Chromatographic Method Research Articles

Simultaneous HPLC determination of remdesivir and dexamethasone in the presence of metformin, sitagliptin, and glimepiride in a synthetic mixture and spiked human plasma

The COVID-19 pandemic has raised many questions regarding the control and therapy of type 2 diabetes and the higher risk of severe disease progression. One of the therapeutic regimens used in moderate and severe cases of COVID-19, endorsed by the World Health Organization, involves the administration of an antiviral medicinal product and a corticosteroid. The present study describes the development of a liquid chromatographic method for the simultaneous separation and quantification of Remdesivir and Dexamethasone in the presence of Metformin, Sitagliptin, and Glimepiride in a synthetic mixture. The developed method also allows determination of Remdesivir, Dexamethasone, and Glimepiride in spiked plasma samples, using Sitagliptin as internal standard. A mixture of acetonitrile and potassium dihydrogen phosphate buffer (pH 3) in a ratio of 45:55 v/v was used as а mobile phase on a C18 column. The recovery percentages from plasma ranged from 85.1 to 108.5%. The developed method can serve in routine quality control and clinical laboratory practice.

Effects of pharmacogenetics on pharmacokinetics and toxicity of doxorubicin in Egyptian breast cancer patients

This study investigates the impact of single nucleotide polymorphisms in genes (SLC22A16 and CBR1) involved in the pharmacokinetics and toxicity of doxorubicin (DOX) in Egyptian female patients with breast cancer. Patients administered DOX (60 mg/m2) for 4 cycles every 3 weeks. The peak DOX plasma concentration was measured using a validated chromatographic method. The genotyping for the selected SNPs, SLC22A16 T > C (rs714368), and CBR1 C > T (rs20572), was performed by RT-PCR. Patients were monitored for hematological and cardiac toxicities. The variant carriers of CBR1 C > T (rs20572) exhibited significantly higher DOX concentration, but no significant association to DOX-induced hematological toxicity. On the other hand, SLC22A16 T > C (rs714368) had no significant influence on DOX plasma concentration, but was significantly correlated with lower risk of neutropenia (OR 0.31, 95% CI 0.12–0.75, p = 0.01) and leukopoenia (OR 0.18, 95% CI 0.07–0.5, p = 0.001). DOX-related cardiotoxicity was correlated with the cumulative dose of DOX (R = 0.238, p = 0.017), but not with any of the two examined SNPs. Genetic polymorphisms in SLC22A16 and CBR1 may explain the inter-individual variations in DOX pharmacokinetics and toxicity. Using pharmacogenetic testing is important to customise drug therapy for cancer patients treated with anthracyclines.

Recent Advances in the Chromatographic Analysis of Emerging Pollutants in Dairy Milk: A Review (2018-2023).

Emerging pollutants (EPs) encompass natural or synthetic substances found in the environment that pose potential risks, but which have only recently been recognized or monitored. EPs consist of various categories, including pesticides, pharmaceuticals, hormones, mycotoxins, and endocrine-disrupting chemicals (EDCs). Through several pathways, EPs can access food, potentially leading to health impacts when safe concentrations are exceeded. Milk, being a highly nutritious food product that is heavily consumed by many consumers of different ages, is a crucial food matrix where EPs should be regularly monitored. In the literature, a large number of studies have been dedicated to the determination of different EPs in dairy milk, employing different analytical techniques to do so. Chromatography-based techniques are the most prevalent means used for the analysis of EPs in milk, demonstrating significant efficiency, sensitivity, and accuracy for this specific purpose. The extraction of EPs from a complex matrix like milk is essential prior to performing chromatographic analysis. This review comprehensively covers relevant research papers on the extraction and subsequent detection and determination of EPs in milk using chromatographic methods from 2018 to 2023.

The algal partnership is associated with quantitative variation of lichen specific metabolites in Cladonia foliacea from Central and Southern Europe

AbstractLichen specific metabolites (LSMs) have interesting biological activities and quantitative variations may be present intraspecifically. For example, variations in medullary fumarprotocetraric acid (FA) and cortical usnic acid (UA) were observed in the lichen Cladonia foliacea, but the mechanism of variation is not well understood. The current study aimed to characterise the quantitative variation of FA and UA and to investigate the association between lichen metabolite content and ecological / biological variables. Fungal and algal trees were constructed using fungal (nrITS, RPB2) and algal (nrITS) loci, respectively. Using a chiral chromatographic method, the contents of (-)-UA were determined in 29 C. foliacea specimens and range from 6.88 to 34.27 mg/g dry wt. The FA contents were lower and varied from 1.44 to 9.87 mg/g dry wt. Although the fungal tree showed two well resolved clades, no significant differences of UA or FA contents were found between the two fungal clades. However, a significantly higher UA/FA ratio as well as a unique habitat were found to be associated with specimens which contained the alga Asterochloris lobophora than those specimens associated other Asterochloris algae. Taking all predictive variables into account (i.e. substrate type, elevation, collection season, photobiont identity), the multivariate data analysis indicated that photobiont identity explained most of the variance of LSM contents in C. foliacea. Thus future LSM biosynthetic studies should take the photobiont into consideration when dealing with intraspecific quantitative variation.

Features of analytical control of glyphosate salts in preparative formulations

Goal. Development of a method of analytical control of glyphosate in preparative forms of herbicides.
 Methods. Determination of glyphosate was carried out by the chromatographic method of thin-layer chromatography (TLC) using Sorbfil plates with a thin layer of silica gel KSK adsorbent. The active substance was identified by the value of Rf, quantitative determination was based on the formalized dependence of the size of the area of the chromatographic zones on the amount of glyphosate.
 Results. Physicochemical properties of herbicides — electrolytes, to which glyphosate belongs, create special problems when developing methods for its determination in various matrices. In herbicides and desiccants, glyphosate (as a salt) usually takes the form of a deprotonated acid residue and a corresponding cation. Therefore, the determination of the active substance is carried out by the acid residue, which is important for the quantitative assessment of its content in the matrix both in the acid equivalent and in the form of the corresponding salt. The density of the preparative form serves, in particular, as a criterion for controlling the form of the glyphosate salt. The method is based on dissolution of the preparation form in 44% ethanol and quantitative determination by TLC method. The determination of the active substance is not hindered by other components of the preparations, derivatives of glycine and amines.The optimal conditions of analysis were chosen using the conceptual designs of the laboratory «Algorithm of chemical-analytical monitoring of pesticides» and «System of multi-quantitative determination of pesticides in matrices», according to which the main stages of the process are extraction/dissolution, purification of extracts, elution (chromatographic process), identification and quantitative analysis . Selective conditions for TLC determination — mobile phase with pH 9: distilled water + ethanol + aqueous ammonia (22 + 20 + 0.25 by volume), developing reagent: a specific complex of ninhydrin with cadmium acetate in an acidic medium. Quantitative determination is carried out taking into account the coefficients of conversion of the corresponding salt form to the acid equivalent.
 Conclusions. The use of the chromatographic method of thin-layer chromatography makes it possible to control the content of the active substance glyphosate in various salt formulations in preparative forms of herbicides and desiccants with high accuracy and reproducibility.

Fluid revisited: An overlook of a method for solubility measurements in supercritical fluids based on chromatography retention

Measuring solubility in supercritical fluids usually represents a technical challenge since it is a highly compressible media sensitive to sampling and processing. Several decades ago, a chromatographic method for measuring solubility in supercritical fluids was presented by Bartle et al based on a linear relationship between solubility and inverse retention coefficient in supercritical fluid chromatography. Despite clear advantages such as high productivity and simplicity of sample preparation the method received only a limited acceptance in the field. In this work, the method was re-evaluated using several model compounds and stationary phases in supercritical fluid chromatography in conditions close to the limits of method applicability. Effects of calibration approaches, flowrates, initial calibration data and chromatography sample solvents were tested. The method appeared to be surprisingly robust and reasonably precise even for compounds which do not fully comply with the requirements for method applicability. It could be considered as an express method of choice when large sets of solubility data at different pressures are required.

Whiter and Greener RP-HPLC Method for Simultaneous Determination of Dorzolamide, Brinzolamide, and Timolol Using Isopropanol as a Sustainable Organic Solvent in the Mobile Phase

A sustainable reversed-phase chromatographic method has been developed and validated for the simultaneous determination of three active pharmaceutical ingredients, dorzolamide, brinzolamide, and timolol, used to treat glaucoma. The eco-friendly solvent isopropanol has been used as an organic mobile phase constituent. According to the Hansen space green solvent selection tool, isopropanol has a G score of 6.5, comparable to ethanol, which has a G score of 6.6. The mobile phase consists of isopropanol: aqueous sodium acetate buffer (0.1 M, pH 4.25) in the ratio of 10:90 (v/v). The flow rate was maintained at 1 mL/min. Dorzolamide and brinzolamide were detected at 254 nm, and timolol was detected at 295 nm. A high-purity silica with a polymeric C18 modification column (150 × 4.6 mm, 5 µm particle size) was used for this separation. The three compounds were eluted within 8 min. The method was validated according to ICH guidelines. The calibration curves were linear in the range of 20–70 µg/mL, 40–140 µg/mL, and 20–70 µg/mL for dorzolamide, brinzolamide, and timolol, respectively. The LODs were found to be 1.61 µg/mL, 1.60 µg/mL, and 3.16 µg/mL for dorzolamide, brinzolamide, and timolol, respectively. Good accuracy and precision were obtained for the three compounds. The greenness and whiteness of the method were indicated using the AGREE, ChlorTox, and RGB12 tools.

Tri-chromic sustainable tools of derivative/discrete Fourier transform HPLC convoluted peaks: Application on LCZ696, an antihypertensive supramolecular complex, with its in process and degradation impurity analysis

The aim of this manuscript is to discuss the use of chemometrics approaches in handling the response data of HPLC to sustainable analytical chemistry procedure development. This was performed on FDA approved LCZ696, an antihypertensive supramolecular complex of sacubitril (SAC) and valsartan (VAL), with the in process and degradation impurity (BMP). Structure similarity between SAC and its in process and degradation impurity (BMP) makes it difficult for their simultaneous determination and need too much time and solvent consumption for their separation. It was found that convolution of the resulting derivative curves using 8-points sin xi polynomials (discrete Fourier functions) after derivative treatment of the chromatographic response data was beneficial in handling one of the most common chromatographic problems which is overlapping of the chromatographic peaks without complete resolution, thus, reducing time of analysis and solvent consumption. The chemometric data handling succeeded in analyzing LCZ696, with its closely related in process impurity in less than 4.5 min. reaching impurity detection limit of 0.5 % in presence of the parent drug, SAC, while the direct chromatographic measurement showed higher LOD and LOQ of BMP with poor recovery, outside the accepted range 98–102 %, ranging from 80.56 to 104.41. Moreover, the proposed chemometric methodology will be compared with other chromatographic methods in terms of greenness, blueness and whiteness (tri-chromic assessment) using Analytical Eco Scale, AGREE, Blue Applicability Grade Index (BAGI) and the up-to-date Chlortox scale in addition to the white RGB 12 model in order to ascertain the influence of chemometric methods in minimizing the amount of time spent conducting the analysis and reducing solvent, energy and waste consumption.

Generic and accurate prediction of retention times in liquid chromatography by post–projection calibration

Retention time predictions from molecule structures in liquid chromatography (LC) are increasingly used in MS–based targeted and untargeted analyses, providing supplementary evidence for molecule annotation and reducing experimental measurements. Nevertheless, different LC setups (e.g., differences in gradient, column, and/or mobile phase) give rise to many prediction models that can only accurately predict retention times for a specific chromatographic method (CM). Here, a generic and accurate method is present to predict retention times across different CMs, by introducing the concept of post–projection calibration. This concept builds on the direct projections of retention times between different CMs and uses 35 external calibrants to eliminate the impact of LC setups on projection accuracy. Results showed that post–projection calibration consistently achieved a median projection error below 3.2% of the elution time. The ranking results of putative candidates reached similar levels among different CMs. This work opens up broad possibilities for coordinating retention times between different laboratories and developing extensive retention databases.

Enantioselective analysis of the pesticide imazamox after in vitro permeability study in Caco-2 cells.

Imazamox (IMX), a chiral herbicide used in cereals and oilseed crops to control weeds, is commonly sold as a racemic mixture. Its enantiomers, being chiral compounds, may exhibit unique properties when exposed to chiral environments. While IMX enantiomers have been reported to degrade differently in soil and be toxic to some species, their effects on human systems remain poorly understood. This study utilized Caco-2 (human colon adenocarcinoma cell line) cells to assess the in vitro permeability of a racemic mixture of IMX and its isolated enantiomers. Additionally, the study aimed to evaluate whether the metabolite imazamox-O-desmethyl (IMX-D) forms during the permeability process. An enantioselective chromatographic method was developed, fully validated, and the apparent permeability values were obtained. The apparent permeability of rac-IMX, (+)-IMX, and (-)-IMX was determined to be 4.15×10-5 , 5.78×10-5 , and 7.33×10-5 cm s-1 , respectively. These findings suggest that IMX exhibits high intestinal permeability, with an enantioselective absorption for (-)-IMX as compared to (+)-IMX. Finally, the permeability study in Caco-2 cells revealed that the metabolite IMX-D was not generated.

Consumer insights into the at‐home liking of commercial beers: Integrating nonvolatile and volatile flavor chemometrics

AbstractConsumer acceptability of beers is influenced by product formulation and processing conditions, which impart unique sensory profiles. This study used multivariate techniques to evaluate at‐home consumer sensory acceptability of six commercial beers considering their style, fermentation type, and chemical composition. Samples included top‐fermented beers (American India Pale Ale and Stout) and bottom‐fermented beers (Pilsner, zero‐alcohol Pilsner, Vienna Lager, and Munich Dunkel). Beer consumers (n = 50) conducted sensory hedonic, check‐all‐that‐apply (CATA) and just‐about‐right (JAR) tests. Chemometric variables included iso‐alpha‐acids, hordenine, and volatile aromatic compounds, quantified by chromatographic methods, whereas bitterness units (IBU) were determined spectrophotometrically. Lager beers had higher acceptability than top‐fermented beer (p < .05) for all attributes. Light‐colored beers and medium‐height foams had the highest liking scores for visual sensory attributes. Higher concentrations of bitter‐tasting molecules, hordenine, and acidity decreased the liking scores of top‐fermented (Ale) beers, as a sensory penalty analysis suggested. In contrast, the most favored beers (Pilsners and Munich Dunkel) contained higher fusel alcohol esters linked to fruity aromatic notes. Although a low conversion rate of fatty acids into fruity esters was noted in nonalcoholic Pilsner, its overall liking score was not statistically different from the alcoholic version. However, consumers perceived the nonalcoholic Pilsner as less bitter than its alcoholic counterpart even when IBUs were nonsignificantly different. This study emphasized the significance of understanding beer chemometrics to comprehend consumer acceptability, highlighting the crucial role of bitter molecules. Hence, hordenine, acidity, and volatile contents provided additional and valuable insights into consumer preferences.

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION FOR THE QUANTITATIVE ESTIMATION OF SODIUM BUTYRATE IN TABLETS

Objective: To develop a simple, accurate, precise, and linear reverse-phase high-performance liquid chromatographic (RP-HPLC) method and verify the quantitative estimation (assay) of sodium butyrate in tablets. Methods: The optimized RP-HPLC method uses a reverse phase stationary phase which is YMC Triart C18 column, having specifications of 250×4.6 mm; 5 μm, a mobile phase composition of pH 8.0 sodium dihydrogen phosphate buffer and acetonitrile in the proportion of 92:8 v/v, flow rate of 0.5 ml/min, injection volume of 10 μL, and detection wavelength of 210 nm using a UV/PDA detector. Results: The developed method gave sodium butyrate eluting at about 6 min. Sodium butyrate exhibited linearity in the range of 119.56–1195.6 μg/ ml. The precision was exemplified by a relative standard deviation of 0.30%. The percentage of individual recovery was found to be in the range of 97.0 and 103.0 during accuracy studies. Conclusion: A simple, specific, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (assay) of sodium butyrate in tablets and hence this method can be explored for the analysis of sodium butyrate in tablets in various pharmaceutical industries.

Ecofriendly bioanalytical validated RP-HPLC method for simultaneous determination of COVID-19 co-prescribed drugs employing quality by design and green chemistry

Molnupiravir, dexamethasone, ertapenem, and remdesivir are among the most frequently co-prescribed drugs used for COVID-19 virus treatment. For the first time, a simple isocratic reversed phase-high performance liquid chromatographic (RP-HPLC) method employing a photodiode array detector has been developed and validated for their simultaneous determination in spiked human plasma using rivaroxaban as an internal standard by the aid of quality by design and in accordance with the criteria of green chemistry. The separation was carried out utilizing a BDS Hypersil ® C18 column (300 × 4.6 mm), 5 μm particle size as the stationary phase using mobile phase consisted of methanol: acidified water; adjusted at pH 4.0 with 0.1 M orthophosphoric acid; with ratio (70:30, v:v) delivered at flow rate 0.6 mL/min, temperature adjusted at 20 °C, photodiode array detector maintained at 240 nm. The method was validated according to bioanalytical FDA guidelines with respect to linearity, accuracy, precision, selectivity, recovery, and stability. The method was found to be linear in the range of 4.00–120.00 μg/mL for molnupiravir, 10.00–140.00 μg/mL for dexamethasone, 10.00–160.00 μg/mL for ertapenem and 4.00–120.00 μg/mL for remdesivir. It is worth mentioning that the previously mentioned ranges covered the Cmax of the drugs. Finally, the green analytical score has been evaluated, and it was found to be an excellent green analytical method based on analytical eco-scale system penalty points. Moreover, it was assessed by analytical GREEnness metric approach and green analytical procedure index tool.

Non-destructive chromatographic determination of surface area in reverse phase chromatographic columns

One of the critical but underutilized parameters that affect chromatographic separation is an adsorbent surface area (S.A.). We introduce a concept of direct determination of the total surface area of chromatographic columns by utilizing a nondestructive measurement of eluent component excess adsorption isotherm measurement and the application of the Standard Excess Adsorption Isotherm from acetonitrile-water and methanol-water binary mixtures for direct determination of the S.A. in chromatographic columns. This nondestructive technique was used to determine the surface area of five commercial columns, including three core-shell particle type columns (Kinetex-C18, Ascentis C18, and Hallo C18) and in-housemade C18 columns. The traditional low-temperature nitrogen adsorption (LTNA) technique was compared to the novel chromatographic method of nondestructive surface area determination for adsorbents modified with octadecylsilanes. The chromatographically determined surface area was in good agreement with the conventional techniques for all columns used. The difference between the three techniques is less than 10%. Columns were characterized in terms of adsorbent geometry (e.g., pore volume, column void volume, interparticle volume), ligands bonding density, and the amount of the packing material in a chromatographic column.

High-throughput method for Peptide mapping and Amino acid sequencing for Calcitonin Salmon in Calcitonin Salmon injection using Ultra High Performance Liquid Chromatography – High Resolution Mass Spectrometry (UHPLC-HRMS) with the application of Bioinformatic tools

BackgroundTandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic peptide drugs, and peptide drug products including side chain modifications in the Peptide drugs. This article explains a step-by-step guide to developing a high-throughput method using high resolution mass spectrometry for characterization of Calcitonin Salmon injection containing high proportion of UV-active excipients. MethodsThe major challenge in the method development of Amino acid sequencing and Peptide mapping was presence of phenol in drug product. Phenol is a UV-active excipient and reacts with both Dithiothreitol (DTT) and Trypsin. Hence Calcitonin Salmon was extracted from the Calcitonin Salmon injection using solid phase extraction after the extraction, Amino acid sequencing and peptide mapping study was performed. Upon incubation of Calcitonin Salmon with Trypsin and DTT, digested fragments were generated which were separated by mass compatible reverse phase chromatography and the molecular mass of each fragment was determined using HRMS. ResultsA reverse phase chromatographic method was developed using UHPLC-HRMS for the determination of direct mass, peptide mapping and to determine the amino acid sequencing in the Calcitonin Salmon injection. The method was found Specific and fragments after trypsin digest are well resolved from each other and the molecular mass of each fragment was determined using HRMS. Sequencing was performed using automated identification of b and y ions annotation and identifications based on MS/MS spectra using Biopharma finder and Proteome discoverer software. ConclusionUsing this approach 100% protein coverage was obtained and protein was identified as Calcitonin Salmon and the observed masses of tryptic digest of peptide was found similar with theoretical masses. The method can be used for both UV and MS based Peptide mapping and whereas the UV based peptide mapping method can be used as identification test for Calcitonin Salmon drug substance and drug product in quality control.

Determination of linearized pDNA template in mRNA production process using HPLC.

The recent clinical success of messenger RNA (mRNA) technology in managing the Covid pandemic has triggered an unprecedented innovation in production and analytical technologies for this therapeutic modality. mRNA is produced by enzymatic transcription of plasmid DNA (pDNA) using polymerase in a cell-free process of in vitro transcription. After transcription, the pDNA is considered a process-related impurity and is removed from the mRNA product enzymatically, chromatographically, or by precipitation. Regulatory requirements are currently set at 10 ng of template pDNA per single human dose, which typically ranges between 30 and 100 µg. Here, we report the development of a generic procedure based on enzymatic digestion and chromatographic separation for the determination of residual pDNA in mRNA samples, with a limit of quantification of 2.3 ng and a limit of detection of less than 0.1 ng. The procedure is based on enzymatic degradation of mRNA and anion exchange HPLC separation, followed by quantification of residual pDNA with a chromatographic method that is already widely adopted for pDNA quality analytics. The procedure has been successfully applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA) and can be considered a generic substitution for qPCR in mRNA in-process control analytical strategy, with added benefits that it is more cost-efficient, faster, and sequence agnostic.

Screening method for the analysis of Rhodamine B in chili powder

Rhodamine B is a synthetic dye known to enhance the visual appearance of chili powder. Due to its toxicity and carcinogenicity, chromatographic methods have been developed to monitor its presence in adulterated chili powder, but their assays are laborious, time consuming and expensive for screening purposes. The present studies propose an alternative for screening Rhodamine B in chili powder samples. The method combines thin layer chromatography (TLC) to solid surface room-temperature fluorescence spectroscopy. The scrape-dissolution procedure common to the instrumental analysis of TLC procedures was replaced with a fiber optic probe coupled to a commercial spectrofluorometer. The determination of Rhodamine B on the chromatographic plate is based on its retardation factor and maximum excitation and emission wavelengths. The limit of detection (1.9 ng.mL−1) and the limit of quantitation (5.2 ng.mL−1) are well below the usual contamination of Rhodamine B in adulterated foods.

Diagnostic Aspects of Paraquat in the Forensic Toxicology: A Systematic Review

Background: Paraquat (N, N-dimethyl-4,4-bipyridinium dichloride) is a nonselective, fast-acting, and contact chemical herbicide used extensively for weed control. It has high acute oral toxicity, the ability to accumulate in the lungs, and a high potential for pulmonary fibrosis after its intoxication. The present systematic review focuses on evaluating diagnostic aspects of paraquat (PQ) in forensic toxicology. Methods: Evaluation of the literature according to the following criteria: only human studies published from February 1971 to March 2022 which are in English on the following databases: 1) Medline/PubMed/MeSH search words: ((Methyl viologen [Title/Abstract]) OR (paraquat [MeSH Terms])) AND (forensic [Title/Abstract]); 2) Scopus Keywords related to the study aim included forensic toxicology, paraquat, Methyl viologen; 3) Web of Science. Keywords related to the study aim included forensic toxicology, paraquat, and Methyl viologen. Results: Thirty full-text articles were included. The results of our review indicate plasma and urine are more used for identifying PQ, and liver, lung, and gastric fluid are important in postmortem cases. Preparation methods, including liquid-liquid extraction (LLE), solid-phase extraction, and acetonitrile-precipitated protein, are often required for removing interfering substances. Chromatographic methods, among other analytical techniques, are more sensitive, specific, and applicable. Conclusion: Our review suggests that plasma, urine, and lungs should be prioritized in sampling. Solid-phase extraction has better recovery than LLE in many samples. Colorimetric methods are not used much today, and radioimmunoassay (RIA) has limited application despite its high sensitivity. Gas and liquid chromatography methods appear to offer the best approach for the analysis of PQ.

Automated determination of 8-OHdG in cells and tissue via immunofluorescence using a specially created antibody

Despite powerful DNA repair systems, oxidative damage/modification to DNA is an inevitable side effect of metabolism, ionizing radiation, lifestyle habits, inflammatory pathologies such as type-2 diabetes or metabolic syndrome, cancer and natural aging.One of the most common oxidative DNA modifications is 8-OHdG (8‑hydroxy-2′-deoxyguanosine), which is the most widely used marker in research and clinical diagnostics. 8-OHdG is easily and specifically detectable in various samples such as urine, plasma, cells and tissues via a large variety of methods like ELISA, HPLC, chromatographic methods, and immunochemistry.Formed by oxidation of guanine and being representative for the degree of DNA damage, 8-OHdG can be also used as biomarker for risk assessment of various cancers as well as degenerative diseases.Here, we present a highly specific, self-developed 8-OHdG antibody in successful comparison to a commercially one, tested in cells (FF95, HCT116, and HT22) and intestinal tissue, focusing on automatized evaluation via fluorescence/confocal microscopy.

Novel HPTLC method for simultaneous estimation from ternary mixture of chlorpheniramine maleate, dextromethorphan hydrobromide and phenylephrine hydrochloride in syrup formulations

A synergic antihistamine, cough suppressant, and decongestant combination of chlorpheniramine, dextromethorphan, and phenylephrine is used to treat acute respiratory infections caused by seasonal viruses. The effective qualitative and quantitative methods require the simultaneous measurement of a ternary combination in the pharmaceutical syrup dosage form. Therefore, a new, simple, fast and robust high performance thin layer chromatographic (HPTLC) method has been developed and validated for chlorpheniramine maleate (CPM), dextromethorphan hydrobromide (DEXO) and phenylephrine hydrochloride (PE). The chromatographic separation was carried out on precoated aluminium plates with silica gel 60 F254 as the stationary phase. Mobile phase used was chloroform: methanol: ammonia (2.5:7.5:0.3, v/v/v) for proper separation. The detection was carried out at 270nm wavelength in absorbance mode. Developed method was validated as per International Council for Harmonization (ICH) Q2 (R1) guideline. The linearity range is 400 to 1400ng/band for CPM, 3000 to 11500ng/band for DEXO and 1000 to 3500ng/band for PE with correlation coefficient ≥0.995. The consistent lower values of relative standard deviation (RSD, %) for precision and robustness study indicate the method reliability. The percent recovery ranged from 97.82 to 102.03% indicates the good accuracy of the method. The proposed method was complying for the analytical method validation parameters suggested by the ICH Q2 (R1) guideline. The method was found to be simple, rapid and reliable for the simultaneous estimation of CPM, DEXO and PE from its pharmaceutical syrup dosage form. The method was successfully applied to quantify these analytes from the several pharmaceutical syrup dosage form.