Methanol-acetic Acid Research Articles

Evaluation of the Physico-chemical and Biological Properties of Fermented Nipa (Nypa fruticans) Sap Under Closed and Open Vessel Fermentation System for Bioethanol Production

Background The potential of Nipa (Nypa fruticans) sap in the Cagayan Region, Philippines, for bioethanol production remains largely underexplored despite its vast expanse. Methods This study investigates the key physico-chemical properties of Nipa sap from Aparri, Cagayan, with a focus on fermentation dynamics and storage conditions to optimize ethanol production. Two fermentation treatments closed and open vessel conditions, were compared to determine the optimal conditions for maximum ethanol yield. Over a 7-day fermentation period, the acetic acid production, yeast activity, sugar content, and ethanol concentration of closed vessel fermented sap (CVFS) and open vessel fermented sap (OVFS) were monitored. Results CVFS demonstrated a higher ethanol yield (7.56%v/v at peak day), characterized by minimized acetic acid accumulation and stable yeast activity. In contrast, OVFS exhibited significant fluctuations in acetic acid and ethanol levels, indicating a lower fermentation efficiency. Conclusion The presence of methanol and high acetic acid content in open-vessel fermentation systems highlights the importance of fermentation conditions to prevent impurities. This study provides valuable insights into optimizing ethanol production from fermenting Nipa sap by employing closed vessel fermentation, which is crucial for sustainable bioethanol production in the Cagayan region.

An eco-friendly smartphone based HPTLC method versus conventional densitometric one for determination of Naltrexone and Bupropion

The rapid uprising technologies of smartphone applications and software introduced a new era for analytical detection techniques. It has transformed bench-top laboratory methods into simpler ones depending on cost-effective, portable, and widely accessible devices. In this work, two high performance thin layer chromatographic (HPTLC) methods were developed based on smartphone’s camera detection and either ImageJ desktop software or Color-Picker smartphone’s application as alternative techniques to conventional densitometric detection. A mixture of Naltrexone hydrochloride (NAL) and Bupropion hydrochloride (BUP) was chromatographed on HPTLC- plates using ethyl acetate, methanol, acetone, and glacial acetic acid (3:6:1:0.5, by volume) as a developing system. The developed plates were scanned at 203 nm for the densitometric analysis, then visualized by modified Dragendorff’s reagent and shot by a smartphone’s camera. The captured images were uploaded to either ImageJ software or Color-Picker application to detect the separated spots. The results derived from the three detection methods were compared over the concentration range of 0.4–24 & 0.6–18 µg/band for the densitometric method, 0.4–24 & 2–24 µg/band for ImageJ built method and 0.8–20 & 5–20 µg/band for Color Picker built method for NAL and BUP, respectively. The methods were found to be appropriate for assaying both active drug substances in pure forms and combined in marketed pharmaceutical formulations. The excellent sustainability of densitometric and ImageJ-based methods enabled also the assessment of their dosage form content uniformity. The greenness and sustainability of the methods were assessed by three metric tools, namely Green Analytical Procedure Index (GAPI), Analytical GREEnness Metric Approach (AGREE), and White Analytical Chemistry (WAC). The assessments results confirmed the sustainability and superiority of the proposed methods in terms of sample treatment, waste mount, energy consumption, cost, and number of analyzed samples per an hour.

Simultaneous determination of five plant hormones in cotton leaves using QuEChERS combined with HPLC‒MS/MS

BackgroundPlant hormones profoundly influence cotton growth, development, and responses to various stresses. Therefore, there is a pressing need for an efficient assay to quantify these hormones in cotton. In this groundbreaking study, we have established QuEChERS-HPLC‒MS/MS method, for the simultaneous detection of multiple plant hormones in cotton leaves, allowing the analysis and quantification of five key plant hormones.ResultsSample extraction and purification employed 0.1% acetic acid in methanol and C18 for optimal recovery of plant hormones. The method applied to cotton demonstrated excellent linearity across a concentration range of 0.05–1 mg·L−1, with linear regression coefficients exceeding 0.99. The limits of quantification (LOQs) were 20 μg·kg−1 for GA3 and 5 μg·kg−1 for the other four plant hormones. Recovery rates for the five plant hormones matrix spiked at levels of 5, 10, 100, and 1000 μg·kg−1 were in the range of 79.07% to 98.97%, with intraday relative standard deviations (RSDs) ranging from 2.11% to 8.47%. The method was successfully employed to analyze and quantify the five analytes in cotton leaves treated with plant growth regulators.ConclusionThe study demonstrates that the method is well-suited for the determination of five plant hormones in cotton. It exhibits excellent selectivity and sensitivity in detecting field samples, thus serving as a robust tool for in-depth research into cotton physiology.

Molecularly imprinted polymers using high-performance liquid chromatography to detect and determine ethion pesticide in apricot, grapes, strawberry and soil samples

ABSTRACT This paper demonstrates the synthesis of molecularly imprinted polymers-solid phase extraction-liquid chromatography method at room temperature using bulk polymerisation of ethion pesticide, by high sensitivity, low costs and high stability. By making MIP for ethion as ethion-MIP, which could be detected with a UV-Vis spectrophotometer at 276 nm, a functional monomer of acrylic acid with cross-linking ethylene glycol dimethyl acrylate. The high-performance liquid chromatography methods developed in this study are accurate, sensitive, and precise to apricot, grapes, strawberry and soil samples. The elution process to the template ethion from the ethion-MIP, caused by minimum volume using solution of 1.0 mL of methanol-acetic acid (9:1) was obtained. Different factors affecting the reaction were studied and optimised. The calibration plot is linear in the concentration range of (0.02 to 1.0 µg mL−1). The relative standard deviations of (±2.0%) and detection limit of the method are 0.02 µg mL−1, and the maximum imprinting factor of ethion-MIP was 6.8 mgg−1. The results obtained the evidence of efficiency and reliability of the method for the ethion pesticide analysis and estimation for the capacitance of molecular imprinted polymer, analytical technical method by molecularly imprinted polymer and high-performance liquid chromatography.

Solvent Replacement Strategies for Processing Pharmaceuticals and Bio-Related Compounds—A Review

An overview of solvent replacement strategies shows that there is great progress in green chemistry for replacing hazardous di-polar aprotic solvents, such as N,N-dimethylformamide (DMF), 1-methyl-2-pyrrolidinone (NMP), and 1,4-dioxane (DI), used in processing active industrial ingredients (APIs). In synthetic chemistry, alcohols, carbonates, ethers, eucalyptol, glycols, furans, ketones, cycloalkanones, lactones, pyrrolidinone or solvent mixtures, 2-methyl tetrahydrofuran in methanol, HCl in cyclopentyl methyl ether, or trifluoroacetic acid in propylene carbonate or surfactant water (no organic solvents) are suggested replacement solvents. For the replacement of dichloromethane (DCM) used in chromatography, ethyl acetate ethanol or 2-propanol in heptanes, with or without acetic acid or ammonium hydroxide additives, are suggested, along with methanol acetic acid in ethyl acetate or methyl tert-butyl ether, ethyl acetate in ethanol in cyclohexane, CO2-ethyl acetate, CO2-methanol, CO2-acetone, and CO2-isopropanol. Supercritical CO2 (scCO2) can be used to replace many organic solvents used in processing materials from natural sources. Vegetable, drupe, legume, and seed oils used as co-extractants (mixed with substrate before extraction) can be used to replace the typical organic co-solvents (ethanol, acetone) used in scCO2 extraction. Mixed solvents consisting of a hydrogen bond donor (HBD) solvent and a hydrogen bond acceptor (HBA) are not addressed in GSK or CHEM21 solvent replacement guides. Published data for 100 water-soluble and water-insoluble APIs in mono-solvents show polarity ranges appropriate for the processing of APIs with mixed solvents. When water is used, possible HBA candidate solvents are acetone, acetic acid, acetonitrile, ethanol, methanol, 2-methyl tetrahydrofuran, 2,2,5,5-tetramethyloxolane, dimethylisosorbide, Cyrene, Cygnet 0.0, or diformylxylose. When alcohol is used, possible HBA candidates are cyclopentanone, esters, lactone, eucalytol, MeSesamol, or diformylxylose. HBA—HBA mixed solvents, such as Cyrene—Cygnet 0.0, could provide interesting new combinations. Solubility parameters, Reichardt polarity, Kamlet—Taft parameters, and linear solvation energy relationships provide practical ways for identifying mixed solvents applicable to API systems.

The Selective Removal of Bisphenol A Using a Magnetic Adsorbent Fused with Bisphenol A-Binding Peptides.

The potential of bisphenol A (BPA)-binding peptides fused to magnetic beads is demonstrated as novel adsorbents that are reusable and highly selective for BPA removal from aqueous environments, in which various interfering substances coexist. Magnetic beads harboring peptides (peptide beads) showed a higher BPA removal capacity (8.6 mg/g) than that of bare beads without peptides (2.0 mg/g). The BPA adsorption capacity of peptide beads increased with the number of peptides fused onto the beads, where monomeric, dimeric, or trimeric repeats of a BPA-binding peptide were fused to magnetic beads. The BPA-adsorbing beads were regenerated using a methanol-acetic acid mixture, and after six regeneration cycles, the adsorption capacity remained above 87% of its initial capacity. The selective removal of BPA was confirmed in the presence of BPA analogs with high structural similarity (bisphenol F and bisphenol S) or in synthetic wastewater. The present work is a pioneering study that investigates the selective affinity of peptides to remove specific organics with high selectivity from complex environmental matrices.

Determination of lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) and domoic acid in mussels by solid-phase extraction and reversed-phase liquid chromatography with tandem mass spectrometry

An accurate and efficient method was developed for the determination of azaspiracid shellfish toxins (azaspiracids-1, -2, and -3), neurotoxic shellfish toxins (brevetoxins-2 and -3), diarrhetic shellfish toxins (okadaic acid and dinophysistoxins-1 and -2), and the amnesic shellfish toxin (domoic acid) in mussels (Mytilus galloprovincialis). Lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) were extracted with 0.5 % acetic acid in methanol under heating at 60°C to improve the extraction efficiency of okadaic acid group toxins and then cleaned up with a C18 solid-phase extraction cartridge. Domoic acid was extracted with 50 % aqueous methanol and then cleaned up with a graphitized carbon solid-phase extraction cartridge. Lipophilic marine biotoxins and domoic acid were quantified by reversed-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The developed method had insignificant matrix effects for the nine analytes and good recoveries in the range of 79.0 % to 97.6 % at three spiking levels for all analytes except brevetoxin-2 (43.8–49.8 %). The developed method was further validated by analyzing mussel tissue certified reference materials, and good agreement was observed between certified and determined values.

Simultaneous determination of levothyroxine and its metabolite liothyronine in human serum by using LC-ESI-MS/MS and its bioequivalence application

Levothyroxine is a synthetic thyroid hormone, used to treat hypothyroidism and related conditions. It has been a widely used and effective medication for many decades. The bioanalytical method is developed for the determination of endogenously present levothyroxine and its metabolite, liothyronine, in both stripped and un-stripped serum, using liquid chromatography-tandem mass spectrometry. Determining an endogenously present analyte is indeed a critical step in pharmacokinetic studies and determining the therapeutic dose of a drug. The term “stripped” typically refers to the removal of endogenous analytes that interfere with the accurate quantification of the analyte of interest. Method employed with, Kinetex® polar C18 (100 × 4.6 mm 2.6 μm) analytical column for chromatographic resolution by using, pump A as acetic acid in water and pump B as acetic acid in methanol. The mass spectrometer used with triple quadrupole operating in the positive ion mode, specifically using multiple reaction monitoring. Multiple reaction monitorin is a highly delicate and discriminating technique for quantifying specific analytes. The transitions are as follows: analyte transition: 777.55→731.65, metabolite transition: 651.70→605.85, IS analyteTransition: 780.60→734.75, and IS metaboliteTransition: 661.60→614.65. A resolution factor of 0.5 was achieved for levothyroxine and liothyronine. Linearity for levothyroxine in a range of 20.0–1000 ng/ml and liothyronine in a range of 0.3–15.0 ng/ml was employed. Method validation was performed to conduct stability studies and demonstrate the reproducibility for accuracy and reliability for LC-ESI-MS/MS assay. The proposed bioanalytical method is highly sensitive and selective, has a minimal processing volume, faster run time, no matrix impact, and the ability to measure analytes in different types of serum (stripped and normal). The method was valuable for both research and clinical laboratories and can provide reliable results.

Advancing PFAS characterization: Enhancing the total oxidizable precursor assay with improved sample processing and UV activation

Per- and polyfluoroalkyl substances (PFAS) encompass over 9000 chemicals utilized in various industrial and commercial applications. However, the quantification of PFAS using standard commercial analytical methods is currently limited to <50 selected compounds. To address this issue, the total oxidizable precursor (TOP) assay was developed, allowing for the oxidative conversion of previously undetectable PFAS precursors into measurable PFAS. This study investigated different sample processing methods to address post-oxidation PFAS loss identified in literature. Using PFOS as a probe molecule, up to 50 % loss of PFOS was identified during sample work-up. It was determined that the use of mass-labelled PFOS and methanolic acetic acid to chemically quench the sample post-oxidation improved PFOS recovery and allowed for correction of any remaining PFOS loss. The use of ultraviolet (UV) light was then investigated as an activator in contrast to the standard thermal activation method. A comparative evaluation was conducted to assess the recovery and conversion of perfluorooctanoic acid (PFOA), PFOS, and 6:2 fluorotelomer sulfonate (6:2 FTS) using both the heat-activated and UV-activated TOP assays. Results demonstrated that the UV-activated TOP assay achieved complete (100 %) oxidation of 6:2 FTS within 7.5 min, resulting in a total yield of generated perfluorinated carboxylic acids (PFCAs) at 108 ± 8 %. The study concluded by investigating the UV-activated TOP assay for its application on various aqueous film forming foam (AFFF) formulations and two AFFF samples drained from military aircraft rescue firefighting vehicles (ARFFVs). Analysis of these AFFF samples were supported by high resolution mass spectrometry and an expanded analytical suite, identifying several fluorotelomer precursors. The findings of this study provide compelling evidence that modifications in sample processing, work-up procedures, expansion of initial PFAS calibration standards, and UV-activation methods enhance the TOP assay, positioning it as a more reliable and quantitative analytical tool for PFAS characterization.

Determination of three plant growth regulators in Dendrobium officinale and Anoectochilus roxburghii by three-phase hollow fiber liquid phase microextraction- high performance liquid chromatography

Dendrobium officinale (D. officinale) and Anoectochilus roxburghii (A. roxburghii) are precious raw materials for traditional Chinese medicine. The growing demand for D. officinale and A. roxburghii cannot be met by current production techniques. Hence, the widespread artificial cultivation of D. officinale and A. roxburghii using substantial amounts of plant growth regulators (PGRs) has emerged. The excessive use of PGRs not only affects the quality and efficacy of medicinal materials but also causes a series of safety issues. Therefore, expanding research on residual PGRs in valuable Chinese medicinal materials is important to avoid the health hazards caused by these substances. Unfortunately, the identification of PGRs is challenging because of their trace and complex matrices. High performance liquid chromatography (HPLC) has become one of the mainstream analytical methods for PGR determination. An important consideration in the application of this technique to the detection of trace acidic PGRs is how to improve its accuracy and sensitivity. Three-phase hollow fiber liquid phase microextraction (3P-HF-LPME) has the advantages of a high enrichment factor, complex sample purification ability, low reagent consumption, low cost, and easy integration with chromatographic systems. Thus, the 3P-HF-LPME method overcomes the many shortcomings of traditional sample pretreatment methods. In this study, a novel, simple, and effective analytical method based on 3P-HF-LPME combined with HPLC was developed to extract, purify, enrich, and detect three trace acidic PGRs (indole-3-acetic acid, naphthyl acetic acid and indolebutyric acid) in D. officinale and A. roxburghii. The chromatographic separation conditions and 3P-HF-LPME model parameters were systematically optimized for this purpose. First, the sample solution was prepared by ultrasonication and low-temperature standing, and then adjusted to pH 3.0 using dilute hydrochloric acid. The sample solution (10 mL) and NaCl (1.50 g) were stored in a 15 mL brown extraction bottle with a built-in magnetic stirrer. Next, 30 μL of NaOH solution (pH 11.0) as the inner phase solution was injected into the inner cavity of a hollow fiber tube, which was subsequently sealed at both ends. The hollow fiber tube was soaked in n-octanol for 5 min and dried naturally to remove excess extraction solvent from its surface. Finally, the fiber tube was placed in a brown extraction bottle and stirred using a thermostatic magnetic stirrer at 40 ℃ and 1600 r/min for 2 h. After extraction, the three target analytes were separated on a Welch Ultimate XB-C18 column (250 mm×4.6 mm, 5 μm) under isocratic elution conditions using acetic acid aqueous solution and methanol (45∶55, v/v) as the eluent. The results indicated that the three PGRs showed good linearity in the range of 0.5-100.0 μg/L (coefficients of determination (r2)=0.9999), with limits of detection (LODs) of 0.02-0.15 μg/L. The method recoveries were 88.5-102.2%, with relative standard deviations (RSDs) of less than 3.7% (n=3). The extraction efficiencies and enrichment factors of the three PGRs in 15 batches of fresh D. officinale and A. roxburghii products were found to be 42.0%-86.8% and 140-289. Full-scan mass spectrometry was used to further identify positive samples to avoid false-positive results and enhance the reliability of the experimental method. In summary, the proposed method is sensitive, accurate, reliable, environment friendly, and capable of high enrichment. It could be used to determine the residues of three acidic PGRs in D. officinale and A. roxburghii. Moreover, it can provide technical support for the residue detection of PGRs in other Chinese medicinal materials.

Process simulation and economic evaluation of an integrated production plant for methanol, acetic acid and DME synthesis via sugarcane bagasse gasification

Sugarcane bagasse is generated in large quantities in Brazil, and it could be gasified for the production of several chemicals, adding value to the sugar and ethanol production chain. The objective of this work is the simulation of an integrated plant for the production of three important chemicals through sugarcane bagasse gasification: methanol, acetic acid and DME. A sensitivity analysis was performed and showed how gasification temperature and steam-to-biomass ratio should be adjusted to favor one or another product (T = 900 °C and SB = 0.9 for methanol and indirect acetic acid and DME production; T = 950 °C and SB = 0.5 for direct DME production), and an economic analysis has shown that the installation of a multipurpose plant is feasible (paybacks of less than 5 years and multipurpose fashion not affecting the investment return) and how the process can be reconfigured depending on the economic scenario.

Solubility and Thermodynamics Profile of Benzethonium Chloride in Pure and Binary Solvents at Different Temperatures.

Benzethonium chloride (BTC) has various applications in several industries. The solubility and solution thermodynamic properties of BTC were measured. The solubility of BTC in methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, water, dimethyl sulfoxide, acetic acid, and dimethyl formamide neat solvents and methanol + water and ethanol + water binary solvents at 298.15-318.15 K over an atmospheric pressure was measured. The solubility data of BTC is positively related to the temperature in all selected solvents. The solubility data was fitted by the Apelblat model, λh model, Yaws model, Van't Hoff equation, CNIBS/R-K model, and modified Jouyban-Acree equation. The RMSD and ARD were chosen to evaluate the fitting of each model. The dissolution thermodynamic parameters, enthalpy of the solution, entropy of the solution, and Gibbs energy of the solution were calculated. The solubility data and dissolution thermodynamic parameters of BTC will provide significant guidance for purification, crystallization, and separation in various areas.

An effective and stability-indicating method development and optimization utilizing the Box-Behnken design for the simultaneous determination of acetaminophen, caffeine, and aspirin in tablet formulation.

Analytical techniques must be sensitive, specific, and accurate to assess the active pharmaceutical ingredients in pharmaceutical dosage forms. The quality-by-design (QbD) application has proven to be a practical method for magnifying HPLC operations. This article discusses the successfully developed QbD-based stability-indicative LC method for evaluating acetaminophen, caffeine, and aspirin (ASP) in tablet dosage form. To achieve the necessary chromatographic separation, Milli-Q water, methanol, and glacial acetic acid were employed in the following ratios: 63:35:2 (v/v/v) for mobile phase A and 18:80:2 (v/v/v) for mobile phase B. The flow rate, column temperature, and detecting wavelength were 1.0ml/min, 40°C, and 275 nm, respectively, and an InertSustain C18 analytical column (150 × 4.6mm, 3μm) was used. Linearity was between 10.0 and 150.0μg/ml for ASP and acetaminophen and between 2.6 and 39.0μg/ml for caffeine. The accuracy findings were more than 97%, and the correlation coefficient for all three components was found to be greater than 0.999. The validated HPLC method yielded reliable and accurate results. ASP was shown to be vulnerable to both acid and alkaline hydrolysis in the forced degradation study. The described method is capable of separating the degradants produced during stress testing and is regarded as stability indicating. The proposed method can be used for a wider range of other formulations with an appropriate diluent selection and sample preparation procedure optimization.

Determination of coumarin-3-carboxylic acid in the liver and heart tissue of Sprague-Dawley rats after intragastric administration of extractive of leaves of F. virens var. sublanceolata by HPLC

AbstractThe study was aimed to validate and optimize high performance liquid chromatographic (HPLC) method for the determination of coumarin-3-carboxylic acid (C3A) in the heart and liver issue of Sprague-Dawley (SD) rats after intragastric administration of extractive of leaves of Ficus virens var sublanceolata. And simple ADME and target prediction analyses were performed for C3A. Ethyl acetate was employed to precipitate protein with appropriate sensitivity and acceptable matrix effects. The satisfactory separation was developed on an ODS2 column (4.6 mm × 250 mm, 5 μm) by gradient elution with a methanol-acetic acid solution (pH = 3.0) as the mobile phase. The flow rate was 1.0 mL min−1, the column temperature was maintained at 30 ± 2 °C, the injection volume was 20 μL, and the detection wavelength was set as 309 nm. The method was fully validated in terms of selectivity, linearity, accuracy, precision, extraction recovery and stability. The results of the ADME analysis found that C3A has excellent characteristics of drug-likeness, consistent with good bio-absorption. And the predicted 12 target protein belongs to the amine oxidoreductase and carbonic anhydrase target class. This method is simple, rapid, sensitive, and accurate for the determination of coumarin-3-carboxylic acid in the heart and liver tissue of SD rats.

Magnetic Molecularly Imprinted Polymers for the Rapid and Selective Extraction and Detection of Methotrexatein Serum by HPLC-UV Analysis.

In this work, novel selective recognition materials, namely magnetic molecularly imprinted polymers (MMIPs), were prepared. The recognition materials were used as pretreatment materials for magnetic molecularly imprinted solid-phase extraction (MSPE) to achieve the efficient adsorption, selective recognition, and rapid magnetic separation of methotrexate (MTX) in the patients’ plasma. This method was combined with high-performance liquid chromatography–ultraviolet detection (HPLC–UV) to achieve accurate and rapid detection of the plasma MTX concentration, providing a new method for the clinical detection and monitoring of the MTX concentration. The MMIPs for the selective adsorption of MTX were prepared by the sol–gel method. The materials were characterized by transmission electron microscopy, Fourier transform-infrared spectrometry, X-ray diffractometry, and X-ray photoelectron spectrometry. The MTX adsorption properties of the MMIPs were evaluated using static, dynamic, and selective adsorption experiments. On this basis, the extraction conditions were optimized systematically. The adsorption capacity of MMIPs for MTX was 39.56 mgg−1, the imprinting factor was 9.40, and the adsorption equilibrium time was 60 min. The optimal extraction conditions were as follows: the amount of MMIP was 100 mg, the loading time was 120 min, the leachate was 8:2 (v/v) water–methanol, the eluent was 4:1 (v/v) methanol–acetic acid, and the elution time was 60 min. MTX was linear in the range of 0.00005–0.25 mg mL−1, and the detection limit was 12.51 ng mL−1. The accuracy of the MSPE–HPLC–UV method for MTX detection was excellent, and the result was consistent with that of a drug concentration analyzer.

Immunoaffinity Cleanup and Isotope Dilution-Based Liquid Chromatography Tandem Mass Spectrometry for the Determination of Six Major Mycotoxins in Feed and Feedstuff.

In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of deoxynivalenol, aflatoxin B1, zearalenone, ochratoxin A, T-2 toxin and fumonisin B1 in feed and feedstuff was established. The sample was extracted with an acetonitrile–water mixture (60:40, v/v), purified by an immunoaffinity column, eluted with a methanol–acetic acid mixture (98:2, v/v), and reconstituted with a methanol–water mixture (50:50, v/v) after drying with nitrogen. Finally, the reconstituted solution was detected by LC-MS/MS and quantified by isotope internal standard method. The six mycotoxins had a good linear relationship in a certain concentration range, the correlation coefficients were all greater than 0.99, the limits of detection were between 0.075 and 1.5 µg·kg−1, and the limits of quantification were between 0.5 and 5 µg·kg−1. The average spike recoveries in the four feed matrices ranged from 84.2% to 117.1% with relative standard deviations less than 11.6%. Thirty-six actual feed samples were analyzed for mycotoxins, and at least one mycotoxin was detected in each sample. The proposed method is reliable and suitable for detecting common mycotoxins in feed samples.

A Non-aqueous Capillary Electrophoresis for Determination of Eugenol in Cloves and Dental Preparations

A total non-aqueous capillary electrophoresis method was developed and applied for the first time for the quantification of eugenol in cloves and dental preparations. The optimized conditions included a buffer consisting of 150 mM sodium acetate and 300 μL 1 M acetic acid methanol solution (30 mM), an applied voltage of 25 kV, and a temperature of 25 °C and an applied wavelength of 214 nm. The developed method of determining the eugenol was characterized by the following parameters: a detection time within 1.97 min, good linearity (R2 = 0.9989–0.9999), detection limit at the level from 0.19 to 0.35 µg mL−1, very good extraction yield of 99.6–100.6% from both methanol standard solutions, clove buds’ matrix, and dental preparations. Limit of quantitation at the level from 0.81 to 0.98 µg mL−1. The method is based on the developed one-step extraction procedure. Moreover, the developed method does not require the use of any eugenol solubility enhancers such as SDS.

Extraction, purification, and quantification of hesperidin from the immature Citrus grandis/maxima fruit Nepal cultivar

Abstract. Manikyam HK, Tripathi P, Patil SB, Lamichhane J, Chaitanya MVNL, Patil AR. 2022. Extraction, purification, and quantification of hesperidin from the immature Citrus grandis/maxima fruit Nepal cultivar. Asian J Nat Prod Biochem 20: 21-26. Hesperidin, a flavanone group of flavonoids and a 7-O-rutinoside of hesperitin is abundantly present in citrus fruits. Pomelo or Citrus grandis (L.) Osbeck/ C. maxima (Burm.) Merr. is grown and cultivated in Nepal as seasonal edible fruit. Pomelo has flavanone and other chemical constituents. Flavanones like naringin, hesperidin, neohesperidin, and naringenin are present in the fruits of C. grandis. Many extraction techniques have been reported in the extraction and purification of hesperidin citrus fruits like sweet lemons, grapefruits, mandarins, etc. However, little information has been provided on hesperidin content in Pomelo fruits. In order to estimate the quantity of hesperidin in the Pomelo fruits of Nepal, we have first time made study. The immature Pomelo fruits of size 2-7 cm were collected from the cultivation field and dried completely. The pulverized fruit powder with moisture content less than 15% was subjected to 5% methanolic acetic extraction and Dimethylformamide (DMF) solvent extraction in Soxhlet apparatus by refluxing at 90°C in 1:15 ration in three subsequent extractions of each 1 hour. The concentrates were crystalized at freezing temperatures in methanol and further purified by repeating the crystallization process. The white amorphous powder was subjected to RP-HPLC analysis method, chromatographic conditions 285 nm wavelength, C18 column 4.6 mm x 15 cm x 5 µm, flow rate 1.2 mL/minute with injection volume 10 µL run time of 30 minutes, column temperature 40°C. Hesperidin yields were 0.12 g/100 g with acetic acid methanol with a purity of 87% and 0.17 g/100 g in DMF extraction with a purity of 90% with recoveries of 87% in acetic acid methanol extraction and 90% in DMF extraction when compared with standard 93%. Further extraction does not yield any hesperidin content indicating a total of 0.15-0.17 g/100 g of hesperidin content in the whole immature fruit of C. grandis/maxima. Thus we found DMF and 5% methanolic acetic solvents as preferable solvents to extract hesperidin.

PB1891: VARIANT PHILADELPHIA CHROMOSOME IN CML

Background: Chronic myeloid leukemia is a clonal malignant disorder of a pluripotent hematopoietic stem cell characterized by the Philadelphia chromosome and the reciprocal translocation t(9:22)(q34;q11). Almost 5 to 10 percent of chronic myeloid leukemia cases demonstrate variant Philadelphia translocations. Aims: To contribute to the description of patients’ clinical characteristics and prognosis with variant Ph chromosome-positive leukemia, we report a Ph chromosome-positive CML in the chronic phase characterized by an unusual variant Ph chromosome. Methods: An elderly male Libyan patient was suspected to have chronic myeloid leukemia. Cytogenetic and molecular exploration were conducted using bone marrow and blood samples. Cytogenetic analysis was performed on lymphocytes and bone marrow cells after short-term culture (48 hours). The cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature. Multiplex reverse transcription-PCR (RT-PCR) was used to identify the BCR-ABL1 transcript. RT-PCR was performed according to European recommendations, as described previously. Results: Clinical assessment confirmed the first chronic phase of a chronic myeloid leukemia. Chromosomal formula showed an homogeneous structural translocation: t(1;9;12;22)(p35-36;q34;p13;q11) involving four chromosomal regions, 1p35-36, 9q34, 12p13 and 22q11.2. Molecular investigation confirmed the Ph nature of the chromosomal abnormality by showing the BCR-ABL1 transcript. The patient was treated by using Imatinib molecule with a favorable therapeutic response. Summary/Conclusion: A new variant Ph chromosome-positive CML in the chronic phase is reported. It is a four-way Ph translocation t(1;9;12;22)(p35-36;q34;p13;q11) involving four chromosomal regions, 1p35-36, 9q34, 12p13 and 22q11.2. Imatinib, the first-generation TKI treatment was efficient.

PB1912: MYELODYSPLASTIC SYNDROME WITH ISOLATED 5Q DELETION AND ASSOCIATED 5Q DELETION TO 13Q DELETION

Background: The 5q deletion is the classic chromosomal abnormality in myelodysplastic syndrome (MDS). Critical regions are 5q31 and 5q33, leading to gene haplodeficiency and other pathogeneic mechanisms. 13q deletion occurs in about 2% of MDS cases. This deletion involved band 13q14. Aims: Here we describe cytogenetic abnormalities in a Tunisian cohort of MDS patients and we shed the light on cases with 5q deletion. Methods: Cytogenetic analyses of bone marrow cells were performed according to standard procedures for 12 Tunisian patients presenting with MDS. Cytogenetic analysis was performed on lymphocytes and bone marrow cells. Cultured cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature. Results: Cytogenetic abnormalities were detected in 9 cases and del5q was recorded in four patients (3 patients with refractory anemia (RA) and a fourth with RA and excess blasts-T (RAEB-T)). The del5q was isolated in two RA cases as a del (5) (q13 q33) in a new diagnosed and a treated patients. It was associated in two patients: a RA patient explored during the evolution, harboring a del (5) (q12 q33) associated to a del (13) (q12 q14) and the RAEB-T patient harbouring a del (5) (q21 q31) associated to a clonal hyperploidy. Summary/Conclusion: The diagnosis of MDS was based on cytomorphology and cytogenetics. However, nowadays, an increasing number of molecular aberrations have been discovered and included in the WHO revised new classification of myeloid neoplasms. The del13q described in MDS seems to be a marker of the evolution of the clonal cells harboring 5q deletion. However, the molecular basis and the implicated genes of the deletion of the long arm of chromosome 13 associated to the 5q deletion clone remain until now not well explored.