Quantitative High-performance Liquid Chromatography Research Articles

A standardized chamuangone enriched extract from Garcinia cowa Roxb. leaves shows acute and sub-acute safety

Ethnopharmacological relevanceThe safety assessment of herbal products is critical for their appropriate pharmacological applications. Garcinia cowa Roxb., commonly known as Cha-muang in Thai, has ethnopharmacological relevance for inflammation, infectious diseases, and diabetes. The leaf extracts of G. cowa have been extensively reported for their anticancer, anti-inflammatory, antimicrobial, and antioxidant effects. Notably, chamuangone is their major active constituent that contributes to various pharmacological properties. Aim of the studyThe current study aims to establish a standardized chamuangone enriched extract (CEE) and assess its acute and sub-acute toxicities in animal models. MethodologyCEE was established from G. cowa leaves using a microwave-assisted extraction (MAE), followed by fractionation and enrichment through silica gel vacuum and column chromatography. The concentration of chamuangone in the extract was quantified using a validated quantitative high-performance liquid chromatography (HPLC) method. The safety profiles of CEE were thoroughly evaluated in rodents according to the Organization for Economic Cooperation and Development (OECD) 425 and 407 guidelines. The effects on oxidative stress markers such as superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT), and malondialdehyde (MDA) levels were also evaluated in various organs. ResultsBased on the quantitative HPLC analysis, the CEE contained 73.0 ± 2.0% w/w of chamuangone. In the acute toxicity study, following up and down procedure the female rats were dosed with CEE at 1750 and 550 mg/kg body weight (b.w.), with CEE 1750 mg/kg b.w. was toxic, causing mortality, while CEE 550 mg/kg b.w. was deemed safe. An LD50 value was calculated according to the standard protocols, resulting in 970 mg/kg b.w. In histopathological examination, 550 mg/kg b.w. of CEE was safe in all the selected organs, while the 1750 mg/kg b.w. CEE treated rats exhibited toxic effects in histological tissues sections in the form of necrosis in the brain, cardiac muscle hypertrophy, liver inflammation, mild untoward effect in the spleen, fibrosis in the lungs, pancreatitis, pyelonephritis, and ovarian cyst. Administration of CEE at doses of 550 mg/kg b.w. (single dose) in the acute and 100 mg/kg b.w. (regularly 28-days) in the sub-acute toxicity studies significantly (p < 0.05) decreased levels of uric acid, triglycerides, and cholesterol. Importantly, the CEE (550 and 100 mg/kg b.w.) also significantly increased the levels of antioxidant enzymes (SOD, GSH, and CAT) and decreased MDA levels. Normal histopathology was observed in the sub-acute toxicity study in all treated groups. ConclusionThis study successfully concludes that CEE at a dose of 100 mg/kg b.w. is safe for therapeutic application or use as a chemopreventive functional food utilizing green extraction methods. However, chronic toxicity studies are further recommended to validate safety concerns over an extended period.

Tetracyclic homoisoflavanoid (+)-brazilin: a natural product inhibits c-di-AMP-producing enzyme and Streptococcus mutans biofilms.

The tenacious biofilms formed by Streptococcus mutans are resistant to conventional antibiotics and current treatments. There is a growing need for novel therapeutics that selectively inhibit S. mutans biofilms while preserving the normal oral microenvironment. Previous studies have shown that increased levels of cyclic di-AMP, an important secondary messenger synthesized by diadenylate cyclase (DAC), favored biofilm formation in S. mutans. Thus, targeting S. mutans DAC is a novel strategy to inhibit S. mutans biofilms. We screened a small NCI library of natural products using a fluorescence detection assay. (+)-Brazilin, a tetracyclic homoisoflavanoid found in the heartwood of Caesalpinia sappan, was identified as one of the 11 "hits," with the greatest reduction (>99%) in fluorescence at 100 µM. The smDAC inhibitory profiles of the 11 "hits" established by a quantitative high-performance liquid chromatography assay revealed that (+)-brazilin had the most enzymatic inhibitory activity (87% at 100 µM) and was further studied to determine its half maximal inhibitory concentration (IC50 = 25.1 ± 0.98 µM). (+)-Brazilin non-competitively inhibits smDAC's enzymatic activity (Ki = 140.0 ± 27.13 µM), as determined by a steady-state Michaelis-Menten kinetics assay. In addition, (+)-brazilin's binding profile with smDAC (Kd = 11.87 µM) was illustrated by a tyrosine intrinsic fluorescence quenching assay. Furthermore, at low micromolar concentrations, (+)-brazilin selectively inhibited the biofilm of S. mutans (IC50 = 21.0 ± 0.60 µM) and other oral bacteria. S. mutans biofilms were inhibited by a factor of 105 in colony-forming units when treated with 50 µM (+)-brazilin. In addition, a significant dose-dependent reduction in extracellular DNA and glucan levels was evident by fluorescence microscopy imaging of S. mutans biofilms exposed to different concentrations of (+)-brazilin. Furthermore, colonization of S. mutans on a representative model of enamel using suspended hydroxyapatite discs showed a >90% reduction with 50 µM (+)-brazilin. In summary, we have identified a drug-like natural product inhibitor of S. mutans biofilm that not only binds to smDAC but can also inhibit the function of smDAC. (+)-Brazilin could be a good candidate for further development as a potent therapeutic for the prevention and treatment of dental caries.IMPORTANCEThis study represents a significant advancement in our understanding of potential therapeutic options for combating cariogenic biofilms produced by Streptococcus mutans. The research delves into the use of (+)-brazilin, a natural product, as a potent inhibitor of Streptococcus mutans' diadenylate cyclase (smDAC), an enzyme crucial in the formation of biofilms. The study establishes (+)-brazilin as a non-competitive inhibitor of smDAC while providing initial insights into its binding mechanism. What makes this finding even more promising is that (+)-brazilin does not limit its inhibitory effects to S. mutans alone. Instead, it demonstrates efficacy in hindering biofilms in other oral bacteria as well. The broader spectrum of anti-biofilm activity suggests that (+)-brazilin could potentially serve as a versatile tool in a natural product-based treatment for combating a range of conditions caused by resilient biofilms.

HPLC and LC-MS/MS-Based Quantitative Characterization of Related Substances Associated with Sotalol Hydrochloride.

In total, three related substances (RS) associated with sotalol hydrochloride (STHCl) were herein identified with a novel gradient high-performance liquid chromatography (HPLC) protocol. Further characterization of these substances was then performed via liquid chromatography-mass spectroscopy (LC-MS/MS) and nuclear magnetic resonance (NMR) approaches. For these analyses, commercial STHCl samples were used for quantitative HPLC studies and the degradation of STHCl under acidic (1M HCl), alkaline (1M NaOH), oxidative (30% H2O2), photolytic (4500 Lx), and thermal stress conditions (100 °C) was assessed. This approach revealed this drug to be resistant to acidic, alkaline, and high-temperature conditions, whereas it was susceptible to light and oxidation as confirmed through long-term experiments. The putative mechanisms governing RS formation were also explored, revealing that RS3 was derived from the manufacturing process, whereas RS2 was generated via oxidation and RS1 was generated in response to light exposure. The cytotoxicity of these RS compounds was then assessed using MTT assays and acute toxicity test. Overall, this study provides details regarding the characterization, isolation, quantification, and toxicological evaluation of STHCl and associated RS compounds together with details regarding the precise, specific, and reliable novel HPLC technique, thus providing the requisite information necessary to ensure STHCl purity and safety.

Quality by Design Based Stability Indicating Quantitative Reverse Phase High Performance Liquid Chromatography Method Development and Validation for Bilastine in Tablet Dosage Form

Quality by Design Based Stability Indicating Quantitative Reverse Phase High Performance Liquid Chromatography Method Development and Validation for Bilastine in Tablet Dosage Form

Influence of Diet on the Bioavailability of Active Components from Zingiber officinale Using an In Vitro Digestion Model

Ginger (Zingiber officinale Rosc.) is a plant known all over the world that is used as a spice and as an ingredient in drinks, dietary supplements, and cosmetics. The growing availability of its fresh rhizomes makes it even more likely to be used in the diet, mainly due to its beneficial health properties and high content of polyphenols (gingerols and shogaols). The main goal and motivation of the authors was to assess the bioavailability of active substances contained in the extract from ginger rhizomes in the presence of various types of diets using the in vitro digestion method, enabling simulation of the processes occurring during the digestion and absorption of metabolites in the small intestine. For the qualitative and quantitative analyses, the HPLC-MS (High Performance Liquid Chromatography–Mass Spectrometry) and HPLC (High Performance Liquid Chromatography) techniques were used, respectively. Based on the obtained results, it was found that the best bioavailability of the selected ginger polyphenols (6-gingerol, 8-gingerdione, 8-shogaol, and 10-gingerdione) was estimated for a high-fiber diet, while the weakest results were obtained for standard and basic diets. In the case of the high-fiber diet, the bioavailability of the mentioned compounds was estimated as 33.3, 21.4, 6.73, and 21.0%, while for the basic diet, it was only 21.3, 5.3, 2.0, and 1.0%, respectively.

Enhancement of Aggregate Formation Through Aromatic Compound Adsorption in Elastin-like Peptide (FPGVG)5 Analogs.

Elastin-like peptides (ELPs) exhibit temperature-dependent reversible self-assembly. Repetitive sequences derived from elastin, such as Val-Pro-Gly-Val-Gly (VPGVG), are essential for the self-assembly of ELPs. Previously, we developed (FPGVG)5 (F5), in which the first valine residue in the VPGVG sequence was replaced with phenylalanine, which showed strong self-aggregation ability. This suggests that interactions through the aromatic amino acid residues of ELPs could play an important role in self-assembly. In this study, we investigated the thermoresponsive behavior of F5 analogs in the presence of aromatic compounds. Turbidimetry, spectroscopy, and fluorescence measurements demonstrated that aromatic compounds interacted with F5 analogs below the transition temperature and enhanced the self-assembly ability of ELPs by stabilizing amyloid-like structures. Furthermore, quantitative high-performance liquid chromatography analyses showed that the F5 analogs could adsorb and remove hydrophobic aromatic compounds from aqueous solutions during aggregate formation. These results suggested that the F5 analogs can be applicable as scavengers of aromatic compounds.

Screening and quantification of antibiotic residues in poultry products and feed in selected areas of Bangladesh.

Antibiotic residues in livestock farming have been identified as a potential cause of antimicrobial resistance in humans and animals. This study aimed to determine whether antibiotic residues were present in the chicken meat, eggs, feces, and feed collected from all four districts in the Mymensingh division of Bangladesh. To detect antibiotic residues in the collected samples, qualitative thin-layer chromatography (TLC) and quantitative high-performance liquid chromatography (HPLC) were used. A total of 230 samples were analyzed for antibiotic residues of commonly used 11 antibiotics. Out of these, 40 meat and 40 feces samples were collected from broilers and layers, 30 egg samples from ducks and layers, and 120 feed samples from broilers and layers from the study area. Thin-layer chromatography was used to screen the presence of antibiotic residues; TLC-positive samples were then subjected to further HPLC analysis to determine the residue concentrations. Thin-layer chromatography analysis revealed that 23.5% of the tested samples contained residues from six different antibiotic classes (tetracyclines, quinolones, beta-lactams, sulfonamides, aminoglycosides, and macrolides). Thin-layer chromatography analysis showed that 35% and 25% of the meat samples were positive for residues from the broiler and layer, respectively. About 15% and 30% of layer and duck egg samples had positive residues, respectively. Out of 120 feed samples analyzed, about 15.8% had various antibiotic residues. In addition, feces samples from broilers and layers had 50% and 35% antibiotic residues, respectively. A total of 2.5% meat and 3.3% egg samples had antibiotic residues above the maximum residue limit (MRL). Based on the findings of this study, the highest percentage of oxytetracycline, followed by doxycycline and ciprofloxacin, were detected in feed samples, and oxytetracycline was detected in meat and egg samples. This study clearly showed the misuse of antibiotics in the poultry sector in Bangladesh. Although antibiotic residues below the MRL level are suitable for human consumption, they may result in antimicrobial drug resistance to pathogens.

Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid – A technical performance study

ObjectiveTo validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples. DesignSynovial fluid from osteoarthritis (OA, n ​= ​25) and knee-injury (n ​= ​13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry. ResultsUnsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in the SF-control sample. For HA and the CS variants in SF-control the intra- and inter-experiment coefficient of variation was between 3–12% and 11–19%, respectively; tenfold dilution gave recoveries between 74 and 122%, and biofluid stability test (room temperature storage and freeze-thaw cycles) showed recoveries between 81 and 140%. Synovial fluid concentrations of the CS variants ΔUA-GalNAc6S and ΔUA2S-GalNAc6S were three times higher in the recent injury group compared to the OA group, while HA was four times lower. Sixty-one different N-glycans were detected in the synovial fluid samples, but there were no differences in levels of N-glycan classes between patient groups. The CS-profile (levels of ΔUA-GalNAc4S and ΔUA-GalNAc6S) in synovial fluid resembled that of purified aggrecan from corresponding samples; the contribution to the N-glycan profile in synovial fluid from aggrecan was low. ConclusionsThe HPLC-assay is suitable for analyzing CS variants and HA in synovial fluid samples, and the GAG-pattern differs between OA and recently knee injured subjects.

English

There have been a growing interest since decades in the medicinal plants in terms of preventive health care and treatment of various diseases. On the other hand, polyphenolic compounds present in medicinal plants are known to have antioxidant potency. In the current work, the quantitative HPLC analysis of the flowers of four plants grown in Bolu-Turkey, Silene compacta, Digitalis lamarckii, Xeranthemum annuum L., and Epilobium hirsutum L was carried out. Prior to HPLC quantification, they were detected by TLC with reference standard compounds, identified by LC-MS and compared for their profiles of phenolic acids and some flavonoids by using high-performance liquid chromatography (HPLC) with diode array detector. The highest contents of benzoic acids were detected in the flowers of E. hirsutum L. as 159 mg in 100 g dry flowers. As for cinnamic acids, the highest content value was found in S. compacta flowers&nbsp; as 292 mg in 100 g dry flowers. On the other hand, E. hirsutum L. flowers contained the highest total flavonoid value as much as 3890 mg. Total polyphenolic content was the highest (4104 mg/100 g dry plant) in E. hirsutum L., however, the lowest value was found as 492 mg/100 g dry flowers for D. lamarcki. Key words:&nbsp; Polyphenols,&nbsp; HPLC,&nbsp; LC-MS,&nbsp; quantitative determination, Silene compacta, Digitalis lamarckii, Xeranthemum annuum L., Epilobium hirsutum L.

Isolation and Characterization of an Unknown Process-Related Impurity in Furosemide and Validation of a New HPLC Method.

Furosemide is a widely used loop diuretic in the treatment of congestive heart failure and edema. During the preparation of furosemide, a new process-related impurity G in the levels ranging from 0.08% to 0.13% was detected in pilot batches by a new high performance liquid chromatography (HPLC) method. The new impurity was isolated and characterized by comprehensive analysis of FT-IR, Q-TOF/LC-MS, 1D-NMR (1H, 13C, and DEPT), and 2D-NMR (1H-1H-COSY, HSQC, and HMBC) spectroscopy data. The possible formation pathway of impurity G was also discussed in detail. Moreover, a novel HPLC method was developed and validated for the determination of impurity G and the other six known impurities registered in the European Pharmacopoeia as per ICH guidelines. The HPLC method was validated with respect to system suitability, linearity, the limit of quantitation, the limit of detection, precision, accuracy, and robustness. The characterization of impurity G and the validation of its quantitative HPLC method were reported for the first time in this paper. Finally, the toxicological properties of impurity G were predicted by the in silico webserver ProTox-II.

Rapid screening of 2-benzylbenzimidazole nitazene analogs in suspect counterfeit tablets using Raman, SERS, DART-TD-MS, and FT-IR.

Developing methods to rapidly screen for novel synthetic 2-benzylbenzimidazole opioids, also known as nitazenes, has become increasingly important due to their high potency. These compounds have potency comparable or exceeding that of fentanyl by up to 10 times and have been implicated in approximately 5% of all drug overdose deaths in the United States in 2021. This paper details the authenticity determination of suspect tablets and the identification of three nitazene analogs (N-pyrrolidino etonitazene, isotonitazene, and etodesnitazene) in suspect tablets seized at a mail facility using Raman and surface-enhanced Raman scattering (SERS) with handheld devices, portable Fourier transform infrared spectrometer (FT-IR), and a direct analysis in real-time ambient ionization coupled to a thermal desorption unit and a mass spectrometer (DART-TD-MS). These methods are rapid and excellent for screening opioids in suspect tablets but could not fully determine the exact structure of some of the nitazene analogs present due to spectral similarities or similar fragmentation patterns. Liquid chromatography-mass spectrometry (LC-MS) confirmed the presence of these nitazene compounds in addition to other opioids/drugs that were in trace quantities. The quantitative high-performance liquid chromatography coupled with ultraviolet (HPLC-UV) detection experiments determined that the suspect tablets contained an average of 0.817 mg of N-pyrrolidino etonitazene per tablet. The results obtained reveal that the simultaneous deployment of these complementary and orthogonal portable analytical techniques as part of a workflow allows suspect tablets to be screened and nitazene-type drugs to be identified in suspect counterfeit tablets at remote sampling sites.

Synthesis and Characterization of Nanoparticles Extracted from &lt;i&gt;Catharanthus roseus&lt;/i&gt; Plant

The Catharanthus roseus plant was extracted and converted to nanoparticles in this work. The Soxhlet method extracted alkaloid compounds from the plant Catharanthus roseus and converted them to the nanoscale. Chitosan polymer was used as a linking material and converted to Chitosan nanoparticles using Sodium TriPolyPhosphate (STPP). The extracted alkaloids were linked with Chitosan nanoparticles CSNPs by maleic anhydride to get the final product (CSNPs- Linker- alkaloids). The synthesized (CSNPs- Linker- alkaloids) was characterized using SEM spectroscopy UV–Vis., Zeta Potential, and HPLC High-Performance Liquid Chromatography. Scanning electron microscope (SEM) analysis shows that the Chitosan nanoparticles (CSNPs) have small dimensions with regular spherical and nanotube shapes of a diameter range of (49 - 70) nm. The final product (CSNPs- Linker- alkaloids) has two shapes (spherical particles and tubes) in nano dimensions and is close to each other compared to normal Chitosan. The absorption peaks for Chitosan (CS), Chitosan nanoparticles (CSNPs), Chitosan nanoparticles (CSNPs), and maleic anhydride revealed that converting Chitosan to Chitosan nanoparticles and mixing it with the plant extract, led to an increase in the absorption value and wavelength range. Also, the appearance of two peaks at 222 nm and 402 nm nano instead of the peak of Chitosan at 289.9 nm. Zeta Potential results of CSNPs- Linker- alkaloids showed that the extract of the nano-alkaloids bound to chitosan nanoparticles carries a positive charge of 54.4 mV. This surface charge is essential in maintaining the colloidal solution's stability in its natural form without changing. High-Performance Liquid Chromatography (HPLC) was used to estimate qualitative and quantitative plants extracted from Catharanthus roseus. Quantitative HPLC results show that Catharanthus roseus contains a good and acceptable concentration of Vinblastine, Vincristine, Vinorelbine, Vincamine, and Vintafolide (66.75, 242.91, 0.7, 83.77, 42.34) ppm respectively. The qualitative results show a good match for the influential groups of pure standard vincristine and alcoholic extract and dry powder of the Catharanthus roseus plant. The successful synthesis of nanoparticles from the Catharanthus roseus plant can be used in biosensors and biomedical applications.

Analysis on quality value transmitting of Qinggusan reference sample of classical prescription based on quantitative fingerprint technology

Qinggusan is the 69th prescription in the first batch of "Catalogue of Ancient Chinese Classic Formulas". In modern clinical practice， Qinggusan is mainly used to treat noninfectious fever. However， because few studies on Qinggusan reference samples and their quality value transfer are available， the development and promotion of its compound preparations are restricted. Therefore， establishing an accurate and comprehensive quality control method to clarify the critical quality attributes of Qinggusan reference samples is of great importance. In this study， 15 batches of Qinggusan reference samples were processed to determine the range of their dry extract ratios. Quantitative high-performance liquid chromatography （HPLC） fingerprint analysis was performed using a Waters Symmetry Shield RP18 column （250 mm×4.6 mm， 5 μm） with acetonitrile-0.1% （v/v） formic acid aqueous solution as the mobile phase in gradient elution mode. The flow rate was 1.0 mL/min， the column temperature was 30 ℃， and the detection wavelength was 254 nm. The HPLC fingerprints of the Qinggusan reference samples were established under these conditions to evaluate their similarity. The established method was systematically validated and found to demonstrate good precision， repeatability， and sample stability. Subsequently， characteristic peaks were identified and attributed by HPLC-quadrupole-time-of-flight-mass spectrometry （HPLC-Q-TOF-MS） analysis. MS was performed in electrospray ionization mode， the data were collected in both positive- and negative-ion modes， and the detection range was m/z 50-2000. The contents and transfer rate ranges of the index components， namely， gentiopicrin， mangiferin， picroside Ⅱ， picroside Ⅰ， and glycyrrhizic acid， were determined to analyze the quality value transfer of the samples. The results demonstrated that the dry extract rate of the 15 batches of Qinggusan reference samples ranged from 24.10% to 26.88% and that their fingerprint similarities were generally greater than 0.95. Twelve common peaks were identified by reference identification， literature comparison， and high-resolution MS analysis. Twelve compounds， including six iridoid glycosides， two flavonoids， one alkaloid， one triterpenoid saponin， and two others. Among them， L-picein， androsin， picroside Ⅳ， picroside Ⅱ and picroside Ⅰ were from Picrorhizae Rhizoma， loganin acid， swertiamarin and gentiopicrin were from Gentianae Macrophyllae Radix， neomangiferin and mangiferin were from Anemarrhenae Rhizoma， dichotomine B was from Stellariae Radix， and glycyrrhizic acid was from Glycyrrhizae Radix et Rhizoma. The five key components presented good linear relationships in their respective linear ranges， and all correlation coefficients were higher than 0.999. The relative standard deviations （RSDs） of precision， stability， and repeatability were less than 1.3%. The average recoveries varied between 95.92% and 102.5%， with RSDs less than 3.9%； these values meet the requirements defined in the 2020 edition of the Chinese Pharmacopoeia. The contents of gentiopicrin， mangiferin， picroside Ⅱ， picroside Ⅰ， and glycyrrhizic acid in the 15 batches of reference samples were in the range of 17.92-27.55， 1.83-4.42， 23.08-36.44， 8.43-15.04， and 0.94-2.39 mg/g， respectively， and their transfer rates from the decoction pieces to the reference samples were 47.91%-63.95%， 22.96%-59.39%， 60.82%-77.82%， 64.25%-99.53%， and 15.30%-39.30%， respectively. In this study， the chemical components of Qinggusan reference samples were comprehensively identified and their quality value transfer was studied through the combination of HPLC fingerprinting and MS. Clarification of the critical quality attributes of Qinggusan reference samples could provide a basis for the quality control of Qinggusan compound preparations.

Determination of Caffeine and Chlorogenic Acid (CGA) in the Methanolic Extracts Coffee (C. arabica. L) To seeds and peels (Unroasted and Roasted) Cultivars Grown in Yemen by High Performance Liquid Chromatography (HPLC)

In this study methanolic extract of one plant namely Arabic coffee, were screened for the presence of analysis Constituents and tested for their of liquid chromatographic separation (HPLC). The quantitative HPLC analysis revealed the results showed presence of Caffeine, chlorogenic acid (CGA) is the concentration of the contents Seeds, peels (unroasted) samples is higher than their contents in the Seeds, and peels (roasted) samples. The highest concentration of Caffeine was 5,334 % in unroasted Arabica coffee peels (Udaini) in Ibb (Kafr) region, while the highest value of chlorogenic acid was 68,460% in unroasted Arabica coffee peels (Tufahi) in Ibb (Kafr). The highest concentration of Caffeine was 9,948% in unroasted Arabic coffee seeds (Udaini) in Ibb (Kafr) region, while the highest concentration of chlorogenic acid was 97,280% in unroasted Arabic coffee seeds (Udaini) in Ibb (Kafr) region. The highest concentration of Caffeine was 1,964 % in roasted Arabica coffee peels (Udaini) in Ibb (Kafr) region, while the highest value of chlorogenic acid was 26,260% in roasted Arabica coffee peels (Bura’ai.) in Sana'a (Haraz). The highest concentration of Caffeine was 2,324% in roasted Arabic coffee seeds (Udaini) in Al- Mahweet (Hufash) region, while the highest concentration of chlorogenic acid was 47,09% in roasted Arabic coffee seeds (Dawairi) in Al- Mahweet (Hufash).

Quantification of atropine sulphate monohydrate and obidoxime dichloride in two-chamber autoinjectors for accessing uniformity of dosage.

In the treatment of organophosphate poisoning atropine sulphate monohydrate (AT) and obidoxime dichloride (OB) play a vital role. Currently, the Austrian Armed Forces use the DOUBLEPEN OA two-chamber autoinjector (ChemProtect) to administer these two drugs. The autoinjector is a part of military standard equipment as a "Basic CBRN-First Aid Kit" and contains OB and AT with a declared concentration of 220mg/2ml and 2mg/2ml, respectively. Especially in the two-chamber autoinjectors, it is highly possible that not all the content of the antidote solution is administered when the autoinjector is triggered. The purpose of the study was to analyze one hundred DOUBLEPEN OA autoinjectors from two different production batches (1707068 and 1707067) for volume loss, drug content and uniformity of dosage unit. Uniformity of dosage units, assessed by the content uniformity method (Chapter 2.9.40 of the European Pharmacopeia), requires the calculation of an acceptable value to quantify the uniformity of the drug product. An acceptance value for the first 10 dosage units of 15.0% or below is considered acceptable. The loss of volume was calculated by determining the density and mass of the solution after triggering the autoinjector. A quantitative high-performance liquid chromatography method has been developed and in-house validated for the determination of the content of two drugs. According to International Council for Harmonisation guidelines, the analytical method was proven to be accurate and repeatable. The obtained results show that the average loss of volume after injection was 5%, and the average content of OB and AT for batch 1707068, was 216.5 and 1.9mg, while for batch 1707067 it was 224.2 and 2.0mg, respectively. Although the loss of volume and content were observed, the calculated acceptance value for both production batches met the requirements of uniformity of dosage unit by the European Pharmacopeia.

Development of an HPLC method for asperulosidic acid quantification in the powder prepared from the spinal degeneration remedy of physician Nguyen Thien Chung

Nguyen Thien Chung’s remedy which consists of 13 medicinal herbs had been demonstrated its analgesic and anti-inflammatory effects on animal experiments. This remedy has further been developed into powder form. This study was conducted to develop a method to quantify asperulosidic acid, the important marker in BT powder, by high-performance liquid chromatography (HPLC). Sample processing conditions and quantitative HPLC conditions were screened. The HPLC method was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines for system suitability, specificity, linearity, precision and accuracy. Methanol was chosen as the solvent for sample preparation and the ratio between BT powder and methanol was 300:25 (mg/ml). The selected HPLC conditions included a C18 Shimpack GIST column (250×4.6 mm, 5 μm), a detection wavelength of 236 nm, a column temperature of 30oC, a flow rate of 1 ml/min, and an injection volume of 10 μl. The mobile phase was a mixture of acetonitrile and 0.1% H3PO4 with a ratio of 9.5:90.5 (v/v). The quantitative method showed a good linear correlation between the peak area and the concentration of asperulosidic acid (r2=0.9987). The Relative standard deviation (RSD) value of the intermediate precision was 1.24%. The recovery was found in a range of 91.22-99.76%. The HPLC method for asperulosidic acid quantification in BT powder met the validation requirements and could be applied to standardise and evaluate the quality of BT powder.

Analysis and Comparison of Rapid Methods for the Determination of Ochratoxin a Levels in Organs and Body Fluids Obtained from Exposed Mice.

Mycotoxins are bioaccumulative contaminants impacting animals and humans. The simultaneous detection of frequent active exposures and accumulated mycotoxin level (s) in exposed organisms would be the most ideal to enable appropriate actions. However, few methods are available for the purpose, and there is a demand for dedicated, sensitive, reliable, and practical assays. To demonstrate the issue, mice were exposed to a relevant agent Ochratoxin A (OTA), and accumulated OTA was measured by fine-tuned commercial assays. Quantitative high-performance liquid chromatography with fluorescence detection, enzyme-linked immunosorbent assay, and flow cytometry assays have been developed/modified using reagents available as commercial products when appropriate. Assays were performed on excised samples, and results were compared. Accumulated OTA could be detected and quantified; positive correlations (between applied doses of exposure and accumulated OTA levels and the results from assays) were found. Dedicated assays could be developed, which provided comparable results. The presence and accumulation of OTA following even a short exposure could be quantitatively detected. The assays performed similarly, but HPLC had the greatest sensitivity. Blood contained higher levels of OTA than liver and kidney. We demonstrate that specific but flexible and practical assays should be used for specific/local purposes, to measure the exposure itself and accumulation in blood or organs.

Determination of 32 oxidative dyes by high performance liquid chromatography and confirmation by high performance liquid chromatography-tandem mass spectrometry

染发类产品中氧化型染料种类多,实际样品测定时干扰多,建立染发类产品中多种常用染料的测定方法,为该类产品的有效监管提供技术手段十分必要。该研究根据染料使用频率分组,采用能够屏蔽硅羟基和金属离子影响的C18柱,优化了《化妆品安全技术规范》(2015年版)中32种染料的高效液相色谱法(HPLC)并建立了高效液相色谱-串联质谱(HPLC-MS/MS)确证方法。样品以10 g/L亚硫酸氢钠水溶液为抗氧化剂,用无水乙醇-水(1∶1, v/v)混合溶液冰浴超声提取10 min。HPLC方法采用甲醇、乙腈和磷酸盐缓冲液为流动相分两个液相色谱条件进行梯度洗脱分离,于280 nm波长下检测,其中一个HPLC条件中的相互干扰组分均在另一个HPLC条件下完全分离,避免了实际样品检测时组分间的干扰,并排除了32种以外的其他15种常用染料的干扰。HPLC-MS/MS方法分别采用5 mmol/L乙酸铵水溶液-乙腈和5 mmol/L乙酸水溶液-乙腈为正离子和负离子模式下的流动相,电喷雾离子模式下用多反应监测(MRM)模式进行定性和定量分析。HPLC和HPLC-MS/MS两个方法中,日内精密度和48 h内稳定性的相对标准偏差(RSD)<10%,回收率为82.6%~114.9%(RSD<10%)。HPLC方法中32种染料在大约10~500 mg/L范围内线性关系良好(r2>0.99),检出限为9.7~40.1 μg/g; HPLC-MS/MS方法中氢醌线性范围为2.0~79.7 mg/L,检出限为8.0 μg/g,其他组分线性范围约为0.1~4 mg/L,检出限为0.01~0.4 μg/g。采用HPLC、HPLC-MS/MS两个方法和《化妆品安全技术规范》方法同时测定实际样品,共检出16种染料,检出含量范围为58~25160 μg/g。3个方法检测结果的RSD为1.9%~10.1%。该研究增加了HPLC-MS/MS确证方法,适应化妆品法定检验中的未知物确认程序;方法简便快速,结果准确,专属性强,具有较好的通用性和可操作性。

Determination of Vitamin C, Vitamin A and Flavonoid Levels in Garcinia cowa Roxb Fruit Flesh Extract

Garcinia cowa Roxb is a plant used as traditional medicine containing antioxidant activity. Plant extracts with antioxidant activity are often used as bioactive ingredients in anti-aging cosmetics. Studies on bioactive compounds and the use of this plant in cosmeceuticals have yet to be widely reported. This study aims to determine and analyze the levels of flavonoids, vitamins C, and vitamin A in G. cowa fruit flesh extract. These three compounds are bioactive ingredients that are beneficial in reducing the symptoms of photo-aging on the skin. The G. cowa used in this study was obtained from Tampunik Kambang, Pesisir Selatan Regency, and was extracted by maceration method using 96% ethanol. The testing of vitamin C levels in the extract of G. cowa fruit flesh was carried out through High-Performance Liquid Chromatography (HPLC) method using a UV detector with a wavelength of 264 nm. Quantitative HPLC analysis was carried out based on the resulting peak area data from the chromatogram. Vitamin A and flavonoid levels were measured using ultraviolet-visible spectrophotometry (UV-VIS) at maximum wavelengths of 286 and 429 nm. The results show that the levels of vitamin C and vitamin A in G. cowa was 151.4 mg/100g extract and 1.23 g 100g extract, while the flavonoid level was 69.5 mg/100g extract. The level of vitamins C, vitamin A, and flavonoids in G. cowa can contribute to natural bioactive substances and can be developed further into anti-aging cosmeceutical products.

Characterization of entrapment behavior of polyphenols in nanostructured lipid carriers and its effect on their antioxidative activity

Polyphenols are widely used as antioxidant agents to protect human health. Resveratrol, kaempferol, and quercetin have been reported to have potent antioxidant activity; however, these compounds have many problems related to their practical application, such as instability and insolubility. Thus, a nanostructured lipid carrier (NLC) was utilized as a drug delivery system (DDS) to overcome these limitations. This study investigated the particle stability, drug loading performance, and antioxidant activity of polyphenols-incorporated NLCs. The particle size and distribution were suitable for DDS applications, and all the samples demonstrated good stability after 2 months of storage. Based on Raman spectroscopy analysis, polyphenols were successfully encapsulated in NLCs. Quantitative high-performance liquid chromatography analysis indicated that NLCs could load resveratrol more than kaempferol and quercetin. In addition, NLCs have successfully improved all the antioxidant activity per unit concentration of polyphenol (specific antioxidant activity) compared to the free polyphenols. Quercetin-incorporated NLCs showed the highest specific antioxidant activity. This result is the opposite of entrapment efficiency and actual antioxidant activity, most likely influenced by the location of entrapped polyphenol molecules. As it was performed, NLCs are highly recommended to be applied as an antioxidant delivery system.