High-performance Liquid Chromatographic Procedure Research Articles

A green perspective on simultaneous HPLC and UV spectrophotometric estimation of Udenafil and Dapoxetine hydrochloride in pharmaceutical formulations

Clinical research has affirmed that the combination of Udenafil and Dapoxetine hydrochloride yields greater improvement in erectile dysfunction compared to the use of individual medications. There is a requirement of precise quantitative method for accurate determination of Udenafil and Dapoxetine when taken together. Consequently, our interest lies in developing and validating analytical methods for the simultaneous estimation of Udenafil and Dapoxetine - Various strategies, such as UV spectrophotometric approaches and an innovative, straightforward, accurate, sensitive, and robust High-Performance Liquid Chromatography (HPLC) procedure, have been explored. An Isocratic HPLC approach was employed using reversed-phase CHEMSIL ODS C18 column (4.6 mm x 250 mm, 5 µm) as a stationary phase and detection wavelength (λ max) 230 nm for the two analytes in the chromatographic column. The mobile phase comprised of (70:30, v/v) mixture of Acetonitrile and 10 mM Ammonium formate buffer having pH 5, adjusted with formic acid. The elution was carried out at a constant flow rate of 1 mL/min. The UV simultaneous equation technique estimated the medicine's quantities and derived a simultaneous equation for DAP and UDN using 2 wavelengths, 230 nm and 298 nm. Furthermore, the two advanced methods underwent rigorous statistical validation, guided by the valuable ICH Q2R1 guidelines, affirming their effectiveness as a successful analytical tool for the concurrent analysis of both tablets in bulk and the synthetic mixture fabricated in the laboratory. To assess the environmental impact of contemporary UV spectrophotometric and HPLC techniques, the Green Analytical Greenness Metric (AGREE) software and the Green Analytical System Index (GAPI) were employed to evaluate the greenness profile. This examination confirmed the method's environmental friendliness concerning solvent usage, chemical ingredients, energy consumption, and waste production. The developed UV method is more sustainable and greener compare to HPLC method for simultaneous estimation of compound, which can be utilised by industry for quantification of each individual drug in their combination products.

Near Infrared Technology Coupled with Different Spectra Correction Approaches for Fast and Non-Destructive Prediction of Chlorogenic Acid on Intact Coffee Beans

Abstract The primary objective of this research was to utilise near-infrared reflectance spectroscopy as a swift, non-destructive method for identifying chlorogenic acid in whole coffee beans. Additionally, this investigation explored the efficacy of different spectral improvement techniques alongside partial least square regression to construct predictive models. NIR spectral data was gleaned from whole coffee beans spanning a wavelength range of 1000–2500 nm, while the chlorogenic acid content was ascertained via high-performance liquid chromatography procedures. Our findings revealed that the highest coefficient of determination reached for chlorogenic acid was 0.97, and the root mean square error for calibration was 0.31% when using the multiplicative scatter correction method. Furthermore, upon testing the model using an external validation dataset, a determination coefficient of 0.91 and a ratio error to range index of 11.56 with a root mean square prediction error at 0.51% was attained. From these results, it can be inferred that the near-infrared technology, coupled with an effective spectral enhancement process, can facilitate quick, non-invasive determination of chlorogenic acid in whole coffee beans.

Imprinted polymer/reduced graphene oxide-modified glassy carbon electrode-based highly sensitive potentiometric sensing module for imidacloprid detection

A sensitive and highly selective potentiometric sensor for imidacloprid detection (IMD) is presented. Reduced graphene oxide (rGO) was used to modify a glassy carbon electrode in the fabrication of this sensor. The rGO is coated with IMD molecularly imprinted polymer (MIP), which serves as a sensing compound for herbicide recognition. Under ideal circumstances, a linear range of 0.5 µM-1.0 mM was measured, with a detection limit of 0.2 µM IMD. The method exhibits great selectivity towards the IMD's determination, according to the examination of the effects of potentially interfering species. For the direct potentiometric detection of IMD in several pesticide formulation samples, the assay method has been validated and shown to be adequate. The obtained recovery ranges from 94.5 to 106.4 % of the nominal or spiked concentration, with a mean relative standard deviation of 1.3 %. The applicability outcomes of using the presented sensor were contrasted with those attained by high-performance liquid chromatography (HPLC). There was no discernible difference between the suggested and HPLC procedures. Due to the sensitive membrane's and the liquid junction's proximity, this module's use permits applications with a small sample volume. The proposed sensor's effectiveness in a complicated matrix points to applications for rapid detection and tests for quality assurance and control in the agro-industry.

Identification and Quantification of the Component Composition of the Active Substance Gentamicin Sulfate by <sup>1</sup>Н and <sup>13</sup>С NMR Spectroscopy

Gentamicin sulfate is a broad-spectrum bactericidal aminoglycoside antibiotic produced by Micromonospora purpurea and used in clinical practice as an active substance containing a mixture of gentamicins C1 and C2. Gentamicin content is a standardised parameter controlled as part of a pharmaceutical product review. Currently, the quality control of the active substance gentamicin sulfate involves the use of high-performance liquid chromatography (HPLC) for the indirect identification of the component composition by comparison with a reference standard and for its quantification by calibration against the reference standard.The aim of the study was to develop a procedure for identifying and quantifying the components of the active substance gentamicin sulfate using nuclear magnetic resonance (NMR) spectroscopy that would not require reference standards.Materials and methods. The authors studied a sample of the active substance gentamicin sulfate using an Agilent DD2 NMR spectrometer operating at 600 MHz to record NMR spectra and an Agilent 1200 HPLC system equipped with a multiple wavelength detector (MWD) to obtain chromatograms.Results. Having determined the spectral properties of the C1, C1A, C2, C2A and C2B gentamicins comprising the active substance, the authors observed characteristic signals of the same type that corresponded to each of the gentamicins and did not overlap with other signals in the 1H and 13C spectra. These characteristic signals provided spectral markers indicative of the gentamicins of interest in the sample, with normalised integrated intensities equal to the mole fractions of the analytes. The authors compared NMR and HPLC measurements of the active substance composition.Conclusions. The authors developed a procedure for identifying and quantifying the constituents of the active substance gentamicin sulfate by 1H and 13C NMR spectroscopy, which requires less effort than HPLC procedures and no reference standards for the intended applications.

Response surface methodology combined Box-Behnken design optimized green kinetic spectrophotometric and HPLC methods to quantify angiotensin receptor blocker valsartan in pharmaceutical formulations

The high-performance liquid chromatographic (HPLC) and kinetic spectrophotometric methods were established to compute valsartan (VAL) in pharmaceutical formulations. The spectrophotometric procedures adopted initial rate, fixed time, and equilibrium strategies to assess VAL. The method was based on the carboxylic acid group of the oxidized VAL with a mixture of potassium iodate (KIO3) and potassium iodide (KI) at room temperature, producing a stable, yellow-coloured absorb at 352 nm. The critical parameters were optimized using green process optimization methodology such as Box-Behnken design (BBD) which belongs to response surface methodology (RSM). After the screening, experiments identified them as significant, and then three crucial factors were optimised: KI volume, KIO3 volume, and reaction time against response as absorbance. The HPLC procedure was also optimized based on the desirability function on RSM-BBD. The parameters such as pH, methanol (%), and flow rate (ml/min) were optimized with the best responses: peak area, symmetry, and theoretical plates. The linearity of spectrophotometric and HPLC methods was within the range of 2–24 and 0.25–11.25 µg/ml, respectively. The developed procedures produced excellent accuracy and precision. The design of the experiment (DoE) setting explained and discussed the individual steps and the importance of independent and dependent variables used to develop the model and optimization. The method was validated as per the International Conference on Harmonization (ICH) guidelines. Furthermore, Youden's robustness study was applied with factorial combinations of the preferred analytical parameters and explored their influence with alternative conditions. The analytical Eco–Scale score was calculated and was found a better option as green methods to quantify VAL. The results were reproducible with the analysis completed with biological fluid and wastewater samples.

Quantitative Determination of Ascorbic Acid in Herbal Medicinal Products by HPLC

Ascorbic acid (vitamin C) is involved in many vital biochemical processes in the human body. Rose hips are a natural source of ascorbic acid. Rose hips are used in decoctions and many vitamin herbal teas. Russian compendial methods for the quantitative determination of ascorbic acid have certain limitations precluding its objective measurement in herbal drugs, herbal drug preparations, and herbal medicinal products.The aim of the study was to develop an analytical procedure for the quantitative determination of ascorbic acid in herbal drugs, herbal medicinal preparations, and herbal medicinal products using high performance liquid chromatography (HPLC).Materials and methods. The study involved two herbal medicinal products, Rose Hips (rose hips) and Vitamin Herbal Tea 2 (rose hips and rowan fruits), and reference standards for ascorbic acid, rutoside, quercetin, gallic acid, caffeic acid, chlorogenic acid, luteolin-7-glucoside, citric acid, DL-malic acid, thiamine, and pyridoxine hydrochloride. The authors used a 1260 Infinity II chromatograph with a diode array detector by Agilent and an Inertsil ODS-3 chromatographic column (250 mm × 4.6 mm, 5 µm) by GL Sciences. The HPLC system was operated in gradient elution mode, and the detector was set at 244 nm.Results. Ascorbic acid content in herbal drugs, herbal medicinal preparations, and herbal medicinal products can be determined by HPLC. Adequate sample preparation and chromatography conditions allow for inhibiting ascorbic acid oxidation in aqueous solutions for a period sufficient to complete testing (not less than 8 h).Conclusions. The authors developed a highly sensitive and selective HPLC procedure for the quantitative determination of ascorbic acid intended for the standardisation of herbal drugs, herbal medicinal preparations, and herbal medicinal products. The procedure is worthy of inclusion in the Rose Hips and Vitamin Herbal Tea 2 monographs of the State Pharmacopoeia of the Russian Federation. Switching to HPLC should not require lowering the limit for ascorbic acid (not less than 0.2%) established in the Rose Hips and Vitamin Herbal Tea 2 monographs for the titration method. The ascorbic acid content was below this limit in the vast majority of study samples. However, this discrepancy may be explained by the presence of dog rose (Rosa canina) species low in vitamin C, which should normally be used only as choleretics.

Spectrophotometric studies of low-cost method for determination of ceftriaxone using a novel charge transfer acceptor, N-(2,4-dinitro-1-naphthyl)-p-toluenesulphonamide

BackgroundThis study reports a new, cost-effective and validated method for the determination of ceftriaxone (CFR). The method involved charge transfer (CT) complexation reaction of ceftriaxone (as n-electron donor) and N-(2,4-dinitro-1-naphthyl)-p-toluenesulphonamide {N-(2,4-DN1NL) PTS} as π-electron acceptor to form a complex. Ultraviolet, infrared and 1H NMR spectra of CFR, N-(2,4-DN1NL) PTS and adduct were then studied to predict the site of interaction between the donor and acceptor.ResultThe complex formed had deep golden-yellow colour, having a new absorption band at 440 nm. Molar absorptivity of 1.667 × 105 L M−1 cm−1 was obtained. The complexation reaction was completed at 30 °C optimal temperature within 10 min. Acetonitrile was found to be the best diluting solvent for optimal detector response and the complex was stable (absorbance unchanged) at room temperature for hours. At concentration of 1.708–11.956 µg mL−1, with low limits of detection of 0.143 µg mL−1, Beer’s law was observed. Between-day recovery statistics of CFR from quality control samples were 102.15 ± 0.062 (% RSD = 0.61, n = 12) over three days. The site of interaction of donor and acceptor molecules, as revealed through infrared (IR) and proton nuclear magnetic resonance studies (1H NMR) and the formation of charge transfer complex is through intermolecular hydrogen bonding between the amino group of the donor and the acidic proton of the acceptor. Common tablet excipients, as observed, did not interfere with the analytical method and no significant difference existed between the results of this new method and the high performance liquid chromatographic procedures (p > 0.05) documented in the USP. The new CT procedure described in this paper is not only simple but also fast, accurate and precise. Also, the reactions were carried out at room temperature compared to previously described procedures.ConclusionsThis novel method could therefore be adopted as a fast but cost-effective alternative for the qualitative and quantitative assessment of CFR in its pure and dosage form. It could find usefulness in on-the-spot detection of counterfeit drugs and in field inspections with reliable accurate results that compares with established methods.

Development of RP-HPLC methods for the analysis of Dexamethasone and Levofloxacin alone and in combinations used in the therapy of Covid-19

In December 2019, the World Health Organization was informed of an outbreak of pneumonia of unknown etiology in Wuhan, Hubei Province, China. On January 7, 2020, a new type of coronavirus was isolated, with the WHO later officially calling it “COVID-19” and the International Committee on Taxonomy of Viruses naming the virus “SARS-CoV-2”. On January 30, 2020, the WHO declared the severe acute respiratory syndrome coronavirus 2 outbreak a public health emergency of international concern, making it an unprecedented global public health challenge. From a scientific and literary reference, it was established that the main drugs in the therapy with Covid-19 are Dexamethasone and Levofluoxetine. For this reason, we pay attention to the analysis of these two medicinal products. In the literature, we did not find an analysis of a combination of Dexamethasone and Levofloxacin. Development and validation of a highperformance liquid chromatographic analytical procedure for simultaneously determining Dexamethasone and Levofloxacin in a synthetic mixture is described in this paper. The separation was made with a LiChrosorb RP 18 (250 × 4.6 mm) column, at 25 °C temperature,with isocratic mode andmobile phase,containingt acetonitrile and woter (70-30v/v). Eluent was monitored at 254 nm and the flow rate was 1.0 ml/min. Dexamethasone and Levofluoxetine were effectively separated with retention time (tr) of 4.69 min and 14.51 min,respectively,with in the selected chromatographic conditions.The method was validated for analytical parameters: specificity, linearity, precision, accuracy,andlimits of detection and quantitation. The calibration curves were linear inthe concentration range of 12.5 to 100.0 μg/ml for Dexamethasone and Levofloxacin, and the regression coefficientswere more than 0.999. For Dexamethasone and Levofloxacin the recovery was 100.01% and 100.04%, respectively. This analytical procedure is applicable for the quality control of drug formulations.

A review of extraction and HPLC methods used for Melastomataceae and Asteraceae leaves in the analysis of flavonoids

HPLC (high-performance liquid chromatography) is one of the accurate chromatographic techniques that is widely used for analysis. This present review submitted the extraction and HPLC methods for determining the compound in Melastomataceae and Asteraceae families that related with insecticidal compounds. This review discusses the standard used for the compound and chromatographic conditions. There are several species under both families. As for the Melastomataceae family, the species are Melastoma malabathricum and Miconia albicans meanwhile, the species under the Asteraceae family are Santolina semidentata, Eupatorium littorale, and Vernonia condensata. This review focus on the flavonoid and phenolic compound as these compounds are categorized as an insecticidal compound. HPLC can be used in conjunction with several detection technologies to identify flavonoids and phenolics in the plant, food, and biological samples because it is a method for separating these molecules. As a result, the HPLC procedure for the chosen molecule was reviewed in this article, which will aid in further analysis.

Quality of Oral Drug Formulations of Artemisinin-Based Combination Therapy Sold in Katsina State, Nigeria

There are increasing reports of substandard antimalarial drugs, and these have been a severe under-recognised public health problem especially in developing countries. For this reason, 21 samples of different brands of artemisinin-based combination therapies (ACTs) comprising of artemether/lumefantrine and artesunate/amodiaquine oral drug formulations that are available on sale in different hospital pharmacies and patent medicine stores in Katsina State, Nigeria were evaluated for microbial and chemical qualities. Microbial limit test (MLT) and assay for the content of the active pharmaceutical ingredients (APIs) using standard high performance liquid chromatography (HPLC) procedures were carried out as described in the official monograph of the United States Pharmacopoeia (USP) and the International Pharmacopoeia (IP). The results obtained had indicated that all the ACTs oral drug preparations were free from microbial contamination except one sample of artesunate-amodiaquine showing viable total combined yeasts/moulds count (TYMC) of 1.0 x 101 colony forming units (CFU)/g. All the samples complied with the USP and IP criteria for the microbiological quality of non-sterile oral dosage forms. On the other hand, 10 (47.6%) out of the 21 samples met the specific chemical quality standards. Moreover, 8 (57.1%) and 3 (42.9%) of the artemether/lumefantrine and artesunate/amodiaquine had active ingredient outside the set pharmacopoeial limit and, therefore, were none compliant to the IP specifications for percentage content. The presence of substandard ACTs may lead to possible therapeutic failure from the use of such kind of formulations, facilitate the development and spread of drug-resistance. There is the need for effective government regulation and adequate enforcement on the production, distribution and sales of good quality medicines.

Isolation and Electrochemical Property of Ho2O@C90 Isomers

Four oxide clusterfullerenes Ho2O@C90 (I–IV) were prepared by a modified arc discharge method and isolated by a multistage high performance liquid chromatography procedure. Based on their vis-NIR spectra, the carbon cage symmetries in Ho2O@C90 (I, III, IV) are confirmed to be C 2(43)-C90, C 2(40)-C90 and C 2(41)-C90, respectively. Electrochemical studies display that the first and second reduction potentials of Ho2O@C90 isomers are close to those of Er2C2@C90 ones, but much lower than those of Sm@C90 ones. Moreover, it is found that the electrochemical band gaps of Ho2O@C90 isomers are also very close to those of Er2C2@C90 isomers. A detailed investigation on the redox potentials of C90 based EMFs reveals that endohedral clusters/atoms have much greater effect than carbon cage symmetries on the electrochemical properties of EMFs.

Preconcentration of sulphonamides in bovine milk by the cloud point extraction method using smartphone-based digital images.

Sulphonamides are a group of synthetic antibiotics used specially in veterinary medicine. Among the procedures employed in the sample preparation for sulphonamide determination are liquid-liquid extraction (LLE) and solid-phase extraction (SPE) that use large volumes of organic solvents. Hence, a clean procedure was developed based on preconcentration and cloud point extraction (CPE) without using organic solvents to quantify total sulphonamides in bovine milk. The procedure was optimized as follows: 2 mL of pre-cleaned milk sample, 2 mL of reagent solution and 1 mL of Triton X-114 7% (m/v) were added to a tube, heated in a water bath at 40 °C for 10 minutes and centrifuged at 2950 rcf for 20 minutes. Digital image acquisition was employed directly at the tube without removing the supernatant/aqueous phase. The linear response was observed between 10 and 400 μg L-1 of total sulphonamides and described by the following equation: S = 2.50 + 0.0514C (μg L-1) and R = 0.999. The LOD and the CV (n = 11) were estimated to be 10 μg L-1 and 1.3%, respectively. The main interferents present at their usual concentrations in the sample did not interfere with the results. Spike and recovery tests of total sulphonamides were carried out in UHT and pasteurized milk with recovery values between 73 and 106% and the results obtained for this kind of sample were in agreement with those achieved by a high performance liquid chromatography (HPLC) procedure at the 95% confidence level. The analytical procedure presents an adequate sensitivity to determine total sulphonamides in bovine milk and does not require organic solvents, being aligned to the principles of green chemistry.

Determination of Vancomycin B and Vancomycin Impurities by Liquid Chromatography

The preferred test methods for control of product-related impurities in medicinal products are high-performance liquid chromatography (HPLC) with a fine sorbent, and ultra-performance liquid chromatography (UPLC), which allow for better chromatographic separation of active substances and related impurities, reduction of time costs, and saving of material resources. The aim of the study was to develop HPLC and UPLC test procedures and assess the chromatographic separation capacity and efficiency in order to improve determination of the main vancomycin component and related impurities. Materials and methods: vancomycin hydrochloride lyophilisate for oral solution and solution for injection, and vancomycin hydrochloride reference standard (USP RS) were used as test objects. Agilent 1290 Infinity liquid chromatography system, and Chromolith® Performance RP-18e, Kinetex C18, Nucleodur C18 Isis, Zorbax RRHD Eclipse Plus C18, and LiChrospher® RP-18 columns were used for the testing. Results: HPLC analysis using a Chromolith® column (100×4.6 mm) reduces the testing time by 10 minutes compared to the USP test procedure, and by 15 minutes compared to the British Pharmacopoeia procedure. The proposed test procedure requires less eluent and increases chromatographic separation efficiency. UPLC analysis using a Kinetex C18 column (50×4.6 mm, 2.6 μm) made it possible to reduce the testing time by two thirds compared to the British Pharmacopoeia procedure. The use of isocratic elution greatly simplified the testing. The testing time under the proposed chromatographic conditions was 10 minutes. Conclusions: the selected HPLC and UPLC test conditions made it possible to significantly reduce the time of testing, minimise the use of expensive reagents, and increase efficiency of chromatographic separation in the determination of vancomycin impurities and the main component Vancomycin B.

PSX-A-27 Late-Breaking: The sulfatase mediated regeneration of androstenone from androstenone sulfate in boar fat

Abstract Boar taint is an off-odour or off-flavour that develops in heated pork products from entire male pigs, which is caused by the accumulation of androstenone, a sex pheromone, in the fat. However, we have previously demonstrated that a significant amount of androstenone undergoes sulfoconjugation upon synthesis in the Leydig cells and circulates in the plasma primarily as a polar steroid sulfate. Therefore, the purpose of this study was to determine if androstenone sulfate can be deconjugated within the adipose tissue by the sulfatase enzyme to return free androstenone and indirectly contribute to the development of boar taint. Backfat was obtained from 6-month-old terminal cross [Duroc x (Landrace x Yorkshire)] boars that had high (n=4) or low (n=4) sulfatase expression as determined by RT-PCR. Sulfatase activity in the fat was measured by quantifying the conversion of androstenone sulfate to free androstenone. Backfat was homogenized and the supernatant was incubated with [3H]-androstenone sulfate for 24-hours. Androstenone was extracted from the incubation using ether and steroid conversion was quantified using high-performance liquid chromatography (HPLC). Additionally, fat androstenone concentrations were quantified using an established HPLC procedure. Statistical analysis was conducted using a Student’s t-test. There was a significant difference (p=0.04) in the expression of sulfatase between the high (2.99 ± 0.67) and low (1.21 ± 0.19) sulfatase boars and the percentage of androstenone sulfate that was converted to free androstenone was proportional to the expression of sulfatase. Interestingly, the expression of sulfatase was positively related to the concentration of androstenone in the fat in boars with high sulfatase expression; however, this relation was not as strong in animals with low sulfatase expression. These preliminary results suggest that the development of boar taint may occur indirectly through the deconjugation of androstenone sulfate in boars with high expression of sulfatase in the fat.

Determination of β-carotene and α-tocopherol Content in Selected Fresh and Dry Vegetables in Butula in Busia County

The study involved the determination of β-carotene and α-tocopherol content in selected fresh and dry vegetables (amaranthus, cowpea leaves, nightshade, slender leaf, pumpkin leaves and frying spider) using High Performance Liquid Chromatography (HPLC) procedure. Fresh blanched vegetables contained high levels of β-carotene; 4000-9700µg/100g and α-tocopherol levels; 3000-7360µg/100g (WW). The solar dried vegetables contained β- carotene levels ranging from 572 to 854µg g-1 dry weight (DW) and α-tocopherol levels ranging from 281 to 673µg g-1 (DW). Solar dried vegetables contained significantly lower (P<0.05) amounts of β-carotene and α-tocopherol which were moderately bioavailable when mixed in good proportion to meet Recommended Dietary Allowance (RDA) of vitamins A and E; which are 750µg retinol equivalent/day and 8mg/day respectively. The results will provide nutritional information on the indigenous vegetables grown in Butula in Busia County.

A Simple Reversed Phase High Performance Liquid Chromatography Method for the Estimation of Related Substances, Assay of Cabozantinib and Nivolumab and its Application to Dissolution Studies

The present work describes the development and subsequent validation of a novel, simple, selective and stability indicating gradient reversed phase high performance liquid chromatography method for the quantitative determination of related substances, assay of cabozantinib and nivolumab and its application to dissolution studies. The chromatographic method was optimized using the impurity-spiked solution. A good resolution between the peaks was achieved under selected chromatographic conditions. The separation was accomplished on an X-Bridge C18, 150×4.6 mm, 3.5 μ column connected to a photo diode array detector using 0.1 % orthophosphoric acid in water as mobile phase A and acetonitrile as mobile phase B, under gradient elution. The mobile phase flow rate was maintained at 1.0 ml/min. The detection of the constituents was done at 216 nm using a ultra-violet detector. Recovery studies were satisfactory and the correlation coefficient for two active pharmaceutical ingredients and their related substances, 0.999 indicates the linearity of the method within the limits. Limit of detection and limit of quantification for all impurities and cabozantinib and nivolumab were established with respect to the test concentration. Specificity, accuracy, precision, ruggedness and robustness were determined as part of the method validation. The performance of the method was validated according to the current International Council for Harmonisation requirements. Moreover, the dissolution study was performed on active pharmaceutical ingredients to estimate the recovery using the same method. Validation of the developed reversed phase high performance liquid chromatography procedure revealed that all the degradation products formed during stress conditions and related impurities were well separated from their active pharmaceutical ingredients and peaks were well resolved from each other with appropriate retention time. The method was characterized by good linearity, specificity, low values of limit of detection and quantisation, accuracy, precision, ruggedness and robustness. All statistical results were within the acceptance criteria and the proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine dissolution analysis and employed for quality control of drug samples during stability studies.

Hepatoprotective effects of Tagetes lucida root extract in carbon tetrachloride-induced hepatotoxicity in Wistar albino rats through amelioration of oxidative stress

Context The roots of Tagetes lucida Cav. (Asteraceae) have antioxidant and antimicrobial properties. Objective This study aimed to examine the hepatoprotective effects of T. lucida roots ethanol extract (TLRE) using carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Materials and methods The active ingredients of TLRE were identified by high-performance liquid chromatography, infra-red spectrum, and mass spectrometric procedures. Ninety rats were distributed into four main groups: positive, therapeutic, protective, and negative group. The therapeutic group was implemented using CCl4 (a single dose of 2 mL/kg) before TLRE or silymarin administration. Meanwhile, the protective group was implemented by administering CCl4 (a single dose of 2 mL/kg) after force-feeding TLRE or silymarin. Each therapeutic and protective group was divided into three subgroups: force-fed with saline, TLRE (500 mg/kg), and silymarin (25 mg/kg). The positive group was split into two subgroups that were force-fed TLRE and silymarin. Positive, therapeutic, and protective groups were compared to the negative group (untreated rats). CCl4, TLRE, and silymarin were orally administrated using a gastric tube. Results In the therapeutic and protective groups, TLRE significantly reduced liver enzymes, i.e., aspartate aminotransferase (12.47 and 6.29%), alanine aminotransferase (30.48 and 11.39%), alkaline phosphatase (17.28 and 15.90%), and cytochrome P450-2E1 (39.04 and 48.24%), and tumour necrosis factor-α (53.72 and 53.72%) in comparison with CCl4-induced hepatotoxicity controls. Conclusions TLRE has a potent hepatoprotective effect with a good safety margin. After a repeated study on another type of small experimental animal, their offspring, and an experiment with a large animal, this study may lead to clinical trials.

Separation and Purification of Glucose in Sake for Carbon Stable Isotope Analysis

An existing procedure for the separation and purification of glucose in sake for compound-specific carbon stable isotope analysis was examined with a view to reducing the sample amount required and process complexity, with a focus on the freeze-drying, solid-phase extraction (SPE), and high-performance liquid chromatography (HPLC) steps. In the revised procedure, the carbon stable isotopic composition, as indicated by δ13C values, of glucose was not significantly affected by glucose concentration in the range 0.25–1.00 mg mL−1, solvent (water or 80% aqueous acetonitrile), or HPLC procedure. Glucose freeze-dried after separation and purification by SPE and HPLC exhibited procedure-induced carbon isotopic discrimination of ≤ 0.1‰. The isolation procedure achieved the same accuracy in glucose δ13C values as that achieved with traditional ion-exchange procedures while requiring a sample volume (1 mL) only 4% that of the usual volume. The improved method enables cost-effective, labor-efficient, small-scale, compound-specific carbon stable isotope analyses of glucose in sake.

Isolation, in vitro and in silico Evaluation of Phenylethanoid Glycoside from Arrabidaea brachypoda as Lipoxygenase Inhibitor

Lipoxygenase (LOX) plays an important role in inflammatory processes and Arrabidaea brachypoda (DC.) Bureau (Bignoniaceae) has been described as presenting anti-inflammatory activity. Therefore, the objective of this study was to develop a high-performance liquid chromatography (HPLC) procedure to directly recognize LOX inhibitor compounds in A. brachypoda crude extract, facilitating the isolation, characterization of bioactive compounds, evaluation of natural compounds using an in vitro 15-LOX assay and prediction of the most probable binding modes of their main constituent through molecular docking simulations. The chemical analysis was performed by ethanol crude extract microfractionation using HPLC-DAD (diode array detector) associated to a fraction collector. The bioactive chromatogram displayed a peak with 50.9% LOX inhibition at 13.6 min retention time. The extract was purified and conandroside was isolated, presenting a LOX inhibitory activity with an inhibitory concentration (IC50) of 7.8 ± 1.1 µM, close to standard quercetin (IC50 7.6 ± 0.3 µM). Additionally, conandroside was not cytotoxic to normal cells (GM07492A). The LOX-conandroside complex displayed a slightly higher docking score (92.7) than quercetin (71.5). These results together suggest that conandroside could be explored as lipoxygenase inhibitor.

Current Approaches to Valerian Tincture Standardisation in Terms of Assay

The quality control of the «Valerian rhizome and roots» herbal substance is carried out using high performance liquid chromatography (HPLC) according to the State Pharmacopoeia of the Russian Federation, XIV edition. The quantitative analysis of the active ingredients in valerian tincture is performed using a non-specific and non-selective spectrophotometric method. Therefore, it is important to introduce in Russia a more modern test procedure for quantitative determination of active ingredients in valerian tincture.The aim of the study was to develop a selective and sensitive HPLC procedure for quantitative determination of the total content of sesquiterpenic acids, expressed as valerenic acid, for the purpose of valerian tincture standardisation.Materials and methods: valerian tincture samples produced by seven Russian manufacturers were used as test samples, and valerenic acid was used as the reference standard. The quantitative analysis of the active ingredients was performed by two methods: spectrophotometry at 512 nm following the reaction of valerenic acid ethylester with hydroxyalamine and ferric chloride, and by HPLC using a Nucleosil C18 column, 125×4.6 mm, 5 µm particle size, in gradient elution mode, with detection at 220 nm.Results: the spectrophotometric technique was shown to be insufficiently specific. The authors of the study validated the chromatographic test procedure, established system suitability criteria, and compared the results obtained by the two test procedures. They also determined a tentative standard of the total content of sesquiterpenic acids, expressed as valerenic acid, obtained by HPLC.Conclusions: the HPLC assay developed for quantitative determination of active ingredients in valerian tincture is more specific as compared to the spectrophotometric technique, as the sum of the peaks of valerenic and acetoxyvalerenic acids and the results for the reference standard are taken into account during calculations. The new test procedure is in line with the cross-cutting standardisation principle and can be recommended for inclusion into the draft monograph «Valerian tincture».