TLC-Densitometry for Determination of Omeprazole in Simple and Combined Pharmaceutical Preparations.

TLC combined with densitometry was used and chromatographic conditions developed to separate omeprazole and diclofenac sodium from their potential impurities. The development of the TLC-densitometry method is based on the elaboration of new chromatographic conditions allowing for the simultaneous determination of omeprazole and diclofenac sodium in a pharmaceutical preparation. Identification and quantification of omeprazole in simple and combined (with diclofenac) pharmaceutical preparations was performed on silica gel 60F254 using one mobile phase: chloroform-methanol-ammonia (36:4:0.60, v/v). Diclofenac sodium was determined in the presence of omeprazole after 2D separation on silica gel using two mobile phases of the first phase of chloroform-methanol-ammonia (36:4:0.60, v/v) and the second mobile phase cyclohexane-chloroform-methanol-glacial acetic acid (6:3:0.5:0.5 v/v). The developed method is simple, economical, specific, precise, accurate, sensitive, and robust, with a good range of linearity for the quantification of omeprazole and diclofenac sodium. TLC in combination with densitometry can be used as an effective analytical tool for quality control and quantitative determination of omeprazole in simple and combined pharmaceutical preparations containing diclofenac sodium. TLC in combination with densitometry can be recommended for the analysis of omeprazole and diclofenac sodium in the absence of HPLC or spectrophotometer in the laboratory or to confirm results obtained with other analytical techniques.

Use of TLC-Densitometric Method for Determination of Valproic Acid in Capsules.

Determination of valproic acid in the drug was carried out on the aluminum silica gel 60F254 plates and using acetone-water-chloroform-ethanol-ammonia at a volume ratio of 30:1:8:5:11 as the mobile phase, respectively. Two methods of detection of valproic acid were used. The first was a 2% aqueous CuSO4×5H2O solution, and the second was a 2',7'-dichlorofluorescein-aluminum chloride-iron (III) chloride system. The applied TLC-densitometric method is selective, linear, accurate, precise, and robust, regardless of the visualizing reagent used for the determination of valproic acid in Convulex capsules. It has low limits of detection (LOD) and limits of quantification (LOQ), which are equal to 5.8 µg/spot and 17.4 µg/spot using a 2% aqueous CuSO4×5H2O solution as visualizing agent and also 0.32 µg/spot and 0.97 µg/spot using a 2',7'-dichlorofluorescein-aluminum chloride-iron (III) chloride system as visualizing reagent, respectively. The described analytical method can additionally be used to study the identity of valproic acid in a pharmaceutical preparation. The linearity range was found to be 20.00-80.00 µg/spot and 1.00-2.00 µg/spot for valproic acid detected on chromatographic plates using a 2% aqueous CuSO4×5H2O solution and the 2',7'-dichlorofluorescein-aluminum chloride-iron (III) chloride system, respectively. A coefficient of variation that was less than 3% confirms the satisfactory accuracy and precision of the proposed method. The results of the assay of valproic acid equal 96.2% and 97.0% in relation to the label claim that valproic acid fulfill pharmacopoeial requirements. The developed TLC-densitometric method can be suitable for the routine analysis of valproic acid in pharmaceutical formulations. The proposed TLC-densitometry may be an alternative method to the modern high-performance liquid chromatography and square wave voltammetry in the control of above-mentioned substances, and it can be applied when other analytical techniques is not affordable in the laboratory.

A cost-effective and sensitive TLC-densitometric identification of meloxicam.

The influence of different chromatographic conditions and the process of spot visualization on determining the limit of detection as well as quantification (LOD and LOQ) of meloxicam by TLC-densitometric technique was estimated. Of all chromatographic conditions tested, the lowest limiting values, thus the best sensitivity, in the NP-TLC system was achieved on silica gel 60F254 and neutral aluminum oxide plates developed with the mobile phase consisting of ethyl acetate/toluene/n-butylamine (2:2:1, V/V/V). In the case of the RP-TLC method, a mixture of methanol/water (8:2, V/V) enabled densitometric detection of meloxicam at the lowest concentration level on RP-8F254 and RP-18F254 plates. Additionally, the smallest LOD value of meloxicam ensured crystalline violet and gentian violet as visualization agents on silica gel 60F254 and neutral aluminum oxide 150F254 plates, resp. Comparison of the densitometrically obtained spectra of meloxicam drug and its standard after the use of appropriate visualization agents could be a good and cheap alternative tool for the identification of meloxicam as an active pharmaceutical ingredient.

The Effect of Cyclosporine A on Dermal Fibroblast Cell - Transcriptomic Analysis of Inflammatory Response Pathway.

BACKGROUND: The first immunosuppressive drug - cyclosporine A (CsA) has many unquestioned merits in maintaining organ transplants in patients, as well as, in the treatment of many inflammatory diseases, also associated with cutaneous manifestations. The main task of this drug is to suppress the inflammatory response at the sites of action, which is not well known. OBJECTIVE: The objective of this study was to evaluate the influence of CsA in therapeutic concentration on the expression of genes associated with the inflammatory response pathway in normal human dermal fibroblasts (NHDF; CC-2511), and this study attempted to determine the mechanism of its action. METHODS: The cytotoxicity MTT test was performed. The expression of the inflammatory response pathway genes was determined using HG-U133A_2.0 oligonucleotide microarrays. Statistical analysis was performed by GeneSpring 13.0 software using the PL-Grid platform. RESULTS: Among the 5,300 mRNA, only 573 were changed significantly in response to CsA compared to the control fibroblasts (P≤0.05). CsA inhibited the expression of most genes associated with the inflammatory response in NHDFs. There were only 19 genes with a fold change (FC) lower than -2.0, among which EGR1, FOS, PBK, CDK1 and TOP2A had the lowest expression, as did CXCL2 which can directly impact inflammation. Furthermore, ZNF451 was strongly induced, and COL1A1, COL3A1, IL33, TNFRSFs were weakly up-regulated (FC lower than 2.0). CONCLUSION: The CsA in therapeutic concentration influences the genes linked to the inflammatory response (in the transcriptional level) in human dermal fibroblasts. The findings suggest that the potential mechanism of CsA action in this concentration and on these genes can be associated with a profibrotic and proapoptotic, and genotoxic effects.

Comparison of the Utility of RP-TLC Technique and Different Computational Methods to Assess the Lipophilicity of Selected Antiparasitic, Antihypertensive, and Anti-inflammatory Drugs.

The aim of this study was to assess the lipophilicity of selected antiparasitic, antihypertensive and non-steroidal anti-inflammatory drugs (NSAIDs) by means of reversed phase-thin layer chromatography (RP-TLC) as well by using Soczewinski-Wachtmeister's and J. Oscik's equations. The lipophilicity parameters of all examined compounds obtained under various chromatographic systems (i.e., methanol-water and acetone-water, respectively) and those determined on the basis of Soczewinski-Wachtmeister's and Oscik's equations (i.e., RMWS and RMWO) were compared with the theoretical ones (e.g., AlogPs, AClogP, milogP, AlogP, MlogP, XlogP2, XlogP3) and the experimental value of the partition coefficient (logPexp). It was found that the RMWS parameter may be a good alternative tool in describing the lipophilic nature of biologically active compounds with a high and low lipophilicity (i.e., antihypertensive and antiparasitic drugs). Meanwhile, the RMWO was more suitable for compounds with a medium lipophilicity (i.e., non-steroidal anti-inflammatory drugs). The chromatographic parameter 0(a) can be helpful for the prediction of partition coefficients, i.e., AClogP, XlogP3, as well as logPexp of examined compounds.

Simple and Accurate HPTLC-Densitometric Method for Assay of Nandrolone Decanoate in Pharmaceutical Formulation.

This study reports the development and validation of a new, simple, and accurate high-performance thin-layer chromatography (HPTLC)-densitomeric method for the determination of nandrolone decanoate in a commercially available injection formulation. Chromatographic analysis was performed on glass CN modified silica gel 60F254 plates developed using n-hexane-ethyl acetate in volume ratio 42.5:7.5 as the mobile phase. Densitometric scanning was carried out at the wavelength of 245 nm. This chromatographic system gave compact spot and a symmetrical peak of nandrolone decanoate with retardation factor (RF) value at 0.57 (±0.02). The linearity of this method with the high correlation coefficient of calibration plot ranges from 0.780 to 12.500 µg/spot. The developed method is characterized by good precision (coefficient of variation CV < 2%) and high accuracy close to 100.3% (R = 99.0%). Values of limits of detection and quantification equal to 0.231 and 0.700 µg/spot, respectively, confirm the sensitivity of the developed method. The analysis of the pharmaceutical formulation of nandrolone decanoate indicates drug content of 50.5 mg/mL and 101.0% in relation to the label claim. This is in good agreement with the recommendation of the International Council for Harmonisation (ICH) guidelines as well as the pharmacopoeial requirements. The low CV value (<1%) of nandrolone decanoate content in the tested injection formulation confirms the suitability of the proposed HPTLC-densitometric method for routine control of this compound in examined pharmaceuticals.

Application of Thin-Layer Chromatography in Combination with Densitometry for the Determination of Diclofenac in Enteric Coated Tablets.

Diclofenac belongs to the drug class non-steroidal anti-inflammatory drugs widely used in Europe as well as all over the world. Thus, it is important to conduct research on its quality control of available pharmaceutical preparations like for example enteric coated tablets. Among various analytical techniques, thin-layer chromatography (TLC) is ideal for this task due to their short time analysis, ease of operation and low cost. Hence, the aim of this study was to develop the optimal conditions of analysis and quantitative determination of diclofenac sodium in enteric tablets by using TLC in combination with densitometry. Of all chromatographic systems tested, the best is the one which consists of silica gel 60F254 and cyclohexane: chloroform:methanol:glacial acetic acid (6:3:0.5:0.5 v/v) as the mobile phase, which allows the successful separation of examined diclofenac sodium as active component and the largest number (twelve) of its degradation products as potential impurities of its pharmaceutical products. This indicates that the newly developed method is more effective than previously reported assays by Starek and Krzek. Linearity range was found to be 4.00-18.00 µg/spot for diclofenac sodium. The results of the assay of enteric tablet formulations equals 98.8% of diclofenac sodium in relation to label claim is in a good agreement with pharmaceutical requirements.

A Simple and Cost-Effective TLC-Densitometric Method for the Quantitative Determination of Acetylsalicylic Acid and Ascorbic Acid in Combined Effervescent Tablets.

A new, simple, and cost-effective TLC-densitometric method has been established for the simultaneous quantitative determination of acetylsalicylic acid and ascorbic acid in combined effervescent tablets. Separation was performed on aluminum silica gel 60F254 plates using chloroform-ethanol-glacial acid at a volume ratio of 5:4:0.03 as the mobile phase. UV densitometry was performed in absorbance mode at 200 nm and 268 nm for acetylsalicylic acid and ascorbic acid, respectively. The presented method was validated as per ICH guidelines by specificity, linearity, accuracy, precision, limit of detection, limit of quantification, and robustness. Method validations indicate a good sensitivity with a low value of LOD and LOQ of both examined active substances. The linearity range was found to be 1.50⁻9.00 µg/spot and 1.50⁻13.50 µg/spot for acetylsalicylic and ascorbic acid, respectively. A coefficient of variation that was less than 3% confirms the satisfactory accuracy and precision of the proposed method. The results of the assay of combined tablet formulation equal 97.1% and 101.6% in relation to the label claim that acetylsalicylic acid and ascorbic acid fulfill pharmacopoeial requirements. The developed TLC-densitometric method can be suitable for the routine simultaneous analysis of acetylsalicylic acid and ascorbic acid in combined pharmaceutical formulations. The proposed TLC-densitometry may be an alternative method to the modern high-performance liquid chromatography in the quality control of above-mentioned substances, and it can be applied when HPLC or GC is not affordable in the laboratory.

Potential Mechanism of Action of Cyclosporin A in Human Dermal Fibroblasts-Transcriptomic Analysis of CYPs.

Effect of cyclosporin A (CsA) in a therapeutic concentration, on the expression of cytochrome P450 genes (CYPs), was investigated in normal human dermal fibroblast cells. The expression of 57 genes, encoding cytochrome P450 isoforms, was estimated using the microarray method. Amongst 396 normalized fluorescence signals related to cytochrome P450 activity, only 91 were strictly connected to CYPs and were analyzed using two methods: a self-organizing feature map of artificial neural networks and typical statistical analysis with significance level at p ≤ 0.05. Comparing the samples from fibroblasts cultured with CsA and those cultured without, up-regulated changes of CYP19A1, 1B1, 7A1, 7F1, 17A1 and down-regulated 2D6 gene expression were observed. The mRNAs with increased changes were in the same neuron of the self-organizing feature map. All distinguished CYPs encode monooxygenases, which plays an important role in steroids biosynthesis and metabolism. Based on the obtained results, we can conclude that CsA in therapeutic concentration changes the expression profile of CYPs in human dermal fibroblasts, especially affecting genes linked to steroids synthesis and/or metabolism. It shows the potential mechanism of action of CsA in human dermal fibroblast cells.

Potential biomarkers for the early diagnosis of colorectal adenocarcinoma - transcriptomic analysis of four clinical stages.

BACKGROUNDS: Colorectal cancer is the third most common cancer in economically developed countries. Molecular studies and, in particular, gene expression have contributed to advances in the diagnosis and treatment of many cancers. Genes can be molecular and therapeutic markers, but because of the large molecular diversity in colorectal cancer the knowledge is not yet fully established. Probably one of the most crucial processes during early cancer development is inflammation. The inflammatory response in the tumor is an important indicator of molecular etiology and later of cancer progression. OBJECTIVE: The aim of this work is to identify potential biomarkers for early stage of colorectal adenocarcinoma in patients' bowel tissues using transcriptomic analysis. METHODS: Expression of the inflammatory response genes of colorectal cancer at all clinical stages (I-IV) and control of the bowel were evaluated by oligonucleotide microarrays. RESULTS: Based on statistical analysis many differentially expressed genes were selected. LCK (LCK Proto-Oncogene, Src Family Tyrosine Kinase), GNLY (granulysin), SLC6A6 (Solute-Carrier Family 6 Member 6) and LAMP2 (Lysosomal Associated Membrane Protein 2) were specific for the early stage of the disease. These genes had the properties of the good biomarkers. CONCLUSIONS: The expression of LCK, GNLY, SLC6A6 and LAMP2 genes could be valuable potential diagnostic biomarkers of the early stage of colorectal adenocarcinoma.

A Rapid and Simple TLC-Densitometric Method for Assay of Clobetasol Propionate in Topical Solution.

A rapid, simple to use and low-cost thin-layer chromatographic procedure in normal phase system with densitometric detection at 246 nm was carefully validated according to the International Conference on Harmonisation (ICH) guidelines for assay of clobetasol propionate in topical solution containing clobetasol propionate in quantity 0.50 mg/mL. The adopted thin-layer chromatographic (TLC)-densitometric procedure could effectively separate clobetasol propionate from its related compound, namely clobetasol. It is linear for clobetasol propionate in the range of 0.188 ÷ 5 µg/spot. The limit of detection (LOD) and limit of quantification (LOQ) value is 0.061 and 0.186 µg/spot, respectively. Accuracy of proposed procedure was evaluated by recovery test. The mean recovery of studied clobetasol propionate ranges from 98.7 to 101.0%. The coefficient of variation (CV, %) obtained during intra-day and inter-day studies, which was less than 2% (0.40 ÷ 1.17%), confirms the precision of described method. The assay value of clobetasol propionate is consistent with the pharmacopoeial requirements. In conclusion, it can be suitable as a simple and economic procedure for routine quality control laboratories of clobetasol propionate in topical solution.

A Validated TLC-Densitometric Method for the Determination of Mesterolone in Bulk Material and in Tablets.

Mesterolone is a synthetic androgenic steroid indicating a weak anabolic activity. A new, simple in use, and economical TLC-densitometric method in normal phase system (NP-TLC) has been developed and validated for the identification and quantitative determination of mesterolone in bulk drug and in tablet formulation. NP-TLC analysis was performed on aluminium plates precoated with silica gel 60F254 as the stationary phase using chloroform-acetone (40 : 10, v/v) as mobile phase. Densitometric analysis was carried out at λ = 745 nm after staining with phosphomolybdic acid. These conditions were found to give visible (dark blue) spot and sharp peak, respectively, for mesterolone at R F 0.75 ± 0.02 and enabled satisfactory separation of mesterolone from its related substance (potential impurity). The proposed NP-TLC-densitometric method was validated for specificity, linearity, precision, accuracy, robustness, and sensitivity according to ICH guideline and other validation requirements. The limit of detection (LOD) and limit of quantification (LOQ) were 61.0 ng · spot(-1) and 184.0 ng · spot(-1), respectively. The percent content of mesterolone in marketed tablet formulation was found to be 99.40% of label claim. The developed TLC-densitometric method can be successfully used in quality control of mesterolone in bulk material and also tablet formulation.