films

A new method of quantitative determination of interfacial copolymers was investigated. Because of the small amount of these copolymers, the measurement is hard and only one technique is available: photoelectron X spectroscopy (XPS). However, as the method is an atomic response, it cannot be executed to any materials: copolymers have to contain different atom from the bulk. In this paper, we show that titration of interfacial copolymers polyamide 6-co-polypropylene-grafted anhydrid maleic (PA6-co-PP-g-AM) can also be done by Fourier Transform Infrared (FTIR). Concerning the quantitative determination of ethylene vinyl alcohol-polypropylene-grafted anhydrid maleic interfacial copolymers (EVOH-co-PP-g-AM), nothing is reported in literature. We show here, that FTIR can measure surface density of those copolymers provided that alcohol functions of EVOH are modified to ester groups.

New method for quantitative determination of water potential of mesophyll cells’ apoplast in substomatal cavity of the leaf

A new method for quantitative determination of water potential of mesophyll cells’ apoplast in substomatal cavity of the leaves of herbaceous (maize, millet, wheat, amaranth, and seepweed) and woody (larch, pine, and birch) plants by means of modern instruments designed to assess photosynthetic(СО2/Н2О) gas exchange is described. The method consists in determination in the light of such a level of humidity above the leaf surface, which would nullify transpiration without a noticeable suppression of photosynthetic assimilation of СО2; this makes it possible to calculate the value of water potential at the interface between aqueous and gaseous phases of mesophyll cells’ apoplast in the substomatal cavity.

Determination of fat content in goat milk using annular photoelectric sensor system

In this paper, a new method for quantitative determination of fat content in goat milk at room temperature (25°C), based on the annular photoelectric sensor system, has been developed. The measurement system consists of an annular photoelectric sensor, thermoelectric cooler (TEC), amplifier, A/D converter, microprocessor and other components. Based on Mie theory, the diffuse reflection light intensity is adopted as the optical parameter representing the fat content in goat milk. In this way, the standard model of the system can be established and loaded into the processor to realize data acquisition, processing and output. The proposed method has been tested on goat milk samples with variable fat content in the prediction experiment. The results show that the fitting equation is y=−0.366 x+0.295 ( R2=0.987) and measurement errors are within ±3%, which indicates that the presented method has high prediction performance and can measure the fat content in goat milk accurately and in real time.

Critical evaluation of a new method for quantitative determination of minerals in solid samples

The new method for the Chemical Quantitative Mineral Analysis (CQMA) is presented and critically evaluated. The percentages of minerals are calculated of the elemental bulk chemical analysis using identified minerals and their crystallochemical formulas. For this calculation an optimization procedure is used. CQMA enables also the feedback calculation of the bulk chemical analysis from CQMA calculated mineral contents. Subsequent comparison of the feedback calculated bulk chemical analysis and actual bulk chemical analysis of the same sample is used for critical evaluation of the input data. If necessary the input data are completed and/or reanalyzed and used for repeated calculation to achieve better match between the feedback calculated and actual chemical analyses, which consequently leads to more accurate mineral analysis. Moreover the feedback bulk chemical analysis can be also separately calculated from any mineral analyses determined e.g. by XRD, FTIR and/or optical method. Six German rock reference samples representing a very wide range of minerals and chemical composition of materials were selected and their mineral analyses carried out by CQMA and Rietveld X‐ray diffraction methods. The results were compared with reference mineral analyses and critically evaluated.

Contributions of ε and α′ TRIP Effects to the Strength and Ductility of AISI 304 (X5CrNi18-10) Austenitic Stainless Steel

The deformation-induced processes by tensile loading of X5CrNi18-10 austenitic stainless steel in the temperature range of 77 K to 413 K (−196 °C to 140 °C) were investigated. The results were presented in the form of stress–temperature-transformation (STT) and strain–temperature-transformation (DTT) diagrams. The thermodynamic stability of the austenite with respect to the e- and α′-martensite transformations was reflected in the STT and DTT diagrams. Furthermore, conclusions could be drawn from the transformation diagrams about the kinetics of stress- and strain-induced martensitic transformations. The diagrams laid foundations for the development of a new method of quantitative determination of strength and elongation contributions by means of induced and often overlapping deformation processes in the austenite. In this context, the plastic strains contributed by the glide and shearing of austenite were quantified and presented in connection with the e and α′ TRansformation-Induced Plasticity effects. Each deformation process was shown to have made a contribution to the strength and ductility, with a magnitude proportional to its dominance. The summation of such contributions provided the tensile strength and the uniform elongation of the steel. In other words, tensile strength and uniform elongation could be derived from a rule of mixtures. The newly proposed method was capable of explaining the anomalous temperature dependence of uniform elongation in the alloy investigated.

New Method of Quantitative Determination of the Carbon Source in Blast Furnace Flue Dust

The unconsumed pulverized coal and coke in flue dusts from two blast furances (BFs) were investigated in this paper. The percentage of unconsumed pulverized coal and coke were determined by means of chemical and petrographical microanalyses, which were time-consuming, requiring special expertise, and cannot be practiced routinely. Therefore, a simplified quantification method was proposed. The percentage of these materials in the BF flue dust samples was established on the basis of a suitable calibration using char and coke standards by Raman spectroscopy. The ratios of ID/IG, IV/IG, and I2D/IG were used to estimate constitutents of the BF flue dust. The amount of unconsumed pulverized coal in 1BF and 2BF was 9.7 and 11.6%, respectively, by means of petrographical microanalysis, and that in 1BF and 2BF established by Raman spectroscopy was in the ranges of 8–10 and 10.5–12%, respectively. The results showed that Raman spectroscopy was a relatively rapid and effective monitoring technique for the purpose of differentitation between coke and uncomsumed pulverized coal in BF flue dust.

Determination of the survival of yeast and bacteria under the influence of gamma or UV radiation in the presence of some scavengers of OH radicals

Two new methods for quantitative determination of the role of scavengers of OH radicals in the radiation protection of various microorganisms were studied. The yeast and bacteria were irradiated with gamma or UVC radiations having quite different effect on living cells. Both methods based on the counting of colonies arising from surviving cells proved to be able to describe quantitatively the protective effect of scavengers. The described method may be helpful for study of the radiation protection of cells, radiation stability of DNA or the permeability of the cell membrane.

Develpoment and evaluation of validation characteristics of the quantitative determination method for loratadine in the syrup

The problem of allergic diseases growth is quite urgent in the world: up to 40% of the adult population and 10-12% of children suffer from this disease. According to the data of epidemiologic studies, the most widespread diseases are bronchial asthma, allergic rhinitis and atopic dermatitis. Among avaliable antihistamine drugs loratadine is one of the most common, it is used for elimination of allergic manifestations both in adults and children. Since for adherence to specifications of the technological process when manufacturing medicines it is necessary to have the developed and validated methods for the quality control, we have proposed a new method for quantitative determination of loratadine in the syrup by the spectrophotometric method. When studying the character of loratadine spectra in alcohol, 0.1 М solution of hydrochloric acid and 0.1 М solution of sodium hydroxide it has been found that the best solvent is 0.1 М solution of hydrochloric acid. The assay was performed by the standard method at the wavelength of 278 nm. The effect of excipients of the syrup was removed by their extraction with ether. The validation characteristics of the method (robustness, accuracy and convergence, linearity, precision) have been determined. The evaluation of such validation characteristics obtained as accuracy (0.18% ≤ 1.54%), convergence (1.6% ≤ 3.2%), intermediate precision (0.62% ≤ 4.80%) allows to control the quality of loratadine in other laboratories according to the method recommended.

Application of Gas Chromatography/Mass Spectrometry to Therapeutic Monitoring of Valproic Acid

A new method for quantitative determination of the anticonvulsant valproic acid that was based on gas chromatography with mass spectrometric detection of three characteristic ionized fragments with m/z = 73, 102, and 115 was developed. The proposed technique was used to solve tasks of therapeutic anticonvulsant drug monitoring and applied to the determination of individual pharmacokinetic characteristics of valproic acid in patients at the Research Center of Neurology of the Russian Academy of Medical Sciences.

A new method for quantitative determination of dimemorfan in human plasma using monolithic silica solid-phase extraction tips

Dimemorfan was extracted from human plasma samples (100 μL) using MonoTip C(18) tips, which were packed with a C(18)-bonded monolithic silica gel attached to the inside of the tip. The samples, which contained dimemorfan and trimeprazine as an internal standard (IS), were mixed with 300 μL of distilled water and 50 μL of 1M glycine-sodium hydroxide buffer (pH 10). The mixture was extracted onto the C(18) phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C(18) phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.25 μm) gave adequate separation of the dimemorfan, IS, and impurities. The recoveries of dimemorfan and the IS spiked into plasma were ≥83%. The regression equation for dimemorfan showed excellent linearity from 0.25 to 32.0 ng/100 μL of plasma, and the limit of detection was 0.125 ng/100 μL of plasma. The maximum intra-day and inter-day relative standard deviations were 13%, while accuracy ranged from 88% to 105%. Dimemorfan was stable for at least 12 h at 4°C, 4 weeks at -80°C, and three freeze-thaw cycles in plasma. This new method is expected to have application as a pretreatment for the rapid, simple, and quantitative determination of dimemorfan in plasma samples.

A new method for quantitative determination of two uronic acids by CZE with direct UV detection

A new method using capillary zone electrophoresis was developed for the rapid quantification of two common uronic acids, galacturonic acid and glucuronic acid, based on utilization of an alkaline background electrolyte with reversed electroosmotic flow (EOF) within 16 min. The method relies on in-capillary reaction and direct UV detection at the wavelength 270 nm. The optimum electrolyte solution was prepared of 130 mm sodium hydroxide, 36 mm disodium hydrogen phosphate dihydrate and 0.5 mm cetyltrimethylammonium bromide. EOF was reversed to detect uronic acids and to improve the separation of neutral sugars. The established method was validated and the results showed good linearity, high precision and satisfactory sensitivity. The newly developed method was successfully applied to analyze galacturonic acid and glucuronic acid content in Forsythia suspensa polysaccharides. The method is fast since only sample hydrolysis and dilution are required in the sample preparation.

Effect of Particle Size of Forage in the Dairy Ration on Feed Intake, Production Parameters and Quantification of Manure Index

The objectives of this study were to measure particle size and evaluate the effect of increasing alfalfa hay particle size on production characteristics in lactating Holstein dairy cows. Ninety multiparous Holstein cows in early to mid-lactation were randomly assigned in a complete randomized design for a 30-day period. Animals were offered one of the three diets, which were identical in energy, protein, and chemical composition, but differed only in particle size of alfalfa hay. The treatments were A) total mixed ration (TMR) in which only fine chopped alfalfa hay was incorporated in the ration, B) the same diet in which half of the alfalfa hay was fine chopped and incorporated in the mixed ration and half was long hay and offered as a top dressing, and C) the same diet with long hay alfalfa offered as a top dressing. Distribution of particle size of rations was determined through 20,000, 8,000 and 1,000 μm sieves. The new method of quantitative determination of manure index was examined for each cow on different treatments. The geometric mean length of particle size in the rations was 5,666, 9,900 and 11,549 μm for treatments A, B and C, respectively. Fat corrected milk (4%), milk fat percentage and production were significantly different (p 0.05). Blood concentration of cholesterol in treatment A was significantly higher (p<0.05) than treatment B and C (181.0 vs. 150.0 and 155.2 mg/dl). Manure index in treatment C was significantly different (p<0.05) from treatment B (15.86 vs. 17.67). Based on these experimental findings, it is concluded that an increase in the ration particle size can increase milk fat percentage due to providing more physically effective fiber, which in turn could effect changes in manure consistency.

Analysis Method Study on 839 Pesticide and Chemical Contaminant Multiresidues in Animal Muscles by Gel Permeation Chromatography Cleanup, GC/MS, and LC/MS/MS

The paper reports the study of gel permeation chromatography (GPC), gas chromatography/mass spectrometry (GS/MS), and column chromatography tandem MS (LC/MS/MS) for 839 pesticides and chemical contaminants, through which a GPC data bank has been established for 744 pesticides, a GC/MS data bank for 541 pesticides, and an LC/MS/MS data bank for 464 pesticides. On the basis of this study, a new method for quantitative determination of 587 pesticide residues in animal muscles such as beef, mutton, pork, chicken, and rabbit has been established using GPC cleanup followed by GC/MS and LC/MS/MS. In the method, 10 g animal samples were mixed with 20 g sodium sulfate and extracted twice with 35 mL cyclohexane-ethyl acetate (1 + 1) by blender homogenization followed by centrifugation, filtration, and concentration. An equivalent of 5 g sample was injected into a 400 x 25 mm S-X3 GPC column, with cyclohexane-ethyl acetate (1 + 1) as the mobile phase at a flow rate of 5 mL/min. The 22-40 min fraction was collected for subsequent analysis. For the 478 pesticides determined by GC/MS, the portions collected from GPC were concentrated to 0.5 mL and exchanged twice with 5 mL hexane. For the 379 pesticides determined by LC/MS/MS, the portions collected from GPC were dissolved with acetonitrile-water (60 + 40) after taking the extract to dryness with nitrogen gas. At the limit of quantification (LOQ) and 10 LOQ fortification levels of 0.1-16 000 microg/kg, recoveries were within 40-130%, among which 563 pesticide recoveries were between 60 and 130%, accounting for 96% of the compounds; 24 analytes were recovered between 40 and 60%, accounting for 4% of the compounds. The relative standard deviation was below 30% for all 587 pesticides. The limits of detection for the method were 0.1-1600 microg/kg. In comparison with GC/MS, LC/MS/MS increased the detection sensitivity 2-1000 times for 236 pesticides.

Quantitative determination of the total content of flavonoids in Flavozid and Immunoflazid syrups

New syrup compositions Flavozid and Immunoflazid that contain 2% Proteflazid (ethanol extract of wild Deschampsia caespitosa L. and Calamagrostis epigeios L.) have been developed. The total content of flavonoids in both syrups is of the level of 4 – 6 gmL, which complicates the development of an analytical method. A new method for quantitative determination of flavonoids in the aforementioned syrups has been developed on the basis of differential UV spectrophotometry of their complexes with aluminum chloride. The proposed method is introduced into the Manufacturer’s Pharmacopoeic Article for the syrups. The existing formulation of Flavozid and Immunoflazid syrups is based on the active pharmaceutical ingredient Proteflazid, which was first developed and registered in Ukraine in 2001 and is the alcohol extract of wild Deschampsia caespitosa L. and Calamagrostis epigeios L. Proteflazid prepared using the original technology was recommended for use in medical practice as a composition with direct antiviral and immunomodulating action with properties of an interferon activator and DNA protease inhibitor in virus-infected cells [1 – 3]. The principal biologically active compounds of Proteflazid are flavonoids similar to quercetin (rutin), the molecules of which are based on a flavone oxygen-containing heterocycle. However, the mixture of Proteflazid flavonoids did not contain those that are frequently used as standards. Therefore, the development of a quantitative determination method for flavonoids in the syrup, where the content of Proteflazid and the corresponding flavonoids is 2% (or 4 – 6 gmL) of the starting extract, is of definite scientific interest. The goal of our work was to develop a method that would enable quantitative determination of total flavonoids, which absorb in the UV and visible spectral regions, using aluminum chloride as a complex-forming reagent. The glycoside species were converted to the aglycons by preliminary boiling of the syrup in the presence of HCl in order to increase the total amount of flavonoids in the analytes. Acid hydrolysis of the glucosides in the syrup was carried out in an excess of conc. HCl taken at 1% of the volume of the investigated syrup. The optimum conditions for acid hydrolysis with time were determined from the degree of conversion of the glycoside component into aglycons with TLC monitoring and UV spectrophotometric data. Aglycons from the syrup were extracted by ethylacetate. Solvent was vacuum distilled until production of a pasty residue that was dissolved in ethanol (96%). Studies of the optimum conditions for complexation using freshly prepared aluminum chloride (5%) in ethanol (96%) showed that increasing the volume of the solution to greater than 1 mL in the analyte did not improve the optical density parameters of the aluminum-chloride complex of the flavonoids. The maximum optical density was retained over 25 – 40 min at wavelength 422 nm. A solution with all components of the test solution with the exception of aluminum chloride was used as a reference to compensate for absorption at 420 – 424 nm of the starting extract before complex formation with aluminum chloride. Considering the complex flavonoid composition of the starting material, known standards from various flavonoid groups were used for the quantitative determination. However, the absorption maximum of the complex with aluminum chloride of none of them agreed with that for the com