DAD Detection Research Articles

The Influence of an Anti-Inflammatory Gluten-Free Diet with EPA and DHA on the Involvement of Maresin and Resolvins in Hashimoto’s Disease

The potential modulation of thyroid inflammatory conditions via a gluten-free diet has been suggested after establishing a link between Hashimoto’s thyroiditis (HT) and celiac disease. However, the majority of targeted studies in this field do not support the general recommendation of prescribing a gluten-free diet (GFD) for all HT patients. This study aims to analyze data regarding the impact of a GFD supplemented with eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), along with vegetables, on the course of inflammation involving long-chain fatty acid mediators. The study cohort consisted of 39 Caucasian female patients with autoimmune HT. Metabolite separations were performed using a liquid chromatograph with a DAD detector. Absorption peaks were read at 210 nm for resolvin E1, protectin DX, and maresin 1 and at 302 nm for resolvin D1. The introduction of a gluten-free diet completed with omega-3, including EPA and DHA, may contribute to a reduction in the inflammatory state in HT patients. This effect is supported by the elevation in the levels of anti-inflammatory mediators derived from long-chain fatty acids with anti-inflammatory properties but not by eliminating gluten. Significant statistical changes in the levels of all derivatives were observed before and after the implementation of the diet. It is worth noting that this effect was not observed in anti-TPO and anti-TG levels. The induction of anti-inflammatory changes can be achieved by supplementing the diet with EPA, DHA and vegetables with increased anti-inflammatory potential.

Effect of gamma irradiation on the degradation rate of various pesticides and active ingredients in dandelion

The levels of 37 pesticides in dandelion were analyzed using gas chromatography-tandem mass spectrometry (GC–MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The dandelion samples were treated with different irradiation doses, pH levels, moisture contents, and initial concentrations. Additionally, the respective contents of chicoric, chlorogenic, and caffeic acids in the dandelion samples were determined using high-performance liquid chromatography (HPLC) with DAD detector. The results showed that the pesticide degradation rate was positively correlated with the irradiation dose and negatively correlated with pesticide concentration. The degradation of most pesticides followed first-order kinetics. The pesticide degradation rate initially decreased and subsequently increased with an increase in the pH. Irradiation below 8 kGy had an insignificant effect on the three active ingredients of dandelion. The chicoric acid content decreased with an increase in water content and pH, whereas the caffeic acid content increased.

UHPLC-DAD/ESI-TOF-MS Phytochemical Characterization and Evaluation of the Impact of Eleutherococcus senticosus Fruit Intractum on Biochemical, Hepatological, and Blood Parameters in Balb/c Mice.

Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. (ES) has gained popularity for its adaptogenic, immunostimulant, and anti-inflammatory properties. Because of overexploitation of the roots, the species is considered to be endangered and has been put on the Red List in some countries (e.g., the Republic of Korea). Therefore, the fruits of E. senticosus might be explored as a new sustainable source of compounds with adaptogenic activity. This study aimed to assess the chemical composition and the safety profile (hepatotoxicity, blood morphology, biochemical parameters of blood plasma) of E. senticosus fruit intractum in Balb/c mice after oral administration of 750 and 1500 mg/kg b.w. UHPLC analysis coupled with DAD and MS detectors was used to quantify the metabolites. For the first time, oleanolic and ursolic acids were quantified in the intractum (16.01 ± 1.3 and 2.21 ± 0.17 µg/g of oleanolic and ursolic acids, respectively). Regarding polyphenols, chlorogenic acid (0.92 mg/g of dried extract), caffeic acid (0.43 mg/g), dicaffeoylquinic acids (in total: 1.27 mg/g), and an unidentified caffeic acid ester (0.81 mg/g) were identified. The results in Balb/c mice revealed that the intractum does not cause significant variations in red blood cells parameters. In turn, a significant decrease in the total number of leukocytes was observed (5.8 × 103 µL), with a percentage increase in lymphocytes among the groups (80.2, 81.8, and 82.6). The ability of the intractum to decrease alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels may indicate its anti-inflammatory activity. Our observations justify that the fruits of E. senticosus are safe in the doses used and do not cause significant changes in the activity of the liver enzymes or in blood parameters.

Stress response in tomato as influenced by repeated waterlogging.

Plants respond to water stress with a variety of physiological and biochemical changes, but their response varies among species, varieties and cultivars. Waterlogging in tomato reduces plant growth, degrade chlorophyll and increase concentration of oxidative parameters. Priming can alleviate stress in plants caused by waterlogging enabling plants to be more tolerant to an additional stress in the current or even subsequent generation. The aim of this study was to evaluate tomato genotypes for their sensitivity to waterlogging stress applied during early vegetative growth and at full flowering stage. The study included two local genotypes, Trebinjski sitni (GB1126) and Žuti (GB1129), and the reference variety Novosadski jabučar (NJ), which is the variety most commonly used in Serbia and Bosnia and Herzegovina. The activity of class III peroxidase (POX), hydrogen peroxide (H2O2) content and malondialdehyde (MDA) content were measured spectrophotometrically, and for quantification of individual phenolic compounds, targeted approach was adopted, using UHPLC/DAD/(-)HESI-MS2 instrument (Dionex UltiMate 3000 UHPLC system with a DAD detector, configured with a triple quadrupole mass spectrometer TSQ Quantum Access Max (Thermo Fisher Scientific, Germany)). Oxidative parameters (H2O2 and MDA) exhibited an increase in content in leaves of tomato plants that underwent waterlogging stress compared to control plants. Moreover, oxidative parameters showed positive correlation with proteins and phenolics content. The obtained correlations can indicate that one of the response strategies of tomato plants to waterlogging is the increased synthesis of proteins and phenolic compounds. The POX activity was not correlated with other parameters except with the polyphenols. A positive correlation was shown between POX activity and the content of phenolic compounds, indicating their independent roles in the removal of ROS. Changes in the phenolic profiles after the exposure of plants to waterlogging stress are recorded, and these changes were more severe in leaves and fruits of GB1129 and NJ genotypes than in GB1126. Thus, genotype GB1126 is the most efficient in maintaining the phenolic profiles of leaves and fruits, and therefore of the nutritive and organoleptic qualities of fruits following the exposure to waterlogging. Also, genotype GB1126 exhibited the ability to maintain the content of oxidative parameters during waterlogging at certain growth stages, implying certain waterlogging tolerance. Waterlogging triggered stress memory but not at all growth stages. The most pronounced stress memory was obtained in fruit samples in the phase of full fruit maturity on the 1st truss. This study shed light on the defense mechanisms of tomato plants to repeated waterlogging stress from the perspectives of the changes in the composition of major phenolics, and pointed to the 5-O-caffeoylquinic acid and rutin as the chemical markers of the waterlogging stress tolerance in tomato. However, it remains to be determined whether this modulation has a positive or negative effect on the overall plant metabolism. Further investigations are needed to fully elucidate the benefits of waterlogging pretreatment in this context.

Derivative spectrophotometry-assisted determination of tryptophan metabolites emerges host and intestinal flora dysregulations during sepsis

Sepsis is a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection. Dysregulated tryptophan (TRP) metabolites serve as significant indicators for endogenous immune turnovers and abnormal metabolism in the intestinal microbiota during sepsis. Therefore, a high coverage determination of TRP and its metabolites in sepsis is beneficial for the diagnosis and prognosis of sepsis, as well as for understanding the underlying mechanism of sepsis development. However, similar structures in TRP metabolites make it challenging for separation and metabolite identification. Here, high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) was developed to determine TRP metabolites in rat serum. The first-order derivative spectrophotometry of targeted metabolites in the serum was investigated and proved to be promising for chromatographic peak annotation across different columns and systems. The established method separating the targeted metabolites was optimized and validated to be sensitive and accurate. Application of the method revealed dysregulated TRP metabolites, associated with immune disorders and NAD + metabolism in both the host and gut flora in septic rats. Our findings indicate that the derivative spectrophotometry-assisted method enhances metabolite identifications for the chromatographic systems based on DAD detectors and holds promise for precision medicine in sepsis.

Identification and Characterization of Rotigotine Degradation Products by HPLC Coupled DAD and CAD Detectors and HRMS Through Q-Orbitrap and Electrospray Ionization

Rotigotine (RTG) is a dopamine agonist used in the treatment of Parkinson's disease. As it is susceptible to oxidation, stability studies must be carefully designed for the identification and characterization of all possible degradation products. Here, RTG degradation was evaluated according to the International Conference on Harmonization guidelines under various stress conditions, including acidic and basic hydrolysis, oxidative, metallic, photolytic, and thermal conditions. Additionally, more severe stress conditions were applied to induce RTG degradation. Significant degradation was only observed under oxidative and photolytic conditions. The samples were analyzed by high performance liquid chromatography coupled to photodiode array detectors, charged aerosol, and high-resolution mass spectrometry. Chromatographic analyses revealed the presence of eight substances related to RTG, four of which were already described and were qualified impurities (impurities B, C, K and E) and four new degradation products (DP-1 – DP-4), whose structures were characterized by high-resolution mass spectrometry through Q-Orbitrap and electrospray ionization. In the stress testing of the active pharmaceutical ingredient in solid form, significant RTG degradation was observed in the presence of the oxidative matrix. The results corroborate the literature that confirm the high susceptibility of RTG to oxidation and the importance of using different detectors to detect degradation products in forced degradation studies.

Determining Antiradical Capacity of Medicinal Plant Extract Individual Constituents Using Post-Column Reaction Method.

The post-column reaction method enables the evaluation of the antiradical capacity of individual components in a mixture by separating the components using HPLC and measuring stable free radical (e.g., DPPH●) scavenging that occurs after the chromatography column. The equipment typically consists of two detectors. The first records signals of the analytes leaving the column. The second records radical scavenging by the analytes, which appears as a negative band. The recorded signals are found on two separate chromatograms, which must be combined to interpret the results. In this study, a single DAD detector was used behind the post-column reactor, enabling the simultaneous recording of the analyte bands and negative signals, indicating radical scavenging. The objective of this study was to evaluate the antiradical capacity of key compounds found in two herbal raw materials used in traditional Chinese medicine. Saposhnikovia divaricata roots contain phenolic acids, chromones, and furanocoumarins. Chlorogenic acid, rosmarinic acid, and imperatorin demonstrated strong radical scavenging, while prim-O-glucoslocimifugin showed a weaker response, both in standards and in root extracts. However, scavenging was not observed for cimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol. Astragalus mongholicus roots contain astragalosides I-IV (triterpene saponins). None of these showed DPPH● scavenging. Furthermore, additional signals were observed, indicating the presence of unidentified radical scavenging compounds.

A Simple, Sensitive, and Greener HPLC-DAD Method for the Simultaneous Analysis of Two Novel Orexin Receptor Antagonists.

The orexin receptor antagonist (ORA) is one of the new psychopharmacological agents used in the treatment of insomnia. There are currently no documented greener high-performance liquid chromatography-diode array detector (HPLC-DAD) methods for the analysis of ORA antagonists, lemborexant (LMB) and suvorexant (SUV) simultaneously. Therefore, in this study, a simple, sensitive, and greener HPLC-DAD method has been developed for the simultaneous quantitative analysis of LMB and SUV in bulk and laboratory-prepared mixture. The developed method was validated for numerous validation parameters and evaluated for greenness. The C18 Waters Spherisorb CN (4.6 × 250 mm2; 5 μm) column was used for the chromatographic separation. The mobile phase composition was ethanol: 10 mM KH2PO4 buffer in a ratio of (60:40 v/v). The DAD detection was performed at 253 nm using a Waters DAD detector. The greenness was evaluated using the analytical Eco-Scale (AES), ChlorTox, and analytical GREEnness (AGREE) techniques. The calibration curves showed excellent linearity for LMB and SUV between the concentration range of 125-5000 ng/mL and 250-10,000 ng/mL, respectively. In addition, the proposed HPLC-DAD method was accurate, precise, robust, highly sensitive, and greener. AES, ChlorTox, and AGREE scales were predicted by the HPLC-DAD method to be 91, 1.14 g, and 0.79, respectively, showing an excellent greenness profile. The greener HPLC-DAD method was successfully used to analyze both medicines quantitatively in bulk and laboratory-prepared synthetic mixtures. The findings of this study indicated that the proposed HPLC-DAD method may be consistently applied to evaluate LMB and SUV in bulk and dosage forms.

DEVELOPMENT AND VALIDATION OF A HPLC/PDA METHOD FOR SIMULTANEOUS QUANTIFICATION OF AMLODIPINE BESYLATE AND VALSARTAN IN DISSOLUTION MEDIA

Background: Valsartan, an angiotensin blocker, is often combined with amlodipine (a calcium channel antagonist) in the current treatment of hypertension. There is a generic brand-name drug Exforge® which has been proven to be clinically effective. However, its price is high, making it quite difficult to access, especially in low- and middle-income countries. Therefore, research and development of generic drugs is essential. In addition, research on simultaneous quantification of amlodipine and valsartan in the dissolution medium must be performed to serve as a basis for comparing in vitro equivalence between the two finished products. Objectives: Investigation of the mobile phase for separating amlodipine and valsartan using the isocratic elution method and validation of the simultaneous quantification of amlodipine and valsartan in three different dissolution media with pH of 1.2, 4.5, and 6.8. Materials and methods: Experimental methods on finished tablets containing amlodipine and valsartan. Results: A procedure for simultaneous quantification of amlodipine and valsartan has been developed and validated in three dissolution media using the HPLC method with DAD detector, ZORBAX Eclipse Plus C18 reversed-phase column (4.6 x 250 mm; 5m), isocratic elution method, detection wavelength of 237 nm, flow rate of 1 mL/min, an injection volume of 20 µL, and mobile phase composed of acetonitrile-triethylamine 0.7% (adjusted to pH 3.0 with 0.05% of phosphoric acid) with a ratio of 40:60. The quantitative method achieves linearity with a correlation coefficient R2>0.999 and the linearity for amlodipine and valsartan was determined in the range at pH of 1.2 (0.25-10 µg/mL and 2-160 µg/mL), at pH of 4.5 and 6.8 (0.5-10 µg/mL and 8-160 µg/mL). The values of amlodipine and valsartan content in the test sample on the same day and between two different days were less than 2.0%. The recovery rate ranged from 98% to 102% with the relative standard deviation (RSD) not exceeding 2.0%. Conclusions: The HPLC method with the above chromatographic conditions can be applied for simultaneous quantification of amlodipine and valsartan in three dissolution media with various pH.

Stability indicating RP-HPLC method for estimation of cariprazine hydrochloride in human plasma

Objective: The objective of the study is to create and validate the easy, dependable, accurate, sensitive, and selective RP-HPLC method for estimating Cariprazine HCl in human plasma. Methodology: The sample was prepared using the protein precipitation extraction method. The chromatographic separation was performed with an AGILENT C18 column (250mm x 4.6ID) as the stationary phase and a mobile phase consisting of a 75:25 v/v solution of Methanol and 0.1% Orthophosphoric acid at a flow rate of 0.7 ml/min. The DAD detector was used to carry out the detection at 253 nm. Cariprazine HCl had a reduced retention duration of 2.46 minutes. Results & Discussion: The calibration curve had a correlation coefficient of 0.998 and was linear over the concentration range of 1–5µg/ml. The method's accuracy was shown at levels between 80%, 100%, and 120% of the specification limit. The developed method exhibited excellent precision, with interday precision ranging from 0.07% to 1.77% and intraday precision from 0.03% to 0.26%. It was discovered that the recovery of Cariprazine HCl was within the 98% range. Cariprazine HCl was discovered to have a Limit of Detection (LOD) of 0.053µg/ml, and the Limit of Quantification was found to be 0.160µg/ml. Conclusion: The solution was injected in duplicate, and the % RSD was measured. The results indicate that the proposed method can be effectively utilized for the routine analysis of Cariprazine HCl in human plasma. The forced degradation studies indicate that the drug is susceptible to Hydrolytic and Photolytic degradation

Development and Validation of Stability indicating RP-HPLC Assay Method for Trospium Chloride in Tablet Dosage Form

A simple, rapid, accurate and sensitive method was developed for quantitative analysis of Trospium Chloride in tablet dosage form using Reverse Phase high performance liquid chromatography (RP-HPLC) with DAD detection. The chromatography separation was achieved Xterra RP18, (250 x 4.6mm, 5µParticle Size)), at column temperature 40°C±2, in the isocratic mobile phase mode using Acetonitrile (ACN): Buffer (65:35; v/v) at a flow rate of 1.0mL/min. The determination was performed using Buffer HPLC with DAD detector set at 215nm. Samples were prepared with diluent [Buffer: ACN (200:100; v/v)], and the volume injected was 50µL. The retention time for Trospium Chloride was 3.275 minute. The analytical curve was linear (r2 0.9993) over a wide concentration range (50.0-150.0 µg/mL). The presence of components of the tablet did not interfere in the results of the analysis. The method showed adequate precision with a relative standard deviation (RSD) less than 2%.

Analysis of bioactive compounds in Cordyceps militaris fruiting bodies and dietary supplements: in vitro bioaccessibility determination in artificial digestive juices

SummaryIn recent years, Cordyceps militaris has garnered increased attention in both scientific research and popular culture, exemplified by references in the entertainment industry, such as ‘The Last Of Us’. This study aims to investigate the qualitative and quantitative composition of bioactive compounds (such as cordycepin, ergothioneine, L‐phenylalanine, lovastatin, sterols, indole and phenolic compounds) in various samples. These samples encompass commercially available dietary supplements (in the form of capsules or powder), featuring standardised extracts or non‐standardised C. militaris material. Additionally, fruiting bodies cultivated in Poland and China were examined. The bioactive compounds were subsequently analysed after extraction into artificial digestive juices. Each sample underwent extraction, evaporation and dissolution in methanol, followed by qualitative and quantitative analyses using RP‐HPLC (with UV and DAD detectors) for the determination of bioactive compounds. The results of this analysis revealed that extracts with cordycepin contents of 7% and 1% exhibited the highest cordycepin concentrations, as well as significant ergothioneine levels (4405 and 928 mg/100 g d.w. and 234 and 77.6 mg/100 g d.w. respectively). However, on analysis of simulated digestive juices, it became evident that the fruiting bodies of C. militaris are superior sources of bioactive compounds. Consequently, this study underscores the efficacy of unprocessed material, such as fruiting bodies, as the preferred form of supplementation for potential consumers of C. militaris.

Evaluation of the RP-LC method for determining the pKa of abiraterone and its assay

AbstractA straightforward, dependable, and quick RP-LC method for the analysis of abiraterone acetate in its dose form and human urine has been devised. With DAD detection, sensitivity was reported to be high. The LOD and LOQ of the procedure were deemed adequate. The suggested approach was exhaustively validated in accordance with ICH requirements, and the findings demonstrated that it was exact, accurate, selective, and sensitive for the analysis of this pharmaceutical. The chromatographic separation was realized using a X-Terra RP-18 (150 × 4.60 mm i.d. × 5 μm) column and a UV detector set at 255 and 267 nm. In addition, pKa values were calculated based on the relationship between the retention factor and the pH of the mobile phase. The influence of the composition of the mobile phase on the ionization constant was investigated by measuring the pKa at various acetonitrile–water combinations ranging from 50 to 70% (v/v).

Determination of cytotoxic, antioxidant activities and LC/MS-MS profiles of three endemic Verbascum L. species

The Verbascum genus includes many species used in folk medicine or the treatment of various diseases. In this study, the cytotoxic, antioxidant activity, and LC/MS-MS profiles of three Verbascum species, which are endemic in Eskişehir and its surroundings, were investigated. The cytotoxic effects of methanol extract of V.detersile, V. eskisehirensis, and V.gypsicola species on the cervical (HeLa) and ovarian cancer (SKOV-3) cells were investigated using a colorimetric assay. The results indicated that cytotoxic effect was not observed after treatment of SKOV-3 cells with Verbascum. On the other hand, the cytotoxic activity of V. detersile was found to be 0.1910 mg/dL and 1.057 mg/dL for HeLa cells after 24 or 48 hours incubation with V.detersile, respectively. The antioxidant activity was determined as 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging activity, trolox equivalent antioxidant capacity (TEAC assay), and also the total phenolic content of the samples was found. Total phenols were estimated as Gallic acid equivalents (GAE), expressed as mg Gallic acid/in 1g extract previously described by Singletton. LC/MS profiles separation and detection of phytochemicals of the extract were performed on a Shimadzu UPLC system consisting of a vacuum degasser, an autosampler (SIL20A Shimadzu Autosampler), a binary pump (LC20AD Shimadzu), an oven (CTO20A Shimadzu Column Oven) and DAD dedector (SPD M20A Shimadzu DAD Detector). In terms of antioxidant activity, V. gypsicola was found to have the least antioxidant activity among the three extracts, which is also correlated with the total amount of phenolic content in its content. In this way, it differs from other species. V. detersile exhibits a different chemical profile from the other two species with the iridoid catalpol derivatives it contains. Apigenin pentoside is the only flavonoid molecule detected in V. detersille.

Simultaneous quantitative and qualitative analysis of three kinds of Jigucao related pharmaceuticals by ultra‐high performance liquid chromatography combined with chemometrics method

AbstractAbri Herba (Jigucao) and Abri mollis Herba (Maojigucao) are derived from dried plants of Abrus cantoniensis Hance (Leguminosae) and Abrus mollis without pods. Abri Herba is commonly used in Jigucao tablets; Abri mollis Herba is usually used in Jigucao capsules and FufangJigucao capsules. Surprising, there are few reports focused on the quantitative control of these mentioned Jigucao related pharmaceuticals. This research endeavour has harnessed the formidable power of cutting‐edge ACQUITY UPLC technology to formulate a groundbreaking methodology for the simultaneous quantification of 10 target compounds in Jigucao capsules, Jigucao tablets and FufangJigucao capsules. The primary aim is to bolster quality control and precise identification within the realm of these pharmaceutical products. Separation was performed on an ACQUITY UPLC HSS T3 Column (2.1 × 100 mm, 1.8 μm), gradient elution was performed with 0.1% formic acid in water and acetonitrile. A column temperature of 40°C acts as our guiding compass, and DAD detection scans the spectral landscape in the wavelength range of 210–400 nm. The concentrations of the 10 target components can be used to identify Jigucao tablets, Jigucao capsules and FufangJigucao capsules through the art of chemometrics. The results showed that the established method for determination of 10 target compounds was accurate and reliable with good linearity (r ≥ .9948) and satisfactory recoveries (82.91%–108.6%). The identification of the three Jigucao related pharmaceuticals can be performed using chemometrics based on the 10 target compounds. Thus, our study not only showcases a pioneering analytical approach but also unveils a new dimension in the identification of these pharmaceutical treasures.

Detection of ustilaginoidins in rice samples by immunoassay based on monoclonal antibodies prepared from hemiustilaginoidin-derived haptens

Ustilaginoidins are a class of bis-naphtho-γ-pyrone mycotoxins to threaten humans, animals and environment. Ustilaginoidins are produced by Villosiclava virens, the rice false smut pathogen. To prepare antibodies for quantitatively analyzing ustilaginoidins in rice samples, hemiustilaginoidins D and F from the laccase gene deficiency mutant of V. virens respectively reacted with diazonium 4-aminobenzoic acid to obtain haptens with a carboxyl group, which further reacted with bovine serum albumin or ovalbumin to get their complete antigens. Two monoclonal antibodies (mAbs) designated as 4A12C6 and 5F4F6 were developed by immunization. The relationships between mAb sensitivity and 20 ustilaginoidins were described. 4A12C6 was chosen for further analysis as it could recognize main ustilaginoidins and was more sensitive than 5F4F6. The achieved indirect competitive enzyme-linked immunosorbent assay (icELISA) based on 4A12C6 had a half maximal inhibitory concentration (IC50) of 0.76 ng/mL and working range of 0.2–2.8 ng/mL to ustilaginoidin A. The results of ustilaginoidins-contaminated rice samples by icELISA detection were consistent with those determined by HPLC‒DAD detection. Therefore, we developed a new strategy to get haptens from the biosynthetic precursors with half structures of ustilaginoidins. The achieved icELISA was demonstrated as a convenient method to monitor ustilaginoidin content in rice samples, and showed that the contents of total ustilaginoidins from the rice cultivars with low resistance to rice false smut were more than those of high resistance cultivars.

Development of a simple, eco-friendly HPLC-DAD method for tulathromycin analysis: Ensuring stability and assessing greenness

The core objective of this analytical study was to develop a simple, green, and efficient high-performance liquid chromatography-diode array detector (HPLC-DAD) method for analyzing Tulathromycin (TUL) in pure powder and injection solution without the need for complex derivatization procedures. A simple liquid mobile solution of 15 mL of phosphate buffer (pH = 7.5) and 85 mL of methyl alcohol was passed through a Hypersil BDS C18 (4.6 mm × 25 cm, 5µ) stationary phase in an isocratic mode. The flow speed was controlled at 1.0 mL/min, and the DAD detector was adjusted at 210 nm because of the extremely weak UV absorption characteristic of TUL. TUL was well separated and quantified in 10 min. The HPLC-DAD forced degradation assay was carried out in dissimilar stressful environments concerning international conference for harmonization (ICH) protocols. TUL was linearly quantified from 1000.0 to 3000.0 µg·mL−1. The moderate sensitivity of the HPLC-DAD is attributed to the aliphatic, less conjugated chemical structure of TUL. The maximum degradation ratios occurred in acidic and oxidative environments with proportions of 20.11 % and 18.87 %, respectively. Furthermore, the eco-friendly merits of the HPLC-DAD method were confirmed via the application of two greenness approaches, specifically the analytical greenness tool (AGREE) and the green analytical procedure index (GAPI). The seven green subdivisions in the GAPI pictogram and the total score of 0.72 in the AGREE pictogram illustrate the greenness appraisal of the RP-HPLC-DAD stability method. The innovative RP-HPLC–DAD method is the first sustainable stability-demonstrating assay for TUL in the co-existence of its degradation products in variable stressful environments.

Sugar-based natural deep eutectic solvent ultrasound-assisted extraction for the determination of polyphenolic compounds from various botanical sources

In this study, a novel ultrasound-assisted extraction method utilizing natural deep eutectic solvent (NADES) was developed for the efficient extraction of 20 polyphenolic compounds from various botanical sources, followed by HPLC–DAD detection. By mixing choline chloride as a hydrogen bond acceptor (HBA) and various sugars, such as arabinose, fructose, galactose, trehalose and raffinose, as well as one sugar alcohol (mannitol), in a range of molar ratios, 21 different DESs were prepared and tested. The important parameters influencing the extraction efficiency were studied and optimized using the one-variable-at-a-time (OVAT) method on Picea abies (L.) Karst. needles as a model sample. The optimal conditions were found to be NADES formed by mixing choline chloride and raffinose in a 1:1 M ratio containing 30% water, extraction temperature 25 °C, extraction time 30 min, ultrasound power 60%. The calibration plots were linear from 0.50 to 50 μg mL−1 from standard for the majority of the target analytes, and the instrumental LOD and LOQ values were found to be in the range 0.02–0.19 μg mL−1 and 0.07–0.64 μg mL−1, respectively. The extraction efficiency of the NADES was compared with the efficiency of classical solvents, such as water or methanol, and the NADES revealed a remarkable increase of extraction yield. The newly developed extraction method was successfully applied for the analysis of diverse range of plant materials, encompassing coniferous plants such as Picea abies (L.) Karst, Pinus sylvestris L., and leafy trees Quercus robur L., Ulmus glabra Hudson and Brassica napus L. thereby revealing the polyphenolic profiles of different botanical species.

Hydrophilic interaction liquid chromatography (HILIC) with DAD detection for the determination of relatively non polar fungicides in orange samples

A new, sensitive, rapid, economic and validated high performance liquid chromatography/ diode array detector (HPLC/DAD) method has been developed for the determination of five fungicides namely; Carbendazim (MBC), Thiabendazole (TBZ), Metalaxyl (MET), Imazalil (IMA) and Propiconazole (PCZ). The analytical method was carried on hydrophilic interaction liquid chromatography (HILIC) column using isocratic elution of acetonitrile and water. Prior extraction of orange samples was done using a mixture of diethyl ether and ethyl acetate as an extraction solvent in liquid liquid extraction. The total run time was 15 min which makes it suitable for routine analysis of MBC, TBZ and IMA in oranges. Procedural standard calibration was applied to compensate the matrix effect. In order to measure the lowest concentration of the investigated fungicides below maximum residue limits (MRL), HPLC parameters were optimized. Mean recoveries were obtained ranging from 99.32% to 101.08% with average %RSD < 5.27%. The selected fungicides' residual quantities were successfully measured using the suggested method in various markets and organic orange samples. The investigation of carbendazim's kinetics of degradation, as well as its half-life and predicted preharvest interval (PHI), in real orange samples obtained from orange trees after being treated with the investigated fungicides, is the first to separate relative nonpolar components using HILIC.

Use of Fe3O4@MPTMS-Dithizone Magnetic Nanoparticles as Solid Phase Sorbent for Sensitive Analysis of Sibutramine Molecules in Herbal Slimming Products

Background: A new enrichment and sensitive determination method, which includes HPLCDAD analysis after Magnetic Solid Phase Extraction (MSPE), has been developed for trace analysis of Sibutramine molecules in herbal slimming products. Sibutramine is one of the most adulterated drug molecules in herbal products. Method: In the proposed method, Sibutramine molecules were pre-concentrated by using Fe3O4@MPTMS-Dithizone magnetic sorbent synthesized in our laboratory. Desorption of Sibutramine molecules from the sorbent phase was carried out by using acetonitrile: methanol (1:1) solvent in the presence of pH 8.0 buffer before chromatographic determinations. Results: Analytical parameters of the method, such as linear range, enrichment factor, and determination limit, were determined after optimizing experimental variables such as interaction time, desorption solvent, pH, etc. The sibutramine molecule was analyzed by isocratic elution of acetonitrile and KH2PO4 (pH 3.0, 0.05 M) (40:60) with a DAD detector at 223 nm wavelength. Limit of detection (LOD) value was calculated as 1.43 ng mL-1. Conclusion: Relative standard deviations (RSD) were below 3.20% for determinations of model solutions, including 100 ng mL-1 of Sibutramine. Finally, the developed method has been applied to herbal slimming tea samples with quantitative recovery experiments.