Development and Validation of an analytical method for the determination of Ofloxacin and Benzyl alcohol in a Pharmaceutical mixture

The aim of the study is to develop a validated, rapid, simple and low-cost method for estimating ofloxacin and benzyl alcohol in a combined dosage. Materials and methods: Materials which were used ofloxacin pure substance and benzyl alcohol pure substance which were determined by UV spectrophotometer using ethanol 95% as a solvent and a blank. Results and discussion: Ofloxacin shows the absorption maxima at 300.0 ± 2.0 nm in ethanol 95% with an apparent concentration absorptivity of (30 to 90) µg/ml and benzyl alcohol shows the absorption maxima at 312.0 ± 2.0 nm in ethanol 95% with an apparent concentration absorptivity of (60 to 200) µg/ml, it was found that in the selected range of application of the method there is a directly proportional relationship between the concentration of ofloxacin and benzyl alcohol respectively, in the measured samples and the analytical signal, linear dependence is characterized by high correlation coefficients (R> 0.999)for both of drugs, which is considered acceptable for establishing strict linearity. Conclusion: The method has been verified as it has been proven simple, fast, accurate and low-cost method and does not require any expensive equipment for the analysis of Ofloxacin and benzyl alcohol separately and in a pharmaceutical mixture.

Rutin and quercetin in common buckwheat and Tartary buckwheat flour / Rutin in kvercetin v moki iz navadne in tatarske ajde

Samples of common buckwheat (Fagopyrum esculentum Moench) and Tartary buckwheat (F. tataricum Gaertn.) were used in milling, sieving and analysing experiments. Rutin and quercetin were analysed in buckwheat samples, in milling and sieving fractions and after the contact of flour particles with water, to simulate conditions in dough. The concentration of rutin in Tartary buckwheat was 1.17–1.75% in dry matter, while it was only 0.003% in dry matter of common buckwheat. Thus it is in Tartary buckwheat in this case 400 times more rutin in comparison to common buckwheat. In buckwheat dough with the time after mixing flour and water, the concentration of rutin diminished, the time needed was different in common and Tartary buckwheat dough, and quercetin appeared instead. Immediately after the direct contact of flour particles of common and Tartary buckwheat with water the rutin concentration changed from 11.7 to 0.79 mg/100 g dry matter (DM), and quercetin appeared (5.7 mg/100 g DM), in comparison in initial flour the concentration of quercetin was only 0.6 mg/100 g DM. In common buckwheat dough the apparent concentration of rutin changed from initial 0.0258 mg/g to 0.0263 mg/g DM, and after one hour after the beginning of contact of flour with water rutin concentration changed to only 0.0005 mg/g DM). Keywords: common buckwheat, Tartary buckwheat, flavonoids, rutin, quercetin, milling, dough Izvleček Raziskovali smo vzorce navadne ajde (Fagopyrum esculentum Moench) in tatarske ajde (F. tataricum Gaertn.). Vzorce smo mleli, presejavali, pripravljali testo (mešanica moke in vode) ter izmerili vsebnost rutina in kvercetina. Tatarska ajda ima bistveno višjo vsebnost rutina kot navadna ajda. Vsebnost rutina v raziskovani tatarski ajdi je 1,17–1,75 % v suhi snovi (SS), v navadni ajdi ´siva´ pa le 0,003 %. V tatarski ajdovi moki smo izmerili okoli 400x več rutina kot v navadni ajdovi moki. Pri neposrednem stiku ajdove moke z vodo težko najdemo vzporednice med tatarsko ajdo in navadno ajdo in dogajanji v povezavi z rutinom v testu. Koncentracija rutina v testu se po določenem času (različen čas pri navadni in tatarski ajdi – 5 minut do 2 uri) močno zniža, pojavi se kvercetin. Pri neposrednem stiku moke z vodo se vsebnost rutina v tatarski ajdovi moki močno zniža že po prvih 5 minutah delovanja (z 11,7 na 0,79 mg/100 g SS), pojavi pa se kvercetin (5,7 mg/100 g SS), v vzorcu moke ga je le 0,6 mg/100 g SS. Pri neposrednem stiku moke iz navadne ajde z vodo vsebnost rutina v moki (vzorec S) naraste v prvi uri z začetnih 0,0258 mg/g na 0,0263 mg/g SS (v začetnem času nekoliko manj enakomerno), v drugi uri stika moke in vode pa koncentracija rutina močno pade (na 0,0005 mg/g SS). Ključne besede: navadna ajda, tatarska ajda, flavonoidi, rutin, kvercetin, mletje, testo

Flavonoid concentration in milling fractions of Tartary and common buckwheat

Common buckwheat (Fagopyrum esculentum Moench) and Tartary buckwheat (F. tataricum Gaertn.) samples were used in milling, sieving and analysing experiments. Flavonoids were analysed in buckwheat samples, in milling and sieving fractions and after the contact of flour particles with water, to simulate conditions in dough. In Tartary buckwheat, there was even more than 100-times higher content of flavonoids flour in comparison to respective fractions of common buckwheat flour. The highest concentration of flavonoids in milling fractions of Tartary buckwheat flour (granulation over 100 |im up to including 1000 |im) was established as 3.5-4.5% flavonoids/DM. Immediately after the direct contact of flour particles of common and Tartary buckwheat with water the apparent concentration of flavonoids rose (even for 100% or more) in the first 5-30 minutes of contact. After one hour, due to the degradation of flavonoids, their concentration decreased. Concentration of flavonoids are after 24 hours of contact of flavonoids with water in all milling fractions lower in comparison to the value after first 5 minutes of contact with water.

Kinetic analysis of transcellular passage of the cobalamin–transcobalamin complex in Caco-2 monolayers

We suggest a novel kinetic approach to quantifying receptor–ligand interactions via the cellular transport and/or accumulation of the ligand. The system of cobalamin (Cbl, vitamin B12) transport was used as a model, because Cbl is an obligatory cofactor, taken up by animal cells with the help of a transport protein and a membrane receptor. Bovine transcobalamin (bTC) stimulated the cellular accumulation and transcytosis of radioactive [57Co]Cbl in polarized monolayers of Caco-2 cells. The bovine protein was much more efficient than human TC. The transport was inhibited in a dose-dependent manner by the unlabeled bTC-Cbl complex, the ligand-free bTC, and the receptor-associated protein (RAP). This inhibition pattern implied the presence of a megalin-like receptor. Quantitative assessment of kinetic records by the suggested method revealed the apparent concentration of receptors in vitro (≈15 nM), as well as the dissociation constants of bTC–Cbl ( Kd = 13 nM) and RAP ( Kd = 1.3 nM). The data were used to estimate the effective luminal concentrations of TC-specific receptors in kidneys (3.8 µM) and intestine (50 nM), the tissues resembling polarized Caco-2 cells.

Quantification of Riboflavin and Pyridoxine in a Mixed Solution at a High Concentration by Fluorescence Spectrophotometry

A new method of quantifying the contents of riboflavin (RF) and pyridoxine (PY) in their mixed solution was introduced in this study. A mathematical model was established to calculate the actual concentration of PY (Z) based on the apparent concentrations of PY (Y) and RF (X), which were quantified directly when RF and PY were mixed together. First, a linear relationship was found between Y and Z with a high coefficient, which defines fluorescence quenching efficiency. Second, a curvilinear equation was established between the apparent concentration of X and the fluorescence quenching efficiency (k) of PY. The actual concentration of PY could be obtained by using the two equations. The established mathematical model was verified, and the relative error of the calculated PY value was below 2.5%. The upper limit of fluorescence spectrophotometry quantification was up to 20 μg/mL for both RF and PY. Compared with RP-HPLC, this method is convenient in terms of sample pretreatment, as well as saves organic solvents and time.

Bioaccumulation and Biomagnification of 2-Ethylhexyl-4-dimethylaminobenzoate in Aquatic Animals

2-Ethylhexyl-4-dimethylaminobenzoate (EHDAB) is a commonly used organic ultraviolet filter. The bioaccumulation and biomagnification of EHDAB were investigated in two aquatic animals, the larvae of midge (Chironomus riparius) and crucian carp (Carassius carassius), and the metabolic enzyme responses in fish liver were determined. EHDAB in the larvae of midge reached a steady state within 10 days of sediment exposure. The biota-sediment accumulation factors ranged from 0.10 to 0.54, and were inversely proportional to the exposure concentrations. The EHDAB-contaminated larvae were used to feed the crucian carp. Within 28 days of feeding exposure, the EHDAB levels in fish tissues gradually increased with the increase of the exposure concentration, exhibiting an apparent concentration-dependence and time-dependence. The liver and kidneys were the main organs of accumulation, and the biomagnification factors of EHDAB ranged from 8.97 to 11.0 and 6.44 to 10.8, respectively. In addition, EHDAB significantly increased the activities of cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase in the fish liver. Our results indicate that EHDAB may pose a risk of biomagnification in an aquatic environment and influence the biological processes of exposed organisms.

Circulating Fragments of N-Terminal Pro–B-Type Natriuretic Peptides in Plasma of Heart Failure Patients

BACKGROUND The use of nonstandardized N-terminal pro–B-type natriuretic peptide (NT-proBNP) assays can contribute to the misdiagnosis of heart failure (HF). Moreover, there is yet to be established a common consensus regarding the circulating forms of NT-proBNP being used in current assays. We aimed to characterize and quantify the various forms of NT-proBNP in the circulation of HF patients. METHODS Plasma samples were collected from HF patients (n = 20) at rest and stored at −80 °C. NT-proBNP was enriched from HF patient plasma by use of immunoprecipitation followed by mass spectrometric analysis. Customized homogeneous sandwich AlphaLISA® immunoassays were developed and validated to quantify 6 fragments of NT-proBNP. RESULTS Mass spectrometry identified the presence of several N- and C-terminally processed forms of circulating NT-proBNP, with physiological proteolysis between Pro2-Leu3, Leu3-Gly4, Pro6-Gly7, and Pro75-Arg76. Consistent with this result, AlphaLISA immunoassays demonstrated that antibodies targeting the extreme N or C termini measured a low apparent concentration of circulating NT-proBNP. The apparent circulating NT-proBNP concentration was increased with antibodies targeting nonglycosylated and nonterminal epitopes (P < 0.05). CONCLUSIONS In plasma collected from HF patients, immunoreactive NT-proBNP was present as multiple N- and C-terminally truncated fragments of the full length NT-proBNP molecule. Immunodetection of NT-proBNP was significantly improved with the use of antibodies that did not target these terminal regions. These findings support the development of a next generation NT-proBNP assay targeting nonterminal epitopes as well as avoiding the central glycosylated region of this molecule.

Analysis of Circulating MicroRNA: Preanalytical and Analytical Challenges

BACKGROUND There is great interest in circulating microRNAs (miRNAs) as disease biomarkers. Translating promising miRNAs into validated clinical tests requires the characterization of many preanalytical and analytical parameters. METHODS miRNAs were extracted from serum and plasma samples of healthy volunteers, and miRNAs known to be present in serum and plasma (miR-15b, miR-16, miR-24, and miR-122) were amplified by reverse-transcription quantitative PCR. Stability and the effects of hemolysis were determined. Assay variation and its components, including the effect of adding control miRNA, were assessed by nested ANOVA. RESULTS miRNA concentrations were higher in plasma than in serum. Processing of plasma to remove subcellular/cellular components reduced miRNA concentrations to those of serum. The miRNAs analyzed were stable refrigerated or frozen for up to 72 h and were stable at room temperature for 24 h. Hemolysis increased the apparent concentration of 3 of the miRNAs. The total variability of replicate miRNA concentrations was <2.0-fold, with most of the variability attributable to the extraction process and interassay imprecision. Normalizing results to those of spiked exogenous control miRNAs did not improve this variability. CONCLUSIONS Detailed validation of the preanalytical steps affecting miRNA detection and quantification is critical when considering the use of individual miRNAs as clinical biomarkers. Unless these causes of imprecision are considered and mitigated, only miRNAs that are extremely up- or downregulated will be suitable as clinical biomarkers.

Analysis of Gold in Solutions Containing Ionic Liquids by Inductively Coupled Plasma Atomic Emission Spectrometry

The analysis of gold by inductively coupled plasma atomic emission spectrometry (ICP-AES) in aqueous solution in the presence of up to 50% w/v of ionic liquid is reported. The ionic liquids investigated contain the 1-butyl-3-methyl-imidazolium (bmIm) cation with the anions Cl–, BF4–, HSO4–, or N(CN)2–. A facile route to the HSO4– salt is also described. The presence of ionic liquids alter the nebulization efficiency and sample transport properties, and the AES signal intensity and apparent concentration of gold in solution is usually suppressed as a result, principally, of increased viscosity of solutions containing an ionic liquid. However, the counterplay between a lower surface tension and a higher viscosity is illustrated by the results for the [bmIm][BF4] ionic liquid. The presence of this liquid at low concentrations causes an enhanced apparent concentration of gold whereas at higher concentrations the apparent concentration is diminished as the viscosity of the solution increases. Comparative data with simple sodium salts is also reported. Use of the standard addition method to compensate for matrix effects in the presence of ionic liquids is effective.