Development and Validation of Analytical Methods for the Detection and Quantification of a Novel Dimeric Ceramide in Stratum Corneum and Other Layers of the Skin

Ceramides (CERs) are integral parts of the intercellular lipid lamellae of the stratum corneum (SC), which is responsible for the barrier function of the skin. Many skin diseases such as atopic dermatitis and psoriasis are associated with depletion or disturbance of the level of CERs in the SC. Administration of an exogenous novel dimeric ceramide (dCER) deep into the SC may help to stabilize the SC barrier substantially and to treat some skin disease conditions and with the help of the existing technology it might be possible to formulate various pharmaceutical dosage forms that can facilitate penetration of dCER into the SC. However, assessment of the rate and extent of permeability of the exogenous dCER involves appropriate analytical techniques which can discriminately quantify the exogenous dCER in the SC and in the other skin layers. Therefore, an attempt was made to develop an AMD-HPTLC and an HPLC/APCI-MS method for the detection and quantification of exogenous dCER in the SC as well as other skin layers. The method involved synthesis of the dCER and development of appropriate HPTLC and LC/ESI-MS methods for the separation and quantification of the dCER in the SC and deeper layers of the skin. The methods developed were optimized for quantification of a novel dCER. In comparison to the AMD-HPTLC method, the HPLC/MS method offers a higher sensitivity. Both methods could be used for the quantification of dCER in presence of a complex matrix (e.g., skin extract). The developed methods are complementary and could be used for the quantification of dCER in any further stage of substance research and industrial application. The methods developed are robust, linear and sensitive with a low limit of detection (LOD) and low limit of quantification (LOQ).

     

Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum

Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR—partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400–1500 cm⁻¹) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R² = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R² (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies.     

表面表征仪器分析方法的新进展

影响表面和界面薄层性能的因素很多，主要有：表面与界面上的化学组成、显微构型及其掺杂物、污染物和缺陷结构的分布状态等等。探测这些表面薄层性能的各种技术目前已日臻完善、成熟，其中以表面表征的仪器分析方法尤为引人注目。本文着重对该研究领域中令人关注的仪器分析技术的应用状况和最新动态进行了简要介绍和评述。包括：扫描隧道显微术、二次离子质谱、Ｒｕｔｈｅｒｆｏｒｄ反向散射光谱、Ｘ射线电子谱、Ａｕｇｅｒ电子谱、     

Lycoperoside H,a Tomato Seed Saponin,Improves Epidermal Dehydration by Increasing Ceramide in the Stratum Corneum and Steroidal Anti-Inflammatory Effect

Tomatoes are widely consumed, however, studies on tomato seeds are limited. In this study, we isolated 11 compounds including saponins and flavonol glycosides from tomato seeds and evaluated their effects on epidermal hydration. Among the isolated compounds, tomato seed saponins (10 µM) significantly increased the mRNA expression of proteins related to epidermal hydration, including filaggrin, involucrin, and enzymes for ceramide synthesis, by 1.32- to 1.91-fold compared with the control in HaCaT cells. Tomato seed saponins (10 µM) also decreased transepidermal water loss by 7 to 13 g/m²·h in the reconstructed human epidermal keratinization (RHEK) models. Quantitative analysis of the ceramide content in the stratum corneum (SC) revealed that lycoperoside H (1–10 µM) is a promising candidate to stimulate ceramide synthesis via the upregulation of ceramide synthase-3, glucosylceramide synthase, and β-glucocerebrosidase, which led to an increase in the total SC ceramides (approximately 1.5-fold) in concert with ceramide (NP) (approximately 2-fold) in the RHEK models. Evaluation of the anti-inflammatory and anti-allergic effects of lycoperoside H demonstrated that lycoperoside H is suggested to act as a partial agonist of the glucocorticoid receptor and exhibits anti-inflammatory effects (10 mg/kg in animal test). These findings indicate that lycoperoside H can improve epidermal dehydration and suppress inflammation by increasing SC ceramide and steroidal anti-inflammatory activity.     

Development and Validation of Chromatographic Methods for the Quantification of Dabigatran Etexilate Mesylate in the Presence of Its Risky Degradation Products

JPC – Journal of Planar Chromatography – Modern TLC - New stability-indicating thin-layer chromatography (TLC)—densitometry and reversed-phase liquid chromatography (RP-LC)...     

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Method for Detection and Quantification of Flunisolide in Tissue Culture Medium

A robust, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of quantifying flunisolide in the tissue culture matrix was developed and validated. Both flunisolide and dexamethasone (internal standard) were extracted from tissue culture medium, with 5% fetal bovine serum, 1% nonessential amino acids, and 1% penicillin/streptomycin by simple liquid–liquid extraction. The analytes were chromatographically separated (10 ng/mL for flunisolide) using a C8 column (4.6 mm × 150 mm; 5 µm particle size). The mobile phase was comprised of methanol:water (80:20 v/v). The analytes were separated at baseline within 2.5 min. using a flow rate of 1 mL/min. Mass spectrometry detection was carried out in negative atmospheric pressure chemical ionization mode. The calibration curves for the analyte were linear over the range of 10–200 ng/mL (R² ≥ 0.9968, n = 6). The inter and intra-batch mean percent accuracy ranged within 97.7–110.6% and the precision range was 3.5–10.4% (% coefficient of variation ≤15%). Stability studies revealed that the analyte was stable in the matrix for at least six hours at room temperature and at the end of three successive freeze and thaw cycles. The method reported here is specific for flunisolide quantification was also used in monolayer efflux assay using CaCo₂ cell lines, which will eventually aid P- glycoprotein transport studies for flunisolide.     

Development of a Bacterial Enzyme-Based Biosensor for the Detection and Quantification of Selenate

An enzymatic biosensor has been developed for the determination of selenate (SeO₄²⁻), in which selenate reductase (SeR) is chemically attached to a gold disk electrode by lipoic acid N-hydroxysuccinimide ester as linker, allowing the catalytic reduction of the SeO₄²⁻ to SeO₃²⁻. Modification of the gold electrode was characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and electrochemistry. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were performed in different buffers for selenate determination. Under optimum conditions, the calibration curve was linear over the range 7.0–3900.0 μg L⁻¹ with limits of detection and quantification of 4.97 and 15.56 μg L⁻¹, respectively. The possible interference of the relevant oxyanions SO₄²⁻, NO₃⁻, NO₂⁻, PO₄³⁻ and AsO₄³⁻ in the determination of SeO₄²⁻ was studied. Finally, the proposed biosensor was used to determine SeO₄²⁻ with recovery between 95.2 and 102.4 % in different real water samples.     

Validation and Comparison of Two Analytical Methods for Imatinib Therapeutic Drug Monitoring

Chromatographia - Our objective was to validate and compare the automated and chromatography methods for determining imatinib levels in plasma, in terms of linearity, precision, accuracy and...     

Development and Validation of a New Analytical Method for the Determination of Related Components in Quetiapine Hemifumarate

A simple, sensitive isocratic rapid resolution liquid chromatographic assay method has been developed for the quantitative determination of quetiapine hemifumarate in bulk active pharmaceutical ingredient, used for the treatment of schizophrenia. The developed method is also applicable for the process related impurities determination. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in a isocratic mode and quantification was by ultraviolet detection at 225 nm at a flow rate of 1.0 mL min^?1. In the developed LC method the resolution between quetiapine hemifumarate and its three potential impurities was found to be greater than 2.0. Regression analysis showed an r value (correlation coefficient) greater than 0.99 for quetiapine hemifumarate and its three impurities. This method was capable to detect all three impurities of quetiapine hemifumarate at a level of 0.003% with respect to test concentration of 1.0 mg mL^?1 for a 3 μL injection volume. The bulk active pharmaceutical ingredient was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RR-LC method was validated with respect to linearity, accuracy, precision and robustness.     

Validation of an Analytical Biomarker Approach for the Detection of Nandrolone Abuse in the Porcine

The use of anabolic steroids as growth promoting agents in food production is prohibited under European Union legislation, but there is currently no internationally accepted method for detecting the abuse of the anabolic steroid nandrolone in the porcine. Therefore, an analytical biomarker approach based on gas chromatography-tandem mass spectrometry (GC-MS-MS) analysis of the major urinary free fraction nandrolone metabolite 19-noretiocholanolone was developed and validated. The lower and upper limits of quantification of the assay were 25 and 3,000 pg mL^?1 respectively. The limit of detection was calculated as 13.2 pg mL^?1, which is significantly lower than previously reported methods. When applied to a population of untreated animals, 19-noretiocholanolone distributions in boars and gilt were bimodal, with a small number of concentrations in each sex at around the 1,000 pg mL^?1 region and the majority of concentrations closer to the lower end of the calibration range. Statistical analysis of the data was carried out in order to suggest screening and confirmatory threshold approaches for this steroid in the urine of boars and gilts. The adopting of particular screening thresholds would be at the discretion of the individual regulating authorities, but at a false non-compliance rate of 1 in 10,000 of the normal population, the suggested confirmatory thresholds (7,501.6 pg mL^?1 for boars and 19,200.4 pg mL^?1 in gilts) are able to detect the abuse of nandrolone for several weeks following administration of this steroid.     

Evaluation of an HPLC Method for the Determination of Natural Moisturizing Factors in the Human Stratum Corneum

The levels of natural moisturizing factors in the skin can be used as a biomarker of hydration, for studying the effect of skin irritants, or as a biomarker of loss-of-function mutations in the filaggrin gene which are the main risk factor for atopic dermatitis. In this study the chromatographic performance and recovery of natural moisturizing factors and proteins from the skin were investigated using different extraction solvents and adhesive tapes. The uppermost layer of the skin stratum corneum, collected by using commercially available D-squame and corneofix adhesive tapes, was extracted by ammonia or potassium hydroxide. Protein levels used to correct for a variable stratum corneum amount on a tape were assessed by measuring optical density of a tape or indirectly by measuring proteins by a spectrophotometric assay. The measured natural moisturizing factors, pyrrolidone-5-carboxylic acid, histidine, tyrosine, trans-urocanic acid, and cis-urocanic acid were determined by ion pair reverse phase HPLC. Sample preparation and chromatographic performance were favorable when ammonia was used as an extraction solvent. Extraction of the natural moisturizing factors with ammonia avoids a time consuming neutralization step as required with extraction procedures using strong base or acid. The only drawback of the ammonia method is incomplete extraction of proteins from the tapes; however this can be avoided by measuring the optical density of stratum corneum-loaded tapes. The sensitivity of the method was sufficiently high to quantify the analytes even in homozygous filaggrin gene carriers. Reduced natural moisturizing factors levels found in the individuals with filaggrin gene mutation or after exposure to a skin irritant sodium lauryl sulfate were consistent with the previously reported studies.     

Validation of a High-Performance Liquid Chromatography with Photodiode Array Detection Method for the Separation and Quantification of Antioxidant and Skin Anti-Aging Flavonoids from Nelumbo nucifera Gaertn. Stamen Extract

Nelumbo nucifera Gaertn., or the so-called sacred lotus, is a useful aquatic plant in the Nelumbonaceae family that has long been used to prepare teas, traditional medicines as well as foods. Many studies reported on the phytochemicals and biological activities of its leaves and seeds. However, to date, only few studies were conducted on its stamen, which is the most important ingredient for herbal medicines, teas and other phytopharmaceutical products. Thus, this present study focuses on the following: (1) the application of high-performance liquid chromatography with photodiode array detection for a validated separation and quantification of flavonoids from stamen; (2) the Nelumbo nucifera stamen’s in vitro and in cellulo antioxidant activities; as well as (3) its potential regarding the inhibition of skin aging enzymes for cosmetic applications. The optimal separation of the main flavonoids from the stamen ethanolic extract was effectively achieved using a core-shell column. The results indicated that stamen ethanolic extract has higher concentration of in vitro and in cellulo antioxidant flavonoids than other floral components. Stamen ethanolic extract showed the highest protective effect against reactive oxygen/nitrogen species formation, as confirmed by cellular antioxidant assay using a yeast model. The evaluation of potential skin anti-aging action showed that the stamen extract has higher potential to inhibit tyrosinase and collagenase compared with its whole flower. These current findings are the first report to suggest the possibility to employ N. nucifera stamen ethanolic extract as a tyrosinase and collagenase inhibitor in cosmetic applications, as well as the utility of the current separation method.     

Analytical Methods for Determination of Phytic Acid and Other Inositol Phosphates: A Review

From the early precipitation-based techniques, introduced more than a century ago, to the latest development of enzymatic bio- and nano-sensor applications, the analysis of phytic acid and/or other inositol phosphates has never been a straightforward analytical task. Due to the biomedical importance, such as antinutritional, antioxidant and anticancer effects, several types of methodologies were investigated over the years to develop a reliable determination of these intriguing analytes in many types of biological samples; from various foodstuffs to living cell organisms. The main aim of the present work was to critically overview the development of the most relevant analytical principles, separation and detection methods that have been applied in order to overcome the difficulties with specific chemical properties of inositol phosphates, their interferences, absence of characteristic signal (e.g., absorbance), and strong binding interactions with (multivalent) metals and other biological molecules present in the sample matrix. A systematical and chronological review of the applied methodology and the detection system is given, ranging from the very beginnings of the classical gravimetric and titrimetric analysis, through the potentiometric titrations, chromatographic and electrophoretic separation techniques, to the use of spectroscopic methods and of the recently reported fluorescence and voltammetric bio- and nano-sensors.     

Determination of Vecuronium Bromide in Pharmaceuticals: Development, Validation and Comparative Study of HPLC and CZE Analytical Methods

Vecuronium bromide is a neuromuscular blocking agent used for anesthesia to induce skeletal muscle relaxation. HPLC and CZE analytical methods were developed and validated for the quantitative determination of vecuronium bromide. The HPLC method was achieved on an amino column (Luna 150 × 4.6 mm, 5 μm) using UV detection at 205 nm. The mobile phase was composed of acetonitrile:water containing 25.0 mmol L^?1 of sodium phosphate monobasic (50:50 v/v), pH 4.6 and flow rate of 1.0 mL min^?1. The CZE method was achieved on an uncoated fused-silica capillary (40.0 cm total length, 31.5 cm effective length and 50 μm i.d.) using indirect UV detection at 230 nm. The electrolyte comprised 1.0 mmol L^?1 of quinine sulfate dihydrate at pH 3.3 and 8.0% of acetonitrile. The results were used to compare both techniques. No significant differences were observed (p > 0.05).     

Development of an Analytical Methodology for Quantification of Strong Acid in Diesel Oil

Many factors can affect the quality of diesel oil, in particular the degradation processes that are directly related to some organosulfur compounds. During the degradation process, these compounds are oxidized into their corresponding sulfonic acids, generating a strong acid content during the process. p‐Toluene sulfonic acid analysis was performed using the linear sweep voltammetry technique with a platinum ultramicroelectrode in aqueous solution containing 3 mol L^?1 potassium chloride. An extraction step was introduced prior to the voltammetric detection in order to avoid the adsorption of organic molecules, which inhibit the electrochemical response. The extraction step promoted the transference of sulfonic acid from the diesel oil to an aqueous phase. The method was accurate and reproducible, with detection and quantification limits of 5 ppm and 15 ppm, respectively. Recovery of sulfonic acid was around 90%.     

Development and Validation of Novel DH-GC-ITMS Methods for the Determination of Freon F-141b in Formulated Polyol and Rigid Polyurethane Foam

Two efficient methods for the determination of 1,1-dichloro-1-fluoroethane (Freon F-141b) in formulated polyol and rigid polyurethane foam by dynamic-headspace-gas chromatography-ion trap-mass spectrometry were developed and validated. Rigid polyurethane foam was efficiently dissolved in dimethylformamide by heating at the temperature of 60 °C for 2 h.Validation was carried out in terms of limits of detection (LOD), limits of quantitation (LOQ), linearity, precision and recovery. LOD values of 4.00 g kg^–1 for rigid polyurethane foam and 0.73 g kg^–1 for formulated polyol were achieved, whereas linearity was statistically verified over one order of magnitude. Precision was evaluated testing two concentration levels. Good results were obtained both in terms of intra-day repeatability and between-day precision: RSD % lower than 4% (n = 6) at the concentration of 15 g kg^–1 were calculated for intra-day repeatability. Extraction recoveries up to 92.6±1.6 % (n = 3) were also calculated by the addition of Freon F-141b to the samples analysed. Both the methods were applied for the analysis of a number of formulated polyol and rigid polyurethane foam samples.     

Development and Validation of a New Analytical Method for the Determination of Related Components in Tolterodine Tartarate Using LC

A novel liquid chromatographic method has been developed, and validated for the determination of tolterodine tartarate, for its potential three impurities in drug substances and drug products. Efficient chromatographic separation was achieved on a C8 stationary phase (150 × 4.6 mm, 3.5 μm particles) with a simple mobile phase combination delivered in an isocratic mode at a flow rate of 0.8 mL min^?1 and quantitation was carried out using ultraviolet detection. Microwave assisted degradation procedure was employed for stress testing studies in addition to the conventional way of a refluxing method. The results of both studies were compared. In the developed LC method, the resolution between tolterodine and its three potential impurities was found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) greater than 0.999 for tolterodine and for its three impurities. This method was capable to detect all three impurities of tolterodine at a level below 0.0038% with respect to a test concentration of 0.5 mg mL^?1 for a 10 μL injection volume. The inter- and intra-day precisions for all three impurities and for tolterodine were found to be within 1.1% RSD at its specification level. The method has shown good, consistent recoveries for tolterodine (98.9–101.6%) and for its three impurities (94.5–103.0%). The test solution was found to be stable in the diluent for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation, as prescribed by ICH. Degradation was found to occur in alkaline stress condition, while the drug was stable to water hydrolysis, acid hydrolysis, oxidative stress, photolytic and thermal stress. The assay of stressed samples was calculated against a qualified reference standard and the mass balance was found close to 99.5%. Microwave degradations were very fast and comparable to the conventional way of the refluxing method. Robustness studies were carried out and suggested that system suitability parameters were unaffected by small changes in critical factors. The validated method was successfully applied for the determination of tolterodine tartarate in drug substances and drug products.     

Development and Validation of a Method for Quantification of Favipiravir as COVID-19 Management in Spiked Human Plasma

In the current work, a simple, economical, accurate, and precise HPLC method with UV detection was developed to quantify Favipiravir (FVIR) in spiked human plasma using acyclovir (ACVR) as an internal standard in the COVID-19 pandemic time. Both FVIR and ACVR were well separated and resolved on the C18 column using the mobile phase blend of methanol:acetonitrile:20 mM phosphate buffer (pH 3.1) in an isocratic mode flow rate of 1 mL/min with a proportion of 30:10:60 %, v/v/v. The detector wavelength was set at 242 nm. Maximum recovery of FVIR and ACVR from plasma was obtained with dichloromethane (DCM) as extracting solvent. The calibration curve was found to be linear in the range of 3.1–60.0 µg/mL with regression coefficient (r²) = 0.9976. However, with acceptable r², the calibration data’s heteroscedasticity was observed, which was further reduced using weighted linear regression with weighting factor 1/x. Finally, the method was validated concerning sensitivity, accuracy (Inter and Intraday’s % RE and RSD were 0.28, 0.65 and 1.00, 0.12 respectively), precision, recovery (89.99%, 89.09%, and 90.81% for LQC, MQC, and HQC, respectively), stability (% RSD for 30-day were 3.04 and 1.71 for LQC and HQC, respectively at −20 °C), and carry-over US-FDA guidance for Bioanalytical Method Validation for researchers in the COVID-19 pandemic crisis. Furthermore, there was no significant difference for selectivity when evaluated at LLOQ concentration of 3 µg/mL of FVIR and relative to the blank.