HPLC-UV Method Research Articles

Development and Validation of HPLC-UV Method for the Determination of Levothyroxine in Orodispersible Minitablets for Pediatric Application

Background: Thyroid hormones play an important role in cognition and brain development. The determination of the content of levothyroxine, as well as related substances and dissolution test analysis, should be carried out by methods that are selective and highly sensitive due to the low concentration used in low dose orodispersible minitablets. Objectives: This study aims to develop and validate an analytical method by HPLC-UV for the quantification of levothyroxine and its related substances in pharmaceutical formulations; looking forward to being this method suitable for a future dissolution test analysis using tandem mass spectrometry detector. Methods: A Hypersil GOLD C18 (100 x 2.1 mm, 3µm) column was used with 25°C column temperature, 5 µL injection volume, 0.3 mL/min flow rate and detection at 225 nm. The mobile phase consisted of methanol: 0.05% formic acid (55:45). The developed method was validated for specificity, linearity, precision, accuracy and robustness. Results: The method is linear within the range of 2-20 µg mL-1 (R2=0.9982), which makes the method suitable for the evaluation of levothyroxine in pharmaceuticals formulations. LOQ was 0.17 µg/mL (0.85 ng on column) and LOD 0.05 µg/mL (0.25 ng on column) of LT4. Therefore, in terms of efficiency (1671), retention factor, k (6.79), Tailing factor, T (1.09) and resolution, Rs (5.11) the chromatographic method was found to be suitable according to USP 43. Conclusion: The HPLC UV method was found to be linear, specific, precise, accurate and robust, therefore it is suitable for the quality control of levothyroxine in pharmaceutical ODMTs.

Optimization of extracting technology of iridoid glycosides and phenylethanoid glycosides from Pedicularis decora Franch. by G1-entropy-coupled response surface methodology and rapid identification by UPLC-Q/TOF-MS

Quality control and clarifying ingredients are the foundation of pharmacodynamic material basis research. Pedicularis decora Franch. (P. decora) is clinically used to treat Alzheimer’s disease and exerts significant antioxidative, anti-inflammatory and neuroprotective effects. However, there is little systematic research on the extraction and composition and the current research on P. decora focused on the quality control and extraction with aucubin as only quality marker. It reported that Iridoid glycosides (IGs) and phenylpropanoid glycosides (PeGs), which are characteristic components of the Pedicularis species, have been reported to have various anti-Alzheimer’s activity. This study focused on the extraction and rapid qualitative analysis of IGs and PeGs in P. decora. First, HPLC-UV method was established to detect five IGs and PeGs analytes simultaneously and assess the effect of the extraction technology. Then, the G1-entropy method with the Box-Behnken response surface design methodology was used to optimized extraction technology. The optimum conditions were determined to be reflux with 55% methanol aqueous solutions, at 75 °C for 50 min. Finally, by establishing UPLC-Q/TOF-MS, 15 IGs and 15 PeGs in P. decora were rapidly identified and characterized for the first time. The results will provide a reverence of a higher quality standard and the pharmacodynamic material basis research.

Ecotoxicological Evaluation of Safener and Antimicrobial Additives in Isoxaflutole-Based Herbicide Formulations

The environmental load by isoxaflutole and its formulated herbicide products has increasingly become apparent because, after the ban of atrazine, isoxaflutole has become its replacement active ingredient (a.i.). Obtaining information regarding the fate of this a.i. in environmental matrices and its ecotoxicological effects on aquatic organisms is essential for the risk assessment of the herbicide. In this study, the effects of Merlin Flexx- and Merlin WG75 formulated isoxaflutole-based herbicide products and two selected additives (cyprosulfamide safener and 1,2-benzisothiazol-3(2H)-one antimicrobial agent) were investigated on Raphidocelis subcapitata in growth inhibition assays. In ecotoxicological tests, two conventional (optical density and chlorophyll-a content) and two induced fluorescence-based (Fv*/Fp: efficiency of the photosystem PSII and Rfd* changes in the observed ratio of fluorescence decrease) endpoints were determined by UV-spectrophotometer and by our FluoroMeter Module, respectively. Furthermore, dissipation of isoxaflutole alone and in its formulated products was examined by an HPLC-UV method. In ecotoxicological assays, the fluorescence-based Rfd* was observed as the most sensitive endpoint. In this study, the effects of the safener cyprosulfamide and the antimicrobial agent 1,2-benzisothiazol-3(2H)-one on R. subcapitata is firstly reported. The results indicated that the isoxaflutole-equivalent toxicity of the mixture of the isoxaflutole–safener–antimicrobial agent triggered lower toxicity (EC50 = 2.81 ± 0.22 mg/L) compared to the individual effect of the a.i. (EC50 = 0.02 ± 0.00 mg/L). The Merlin Flexx formulation (EC50 = 27.04 ± 1.41 mg/L) was found to be approximately 50-fold less toxic than Merlin WG75, which can be explained by the different chemical characteristics and quantity of additives in them. The additives influenced the dissipation of the a.i. in Z8 medium, as the DT50 value decreased by approximately 1.2- and 3.5-fold under light and dark conditions, respectively.

Investigation of Chemical Compounds and Effect of Astragalus Galactites (Pall.) on MSU Crystal-Induced Acute Gouty Arthritis in Rats

The objective of the present investigation is to identify the biologically active components of Astragalus galactites (AG) and examine their effect on rats of acute gouty arthritis induced by crystals of the compound monosodium urate (MSU). Analyses of AG chemical constituents and their antioxidant activity were performed using both UV spectrophotometric and HPLC methods. Twenty-five adult Wistar rat males in total were assigned at random to a choice of five groups: AG 160 mg/kg, AG 330 mg/kg, MSU group, control group, and normal group. We reported the contents of Formononetin 1.97 µg/mL, Astragaloside IV 0.56 mg/mL, and total flavonoid 0.22±0.07% in the aerial part of our sample AG. In the DPPH scavenging assay, methanol and ethanol extracts established antioxidant properties with an IC50 concentration of 91.04 µg/mL and 93.13 µg/mL, respectively. In the ABTS scavenging assay, IC50 concentrations of 387.2 µg/mL (methanol extract) and 436.2 µg/mL (ethanol extract) were also shown. This investigation also looked at the histopathological characteristics related to MSU-induced gouty arthritis in order to assess the preventive effects of the AG plant on inflammatory mediator levels. In the AG 330 mg/kg group, the level of PGE2 significantly reduced (p<0.001). Our results showed that the AG 330 mg/kg group was relatively effective in the treatment of gouty arthritis compared to other groups, which appears to be mediated by inhibiting the release of cytokines that pro-inflammation. The main compounds of the AG medicine plant, flavonoids and saponins, are acutely anti-gout due to the resulting decrease in PGE2 levels. We have hypothesized that it is able to treat acute gouty arthritis by reducing levels of UA and PGE2, providing an anti-inflammatory effect. This study contributes to the body of evidence that AG can be utilized in preventing and treating hyperuricemia.

Decrease of cholesterol content in cereal products containing animal-based components

AbstractThe daily intake of excessive cholesterol plays an important role in the incidence of cardiovascular diseases (CVD), while the main contributors are foods of animal origin. However, animal-based foods are also common components of many cereal products, which can then play a role of hidden cholesterol source. Thus, this study aims to decrease cholesterol content in three types of commonly consumed cereal products (biscuits, pasta, and muffins) by replacing original animal-based components (milk and eggs) with the same components, from which the cholesterol was removed using β-cyclodextrin treatment procedure. The cholesterol content in the original and treated samples was then analyzed using the validated HPLC–UV method. So, while the cholesterol content in the original biscuits, pasta, and muffins was observed at 329.2, 1117.1, and 487.2 mg/kg levels, the same products containing low cholesterol animal-based components exhibited a considerable decrease in cholesterol content by 93.1, 91.6, and 89.5%, respectively. The optimum β-cyclodextrin level was observed at 4.0, 10.0, and 2.0% (w/w) for butter, egg melange, and milk, respectively. Furthermore, it is notable that the β-cyclodextrin treatment procedure itself did not significantly change the organoleptic profiles of the cereal products, such as colour, culinary, and texture properties. Therefore, it can be concluded that the application of animal-based low cholesterol components in cereal food products could be one of the effective tools in the overall strategy to decrease the total daily cholesterol intake. Thus, this study presents a novel strategy that could have substantial health benefits in relation to the incidence of CVD.

Determination of α-Dicarbonyl compounds in traditional Chinese herbal medicines

α-DCs (α-dicarbonyls) have been proven to be closely related to aging and the onset and development of many chronic diseases. The wide presence of this kind of components in various foods and beverages has been unambiguously determined, but their occurrence in various phytomedicines remains in obscurity. In this study, we established and evaluated an HPLC-UV method and used it to measure the contents of four α-DCs including 3-deoxyglucosone (3-DG), glyoxal (GO), methylglyoxal (MGO), and diacetyl (DA) in 35 Chinese herbs after they have been derivatized with 4-nitro-1,2-phenylenediamine. The results uncover that 3-DG is the major component among the α-DCs, being detectable in all the selected herbs in concentrations ranging from 22.80 μg/g in the seeds of Alpinia katsumadai to 7032.75 μg/g in the fruit of Siraitia grosuenorii. The contents of the other three compounds are much lower than those of 3-DG, with GO being up to 22.65 μg/g, MGO being up to 55.50 μg/g, and DA to 18.75 μg/g, respectively. The data show as well the contents of the total four α-DCs in the herbs are generally in a comparable level to those in various foods, implying that herb medicines may have potential risks on human heath in view of the α-DCs.

A simple and rapid HPLC-UV method for the determination of valproic acid in human plasma using microwave-assisted derivatization with phenylhydrazine hydrochloride

This study presents an efficient high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for monitoring valproic acid (VPA) level in human plasma. This method is distinguished by its simplicity, cost-effectiveness, and rapid execution, addressing the limitations associated with other advanced analytical techniques like liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and immunoassays, which are generally complex and costly for routine application. A challenge in analyzing VPA is its non-linear protein binding profile and the absence of a chromophore in its structure, making direct detection difficult. To overcome this, the study developed an efficient HPLC-UV for VPA determination in human plasma, utilizing a simplified and rapid microwave-assisted derivatization process. Due to the lack of a chromophore in VPA structure, this work developed a microwave-assisted derivatization of VPA using phenylhydrazine hydrochloride (PH HCl). The process optimization was achieved at 450 W for 50 s, facilitating effective HPLC-UV detection. The derivatized product was characterized using 1H nuclear magnetic resonance (NMR) and Fourier transform infrared spectrometer (FT-IR). The derivative, identified as (Z)-N-phenyl-2-propylpentanehydrazonic acid, demonstrated specificity in plasma analysis with no detectable interference. The method exhibited a linear response for VPA concentrations ranging from 30 to 150 μg/mL, with a correlation coefficient exceeding 0.99. Recovery varied between 86.7% and 107%, with a maximum coefficient of variation (CV) of 10.0%. The findings suggest that the microwave-assisted derivatization technique substantially improves the feasibility and cost-effectiveness of HPLC-UV for the analysis of VPA in plasma. This method provides a viable alternative to conventional HPLC methodologies, offering a balance of efficiency and economic practicality for VPA quantification.

Impact of Perinatal Arsenic Exposure on Amino Acid Neurotransmitters and Bioenergetics Molecules in the Hippocampus of Rats

Developmental neurotoxicity of Arsenic (As) is a major concern worldwide. High level As exposure is associated with several chronic diseases including adverse pregnancy and birth outcomes. However, very a lack of information on its ability to impair neurodevelopment at lower exposure. To date, there are very few animal studies during the perinatal period of As exposure. Although exposure to As induces developmental neurotoxicity, there is a lack of data regarding its specific effects on amino acid neurotransmitters and bioenergetics biomolecules in the hippocampus of developing rats exposed to As during the perinatal period (GD6-PD21). In continuation of previous studies, rats were exposed to As from gestational day (GD 6) through PD 21 with targeted doses of 0, 2.0, and 4.0 mg/kg/day, respectively. HPLC-UV method was used to estimate the level of amino acid neurotransmitters (aspartate, glutamate, homocysteine, glutamine, serine, and glycine) and the level of Adenosine 5’-Triphosphate (ATP), Adenosine Diphosphate (ADP), Adenosine Monophosphate (AMP), Nicotinamide Mononucleotide (NMN), Nicotinamide Adenine Dinucleotide (NAD+), reduced Nicotinamide Adenine Dinucleotide (NADH) in the hippocampus of rats after the exposure of As. Amino acid neurotransmitter levels, a predictive biomarker of As-induced developmental neurotoxicity were found to be altered. ATP, ADP, and AMP were also significantly impaired in the hippocampus of As-exposed rats. We have observed that the hippocampus is susceptible to As toxicity, both because of the high energy depletion and the alterations in the levels of selected amino acid neurotransmitters. Taken together, our results indicate that perinatal As exposure appears to be critical and vulnerable.

Identification, trace level quantification, and in silico assessment of potential genotoxic impurity in Famotidine

Potential genotoxic impurities in medications are an increasing concern in the pharmaceutical industry and regulatory bodies because of the risk of human carcinogenesis. To prevent the emergence of these impurities, it is crucial to carefully examine not only the final product but also the intermediates and key starting material (KSM) used in drug synthesis. During the related substances analysis of KSM of Famotidine, an unknown impurity in the range of 0.5–1.0% was found prompting the need for isolation and characterization due to the possibility of its to infiltrate into the final product. In this study, the impurity was isolated and characterized as 5-(2-chloroethyl)-3,3-dimethyl-3,4-dihydro-2H-1,2,4,6-thiatriazine 1,1-dioxide using multiple instrumental analysis, uncovering a structural alert that raises concern. Considering the potential impact of impurity on human health, an in silico genotoxicity assessment was established using Derek and Sarah tool in accordance with ICH M7 guideline. Furthermore, molecular docking and molecular dynamics simulation were performed to evaluate the specific interaction of the impurity with DNA. The findings reveal consistent interaction of the impurity with the dG-rich region of the DNA duplex and binding at the minor groove. Both in silico prediction and molecular dynamic study confirmed the genotoxic character of the impurity. The newly discovered impurity in famotidine has not been reported previously, and there is currently no analytical method available for its identification and control. A highly sensitive HPLC-UV method was developed and validated in accordance with ICH requirements, enabling quantification of the impurity at trace level in famotidine ensuring its safe release.

Development and Validation of an HPLC-UV Method for the Quantification of Acyclovir and Ganciclovir in the Plasma of Pediatric Immunocompromised Patients.

Acyclovir and ganciclovir comprise the prophylaxis and treatment of herpesvirus and cytomegalovirus infections occurring in immunocompromised patients. Their therapeutic drug monitoring is fundamental because of interindividual variability leading to side effects and drug resistance and is performed through several techniques, such as liquid chromatography coupled with UV spectrophotometry (HPLC-UV) or mass spectrometry (LC-MS/MS). Therefore, we developed and validated a low-cost, non-time-consuming, and low-sample-consuming HPLC-UV method. Briefly, 100 µL of sample was used for sample preparation, mainly consisting of precipitation through organic solvent. In total, 20 µL was injected into the instrument. Chromatographic separation was obtained eluting mobile phases A (10 mM ammonium formiate 0.01% formic acid) and B (acetonitrile) on a Poroshell 120 SB-C8 2.1 × 150 mm, 2.7 µm for 12 min isocratically (97:3; A:B) at a flow rate of 0.2 mL/min. The linearity range (0.5-40 mg/L) of the method allowed us to quantify both the Cmin and Cmax of acyclovir and ganciclovir. Plasma concentrations measured on a small cohort of patients undergoing acyclovir (31) and ganciclovir (9) treatment by the proposed method and the LC-MS/MS methods, already in use, were significantly correlated. The proposed HPLC-UV method may be implemented in diagnostics as an alternative method in case of the unavailability of the LC-MS/MS system.

Chemical Stability of Lorazepam Oral Solution Repackaged in Plastic Oral Syringes at Room and Refrigerated Temperatures

Purpose: Generic lorazepam oral solution is supplied in a 30 mL multi-dose bottle requiring protection from light and refrigeration, with a beyond use date of 90 days once the bottle is opened. The repackaging of 1 mL doses of lorazepam oral solution into oral syringes allows for facilitated dispensing, yet no available data supports repackaging and storing lorazepam oral solution in syringes. The validation and application of a stability-indicating high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the quantification of lorazepam allowed for the determination of the stability of lorazepam oral solution when stored in oral syringes. Methods: A stability-indicating HPLC-UV method was developed for the quantification of lorazepam in oral solution. The method was validated using guidance from USP < 1225 >. For the stability investigation, 2 mg/mL lorazepam oral solution was aliquoted into clear plastic oral syringes in 1 mLmilliliter doses from 2 multi-dose stock bottles and randomly allocated for storage in room temperature or refrigerated environment. Baseline lorazepam concentrations were measured on the day the study was initiated and designated as 100% initial concentration samples. Subsequent samples were analyzed in triplicate at time points of 24, 48, and 96 hours and 7, 10, 14, 21, 30, and 60 days. Results: The calibration curves on three non-consecutive days met the linearity criteria of R2 > 0.99. Inter- day and intra-day precision and accuracy (percent relative standard deviation and percent error) were ≤2% over three days. During the stability investigation, percent initial concentration of lorazepam from room and refrigerated syringes remained above 90% for the duration of the study. Conclusion: The stability-indicating HPLC-UV method was successfully applied to the investigation of lorazepam oral solution stability when stored in syringes at room and refrigerated temperatures. The emergent need for use of lorazepam concentrate for inpatients and the restrictions of how the medication is supplied necessitated a need for the evaluation of repackaging into unit dose syringes for immediate availability from automated dispensing cabinets. Lorazepam oral solution stored in clear plastic syringes maintained greater than 90% initial concentration at both room and refrigerated temperatures for 60 days.

CBD products: Labeling accuracy of an obscure niche market

In recent years, there has been an increased availability of cannabis products in Europe, despite strict regulations. Numerous manufacturers of niche products promote cannabidiol (CBD) and Cannabis sativa (C. sativa) as ingredients, often in ambiguous ways leading to consumer confusion. The aim of this study was to analyze a diverse range of foods, food supplements, health related products and other consumer products (e.g. vapes) associated with C. sativa/CBD in order to determine whether the declared and promised ingredients are present in the products and how these comply to current legal regulations. We have established a HPLC-UV method to quantify the most important cannabinoids, namely the legally important THC and THCA, and the CBD/CBDA. All of the 35 analyzed products met the legally required limit for THC/THCA. A total of 27 of the samples explicitly listed CBD contents. In 33% of the samples, the analyzed value matched the content given on the label, 26% exceeded the declared CBD content, while 41% contained lower amounts. CBD products with an unclear or not defined CBD content, were mostly below the limit of detection (LoD). Products promising “full spectrum C. sativa extracts” were further screened by GC-MS method to detect additional naturally-occurring cannabinoids, to verify the use of C. sativa plant material/extracts as the source of CBD. No further cannabinoids could be detected in one out of eight products claiming the use of C. sativa extracts.This study shows, that this niche market is still a constantly evolving product sector, depending on the type and origin of the products, there may be significant discrepancies between the marketing and the actual ingredients contained, making the market less transparent for consumers.

Determination of urea in pet feed: assessing the suitability of different analytical techniques using proficiency test data

The determination of urea in pet feed at contaminant levels using the spectrophotometric method described in Commission Regulation (EC) No 152/2009 has been reported by several EU laboratories to lack the required selectivity. Whilst urea is not authorised as an additive in pet feed, the control of urea in pet feed is of economic importance, because the addition of urea may unlawfully increase the apparent protein content. To investigate the capabilities of different analytical techniques, a proficiency test was organised where the participants (EU official control laboratories, laboratories from the academia and private laboratories) were free to use their method of choice for analysing three dog feed test materials, two samples of which were spiked with urea. Twenty-one laboratories submitted results using the following techniques: spectrophotometry (Implementing Regulation (EC) No 152/2009), LC-MS/MS, HPLC-UV, enzymatic-colorimetry, gravimetry and an ‘in-house photometric’ method. Only two laboratories that used LC-MS/MS were able to quantify urea accurately in the test material containing a mass fraction of 18.9 mg kg−1 whereas satisfactory results at the level of 258.9 mg kg−1 were obtained by one participant that used an ‘in-house photometric method’ and one that used the enzymatic method, in addition to the five participants using LC-MS/MS. The technique that provided the highest success rate across the three test materials was LC-MS/MS, whereas spectrophotometry, the enzymatic-based and HPLC-UV methods led to overestimated results in addition to a dispersion of results not suitable for compliance analysis. To address the determination of urea in pet feed at low levels, a better performing method than the one described in the legislation is required.

A Novel Antibiotic, Rhodomyrtone: Pharmacokinetic Studies in a Murine Model and Optimization and Validation of High-Performance Liquid Chromatographic Method for Plasma Analysis.

Rhodomyrtone has indisputable and undeniable potential as a new antibiotic for antibiotic-resistant Gram-positive bacteria. Therefore, the main objective of this study was to determine the pharmacokinetics profiles of orally administered rhodomyrtone in rats. A reverse-phase HPLC-UV method was developed, optimized and validated for the analysis of rhodomyrtone concentrations in rat plasma. The retention time of papaverine and rhodomyrtone was 3.928 and 5.937 min, with no interference with the excipients used. The lower limit of quantification (LLOQ) of rhodomyrtone in the plasma sample was 0.04 μg/mL, the accuracy of rhodomyrtone at the LLOQ level ranged from 93.64 to 106.36%, precision was 6.59%, 80-120% for accuracy and <20% CV for precision. The calibration curve was linear at concentrations ranging from 0.04 to 128 µg/mL with a correlation coefficient (r) value of equal to or greater than 0.999. Sprague Dawley rats received a single dose of rhodomyrtone at 50 and 100 mg/kg. Blood samples were collected from tail veins. The peak plasma concentration was observed at 2 h, and the area under the curve of rhodomyrtone at 50 mg/kg and 100 mg/kg was 3.41 ± 1.04 and 7.82 ± 1.53 μg·h/mL, respectively. The results demonstrated linear pharmacokinetics characteristics at the studied dosage range. The plasma concentration of rhodomyrtone was above the minimal inhibition concentrations of several common pathogenic bacteria of medical importance. The proposed HPLC-UV method is fast, cost-effective, reliable and reproducible, and it is proposed for the routine analysis of rhodomyrtone.

HPLC-UV Analysis of Flavonoid Glycosides in the Herb of &lt;i&gt;Astragalus&lt;/i&gt; (&lt;i&gt;A. dasyanthus&lt;/i&gt;, &lt;i&gt;A. varius&lt;/i&gt;, &lt;i&gt;A. testiculatus&lt;/i&gt;, &lt;i&gt;A. henningii&lt;/i&gt;), Growing in the Volga Region

Introduction. The search for new promising sources of biologically active substances is one of the tasks of modern pharmacognosy. Among the huge variety of species of the genus Astragalus L., only a few of them have been studied. In addition, none of them is included in the current edition of the State Pharmacopoeia of the Russian Federation. In our opinion, the study of four species of this genus of Astragalus L. (A. dasyanthus, A. varius, A. testiculatus, A. henningii) growing in the Volga region is of interest.Aim. Study of the glycoside composition of flavonoids in the astragalus herb of four species (A. dasyanthus, A. varius, A. testiculatus, A. henningii) by HPLC-UV using external standards.Materials and methods. The material was samples of astragalus grass (A. dasyanthus, A. varius, A. testiculatus, A. henningii), collected on the territory of the Saratov region in accordance with the basic rules of collection and preparation and dried to an air-dry state. Aqueous-alcoholic extracts (1 : 50) were analyzed. Qualitative and quantitative analysis of flavonoid glycosides was carried out on an Agilent 1260 chromatograph (Agilent Technologies, USA) equipped with a diode array detector, manual sample injection (Agilent G1328C manual injector, Agilent Technologies USA) and an Agilent Open Lab CDS system for collecting and processing chromatographic data. The elution gradient was selected individually using 0.1 % phosphoric acid/acetonitrile solution as solvents. Compounds were identified by retention times and UV spectra of reference standards (RS). The quantitative content of each detected component was expressed in mg/g of dry raw material. All studies were repeated five times, the results were statistically processed using MS Exсel 2010.Results and discussion. The chromatographic profiles of aqueous-alcoholic extracts from the herbs of four types of astragalus were studied. Differences were found in both the qualitative composition and quantitative content of flavonoid glycosides in the grass of the analyzed species. The largest number of compounds was found in the herb of A. varius (6 compounds), 4 compounds were identified in the herb of A. dasyanthus and A. henningii, and 3 compounds were identified in the herb of A. testiculatus. The dominant components in the herb of A. henningii are orientin and rutin, in the herb of A. testiculatus and A. varius it is cynaroside, and in A. dasyanthus it is rutin.Conclusion. A HPLC-UV method was developed for determining the quantitative content of flavonoid glycosides in extracts from the herbs of four species of astragalus (A. dasyanthus, A. varius, A. testiculatus, A. henningii) and the chromatographic profiles of the analyzed samples were studied.

Synthesis and research of polyurethane urea with 2-(2-aminoethoxy)ethan-1-amine and 3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propane-1-amine as macrochain extenders, and compositions with ifosphamide based on them

Polyurethane urea (PUU) was synthesized using macrochain extenders of 2-(2-aminoethoxy)ethan-1-amine (DA1) and 3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propan-1-amine (DA3) at a molar ratio of 4,4'-diaminodiphenylmethane (DADP) to DA1 and DA3 as 30:70; 50:50; and 70:30. The tensile strength () of PUU is in the range of 0.74–2.21 MPa, and the relative elongation at break () is 27.4–1003%. According to the DSC method, the studied PUUs are single-phase systems. The glass transition temperature (Tg) of the PUU DA1 series is from 21.920C to –39.480C, and the glass transition temperature for the PUU DA3 is from –20.040C to –33.960C. Based on the synthesized PUUs, composite materials were prepared containing 1 wt.% of ifosfamide (IFO) (=0.74–2.21 MPa, and =31.8–276.3%). According to the results of thermophysical studies, the compositions with IFO are single-phase systems with Tg of –24.80C to –36.220C. Thus, the introduction of IFO into the composition leads to an increase in the packing density of the macrochain, a decrease in the size of the free volume and, consequently, a decrease in the mobility of the blocks in space and a decrease in Сp. Compositions with IFO are resistant to temperatures of ~2500C, which allows thermal sterilization before use. Using the HPLC-UV method, the dynamics of IFO release from samples of PUU compositions synthesized at a molar ratio of DADP:DA1 as 0.5:0.5 and DADP:DA3 as 0.7:0.3 was investigated. It was established that IFO is released within 60 days from compositions based on PUU DA3 in the amount of 29.6%, and from compositions based on PUU DA1 in the amount of 42% with respect to the total amount of IFO introduced. The resulting composites are promising materials for medicine as means of local prolonged therapeutic action.

Neuroprotective Effect of Boswellia serrata against 3-NP Induced Experimental Huntington’s Disease

Objectives: The study aimed to assess the neuroprotective effect of Boswellia serrata against 3-NP-induced experimental Huntington’s disease. Background: Previous studies have shown Boswellia to have sedative, analgesic, and anti-tumour effects. Boswellia serrata yields four pentacyclic triterpene acids and boswellic acid, a bioactive substance that prevents leukotriene biogenesis. Methodss: The potential neuroprotective effect of Boswellia serrata against 3-nitro propionic acid (3-NP)-induced Huntington's disease (HD) was examined at oral doses of 45 mg/kg, 90 mg/kg, and 180 mg/kg. In this study, HD was induced by 3-NP at a dose of 10 mg/kg in Wistar rats. The study used 56 Wistar rats (8 per group) for biochemical (inflammatory markers, acetylcholinesterase activity) and behavioural (elevated plus maze, Y-maze, open-field, tail suspension tests, etc.) assessments. Additionally, a histological examination of the brain was carried out. In addition, the analysis of Boswellia serrata extract was performed by different analytical techniques, like UV spectrophotometer, FTIR, and HPLC methods. Results In the brain, succinate dehydrogenase is a mitochondrial enzyme irreversibly inhibited by 3-NP. Administration of 3-NP resulted in HD with altered behavioural and motor changes in rats. Treatment with Boswellia serrata resulted in remarkable protection of rats against 3-NP-induced behaviour and motor deficits in a dose-dependent manner. Moreover, in rats administered with 3-NP, Boswellia serrata improved memory performance and lowered levels of inflammatory biomarkers. These results have also been supported by histopathological analysis. Acetyl-11-keto-p-boswellic acid was found to be the main active component of Boswellia serrata extract. Conclusion:: Boswellia serrata at a dose of 180 mg/kg exhibited better protection compared to the other doses against HD induced by 3-NP. More detailed studies based on molecular targets are needed for the Boswellia serrata to transition from the bench to the bedside for use as an adjuvant in HD patients.

[Simultaneous determination of four N-lauryl amino acid surfactants in facial cleansers by high performance liquid chromatography-ultraviolet detection].

Significant developments have recently been achieved in the field of N-lauryl amino acid (NLAA) surfactants derived from renewable resources. Compared with conventional surfactants, NLAAs exhibit remarkable surfactant properties, exceptional biodegradability, good biocompatibility, and high safety profiles. These attributes have led to the widespread use of NLAAs in personal-care products. The detection methods employed for NLAAs include two-phase titration (TT), spectrophotometric analysis (SA), and high performance liquid chromatography (HPLC). However, because both TT and SA measure the total concentration of anionic active matter, identifying and quantifying individual compounds in a sample containing multiple anionic surfactants is impossible. The presence of cationic surfactants in the sample also introduces interferences, which lead to significant errors. Compared with TT and SA, HPLC offers direct and rapid testing procedures. However, compounds with no or weak UV-visible light absorption exhibit low sensitivity when detected by UV, necessitating the use of detectors such as differential refractive index detectors (RIDs), evaporative light scattering detectors (ELSDs), or charged aerosol detectors (CADs). Most HPLC users consider UV light as the fundamental configuration of the instrument, and other detectors are less commonly employed. Therefore, establishing a new HPLC method suitable for the UV detection of NLAAs is of practical significance. In this study, a novel HPLC-UV method was developed for the simultaneous detection of N-lauryl glutamine (LG), N-lauryl glycine (LC), N-lauryl alanine (LA), and N-lauryl sarcosine (LS) by optimizing the mobile-phase composition and selecting an appropriate chromatographic column and detection wavelength. The samples were mixed with acetonitrile-0.10% H3PO4 aqueous solution (60∶40, v/v) and sonicated for 10 min, then stayed at room temperature for 5 min. Subsequently, the mixture was filtered through a 0.22 μm filter membrane and separated on an Agilent Eclipse Plus C18 column (150 mm×4.6 mm, 5 μm). The mobile phase used for separation consisted of acetonitrile-0.10% H3PO4 aqueous solution at a flow rate of 1.0 mL/min. The detection wavelength was set at 205 nm, and the injection volume was 10 μL. The results demonstrated that the four NLAAs exhibited good linearity in the range of 2.0-800.0 mg/L, with correlation coefficients (r)≥0.9995. The limits of detection (LODs) ranged from 0.17 to 0.49 mg/L, and limits of quantification (LOQs) ranged from 0.57 to 1.63 mg/L. The relative standard deviations (RSDs) for precision, repeatability, and stability over 24 h were all below 2.0%. Using this method, the NLAA contents of five facial-cleanser products were determined. The results demonstrated that all five samples contained one or more NLAAs, and the total NLAA contents ranged from 64.58 to 97.01 mg/g. The five spiked-sample recoveries of the NLAAs at four different spiked levels (0.60, 4.50, 15.00, 24.00 mg/g) ranged from 94.3% to 107.4%, indicating satisfactory accuracy. However, the actual NLAA composition and label for one facial-cleanser product were not consistent with our test results. This finding demonstrates the necessity of strengthening market monitoring through testing. The proposed method has the advantages of simple pretreatment, rapid testing, good precision, high accuracy, and appropriate stability. Thus, it is suitable for the determination of NLAA contents in facial cleansers and provides an effective technical reference for the raw-material purity assessment, synthetic yield detection, and product quality control of this type of surfactant.

Investigations of potential non-amino acid SNAT2 inhibitors.

The sodium-coupled neutral amino acid transporter 2 (SNAT2, SLC38A2) has been implicated in cancer for its ability to supply cancer cells with glutamine and sarcosine. A recent high-throughput screen published by Gauthier-Coles et al. identified the non-amino acid 3-(N-methyl (4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide (MMTC or 57E) as a potent and selective SNAT2 inhibitor. Here we have investigated the ability of MMTC and four other compounds selected from the screen by Gauthier-Coles et al. to decrease 3H-Gly uptake in hyperosmotically treated human prostate cancer PC-3 cells. In these cells, SNAT2 is highly upregulated when the cells are hyperosmotically stressed for 24h and is the primary contributor to glycine uptake. The five compounds were investigated at concentrations of 1-50µM based on their equilibrium solubility. At 37°C the equilibrium solubility in HEPES buffered HBSS at pH 7.4 was measured to be 24.9 (53B), 56.1 (54F), 13.3 (55B), and 27.5 (57B)µM, respectively. The equilibrium solubility of MMTC was below the detection limit of the HPLC-UV method, thus less than 1.8µM. However, a kinetic solubility of approximately 2.5-10µM could be achieved during the course of the uptake study. In contrast to the previous publication, MMTC showed no inhibition of SNAT2-mediated 3H-Gly uptake in PC-3 cells at a concentration of 1 or 5μM, despite a published IC50 of 0.8µM. Similarly, 53B, 55B, and 57B showed no inhibition at soluble conditions, whereas 54F showed approximately 20% inhibition at 50µM. In our experimental setup, the investigated compounds showed limited potential as SNAT2 inhibitors.

Eco-friendly based HPLC and UV-spectrophotometric methods for simultaneous estimation of Efonidipine hydrochloride Ethanolate and Chlorthalidone in their dosage form

ObjectiveEfonidipine hydrochloride Ethanolate and Chlorthalidone combination was approved by CDSCO for Phase III clinical trial. This combination is utilized to pharmacological therapy that can effectively control hypertension situations. The present research includes an RP-HPLC and Simultaneous equation UV methods for the determination of drug combination in tablet dosage form. Furthermore, greenness assessment and statistical evaluation of developed methods. MethodsThe HPLC chromatography was performed using a gradient technique on a reversed-phase C18 column with mobile phase based and optimized depending on the polarity of the molecules. The Unesphere C18 column Agela Tech., (250 mm x 4.6 mm i.d, 5 μm,) as stationary phase and Acetonitrile: 0.2 M Potassium dihydrogen orthophosphate pH 4 (68: 32% v/v) as mobile phase. The Simultaneous equation UV method was developed and validated at 220 nm and 250 nm in methanol: distilled water (20:80, v/v). The greenness assessment tools were used to assess the ecological impact of the new UV spectrophotometric method and HPLC method. ResultsUV detection in RP-HPLC method at 220 nm for gradient elution was used to achieve chromatographic separation over the linearity 3.0–60.0 μg/mL and 10.0 - 200.0 μg/mL and for UV method 3.0–15.0 μg/mL and 10.0–50.0 μg/mL for Chlorthalidone (CLT) and Efonidipine (EFD) respectively.The value of% RSD within the limit indicated that the method is precise, accurate and% recovery shows that there is no interference from the excipients.Greenness assessment data confirmed the method's environmental friendliness in terms of solvent usage, chemical compounds, energy consumption, and trash creation. There were no spectral or chromatographic interferences from formulation excipients discovered. ConclusionThe analytical method validation parameters were found to be as per acceptance criteria. In terms of accuracy and precision, statistical testing employing an analysis of variance test did not find any significant differences between the two methods. To evaluate the greenness and whiteness of an analytical method, a reliable assessment of the health and environmental risk associated with chemical use is essential.