Peak Retention Time Research Articles

TAILOR-MS, a Python Package that Deciphers Complex Triacylglycerol Fatty Acyl Structures: Applications for Bovine Milk and Infant Formulas.

Liquid chromatography tandem mass spectrometry (LC/MS) and other mass spectrometric technologies have been widely applied for triacylglycerol profiling. One challenge for targeted identification of fatty acyl moieties that constitute triacylglycerol species in biological samples is the numerous combinations of 3 fatty acyl groups that can form a triacylglycerol molecule. Manual determination of triacylglycerol structures based on peak intensities and retention time can be highly inefficient and error-prone. To resolve this, we have developed TAILOR-MS, a Python (programming language) package that aims at assisting: (1) the generation of targeted LC/MS methods for triacylglycerol detection and (2) automating triacylglycerol structural determination and prediction. To assess the performance of TAILOR-MS, we conducted LC/MS triacylglycerol profiling of bovine milk and two infant formulas. Our results confirmed dissimilarities between bovine milk and infant formula triacylglycerol composition. Furthermore, we identified 247 triacylglycerol species and predicted the possible existence of another 317 in the bovine milk sample, representing one of the most comprehensive reports on the triacylglycerol composition of bovine milk thus far. Likewise, we presented here a complete infant formula triacylglycerol profile and reported >200 triacylglycerol species. TAILOR-MS dramatically shortened the time required for triacylglycerol structural identification from hours to seconds and performed decent structural predictions in the absence of some triacylglycerol constituent peaks. Taken together, TAILOR-MS is a valuable tool that can greatly save time and improve accuracy for targeted LC/MS triacylglycerol profiling.

Bioseparation and Purification of Hepatotoxins Microcystin–LR from Cyanobacteria species Westiellopsis prolifica.

The presents study included bioseparation and purification of hepatotoxins microcystin-LR from cyanobacteria species westeillopsis prolifica the concentration of toxin was determined by preparative high performance liquid chromatography by comparing peak area and retention time of analytical standard of microcystin-LR with peak area and retention time of extraction of each species of cyanobacteria, the retention time of analytical standard of microcystin-LR were 9.55 min and it’s concentration was 10µg/ml, W. prolifica retention time was 9.5 min, it’s concentration was 28.385 µg/ml.

DEVELOPMENT OF METHODS FOR QUANTITATIVE DETERMINATION OF ALBENDAZOLE AND ITS METABOLITES IN BIOLOGICAL TISSUES USING HPLC / FLD

This manuscript presents the results of developed method is intended for clinical and pharmaceutical studies of veterinary drugs based on the active substances albendazole ([5-(propylthio)-1H-benzimidazol-2yl]carbamic acid methyl ester) and its main metabolites, albendazole sulfoxide, albendazole sulfone, and albendazole-2- aminosulfone in sheep muscles. Tissue samples were made alkaline with sodium carbonate, extracted twice with acetonitrile and degreased with hexane. The extracts are further purified using a series of liquid-liquid extraction and solid phase extraction. After concentration and drying, the dry residue was recovered in the mobile phase. Separation was performed on an inverted phase Acclaim 120 C18 column using acetonitrile and phosphate buffer as the mobile phase. The gradient mode of eluents was used during 12 min at a flow rate of 1,8 ml/min. The peak retention time of albendazole 2-aminosulfoam is 3,0 min, albendazole sulfoxide is 3,9 min, albendazole sulfone is 4,8 min, and the retention time of albendazole peak is 6,6 min. The specificity of the analytical method was checked by comparing the chromatographic separation of a sample of muscle tissue enriched with a standard solution of a mixture of albendazole and its metabolites at the level of MDR (100 μg/kg) and a sample of muscle tissue placebo. The validation parameters of the method “recovery” and “coefficient of variation” were considered in accordance with the criteria of Council Directive 2002/657/EC. The procedure of sample preparation of fortified tissues to construct calibration graphs is described in the manuscript. The mean recovery from fortified muscle tissue in the range of 50-150 μg/kg albendazole, albendazole sulfoxide, albendazole sulfone and albendazole 2-aminosulfon was 100.2; 100.9; 100.7 and 100.2%, respectively. The average coefficient of variation for each compound was ≤ 10%.
 The method is linear in the concentration range of 25.0 - 200.0 μg/kg of each analyte. The results obtained in the study of the linearity of this technique were used to estimate the correctness and convergence. The accuracy of the measurements was evaluated by examining the known amounts of analytes added to the control muscle tissue. Recovery data are acceptable because they are within ± 10% of the target value. The method has sufficient convergence (accuracy). The evaluation of the intermediate accuracy of albendazole and its metabolites was assessed on three different days of analysis. The limit of detection for albendasole is 0.4 μg/kg. The average CV for each compound was <10%.
 The procedure was confirmed and then applied to determination albendazole and its metabolites in the sheep muscle tissue obtained after feeding animals with the veterinary drug albendazole. The HPLC/FLD method can be used for withdrawal time albendazole and its metabolites.

Chromato-mass spectroscopy of tinctures of Valeriana officinalis aerial part

Underground Valerian roots (Vаlеrіаnае radix) are officinal raw materials. More than 1000 compounds were detected in Valeriana officinalis L. s.l., of which no less than 500 natural substances of organic and inorganic origin were identified, among them – carbohydrates, amino acids, lipids, vitamins, enzymes, phenolic compounds, essential oil, iridoids, alkaloids, steroids. The aerial part of Valerian is a source of various flavonoids with pronounced antioxidant properties and specific pharmacotherapeutic activity.
 The aim of the research is to determine, by means of gas chromatography, component composition of Valerian tinctures, made of the aerial part of V. tuberosa L., V. stolonifera Czern (Kantserivska balka, Zaporizhzhia region, Ukraine) and V. officinalis L. (Łódź, Bydgoszcz, Poland).
 Materials and methods. Tincture samples of the aerial part of wild species V. tuberosa L., V. stolonifera Czern (Kantserivska balka, Zaporizhzhia region), and V. officinalis L. (the experimental site of ZSMU; Łódź and Bydgoszcz in Poland) were selected for experimental studies according to the traditional production recipe for making tinctures (1 : 5). Component composition of the tinctures was studied using a 7890B gas chromatograph with a 5977B mass spectrometer detector.
 Results. By means of chromato-mass spectrometry, component composition of the Valerian tinctures has been identified, namely: in the tincture prepared from the aerial part of V. officinalis (Łódź, Poland) 54 components have been identified, V. tuberosa – 50; V. stolonifera – 46 and V. officinalis (Bydgoszcz, Poland) – 30. The chromatogram analysis of four Valerian tinctures helped to identify 118 components, 5 of which were present in all studied tinctures; 12 components were present in three tinctures; 22 components have been found in two tinctures. It should be noted that 10 components have been found in the tinctures in large quantities, their content ranging from 10.91 % to 30.02 %.
 Conclusions. Analyzing the obtained data of the GC, it can be seen that Valerian tinctures differ both in quantitative and qualitative composition. From the aerial part of Valeriana officinalis, prepared in Łódź and Bydgoszcz in Poland, 17 components coincide. In the tinctures, prepared from herbs collected in Ukraine, 10 components coincide. The highest content of Butanoic acid, 3-methyl – from 4.01 % to 16.78 %; 9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)- – from 0.47 % to 7.09 %; Dihydroxyacetone – from 0.37 % to 5.69 % and Quinic acid – from 0.30 % to 4.66 % in all four tinctures by peak areas and retention time should be marked. In three tinctures Cyclododecane, 1,5,9-tris(acetoxy)- has been identified in amount of 30.56 % to 14.56 %. The obtained results confirm the expediency of using the aerial part of Valeriana officinalis as raw material with a large commodity yield and various contents of biologically active substances for further pharmacognostic researches for the purpose of creation of new medicines and phytopreparations

Effect of 18-Crown-6 on Oxytocin Stability in Aqueous Buffer Solutions.

In this study, the effect of 18-crown-6 on the stability of oxytocin in aqueous solution was explored. The study found that while 12-crown-4 and 15-crown-5 do not stabilize oxytocin, 18-crown-6 does have a stabilizing effect in citrate/phosphate buffer at pH 4.5. However, in acetate buffer at the same pH, the presence of 18-crown-6 had a destabilizing effect, possibly leading to a different degradation pathway. Both the stabilizing and destabilizing effects, depending on the buffer used, are concentration dependent where a higher concentration of 18-crown-6 is linked to a stronger effect. It is hypothesized that this effect may be linked to 18-crown-6 binding to the protonated ammonium group of oxytocin. Upon changing the mobile phase used in high-performance liquid chromatography experiments, we observed evidence supporting this binding hypothesis. When an acidic mobile phase was used (0.01% trifluoroacetic acid (TFA)), a partial shift in oxytocin retention time was observed for samples in acetate buffers in the presence of 18-crown-6 when using a 150 mm column (C18). The amount of the peak that shifted depended on the 18-crown-6 concentration used. A similar shift in oxytocin peak retention time was observed for samples in both acetate and citrate/phosphate buffers when using a 250 mm column (C18), but the peak completely shifted in those samples. When using an even more acidic mobile phase (0.1% TFA), the oxytocin peaks all had the same retention time again. Ultraviolet and nuclear magnetic resonance spectroscopy experiments also showed that the presence of 18-crown-6 has an observable effect on the resulting oxytocin spectra.

Extraction and quantitative determination of bile acids in feces

With rapid advances in gut microbiome research, fecal bile acids are increasingly being monitored as potential biomarkers of diet related disease susceptibility. As such, rapid, robust and reliable methods for their analysis are of increasing importance. Herein is described a simple extraction method for the analysis of bile acids in feces suitable for subsequent quantification by liquid chromatography and tandem mass spectrometry. A C18 column separated the analytes with excellent peak shape and retention time repeatability maintained across 800 injections. The intra-day and inter-day precision and accuracy was greater than 80%. Recoveries ranged from 83.58 to 122.41%. The limit of detection and limit of quantification were in the range 2.5–15 nM, respectively. The optimized method involved extracting bile acids from wet feces with minimal clean up. A second aliquot of fecal material was dried and weighed to correct for water content. Extracting from dried feces showed reduced recovery that could be corrected for by spiking the feces with deuterated standards prior to drying. Storage of the extracts and standards in a refrigerated autosampler prior to analysis on the LC-MS is necessary. Multiple freeze-thaws of both extracts and standards lead to poor recoveries for some bile acids. The method was successfully applied to 100 human fecal samples.

Phenolic Profile: Antimicrobial Activity and Antioxidant Capacity of Colocasia esculenta (L.) Schott.

The aim of the study was to investigate the presence of bioactive compounds in the ethanolic extract of C. esculenta by HPLC and GC methods. The identification of phytochemical compounds is based on the peak area, retention time and pharmacological actions. The HPLC chromatogram of ethanol extract showed the presence of 13 compounds namely, 6,2',4'-trimethoxyflavanone, benzoic acid, gallic acid, luteolin-6,8-C-diglucoside, catechin, chlorogenic acid , astilbin, quercitin, kampferol, and major compounds with high concentration of absorbance are , rutin, , vitexin, ellagic acid and caffeic acid. The GC analysis of petroleum ether of C. esculenta extract showed presence 31 saturated fatty acid ,volatile organic hydrocarbons and sesquiterpenes .The major components were determined as follows, saturated fatty acid, Stearic acid, Arachidonicacid, 8-Heptadecene, monounsaturated fatty acid, (S)-citronellic acid and volatile organic hydrocarbons, specifically, sesquiterpenes as D-Germacrene and Isoledene, The antimicrobial activity of ethanol extract showed characteristic zone of inhibition against six clinical pathogens. The highest zone of inhibition was observed at 100mg/ml concentration against , the (Gram Negative Bacteria) Proteus vulgaris (ATCC13315) at 15mm, while Escherichia coli (ATCC25955), at 13 mm and Pseudomonas aeruginosa (ATCC27853) at 12 mm , while the zone of inhibition against (Gram positive Bacteria ) Staphylococcus aureus (RCMB010010), at 14 mm, Enterococcus faecalis (ATCC 29212), at 12 mm ,and no effect against Bacillus subtitis (RCMB 015 (1) NRRL B-543) , when compared with the standard drugs Gentamicin . The antifungal activity against six Fungi, was Cryptococcus neoformans ( RCMB 0049001) at 16mm, Aspergillus fumigatus (RCMB 002008 ) at 11mm, Syncephalastrum racemosum RCM 016001 (1) at 10 mm, and Candida albicans (RCMB 005003 ATCC 10231 ) at 9 mm . The zone of inhibition was observed at 100 mg/ml concentration when compared with the standard drugs Ketoconazole with (MIC) 100 mg/ml . The antioxidant activities of the extracts were determined using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging . ethanol extract of C. esculenta exhibited with IC50 value (22 µg/mL), while the standard ascorbic acid with IC50 (11.4 µg/mL) .This result is promising The above findings may justify the medicinal uses of the plants. The results suggest that the leaves of C. esculenta are a good sources of bioactive compounds with potent antimicrobial and antioxidant substances.

Antibacterial effects of Pheretima javanica extract and bioactive chemical analysis using Gas Chromatography Mass Spectrum

Pheretima sp is an earthworm from the Oligochaeta group found mostly in Java. The characteristics has segments reaching 95-150 segments. Clitellum is located in segment 14-16. The body fluids contain protein, amino acids and various enzymes. The purpose of this study was to determine the composition of bioactive compounds and evaluate antibacterial activity. The method used was maceration, antibacterial test against Salmonella typhi and GCMS analysis to identify bioactive compounds. Antibacterial test showed the inhibition zone diameter ranged from 15 to 20 mm. The identification of bioactive compounds is based on the percentage area, percentage peak height, retention time, molecular weight and pharmacological action. GC-MS analysis showed the presence of 50 peaks of compounds. Bioactive compounds which are antibacterial are 1) Nitrogen oxide (N2O) (CAS) Nitrous oxide with an area 2.03%, height 7.36%, retention time 1.361, molecular weight 44.013 g/mol; 2) Acetic acid (CAS) Ethylic acid with an area 17.02%, height 29.03%, retention time 1.789, and molecular weight 60.05 g/mol; 3) Butanoic acid, 3-methyl-(CAS) Isovaleric acid with an area of 3.27%, height 2.04%, 3.456, molecular weight 102.13 g/mol; 4) 1,2-Benzenedicarboxylic acid, diethyl ester (CAS) with an area 0.95%, height 1.32%, retention time 36.306 and molecular weight 222.24 g/mol.

Spectral studies of Amaranthus tristis Linn. in Bioremediated Silk dyeing effluent with mixed biofertilizer inoculants.

Microbial degradation as a treatment, with the combination of mixed inoculants of the Biofertilizer of Pseudomonas sp., Azospirillium sp. and Rhizobium sp., was employed for the remediation of Silk dyeing effluent. Remediating studies was undertaken to assess the feasibility of the mixed biofertilizer inoculant source for degradation of the Azodye effluent from the Silk dyeing Industry. The Green leafy vegetable (GLV), Amaranthus tristis Linn used as investigational prototypical plant species is selected for examining the phytochemicals, functional groups and its compounds grown in the effluent and biotreated environment and compared. The laboratory scale investigation showed that leaves, stem and root of the Amaranthus tristis Linn was qualitatively analysed for 20 phytochemicals which was grown in the different treatments of raw effluent and the biotreated effluent and the results showed the phytochemicals on the effluent’s influence reduced from strong positive to trace amounts while recovered on the biotreated environment. The FTIR analysis of the GLV grown in effluent and biotreated environments on comparison resulted in the functional group Alkene rescued in the biotreated effluent environment compared to the effluent contaminated area. The HPLC analysis of methanolic extracts of A. tristis grown in fresh water has 6 peaks of retention time of 2.6, 3, 3.9, 4, 4.2, and 4.6 RT whereas GLV effluent had only one peak of retention time of 4.1 RT. In the GLV from biotreated environment have 4 peaks were found with the maximum percentage area of 95.2% which proves that the compounds are rescued in the biotreated environment and few active compounds were confirmed in GCMS analysis. The Soil analysis results also indicate that the biotreatment of mixed inoculant of biofertilizers in the biotreated soil had influence resulting in improved levels of Ca, N, P and K with 114, 213, 10.5, 268 kg/ha respectively in the mixed inoculant biotreated soil. Similarly the micronutrients suchas Fe, Mn, Cu and Zn ranges to 4.1, 20.22, 2.13, 1.13 ppm respectively in the mixed inoculant biotreated soil within the optimal range. The study revealed that mixed biofertilizer inoculant has the recovery effect on the Silk dyeing (Azodyes) effluents effective reducing the pollutant capacity thereby meeting the discharged standards.

Rapid Screening α-Glucosidase Inhibitors from Natural Products by At-Line Nanofractionation with Parallel Mass Spectrometry and Bioactivity Assessment

In this study, a novel at-line nanofractionation screening platform was successfully developed for the rapid screening and identification of α-glucosidase inhibitors from natural products. A time-course bioassay based on high density well-plates was performed in parallel with high resolution mass spectrometry (MS), providing a straightforward and rapid procedure to simultaneously obtain chemical and biological information of active compounds. Through multiple nanofractionations into the same well-plate and comparisons of the orthogonal separation results of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC), the α-glucosidase inhibitors can be accurately identified from co-eluates. The screening platform was comprehensively evaluated and validated, and was applied to the screenings of green tea polyphenols and Ginkgo folium flavonoids. After accurate peak shape and retention time matching between the bioactivity chromatograms and MS chromatograms, ten α-glucosidase inhibitors were successfully screened out and identified. The proposed screening method is rapid, effective and can avoid ignoring low abundant/active inhibitors.

Development and Validation of Simple, Rapid and Sensitive High- Performance Liquid Chromatographic Method for the Determination of Butenafine Hydrochloride

Aims: The current paper reports a simple, rapid, sensitive, accurate, and precise Reverse-phase high performance liquid chromatography (RP-HPLC) method with wide range of estimation to determine butenafine hydrochloride in nanosponges. This method has been validated as per ICH norms.
 Study Design: Experimental design with influence of variables such as mobile phase composition, flow rate, temperature and wavelength on the chromatographic peaks.
 Place and Duration of Study: Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj, Saudi Arabia between Jan 2020 and March 2020.
 Methodology: Separation was achieved by utilizing the most commonly used reverse phase column (C-18, 5 μm, 150 mm x 4.6 mm) set at 30ºC and quantified by UV detection at 280 nm after isocratic elution from a mobile phase (70:30 v/v of methanol: phosphate buffer pH 3.0) flowing at 1 ml/min.
 Results: A sharp and symmetrical peak was observed at 4.08 ± 0.01 minutes. The low variation in peak area and retention time (1.12% and 0.29%, respectively) and a high number of theoretical plates (>2000) indicated this method’s efficiency and suitability. The least square linear regression analysis (Y = 9265.5 X + 1961.4) showed excellent correlation (r2 = 0.999 ± 0.0003) between concentration and peak area of butenafine hydrochloride through a wide concentration range of 1–50 µg/ml. The limits of detection and quantification (LOD and LOQ) were 0.18 µg/ml and 0.57 µg/ml, respectively. The assay or determinations were accurate, precise and reproducible with mean accuracy and mean relative standard deviation of precision of 101.53 ± 0.43% and 0.51 ± 0.11% respectively.
 Conclusion: The developed RP-HPLC method was simple, sensitive, reproducible with wide range of estimation of butenafine hydrochloride in the nanosponges. The proposed method could be used for the analysis of butenafine hydrochloride in the conventional pharmaceutical formulations such as tablets, syrup, creams including novel formulations such as nanoparticles, nanosponges, nanoemulsions. The proposed method overcomes the specificity, sensitivity and reproducibility related issues of ultraviolet-visible spectroscopy.

A gas chromatography-flame ionization detection method for direct and rapid determination of small molecule volatile organic compounds in aqueous phase.

It is still difficult to directly detect low content of volatile organic compounds (VOCs) in water samples by gas chromatography (GC) because when water is the only solvent, it would result in the instability and poor repeatability of peak retention time and peak shape. The adverse effects of water on direct GC analysis of VOCs cannot be significantly reduced or eliminated by simply changing the detection condition of GC. However, it was found that the addition of methanol in samples to a certain final proportion, such as 50 or 75% (v/v), could greatly reduce or eliminate the adverse effects of water. By using 75% (v/v) methanol as a solvent, the standard curves of ethanol, acetic acid, acetone, and isopropanol with correlation coefficient (R 2) over 0.99 were successfully plotted by gas chromatography-flame ionization detection (GC-FID) in a certain concentration range, respectively. The results showed that the retention time and peak shape stability of ethanol, acetic acid, acetone, and isopropanol in aqueous solution were greatly improved by the addition of methanol to final concertation of 75% (v/v). To verify the practical application potential of this method, the method was applied to the detection of components in isopropanol fermentation wastewater. The results showed that the method has well applicability and reliability. The key points in the application of this method were also summarized. This GC analysis method would have a wider and better application prospect in the detection of water-soluble organic matters.

Exploring actinomycetes and endophytes of rice ecosystem for induction of disease resistance against bacterial blight of rice

Biocontrol agents and plant growth promoting microbes have emerged as promising tools for the management of plant diseases and in sustainable crop production. The present study was made to explore the potential metabolites of actinomycetes and endophytes of rice (Oryza sativa L.) and to assay the induced defence reactions in rice plants against bacterial blight (BB) of rice (Xanthomonas oryzae pv. oryzae). In-vitro studies revealed that few promising rhizospheric Streptomyces (S. fimicarius and S. laurentii) and endophytes (Pseudomonas putida and Metarhizium anisopliae) could suppress Xoo effectively in dual culture assay. P. putida + S. fimicarius + S. laurentii showed the highest (58.71%) inhibition of BB. Multiple growth promoting characteristics were assayed for these effective isolates. The effectiveness of the cell suspension (107 cfu/ml) of P. putida, M. anisopliae, S. fimicarius and S. laurentii in suppressing BB of rice was tested in planta by applying as seed treatment, root treatment, soil treatment and spray application. Lowest disease incidence was observed in plants treated with the combination of P. putida + S. fimicarius + S. laurentii (10.29%) as compared to other treatments. To understand the resistance mechanisms of rice plants against Xoo, few effective secondary metabolites were assayed where the total phenol content of the treated plants showed higher concentration (2.52%). Antibiotics were recorded at a higher peak of retention time, such as pyrisulfoxin B (12.26 min), APHE 4 (13.42 min), kanamycin C (22.22 min), nitracidomycin B (15.40 min), clavulanic acid (7.89 min), neothramycin A (16.22 min), nitracidomycin A (17.78 min) and furaquinocin E (24.74 min).

Zinc-protoporphyrin determination by HPLC with fluorescence detection as a biomarker of lead effect in artisanal pottery workers.

Lead (Pb) exposure compromises heme synthesis by inhibiting ferrochelatase, forming zinc-protoporphyrin (ZnPP). This study aims to validate a method for the determination of ZnPP by HPLC with fluorescence detection and apply this method to evaluate the extent of Pb exposure of artisanal pottery workers. Extraction procedures were tested using both nonacid and acid liquid-liquid extraction. The former presented a better chromatogram and recovery results. The validated method yielded a good resolution of ZnPP and its free form peaks with acceptable precision and accuracy. Total run time was 15 min and ZnPP peak retention time was 5.6 min. We applied this method to evaluate 39 potters (90% male), mean age 40 years (9-80). The medians (ranges) of blood lead, ZnPP and hemoglobin were 16.0 μg/dl (2.2-71.5), 12.6 μg/dl (4.6-279.8) and 15.1 g/dl (11.0-17.8), respectively. Significant differences were observed for blood lead according to gender, age range (>40 years), direct handling of lead oxide and years of occupation. Significantly higher levels of ZnPP were observed in male potters involved with lead glazing activity. The validated method was shown to be simple with one-step nonacid extraction, good sensitivity, reproducibility and accuracy. Our data shows that these laborers are dangerously exposed to Pb, reflecting the effect on the heme synthesis.

Exosomes derived from smooth muscle cells ameliorate diabetes-induced erectile dysfunction by inhibiting fibrosis and modulating the NO/cGMP pathway.

Erectile dysfunction (ED) is a major health issue among men with diabetes, and ED induced by diabetes mellitus (DMED) is particularly difficult to treat. Therefore, novel therapeutic approaches for the treatment of DMED are urgently needed. Exosomes, nanosized particles involved in many physiological and pathological processes, may become a promising tool for DMED treatment. In this study, we investigated the therapeutic effect of exosomes derived from corpus cavernosum smooth muscle cells (CCSMC‐EXOs) on erectile function in a rat model of diabetes and compared their effect with that of exosomes derived from mesenchymal stem cells (MSC‐EXOs). We incubated labelled CCSMC‐EXOs and MSC‐EXOs with CCSMCs and then observed uptake of the exosomes at different time points using laser confocal microscopy. CCSMC‐EXOs were more easily taken up by CCSMCs. The peak concentration and retention time of labelled CCSMC‐EXOs and MSC‐EXOs in the corpus cavernosum of DMED rats after intracavernous injection were compared by in vivo imaging techniques. Intracavernous injection of CCSMC‐EXOs was associated with a relatively high peak concentration and long retention time. Our data showed that CCSMC‐EXOs could improve erectile function in DMED rats. Meanwhile, CCSMC‐EXOs could exert antifibrotic effects by increasing the smooth muscle content and reducing collagen deposition. CCSMC‐EXOs also increased the expression of eNOS and nNOS, followed by increased levels of NO and cGMP. These findings initially identify the possible role of CCSMC‐EXOs in ameliorating DMED through inhibiting corporal fibrosis and modulating the NO/cGMP signalling pathway, providing a theoretical basis for a breakthrough in the treatment of DMED.

Determination of monosaccharides hydrolyzed from polysaccharides in activated sludge by ion chromatography-mass spectrometry with online pretreatment of column switching technique.

The simultaneous determination of monosaccharides present in the activated sludge would be crucial to understand the water treatment mechanism. Herein, an ion chromatography-mass spectrometry (IC-MS) with online pretreatment of column switching technique was proposed to analyze monosaccharides hydrolyzed from extracellular polysaccharides in the activated sludge. When the matrix was eliminated in the first dimension, monosaccharides were immediately identified by IC-MS. The improved ionization efficiency was achieved with the addition of T-joint prior to MS. During the performance test, our established method showed excellent detection limits ranging from 0.34 to 2.15μg/L for all sugar targets. Great linearity (R ≥ 0.9955) was also achieved using this method in the range from 0.01 to 5mg/L. Furthermore, the average recoveries were obtained between 84.82 and 113.46%. RSDs for peak areas and retention times were determined as 3.76% and 0.27%, respectively. Finally, this approach provided a rapid, convenient, and practical determination of monosaccharides in the activated sludge, which would be helpful for the analysis of monosaccharides derived from other biological samples.

Box-Behnken Design-Desirability Function Approach in Optimization of HPLC Method for Simultaneous Determination of Ibuprofen Along with Additives in Syrup Formulation.

The advantages of simultaneous analyses are a decrease in analysis time and a more economical use of solvents and reagents. It is desirable that HPLC analyses possess both short term and good resolution. The aim of the current study is to develop an HPLC method for the simultaneous determination of ibuprofen, sodium benzoate, methyl paraben and propyl paraben as preservatives, and sunset yellow as a colorant, in syrup formulation. To optimize chromatographic separation conditions, multi-response optimization using the Derringer's desirability function was employed for the development of a rapid and efficient HPLC method. The ranges of independent variables used for the optimization process were 50-60% (v/v) for acetonitrile, 5.0-7.0 for pH, and 1.0-2.0 mL/min for flow rate of the mobile phase. The effects of these variables on the output responses, such as critical resolution between sunset yellow and sodium benzoate and retention time of the last peak indicating analysis time of the method, were evaluated by statistical experimental design. Optimum conditions fixed for the simultaneous analyses were acetonitrile:phosphate buffer (60:40, v/v), pH 5.0, and a flow rate of 1.8 mL/min. The eluate was monitored using a photo diode detector set at 220 nm. Total chromatographic analysis time was approximately 3 min. The developed method validated as per International Conference on Harmonization guidelines was successfully applied for the determination of five compounds in their pharmaceutical formulation. This efficient method has isocratic elution system and can be used for routine analyses of these compounds in similar pharmaceutical products.

Analytical Method Development and Validation for the Estimation of Silodosin and Dutasteride in Capsule Dosage Form by RP-HPLC Method

A simple, rapid, accurate, efficient and reproducible reverse phase high performance liquid chromatography (RPHPLC) method was developed and subsequently validated for simultaneous estimation of silodosin and dutasteride capsules. The analysis was carried out using Unison C8 column (250 mm x 4.6, 5 μm particle size) at 40°C temperature using mobile phase buffer:Methanol:Acetonitrile (20%:40%:40% v/v/v) at flow rate 1.0 ml/min. Quantification was achieved with PDA detector at a wavelength of 275nm. The silodosin peak retention time was 2.4 mins and the dutasteride retention was 10.9 mins, so the total run time was set to 14mins. The linearity range of Silodosin and Dutasteride is 20-120 μg/ml and 1.25-7.5 μg/ml respectively. Linear regression found to be 0.999. The proposed method was validated according to International Conference on Harmonisation (ICH) guidelines. The results shown in the method was accurate, precise, sensitive and economical. Thus, the current study showed that the developed reverse-phase liquid chromatography method is sensitive and selective for the estimation of Silodosin and Dutasteride in Capsule Dosage Form

Integrated Analytical Quality by Design (AQbD) Approach for the Development and Validation of Bioanalytical Liquid Chromatography Method for Estimation of Valsartan.

The present studies describe the systematic development and validation of a simple, rapid, sensitive and cost-effective reversed-phase high-performance liquid chromatographic bioanalytical method for the estimation of valsartan in rat plasma employing analytical quality by design (AQbD) principles quality risk management was applied for identifying the critical method parameters (CMPs) and subsequently method optimization was performed employing Box-Behnken design by selecting mobile phase pH, flow rate and % organic modifier as the CMPs and evaluated for critical analytical attributes (CAAs) such as peak area, retention time, peak tailing and number of theoretical plates. The developed method was then transferred to bioanalysis, where liquid-liquid extraction process was used for separating the drug from rat plasma. The optimization of extraction process was performed with the help of face-centered cubic design by selecting centrifugation speed and centrifugation time as the CMPs for maximizing % recovery, signal-to-noise ratio and purity threshold of the drug peak after extraction as the CAAs. Optimum chromatographic solution was chosen by mathematical and graphical search techniques, and design space was demarcated. Validation studies performed for the developed method indicated linearity ranging between 5 and 100ng.mL-1, whereas accuracy and precision study showed good percent recovery (99-102%) along with % relative standard deviation within ±2%. Sensitivity evaluation revealed limit of detection and limit of quantification were found to be 0.76ng.mL-1 and 2.29ng.mL-1, respectively. In a nutshell, the present work demonstrates significant merits of AQbD approach for holistic process understanding and analytical method development and validation with enhanced robustness and performance.

Chromato-mass spectrometric characteristics of red clover and motherwort tinctures

Motherwort has a cardiotonic effect – slows the heart rate and increases the strength of heart contractions, antihypertensive – lowers blood pressure, has a calming and antispasmodic effect in disorders of the cardiovascular system: in the early stages of hypertensive heart disease, mild forms of angina pectoris, heart defects and Graves' disease.Red clover tincture is used for atherosclerosis, which is accompanied by headaches and tinnitus, but with normal blood pressure; it provides detoxification of the liver and the body as a whole by cleansing blood and lymph; improves bile flow; normalizes bowel activity.The purpose of the work is to determine, by means of gas chromatography, the component composition of red clover and motherwort tinctures.Materials and methods. Red clover grass (Trifolii pretense herba) was collected in Zakarpattia, Chynadiyovo village, in July 2019; motherwort grass (Leonuri herba) was collected on the experimental site of ZSMU in June 2019. The tinctures were prepared from fresh raw materials (1:5) by maceration, using 70 % ethanol as the extractant. The tinctures were examined using an Agilent 7890B gas chromatograph with a 5977B mass spectrometer detector.Results. 20 Components were identified with gas chromatography in red clover tincture, of which: 1 aldehyde, 2 ketones, 3 esters, 3 heterocyclic compounds, 3 acids, 4 alcohols, and 4 aliphatic carbohydrates.In the motherwort tincture 30 characteristic components were identified, related to: organic acids (3 compounds); ketones (4); esters (6); aliphatic carbohydrates (1); alcohols (2); heterocyclic compounds (2); glycosides (1); nitrogen-containing compounds (1); aromatic compounds (3); sesquiterpenoids (3); phenolic compounds (2); aldehydes (1); undetermined compounds (2).Conclusions. It was used a gas chromatograph with a mass spectrometric detector in this work. 30 components were identified in motherwort tincture, of which, considering the quantitative analysis of the total peak area and retention time, the following should be distinguished: RT 16.26 – n-hexadecanoic acid – 15.2 %; RT 17,695 – phytol – 13.66 %; RT 12,835 – ethyl-d-glucopyranoside –10.99 %; RT 6.451 – 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl – 3.98 %; RT 20.7581 – penten-3-one, 1-(2,6,6-trimethyl-1-cyclohexen-1-yl) – 3.42 %; RT 10.43 – benzaldehyde, 2-hydroxy-6-methyl – 3.04 %; RT 3.664 –1,2-cyclopentanedione –1.22 %.In red clover tincture 20 components were identified, of which, considering the quantitative content, the following should be distinguished: RT 13.921 – myo-inositol, 4-C-methyl – 50.03 %; RT 12.742 – ethyl-d-glucopyranoside – 4.83 %; RT 22,435 – γ-sitosterol – 3.84 %; RT 16.261 – n-hexadecanoic acid – 3.72 %; RT 17,696 – phytol – 1.56 %; RT 8.764 – ethanone, 1- (2-hydroxy-5-methylphenyl) – 1.39 %.The two components coincide – n-hexadecanoic acid and phytol. In quantitative terms, there were more of them in the motherwort tincture.The results of the study can be used to develop the technology of herbal preparations, which include the raw material of red clover and motherwort. The study also has practical importance in the identification of raw materials that are included in the herbal products.