Development and validation of an LC-MS/MS method for simultaneous determination of three organic azido impurities in tetrazole-containing sartans

This study aimed to develop a sensitive and simple liquid chromatography-tandem mass spectrometry for the simultaneous determination of organic azido impurities (AZBC, AZBT, and AZTT) in sartan active pharmaceutical ingredients and drug products. The method employed a HALO C8 column for chromatographic separation using gradient elution, with a mobile phase composed of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Quantification of analytes was achieved using a triple quadrupole mass spectrometer, operating in multiple reaction monitoring mode with positive electrospray ionization. The method was fully validated following the ICH Q2 (R1) guideline, and the validation parameters met the criteria for specificity, carryover, and robustness. The developed method was found to be sufficiently sensitive, with LOD and LOQ below the acceptable limits of azido impurities in pharmaceuticals as per the ICH M7 guideline. The assay had dynamic ranges of 1.00–20.00 ng/mg for AZBC and 0.10–15.00 ng/mg for AZBT and AZTT in sartans. In conclusion, the established method was effectively utilized for determining azido impurities in sartan active pharmaceutical ingredients and drug products, achieving high accuracy and precision

Chlorogenic acid content, essential oil compositions, and in vitro antioxidant activities of Chromolaena odorata leaves

Chromolaena odorata (L.) R. M. King and H. Rob. is a Thai medicinal plant used for the treatment of wounds, rashes, diabetes, and insect repellent. The leaves of C. odorata were collected from 10 different sources throughout Thailand. The chemical constituents of essential oils were hydro-distilled from the leaves and were analyzed by gas chromatography-mass spectrometry. Chlorogenic acid contents were determined by thin-layer chromatography (TLC) - densitometry with winCATS software and TLC image analysis with ImageJ software. The TLC plate was developed in the mobile phase that consisted of ethyl acetate:water:formic acid (17:3:2). Antioxidant activities were examined by 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging and β-carotene bleaching assays. C. odorata essential oil has shown the major components of pregeijerene, dauca-5, 8-diene, (E)-caryophyllene, β-pinene, and α-pinene. The chlorogenic acid content of C. odorata leaves was determined by TLC-densitometry and TLC image analysis. Results have shown that TLC-densitometry and TLC image analysis method were not statistically significantly different. DPPH radical scavenging and β-carotene bleaching assays of ethanolic extract of C. odorata leaves showed its antioxidant potential

A validated TLC-image analysis method for detecting and quantifying bioactive phyllanthin in Phyllanthus amarus and commercial herbal drugs

Phyllanthus amarus Schum. and Thonn. has long been used for the treatment of liver diseases. The hepatoprotective compound presented in P. amarus was phyllanthin. In this study, the fast determination and quantitation of bioactive phyllanthin in P. amarus and its commercial herbal drugs were developed using a simple thin-layer chromatographic (TLC)- image analysis method. Chromatographic separation of phyllanthin was carried out and the intensity of phyllanthin was examined by TLC-image analysis. The linear equation line showed good relationship for the calibration curve in the various concentrations of phyllanthin. The limits of detection and limits of quantitation were 0.16 and 0.49 µg/spot, respectively. The contents of phyllanthin from fifteen plant materials collected from different locations and twelve commercial herbal drugs determined using the TLC-image analysis method were not significantly different from those determined using the TLCdensitometric method. These results suggest that the proposed TLC-image analysis method can be used as an effective method for quantitating phyllanthin in P. amarus plants and commercial products

A Stability-Indicating Ultra Performance Liquid Chromato-Graphic (UPLC) Method for the Determination of a Mycophenolic Acid-Curcumin Conjugate and Its Applications to Chemical Kinetic Studies

A simple, precise, and accurate reversed-phase ultra-performance liquid chromatographic (UPLC) method was developed and validated for the determination of a mycophenolic acid-curcumin (MPA-CUR) conjugate in buffer solutions. Chromatographic separation was performed on a C18 column (2.1 × 50 mm id, 1.7 µm) with a gradient elution system of water and acetonitrile, each containing 0.1% formic acid, at a flow rate of 0.6 mL/min. The column temperature was controlled at 33 °C. The compounds were detected simultaneously at the maximum wavelengths of mycophenolic acid (MPA), 254 nm, and curcumin (CUR), or MPA-CUR, at 420 nm. The developed method was validated according to the ICH Q2(R1) guidelines. The linear calibration curves of the assay ranged from 0.10 to 25 μg/mL (r2 ≥ 0.995, 1/x2 weighting factor), with a limit of detection and a limit of quantitation of 0.04 and 0.10 μg/mL, respectively. The accuracy and precision of the developed method were 98.4–101.6%, with %CV < 2.53%. The main impurities from the specificity test were found to be MPA and CUR. Other validation parameters, including robustness and solution stability, were acceptable under the validation criteria. Forced degradation studies were conducted under hydrolytic (acidic and alkaline), oxidative, thermal, and photolytic stress conditions. MPA-CUR was well separated from MPA, CUR, and other unknown degradation products. The validated method was successfully applied in chemical kinetic studies of MPA-CUR in different buffer solutions

The interaction of Suk-Saiyasna remedy with GABAA and CB1 receptor-targeting drugs: Enhancing hypnotic and sedative effects in in vivo models

The Suk-Saiyasna remedy, an herbal treatment, was historically used but ceased due to its cannabis content. After a relaxation of drug control laws in Thailand, its use re-emerged. This study examines the Suk-Saiyasna remedy's impact on rodent behavior and its receptor effects. This study was conducted to assess the sedative-like effects of the remedy on mice. The mice were divided into groups receiving 0.6, 3, 30, and 60 mg/kg extracts, with negative controls for comparison. We also investigated the impact on receptors, utilizing negative controls and pretreatment with receptor blockers, followed by either a negative control or a 60 mg/kg extract. Furthermore, this study investigated the behavioral aspects of mice, including anxiolytic effects, antidepressant-like effects, and motor coordination, utilizing the elevated plus-maze, open-field, and rotarod performance tests. The Suk-Saiyasna remedy (P < 0.05) decreased significantly in the latent period and increased sleeping time in the treated groups. Moreover, the Suk-Saiyasna remedy also showed efficacy in reaction to GABAA receptors and cannabinoid CB1 receptors (P < 0.05). In addition, positive effects were observed regarding the animal behavior in the arena, as the animal activity, behavior, and muscle coordination were reduced (P < 0.05). The Suk-Saiyasna remedy may be involved in a sedative–hypnotic-like effect in rodents under normal conditions through the modulation of GABAergic neurons and induction of the cannabinoid CB1 receptor mechanism

A Stability-Indicating Assay for Tetrahydrocurcumin-Diglutaric Acid and Its Applications to Evaluate Bioaccessibility in an In Vitro Digestive Model

A simple and reliable ultra-high-performance liquid chromatographic (UHPLC) method was developed and validated for determination of tetrahydrocurcumin diglutaric acid (TDG) and applied for evaluation of its bioaccessibility. The analytical method was validated to demonstrate as a stability-indicating assay (SIA) according to the ICH Q2(R1) guidelines under various force degradation conditions including thermal degradation, moisture, acid and base hydrolysis, oxidation, and photolysis. The developed chromatographic condition could completely separate all degradants from the analyte of interest. The method linearity was verified in the range of 0.4–12 μg/mL with the coefficient of determination (r2) > 0.995. The accuracy and precision of the method provided %recovery in the range of 98.9–104.2% and %RSD lower than 4.97%, respectively. The limit of detection and quantitation were found to be 0.25 μg/mL and 0.40 μg/mL, respectively. This method has been successfully applied for the bioaccessibility assessment of TDG with the bioaccessibility of TDG approximately four fold greater than THC in simulated gastrointestinal fluid. The validated SIA method can also benefit the quality control of TDG raw materials in pharmaceutical and nutraceutical development

Discovery of Natural Lead Compound from Dendrobium sp. against SARS-CoV-2 Infection

Since the pandemic of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in December 2019, the infection cases have quickly increased by more than 511 million people. The long epidemic outbreak over 28 months has affected health and economies worldwide. An alternative medicine appears to be one choice to alleviate symptoms and reduce mortality during drug shortages. Dendrobium extract is one of the traditional medicines used for COVID-19 infection. Several compounds in Dendrobium sp. had been reported to exert pharmacological activities to treat common COVID-19-related symptoms. Herein, in silico screening of 83 compounds from Dendrobium sp. by using the SARS-CoV-2 spike protein receptor-binding domain (RBD) as a drug target was performed in searching for a new lead compound against SARS-CoV-2 infection. Four hit compounds showing good binding affinity were evaluated for antiviral infection activity. The new lead compound DB36, 5-methoxy-7-hydroxy-9,10-dihydro-1,4-phenanthrenequinone, was identified with the IC50 value of 6.87 &plusmn; 3.07 &micro;M. The binding mode revealed that DB36 bound with the spike protein at the host receptor, angiotensin-converting enzyme 2 (ACE2) binding motif, resulted in antiviral activity. This study substantiated the use of Dendrobium extract for the treatment of SARS-CoV-2 infection and has identified new potential chemical scaffolds for further drug development of SARS-CoV-2 entry inhibitors

Diversity, astaxanthin production, and genomic analysis of Rhodotorula paludigena SP9-15

Astaxanthin is a carotenoid known for its powerful antioxidant properties. This study focused on isolating yeast strains capable of producing astaxanthin from flower and fruit samples collected in Thailand. Out of 115 isolates, 11 strains were identified that produced astaxanthin. Molecular identification techniques revealed that these isolates belonged to two species: Rhodotorula paludigena (5 isolates) and Rhodosporidiobolus ruineniae (6 isolates). Whole-genome analysis of one representative strain, R. paludigena SP9-15, identified putative candidate astaxanthin synthesis-associated genes, such as CrtE, CrtYB, CrtI, CrtS, CrtR, CrtW, CrtO, and CrtZ. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) confirmed astaxanthin production. Further optimization of astaxanthin production was carried out by investigating the effects of various factors on the growth rate and astaxanthin production. The optimal conditions were 40 g/L glucose as a carbon source, pH 7.5, and cultivation at 25 °C with 200 rpm for 3 days. Under these conditions, R. paludigena SP9-15 synthesized biomass of 11.771 ± 0.003 g/L, resulting in astaxanthin with a content of 0.558 ± 0.018 mg/g DCW (dry cell weight), an astaxanthin yield of 6.565 ± 0.238 mg/L, and astaxanthin productivity of 2.188 ± 0.069 g/L/day. These findings provide insights into astaxanthin production using red yeast strains from Thailand and highlight the potential of R. paludigena SP9-15 for further application

Genomic Insight and Optimization of Astaxanthin Production from a New <i>Rhodotorula</i> sp. CP72-2

Astaxanthin is a carotenoid pigment extensively used in various industries. Rhodotorula sp. CP72-2, isolated from Calotropis gigantea, showed potential astaxanthin production. In this study, strain CP72-2 was identified as a putative new species in the genus Rhodotorula based on the 26S rRNA gene sequence (98% identity). It was first used as the microbial source for producing astaxanthin. Strain CP72-2 was screened for its astaxanthin production and was identified and quantified by High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and UV-Vis spectrophotometer. After a screening of astaxanthin production, various carbon sources, pH, temperature, and incubation period were evaluated for their effect on the astaxanthin production of strain CP72-2. Among the several experimental factors, the most efficient conditions for astaxanthin production were glucose (50 g/L), pH 4.5, 25 °C, and three days of cultivation. The assembly genome of strain CP72-2 has a total length of 21,358,924 bp and a GC content of 64.90%. The putative candidate astaxanthin biosynthesis-associated genes (i.e., CrtE, CrtYB, CrtI, CrtS, CrtR, CrtW, CrtO, and CrtZ) were found. This research presents the first report on the production and optimization of astaxanthin from strain CP72-2 and its genome analysis, focusing on the biotechnological potential of the astaxanthin producer