Separation Of Analytes Research Articles

Forced Degradation and Stability-Indicating Study for the Binary Mixture of Allopurinol and Thioctic Acid Using Validated HPLC-DAD Method.

Careful review of the scientific databases revealed that no stability-indicating analytical method is available for the binary mixture of allopurinol (ALO) and thioctic acid (THA). A comprehensive stability-indicating HPLC-DAD procedure has been executed for concurrent analysis of ALO and THA. Successful chromatographic separation of the cited drugs was reached using a Durashell C18 column (4.6 × 250 mm, 5 µm particle size). The mobile phase consisted of a mixture of acidified water (pH 4.0) using phosphoric acid and acetonitrile pumped in gradient elution mode. For quantification of ALO and THA, their respective peak areas were recorded at 249 and 210 nm. A systematic validation of analytical performance was investigated in terms of system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. ALO and THA peaks emerged at retention times 4.26 and 8.15 min, respectively. Linear ranges for ALO and THA were 5-100 and 10-400 µg/mL, respectively, with correlation coefficient values exceeding 0.9999. Both drugs were exposed to conditions of neutral, acidic, and alkaline hydrolysis, oxidation, and thermal decomposition. Stability-indicating features have been demonstrated by resolution of the drugs from their forced degradation peaks. For verification of peak identity and purity, the diode-array detector (DAD) was used. In addition, degradation pathways for the cited drugs were postulated. Furthermore, separation of both analytes from about 13 medicinal compounds of different therapeutic classes disclosed optimum specificity of the proposed method. Advantageous application of the validated HPLC method for the concurrent analysis of ALO/THA in their tablet dosage form was accomplished. So far, the described HPLC-DAD method is considered the first detailed stability-indicating analytical study for this pharmaceutical mixture.

Development of HPLC system that uses phase-separation multiphase flow as an eluent.

We developed a new type of HPLC system that uses phase-separation multiphase flow as an eluent. A commercially available HPLC system with a packed separation column filled with octadecyl-modified silica (ODS) particles was used. First, as preliminary experiments, 25 kinds of mixed solutions of water/acetonitrile/ethyl acetate and water/acetonitrile were supplied to the system to act as eluents at 20°C. 2,6-Naphthalenedisulfonic acid (NDS) and 1-naphthol (NA) mixture was used as a model and mixed analyte was injected into the system. Roughly speaking, they were not separated in organic solvent-rich eluents and well separated in water-rich eluents, in which NDS eluted faster than NA. This means that HPLC worked under a reverse-phase mode for separation at 20°C. Next, the separation of the mixed analyte was examined on HPLC at 5°C, and then after judging the results, four kinds of ternary mixed solutions were in detail as eluents on HPLC at 20°C and 5°C. Based on their volume ratio, the ternary mixed solutions acted as a two-phase separation mixed solution, leading to a phase-separation multiphase flow. Consequently, the solutions flowed homogeneously and heterogeneously in the column at 20°C and 5°C, respectively. For example, the ternary mixed solutions containing water/acetonitrile/ethyl acetate at volume ratios of 20:60:20 (organic solvent-rich) and 70:23:7 (water-rich) were delivered into the system as eluents at 20°C and 5°C. In the organic solvent-rich eluent, the mixture of NDS and NA was not separated at 20°C but was separated at 5°C, the elution of NA being faster than the one of NDS (phase-separation mode). In the water-rich eluent, the mixture of analytes was separated at both 20°C and 5°C, the elution of NDS being faster than the one of NA. The separation at 5°C was more effective than at 20°C (reverse-phase mode and phase-separation mode). This separation performance and elution order can be attributed to the phase-separation multiphase flow at 5°C.

Rapid determination of malondialdehyde in serum samples using a porphyrin-functionalized magnetic graphene oxide electrochemical sensor

An electrochemical sensor based on a screen-printed carbon electrode (SPCE) modified with porphyrin-functionalized magnetic graphene oxide (TCPP-MGO) was developed for the sensitive and selective determination of malondialdehyde (MDA), an important biomarker of oxidative damage, in serum samples. The coupling of TCPP with MGO allows the exploitation of the magnetic properties of the material for separation, preconcentration, and manipulation of analyte, which is selectively captured onto the TCPP-MGO surface. The electron-transfer capability in the SPCE was improved through derivatization of MDA with diaminonaphthalene (DAN) (MDA-DAN). TCPP-MGO-SPCEs have been employed to monitor the differential pulse voltammetry (DVP) levels of the whole material, which is related to the amount of the captured analyte. Under optimum conditions, the nanocomposite-based sensing system has proved to be suitable for the monitoring of MDA, presenting a wide linear range (0.01–100 µM) with a correlation coefficient of 0.9996. The practical limit of quantification (P-LOQ) of the analyte was 0.010 µM, and the relative standard deviation (RSD) was 6.87% for 30 µM MDA concentration. Finally, the developed electrochemical sensor has demonstrated to be adequate for bioanalytical applications, presenting an excellent analytical performance for the routine monitoring of MDA in serum samples.Graphical abstract

Two-dimensional gas chromatography coupled to isotope ratio mass spectrometry for determining high molecular weight polycyclic aromatic hydrocarbons in sediments

The accuracy of compound-specific isotope analysis (CSIA) of trace-level pollutants in complex environmental samples has always been limited by two main challenges: poor chromatographic separation and insufficient amounts of analytes. In this study, a two-dimensional gas chromatography-isotope ratio mass spectrometry (2DGC-IRMS) system was constructed for compound-specific δ13C analysis of high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) in estuarine/marine sediments. This construction occurred through hyphenating an extra gas chromatography system (GC) to a conventional GC-IRMS using a commercially available multi-column switching-cryogenic trapping system (MCS-CTS). Compared with the previous 2DGC-IRMS strategy, which utilizes a Deans Switch device, the newly implemented 2DGC-IRMS scheme resulted in online purification of target analytes as well as enriched them online via duplicate injection and cryogenic trapping in CTS; this resultingly lowered the limits of detection (LOD) of CSIA. To improve the sample transfer efficiency to the IRMS, a broader-bore and longer fused-silica capillary was utilized to replace the original sample capillary running from the sample open split to the IRMS. A ẟ13C analysis of PAH standards showed accurate ẟ13C values, and high precisions (standard deviations 0.13–0.37%) were achieved, with the LOD of HMW-PAHs reduced to at least 1.0 mg/L (i.e., 0.07 to 0.09 nmol carbon per compound on-column). The successful application of this newly developed 2DGC-IRMS scheme provides a practical solution for the reliable CSIA of trace-level pollutants in complex environmental samples that cannot be measured using the conventional GC-IRMS system.

Comprehensive lipidome of human plasma using minimal sample manipulation by liquid chromatography coupled with mass spectrometry.

The present work shows comprehensive chromatographic methods and MS conditions that have been developed based on the chemical properties of each lipid subclass to detect low-abundance molecular species.This study shows that the developed methods can detect low- and/or very-low-abundant lipids like phosphatidic acid (PA) in the glycerophospholipid (GP) method; dihydroceramide (dhCer) and dihydrosphingosine/sphinganine (dhSPB) in the sphingolipid (SP)method; and lysophosphatidic acid (LPA), LPI, LPG and sphingosine-1-phosphate (SPBP) in the lysolipid method. An optimised method for the extraction of lysolipids in plasma is used in addition to Folch extraction. Then, four chromatographic methods coupled with mass spectrometry using targeted and untargeted approaches are described here. Three of the methods use a tertiary pumping system to enable the inclusion of a gradient for analyte separation (pumps A and B) and an isocratic wash (pump C).This wash solution elutes interfering compounds that could cause background signal in the subsequent injections, reducing column lifetime. Semi-quantitative values for 37 lipid subclasses are reported for a plasma sample (NIST SRM 1950). Furthermore, the methods presented here enabled the identification of 338 different lipid molecular species for GPs (mono- and diacyl-phospholipds),SPs, sterols and glycerolipids. The methods have been validated, and the reproducibility is presented here. The comprehensive analysis of the lipidome addressed here of glycerolipids, GPs, sterols and SPs is in good agreement with previously reported results, in the NIST SRM 1950 sample, by other laboratories.Ten lipid subclasses LPS, LPI, alkyl-lysophosphatidic acid/alkenyl-lysophosphatidic acid, alkyl-lysophosphatidylethanolamine/alkenyl-lysophosphatidylethanolamine, dhCer (d18:0), SPB (d18:1), dhSPB (d18:0) and SPBP (d18:2) have been detected using this comprehensive method and are uniquely reported here.

Simultaneous Quantification of Avermectins in Six Aquatic Foods by UHPLC/FLD with Precolumn Derivatization

In this study, a fast, concise and reliable ultra-high performance liquid chromatography-fluorescence (UHPLC/FLD) detection method for simultaneous quantification of avermectins (AVMs), including avermectin (AVM), ivermectin (IVM), emamectin (EMM), moxidectin (MOX) and doramectin (DOR) in six aquatic foods was established. Based on the QuEChERS pretreatment method, the samples were extracted with 0.2% (v/v) ammonia acetonitrile. N-methyl imidazole mixed with acetonitrile (1:1, v/v) and trifluoroacetic anhydride with acetonitrile (1:2, v/v) were used as derivatization reagents. The mobile phase consists of acetonitrile and water with a flow rate of 1.0 mL/min. An Infinity Lab Poroshell 120 EC-C18 column was used for optimum chromatographic separation of target analytes at 40 °C; the excitation and emission wavelengths were set at 365 nm and 465 nm, respectively. In six kinds of aquatic foods, the limits of detection (LODs) of AVM, IVM, EMM, MOX, and DOR were 2.7 μg/kg, 1.8 μg/kg, 2.1 μg/kg, 1.2 μg/kg, and 2.7 μg/kg, respectively, and the limits of quantification (LOQs) of AVM, IVM, EMM, MOX, and DOR were 5 μg/kg, 4.5 μg/kg, 4.5 μg/kg, 3.5 μg/kg and 5.0 μg/kg, respectively. The recoveries were all above 85.38% when the samples were spiked with the target compounds at the concentration level of 5, 10, 50, and 100 μg/kg. The intra-day and inter-day relative standard deviations (RSDs) were all less than 15%. This method considers the requirements of sensitivity, accuracy, and economics of the instrument.

Analytical quality by design oriented development of the UPLC method for analysing multiple pharmaceutical process intermediates: A case study of Compound Danshen Dripping Pills

This work presented the integration of the Analytical Quality by Design concept in developing a UPLC method for the quantification of saponins and phenolic acids in two Compound Danshen Dripping Pills intermediates of a concentrated extract and a supernatant. Prior knowledge and systematic risk analysis were conducted to identify potential critical method parameters (CMPs) that affected critical quality attributes (CQAs). A definitive screening design was employed to investigate the quantitative relationship among the intermediate type, CPPs, and CQAs. Intermediate type was added in the experimental design table as a categorical variable. After the models were built, the method operable design region (MODR) was calculated with an exhaustive search-Monte Carlo method. The CPP combinations with a 95 % or higher probability of reaching the preset standard were collected in MODR. The MODR robustness test was conducted with a Plackett–Burman design. The separation of analytes was achieved with a Waters Acquity HSS T3 (100 × 2.1 mm, 1.8 µm) column at 30 °C, using 0.05 % phosphoric acid and acetonitrile as the mobile phases in gradient elution at a flow rate of 0.40 mL/min. The elution was monitored at UV detection wavelengths of 280 nm and 203 nm. The optimized method was also validated. Good robustness, accuracy, precision, stability and repeatability, and linear regression (R > 0.9997) were obtained. All recoveries of the 9 compounds ranged from 97.8 % to 101.7 %, with an RSD of less than 3.49 %. Nine batches of Danshensu, protocatechualdehyde, rosmarinic acid, salvianolic acid B, salvianolic acid A, notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 were analysed. Danshensu was found to be the compound with the highest content.

Development and Validation of a Quantitative LC-MS/MS Method for CDK4/6 Inhibitors Palbociclib, Ribociclib, Abemaciclib, and Abemaciclib-M2 in Human Plasma.

The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, palbociclib, ribociclib, and abemaciclib, are standard-of-care agents for patients with hormone receptor-positive human epidermal growth factor receptor 2-negative metastatic breast cancer. In support of therapeutic drug monitoring and clinical pharmacokinetic studies, a liquid chromatography coupled with tandem mass spectrometry assay for the simultaneous quantitation of CDK4/6 inhibitors and the major active metabolite M2 of abemaciclib in human plasma has been developed. Analytes were extracted from 50 μL of human plasma by precipitating proteins with methanol and then collecting the supernatant. Reversed-phase high-performance liquid chromatography was performed for analyte separation using a biphasic gradient at a flow rate of 0.25-0.5 mL/min. The total run time was 9.5 minutes. The analytes were detected using MS/MS with electrospray ionization operating in positive ion mode. Validation according to the US Food and Drug Administration's guidance showed that the new assay produced accurate (94.7%-107%) and precise (within-run: 1.2%-8.2%; between-run: 0.6%-7.5%) measurements of all analytes over a concentration range of 5-2000 ng/mL. Overall, analyte recoveries were consistent (mean values: 110%-129%). The analytes were also stable in human plasma and the final extract under various storage conditions. Finally, the clinical applicability of the assay was confirmed by quantitation of all analytes in plasma samples obtained from patients treated with CDK4/6 inhibitors. Reproducibility of the measured analyte concentrations in study samples was confirmed successfully by incurred sample reanalysis. A sensitive liquid chromatography coupled with tandem mass spectrometry method to measure CDK4/6 inhibitors was developed and validated according to the Food and Drug Administration criteria. Quantitation of all analytes in clinical plasma samples confirmed that the assay is suitable for therapeutic drug monitoring and clinical pharmacokinetic studies of CDK4/6 inhibitors.

Analytical quality by design-based RP-HPLC method for quantification of pioglitazone and candesartan cilexetil in bilayer tablet and its forced degradation studies

Aim: The current project involves developing an RP-HPLC method for simultaneous quantification of Candesartan Cilexetil and Pioglitazone based on analytical quality by design (AQbD). Materials and methods: When analysed in the Design Expert application, the critical method parameters were systematically refined using Central Composite Design and contours were derived for significant variables. A contour plot has been used to discover the technique operable design region that governs response variation, which is then empirically tested. Results: Successful chromatographic separation of title analytes was achieved on kromasil C18 (150 × 4.6 mm, 5 µm) column at 30 °C with mobile phase comprising 60% 20 Mm Potassium dihydrogen orthophosphate and 40% acetonitrile (v/v), isocratic elution pattern, 0.9 mL/min flow rate, and UV detection at 220 nm. The linear model for Candesartan Cilexetil was from 4 to 24 µg/ mL and Pioglitazone at 7.5–45 µg/ mL, respectively. Conclusion: The method met all the ICH Q2 (R1) validation criteria. The current approach aided for analysing simultaneous drugs can be expanded into quantifying drugs in biological matrix predominance with maximum recovery.

Chemical analysis of electronic cigarette liquids (e-liquids) and direct nicotine quantitation using surface-assisted flowing atmospheric-pressure afterglow desorption/ionization mass spectrometry (SA-FAPA-MS).

Ambient desorption/ionization mass spectrometry (ADI-MS) has been widely used for direct analysis of real samples without sample preparation or separation. Studies on the quantification of low molecular weight compounds in complex matrices with ADI-MS remain scarce. In this paper, we report the application of surface-assisted flowing atmospheric-pressure afterglow mass spectrometry (SA-FAPA-MS) for fast qualitative screening of electronic cigarette liquid (e-liquids) ingredients and direct quantification of nicotine. The quantification approach is rapid, uses a deuterated D4-nicotine standard spike, and does not require a preceding chromatography step or other methods to remove the complex sample matrix. Selected e-liquids were directly applied on thin-layer chromatography (TLC) plate surfaces (normal phase (NP) silica, reversed phase (RP) modified silica, cyano (CN) modified silica, and dimethyl (RP2) modified silica) after dilution and internal standard spiking. The plates served purely as sample carriers and no analyte separation was performed. Promising qualitative results were obtained, demonstrating the ability to detect nicotine alkaloids using this approach and the ability to differentiate e-liquids based on their flavor variations. In addition, dimethyl- (RP2-) and cyano-modified (CN-) silica surfaces were selected for quantification based on performance results of previous studies. It was shown that results were in high accordance with high-performance liquid chromatography (HPLC) experiments with lowest deviations <3% on dimethyl surfaces. Additional quantitative experiments including a certified reference material achieved equally satisfying results with lowest deviations of -1.1% from the certified nicotine content. For nicotine, detection limits down to the fmol range (96 fmol on CN and 20 fmol on RP2) were obtained. A detailed comparison of glass surfaces with functionalized surfaces showed that the functionalized surfaces were superior in terms of sample application reproducibility, mass spectra quality, sensitivity, and information density. Thus, functionalized thin-layer surfaces are considered promising tools for both qualitative and quantitative ADI-MS analysis of complex samples.

Gel-Electrophoretic Separation of a Number of Synthetic Food Dyes with Following Determination by Spectrophotometric and Visual Method: Simply and Economically

An aim of investigation was separation of synthetic food dyes E 102, E 110, E 122, E 124, E 129, E 132 and E 133 by method of planar gel-electrophoresis with following detection and determination of analytes directly on gel plate. Agar-agar gel or polyacrylamide gel was used as carrier. The influence of electrophoretic buffer pH, amperage, voltage as well as time of electrophoresis on dyes separation in agar-agar gel was investigated. Changing of dyes mobility with pH changing was explained by analysis of their ionization constants. The results of dyes electrophoretic separation were evaluated directly on gel plate by spectrophotometric and visual methods. The metrological characteristics of dyes spectrophotometric quantification and visual semiquantification after analytes separation were evaluated. The metrological characteristics of visual detection and semi-quantification of dyes were evaluated on the basis of statistical approach and investigation of analyte detection probability distribution in the range of reaction unreliability. The suggested technique of electrophoretic separation and following spectrophotometric or visual determination of dyes was successfully checked in analysis of pharmaceuticals.

Flash Chromatography and Semi-Preparative HPLC: Review on the Applications and Recent Advancements over the Last Decade

Flash chromatography and Semi-Preparative High Performance Liquid Chromatography have showed themselves as promising separation tools. These techniques assist in the process of isolation, fractionation and purification of chemical, biological and pharmaceutical substances. The present article describes the recent advancements made in flash chromatography and semi-preparative HPLC techniques in the last decade with specific focus on natural products. This article highlights the basics, instrumentation, current advancements made to facilitate the separation, advantages and applications of these two techniques. Flash chromatography is a versatile tool for the rapid but efficient separation and purification of analytes in relatively pure form. Nowadays, there are even functionalized silica pre-packed cartridges with different silica weights and particle sizes that are available to use. Flash separation using dual columns can be used to enhance separation of complex mixtures and can be applied to numerous classes of compounds. The semipreparative techniques, despite their high cost, can be helpful in high purity separation and production of reference standards for the pharmaceutical industry. This comprehensive review presents a brief analysis of all the recent research employing these two techniques for varied applications. This review will help chromatography specialists to make the decision of applying these two techniques in order to accelerate their research and development journey.

Tuning the electrophoretic separations on a surface-accessible and flexible fibre-based microfluidic devices.

Electrophoresis on textile fiber substrates provides a unique surface-accessible platform for the movement, separation and concentration of charged analytes. The method employs the inherently inbuilt capillary channels existing within textile structures, which can support electroosmotic and electrophoretic transport processes upon applying an electric field. Unlike confined microchannels in classical chip-based electrofluidic devices, the capillaries formed by the roughly oriented fibers within textile substrates can impact the reproducibility of the separation process. Here, we report an approach for precise experimental conditions affecting the electrophoretic separation of two tracer solutes, fluorescein (FL) and rhodamine B (Rh-B) on textile-based substrates. A Box-Behnken response surface design methodology has been used to optimise the experimental conditions and predict the separation resolution of a solute mixture using polyester braided structures. The magnitude of the electric field, sample concentration and sample volume are of primary importance to the separation performance of the electrophoretic devices. Here, we use a statistical approach to optimise these parameters to achieve rapid and efficient separation. While a higher potential was shown to be required to separate solute mixtures of increasing concentration and sample volume, this was counteracted by a reduced separation efficiency due to joule heating, which caused electrolyte evaporation on the unenclosed textile structure at electric fields above 175 V cm-1. Using the approach presented here, optimal experimental conditions can be predicted to limit joule heating and attain effective separation resolution without compromising the analysis time on simple and low-cost textile substrates.

Gold nanoshells with magnetic cores and a urea-based receptor for SERS sensing of fluoride anions: experimental and computational study.

The study demonstrates that a combination of plasmonic nanostructures and artificial receptors can be applied for sensing small molecular species. Gold nanoshells containing magnetic cores are used as the SERS-active substrates, which opens the way for the development of multimodal contrast agents with applicability extended to sensing or for the separation of analytes by magnetic solid-phase extraction. Disubstituted ureas forming hydrogen-bonded complexes with certain anions can be employed as molecular sensors. In this case study, gold nanoshells with silica-coated Mn-Zn ferrite cores were prepared by a multistep procedure. The nanoshells were co-functionalized with an N-(4-mercaptophenyl)-N'-(4-nitrophenyl)urea sensor synthesized directly on the gold surface, and with 4-nitrothiophenol, which is adopted as an internal standard. SERS measurements were carried out with acetonitrile solutions of tetrabutylammonium fluoride (Bu4NF) over a concentration range of 10-10-10-1 mol L-1. The spectral response of the sensor is dependent on the fluoride concentration in the range of 10-5-10-1 mol L-1. To investigate further the SERS mechanism, a model sensor, N-(4-bromophenyl)-N'-(4-nitrophenyl)urea, was synthesized and used in Raman spectroscopy with solutions of Bu4NF, up to a molar ratio of 1 : 20. The spectra and the interactions between the sensors and fluoride anions were also studied by extensive DFT computations.

Florfenicol and Florfenicol Amine Quantification in Bull Serum and Seminal Plasma by a Single Validated UHPLC-MS/MS Method.

Florfenicol is a broad-spectrum antibiotic belonging to the amphenicols class that inhibits protein synthesis by binding to bacteria's ribosomal subunits. This drug is commonly used in veterinary medicine to treat bacterial infectious diseases in cattle, swine, poultry, and fish. The proposed method uses a quick protein precipitation with acetonitrile for the extraction of florfenicol and florfenicol amine in serum and seminal plasma, followed by analysis in UHPLC-MS/MS for their simultaneous quantification. A BEH C18 reversed-phase column was chosen for analyte separation, allowing to obtaining sharp and symmetrical peak shapes in a chromatographic run of just 3.5 min under programmed conditions. Two specific transitions were observed for each analyte, and florfenicol-d3 was used as the internal standard. The approach was fully validated in each matrix over ranges suitable for field concentrations of florfenicol and florfenicol amine, showing good linearity during each day of testing (R2 always >0.99). Excellent accuracy and precision were demonstrated, for both analytes, by calculated bias always within ±15% and CV% always below 15% at all QC levels tested. The satisfactory outcomes obtained during recovery, matrix effect, and process efficiency investigations in serum and seminal plasma confirmed the strength of the method for the quantification of target compounds. To our knowledge, this is the first LC-MS/MS-validated approach for the quantification of florfenicol and florfenicol amine in serum and seminal plasma and was successfully applied for the determination of their concentration-time profiles in bulls. This paves the way to understanding the pharmacokinetics of this antibiotic and its active metabolite in bull's seminal plasma, which will enable the design of more appropriate treatment protocols.

Performance of global retention models in the optimisation of the chromatographic separation (I): Simple multi-analyte samples

Conventional retention models lead to accurate descriptions of the elution behaviour from the fitting of data for single solutes or from a set of solutes, one by one. However, the simultaneous fitting of several solutes through a regression process that separates the contributions of column and solvent from those of each solute is also possible. The result is a global retention model constituted by a set of equations with some common parameters (those associated with column and solvent), whereas others, specific to each solute, differ for each equation. This work explores the possibilities, advantages, and limitations of global models when they are applied to the optimisation of chromatographic resolution. A set constituted by 13 drugs (diuretics and β-blockers) and a training experimental design of seven multi-linear gradients are considered. Since standards for all compounds were available, the optimisation based on global models could be compared with the conventional optimisation, which is based on individual models. In their current state, global models do not predict changes in elution order, but they do allow for incorporating additional solutes (e.g., new analytes or matrix peaks) with only one new experiment. This possibility is explored by extending the model for the 13 analytes to include 26 peaks associated with a contamination in the injector. The combination of individual and global models allows an optimisation where the effects of matrix peaks on the separation of analytes can be integrated.

A Simple and Validated Stability Indicating HPLC Method for Simultaneous Quantification of Brimonidine, Timolol and Benzalkonium Chloride in Anti-Glaucoma Ophthalmic Formulations

The combined dosage form of brimonidine, timolol and benzalkonium chloride is prescribed for lowering intraocular pressure in patients with ocular hypertension. As no analytical HPLC method reported for the estimation of brimonidine, timolol and benzalkonium chloride together in ophthalmic formulations, the present study was aimed to fulfill the gap identified in literature. The separation of analytes was achieved on Cosmosil C18 (250 × 4.6 mm; 5 μ id) as stationary phase, pH 4.3 acetate buffer and methanol in 30:70 (v/v) as mobile phase at 1.0 mL/min and UV detection at 227 nm. In this condition, well resolved, retained peaks were identified at 3.11 min for brimonidine, 4.86 min for timolol and 5.57 min for benzalkonium chloride. The method reports 0.5, 1.25 and 0.0125 μg/mL, respectively for brimonidine, timolol and benzalkonium chloride as LOD, which proves that the method have enough sensitivity levels for the detection analytes in samples. The method passes all the validation parameters as per the guidelines proved that the method was valid. The method shows less % degradation in various stress studies such as acidic, base, peroxide, thermal and UV light conditions and can effectively separate various stress degradation compounds and confirms the stability indicating nature of the method. The method applicability was assessed by analyzing the drug content in Combigan® ophthalmic drops and method reports the assay of 98.92%, 99.36% and 98.46% for brimonidine, timolol and benzalkonium chloride, respectively. Based on the results, it can be concluded that the method can adequately suitable for the separation and quantification of brimonidine, timolol and benzalkonium chloride and hence can be applicable for the routine analysis of brimonidine, timolol and benzalkonium chloride in ophthalmic formulations.

Глубокие эвтектические растворители в химическом анализе. Возможности и ограничения

Deep eutectic solvents (DES) are a mixture of two or more components, the melting point of which is significantly lower than the melting points of the starting materials. Environmental safety, ease of preparation in the laboratory, and low cost of DES have led to the fact that they have recently found increasing use in analytical chemistry. DES are most widely used in separation and concentration methods as effective extractants for the isolation of both hydrophobic and hydrophilic analytes from various objects of analysis. However, the possibility of their application is not limited to use only as extractants. This paper presents the classification of DES according to the criterion of their stability in the aqueous phase, the possibilities and examples of the separation of analytes from liquid, solid-phase and gaseous samples into the DES phase are also considered. In addition, various schemes for the implementation of liquid microextraction based on the principles of the formation and destruction of DES and methods for their automation based on the principles of flow methods are considered. The possibility of using eutectic compounds as effective dispersants in dispersive liquid extraction has been demonstrated. The possibility of simultaneous use of eutectic solvents both as extractants and as reagents for the derivatization of analytes to convert them into an analytical form directly in the extraction process, is shown. The possibility of modification of various sorption materials with eutectic solvents for the method of solid-phase microextraction, is shown. The paper touches upon the problems and limitations associated with the use of DES in chemical analysis, as well as the main prospects for its development.

Aptamer affinity-based microextraction in-line coupled to capillary electrophoresis mass spectrometry using a porous layer/nanoparticle -modified open tubular column

An aptamer affinity based microextraction column is developed to be directly in-line coupled to capillary electrophoresis-mass spectrometry (CE-MS) for analyzing mycotoxins in food samples. Single-stranded DNA aptamers for selective recognition of aflatoxin B1 (AFB1) and ochratoxin A (OTA) targets are co-immobilized via covalent bonds on the surface of the inlet end of a capillary, which is pre-modified with three-dimensional porous layer and gold nanoparticles to enhance the specific surface area and loading capacity. The outlet of the capillary is designed as a porous tip to serve as the spray source for injection to the mass spectrometry. All the necessary processes for pretreatment and analysis of a sample are accomplished in one injection, including aptamer affinity-based microextraction, CE separation and MS detection of analytes. AFB1 and OTA are simultaneously determined in a wide linear range with sample consumption of only 1 μL and the limit-of-detection as low as 1 pg/mL. The microextraction column exhibits excellent repeatability and stability, which can be used over 45 runs within a month with CE separation efficiency and only MS intensity slightly decreased. Mycotoxins in three kinds of cereal based infant foods are accurately analyzed using the proposed method. The study provides a robust and universal approach that would have potential applications in a variety of analytical fields based on selective molecular recognition coupling to CE-MS analysis.

Soluble Carbohydrates in Several Transylvanian Potato Cultivars

This paper is the first to report the soluble carbohydrate content at harvest for eight Transylvanian potato cultivars: Christian, Cumidava, Kronstadt, Riviera, Roclas, Rustic, Tampa and Zamolxis. The aim of this study is to explore the soluble carbohydrate composition of the above-mentioned cultivars, since such quantitative information is important for breeding programs, consumers and processing units. High performance liquid chromatography was used for analysis, separations being achieved using a Prominence Shimadzu system with a refractive index detector, under isocratic conditions with a mobile phase consisting of acetonitrile: water (80:20%) delivered at 1 mL/min; baseline separations of the target analytes were accomplished with an EC 250/4 Nucleodur 100-5 NH2 RP column in less than 10 min. The carbohydrate concentrations were found to range from 24.03 mg/100 g (Zamolxis) to 76.58 mg/100 g (Riviera) for fructose, while the corresponding range was from 52.78 mg/100 g (Zamolxis) to 232.97 mg/100 g (Riviera) for glucose and from 238.41 mg/100 g (Zamolxis) to 378.45 (Cumidava) for sucrose. Chromatographic data were then subjected to chemometric analysis; the association of these complementary techniques allowed a fast selection of cultivars with low-reducing carbohydrate content for food processing purposes-the cultivars Zamolxis, Kronstadt, Christian and Roclas were outlined exhibiting both the lowest reducing carbohydrate content and the lowest sucrose content.