A Pair of Epimers of Lignan Alleviate Neuroinflammatory Effects by Modulating iNOS/COX-2 and MAPK/NF-κB Signaling Pathways.

Neuroinflammation is a causative factor in neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Previous studies have shown that Artemisia mongolica has anti-inflammatory properties. Aschantin (AM3) has been shown to have anti-inflammatory effects. However, the mechanism of AM3 and its epimer epi-aschantin (AM2) remains controversial. Therefore, the present study explored the mechanism of neuroinflammation by AM2 and AM3 and attempted to reveal the relationship between the structure of AM2 and AM3 and anti-neuroinflammatory activity. We isolated for the first time 12 lignans from A. mongolica that inhibited NO content at 10 µM in LPS-stimulated BV2 cells. Among them, epi-aschantin (AM2) and Aschantin (AM3) showed significant inhibition in NO screening. With further studies, we found that both AM2 and AM3 effectively inhibited the overproduction of NO, PGE2, IL-6, TNF-α and MCP-1, as well as the overexpression of COX-2 and iNOS. Mechanistic studies have shown AM2 and AM3 significantly inhibited the phosphorylation of ERK, JNK and P-38 in the MAPK signaling pathway and p-IκBα,p-p65 and blocked p65 entry into the nucleus. The results suggested that the pair of epimers (AM2 and AM3) can be used as potential therapeutic agents in the treatment of various brain disorders and that structural differences do not differ in anti-neuroinflammatory effects.

HPLC with chiral stationary phase for separation and kinetics study of aspartic acid epimerization in Peroxiredoxin 2 active site peptide.

Amino acid epimerization, a process of converting L-amino acids to D-amino acids, will lead to modification in the protein structure and, subsequently, its biological function. This modification causes no change in protein m/z and may be overlooked during protein analysis. Aspartic Acid Epimerization (AAE) is faster than other amino acids and could be accelerated by free radicals and peroxides. In this work, a novel and site-specific HPLC method using a chiral stationary phase for determining the AAE in the active site model peptide (AP) of Peroxiredoxin 2 has been developed and validated. The developed method showed good linearity (1 - 200 µg/mL) and recoveries of the limit of quantification (LOQ), low, medium, and high concentrations were between 85% and 115%. The Kinetics of AAE in AP were studied using the developed method, and the results showed that when ascorbic acid and Cu2+ coexisted, the AP epimerized rapidly. The AAE extent increased with time and was positively correlated with hydrogen peroxide generation.

Unraveling the complex interplay between obesity and vitamin D metabolism.

Vitamin D deficiency and obesity are a worldwide health issue. Obesity refers to the accumulation of excessive fats in the body which could lead to the development of diseases. Obese people have low vitamin D levels for several reasons including larger volume of distribution, vitamin D tightly bound in fatty tissues, reduced absorption, and diets with low vitamin D. Accurately measuring vitamin D metabolites is challenging. The Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method was developed and validated for the analysis of vitamin D metabolites in the serum. Blood samples were collected from 452 subjects which consisted of baseline (vitamin D deficient obese subjects), follow-up (supplemented obese subjects), and healthy volunteers. The vitamin D metabolites were separated adequately by the developed UHPLC-MS/MS method. Moreover, the validation criteria for the method were within an acceptable range. The baseline, follow-up and even healthy volunteers were deficient in 25OHD3 and 25OHD2. The baseline and healthy subjects had comparable concentration of vitamin D2 and D3. However, healthy subjects had a higher concentration of 25OHD and its epimer compared to the baseline subjects. The vitamin D3 was increased significantly in the follow- up subjects; therefore, the 25OHD3 was increased significantly compared to the baseline as well; however, the increase was insufficient to achieve the optimal range. The UHPLC-MS/MS method test was applied successfully on estimation of vitamin D metabolites in subjects. This study indicates the significance of taking into account the metabolic and storage effects when evaluating the vitamin D status in obese subjects.

Disposition of Hexahydrocannabinol Epimers and Their Metabolites in Biological Matrices following a Single Administration of Smoked Hexahydrocannabinol: A Preliminary Study.

In 2023, hexahydrocannabinol (HHC) attracted the attention of international agencies due to its rapid spread in the illegal market. Although it was discovered in 1940, less is known about the pharmacology of its two naturally occurring epimers, 9(R)-HHC and 9(S)-HHC. Thus, we aimed to investigate the disposition of hexahydrocannabinol epimers and their metabolites in whole blood, urine and oral fluid following a single controlled administration of a 50:50 mixture of 9(R)-HHC and 9(S)-HHC smoked with tobacco. To this end, six non-user volunteers smoked 25 mg of the HHC mixture in 500 mg of tobacco. Blood and oral fluid were sampled at different time points up to 3 h after the intake, while urine was collected between 0 and 2 h and between 2 and 6 h. The samples were analyzed with a validated HPLC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC and eight metabolites. 9(R)-HHC showed the highest Cmax and AUC0-3h in all the investigated matrices, with an average concentration 3-fold higher than that of 9(S)-HHC. In oral fluid, no metabolites were detected, while they were observed as glucuronides in urine and blood, but with different profiles. Indeed, 11nor-9(R)-HHC was the most abundant metabolite in blood, while 8(R)OH-9(R) HHC was the most prevalent in urine. Interestingly, 11nor 9(S) COOH HHC was detected only in blood, whereas 8(S)OH-9(S) HHC was detected only in urine.

Dietary 25 Hydroxyvitamin D3 Improved Serum Concentration Level and Alkaline Phosphatase Activity during Lactation but Had Meager Impact on Post-Farrowing Reproductive Performance in Sows.

Dietary 25 hydroxyvitamin D3 (25(OH)D3) promotes serum 25(OH)D3 concentration and alkaline phosphatase activity (ALP); however, post-farrowing reproductive performance of lactating sows fed with 14-epimer of 25(OH)D3 is uncertain. This study investigated post-farrowing reproductive performance, serum ALP activity, and serum 25(OH)D3 concentration in sows fed VD3, 25(OH)D3, or 14-epi 25(OH)D3. Weaned sows (n = 203) in parities 2 and 3 were blocked weekly and treated with 2000 IU/kg VD3 (T1), 25 µg/kg 25(OH)D3:14-epi 25(OH)D3 (T2), or 50 µg/kg 25(OH)D3 (T3) diets, all equilibrated to 2000 IU/kg as fed. Sow performance, treatment, and sampling period effects were analyzed. Environmental conditions were analyzed as covariates. The number of piglets weaned (p = 0.029), pre-weaning mortality (p = 0.029), sampling period (p < 0.001), and treatment and period interaction (p = 0.028) differed significantly. There was an increase in 25(OH)D3 during lactation due to physiological demands for milk calcium and milk production. Supplementing twice the concentration of 25(OH)D3 compared to its epimer, 25(OH)D3:14-epi 25(OH)D3, had no significant effect on the post-farrowing reproductive performance of lactating sows. The effect of 25(OH)D3 on post-farrowing reproductive performance and ALP activity in sows was influenced by metabolic demand for calcium due to physiological changes during lactation as well as epimer conformation.

Accurate Clinical Detection of Vitamin D by Mass Spectrometry: A Review.

Vitamin D deficiency is thought to be associated with a wide range of diseases, including diabetes, cancer, depression, neurodegenerative diseases, and cardiovascular and cerebrovascular diseases. This vitamin D deficiency is a global epidemic affecting both developing and developed countries and therefore qualitative and quantitative analysis of vitamin D in a clinical context is essential. Mass spectrometry has played an increasingly important role in the clinical analysis of vitamin D because of its accuracy, sensitivity, specificity, and the ability to detect multiple substances at the same time. Despite their many advantages, mass spectrometry-based methods are not without analytical challenges. Front-end and back-end challenges such as protein precipitation, analyte extraction, derivatization, mass spectrometer functionality, must be carefully considered to provide accurate and robust analysis of vitamin D through a well-designed approach with continuous control by internal and external quality control. Therefore, the aim of this review is to provide a comprehensive overview of the development of mass spectrometry methods for vitamin D accurate analysis, including emphasis on status markers, deleterious effects of biological matrices, derivatization reactions, effects of ionization sources, contribution of epimers, standardization of assays between laboratories.

The C-8-S-isomers of ergot alkaloids - a review of biological and analytical aspects.

Ergot alkaloids are secondary metabolites that are produced by fungi and contaminate cereal crops and grasses. The ergot alkaloids produced by Claviceps purpurea are the most abundant worldwide. The metabolites exist in two configurations, the C-8-R-isomer (R-epimer) and the C-8-S-isomer (S-epimer). These two configurations can interconvert to one another. Ergot alkaloids cause toxic effects after consumption of ergot-contaminated food and feed at various concentrations. For bioactivity reasons, the C-8-R-isomers have been studied to a greater extent than the C-8-S-isomer since the C-8-S-isomers were considered biologically inactive. However, recent studies suggest the contrary. Analytical assessment of ergot alkaloids now includes the C-8-S-isomers and high concentrations of specific C-8-S-isomers have been identified. The inclusion of the C-8-S-isomer in regulatory standards is reviewed. This review has identified that further research into the C-8-S-isomers of ergot alkaloids is warranted. In addition, the inclusion of the C-8-S-isomers into regulatory recommendations worldwide for food and feed should be implemented. The objectives of this review are to provide an overview of historic and current studies that have assessed the C-8-S-isomers. Specifically, this review will compare the C-8-R-isomers to the C-8-S-isomers with an emphasis on the biological activity and analytical assessment.

Variable fragmentation and ionization of amyloid-beta epimers and isomers.

While the existence of D-amino acids in peptides and proteins has recently been accepted in higher forms of life, their roles and importance are yet to be understood. The lack of analytical methods present for such epimeric and/or isomeric analyses often limits developments in the field. Studies have shown the elevated presence of epimeric and isomeric modifications to amyloid-beta (Aß) peptides extracted from Alzheimer's disease patients. These modifications most frequently occur through aspartic acid and serine residues. Because such peptides are indistinguishable by mass alone, selective liquid chromatography tandem mass spectrometry analysis is required to differentiate such peptides. Herein, we examine MS/MS of tryptic fragments of Aß peptides containing D-Asp, L-iso-Asp, D-iso-Asp, and/or D-Ser modifications. Peptide ionizability and fragmentation are explored through selected reaction monitoring, selected ion monitoring, and product ion scan. The results show the variability of ionization and fragmentation for many "identical mass peptides" and how these differences can affect the analysis of isomeric and epimeric peptides.

Insight into the photolytic degradation products of Rosuvastatin: Full chiral and structural elucidation and conversion kinetics by a combined chromatographic, spectroscopic and theoretical approach.

Rosuvastatin (RSV) is a well-established lipid-lowering drug. RSV is susceptible to degradation under various stress conditions and forms two cyclic derivatives by a radical-mediated photolytic mechanism. On a structural basis, these epimeric compounds (reported as FP-B in the European Pharmacopeia monograph Rosuvastatin tablets) retain the configuration of the stereogenic carbons of RSV (3R,5S) and have opposite absolute configurations at the third stereogenic center. Herein, we report the kinetics of formation and the complete structural characterization, including the assignment of the absolute configuration, of each epimer collected after HPLC separation on a chiral stationary phase. The stereochemistry of the epimers was determined by comparison of the experimental circular dichroism data with the corresponding theoretical values. Kinetic studies revealed that RSV degrades completely to FP-B within 3 h at room temperature. Furthermore, through a multi-disciplinary approach involving chromatography (HPLC and UHPLC), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), it was demonstrated that FP-B in turn degrades to the lactones under the mild acidic conditions of the chromatographic mobile phase. The ability of RSV to form multiple degradation products may affect the quantification of RSV-related substances and draw attention to potentially toxic RSV-like species in the environment.

The microbial-derived bile acid lithocholate and its epimers inhibit Clostridioides difficile growth and pathogenicity while sparing members of the gut microbiota.

Clostridioides difficile is a Gram-positive, spore-forming anaerobe that causes clinical diseases ranging from diarrhea and pseudomembranous colitis to toxic megacolon and death. C. difficile infection (CDI) is associated with antibiotic usage, which disrupts the indigenous gut microbiota and causes the loss of microbial-derived secondary bile acids that normally provide protection against C. difficile colonization. Previous work has shown that the secondary bile acid lithocholate (LCA) and its epimer isolithocholate (iLCA) have potent inhibitory activity against clinically relevant C. difficile strains. To further characterize the mechanisms by which LCA and its epimers iLCA and isoallolithocholate (iaLCA) inhibit C. difficile, we tested their minimum inhibitory concentration against C. difficile R20291 and a commensal gut microbiota panel. We also performed a series of experiments to determine the mechanism of action by which LCA and its epimers inhibit C. difficile through bacterial killing and effects on toxin expression and activity. Additionally, we tested the cytotoxicity of these bile acids through Caco-2 cell apoptosis and viability assays to gauge their effects on the host. Here, we show that the epimers iLCA and iaLCA strongly inhibit C. difficile growth in vitro while sparing most commensal Gram-negative gut microbes. We also show that iLCA and iaLCA have bactericidal activity against C. difficile, and these epimers cause significant bacterial membrane damage at subinhibitory concentrations. Finally, we observe that iLCA and iaLCA decrease the expression of the large cytotoxin tcdA, while LCA significantly reduces toxin activity. Although iLCA and iaLCA are both epimers of LCA, they have distinct mechanisms for inhibiting C. difficile. LCA epimers, iLCA and iaLCA, represent promising compounds that target C. difficile with minimal effects on members of the gut microbiota that are important for colonization resistance. IMPORTANCE In the search for a novel therapeutic that targets Clostridioides difficile, bile acids have become a viable solution. Epimers of bile acids are particularly attractive as they may provide protection against C. difficile while leaving the indigenous gut microbiota largely unaltered. This study shows that LCA epimers isolithocholate (iLCA) and LCA epimers isoallolithocholate (iaLCA) specifically are potent inhibitors of C. difficile, affecting key virulence factors including growth, toxin expression, and activity. As we move toward the use of bile acids as therapeutics, further work will be required to determine how best to deliver these bile acids to a target site within the host intestinal tract.

Isomeric eremophilane lactones from the whole plant of Parasenecio albus and their cytotoxic and immunosuppressive activities.

Ten previously unreported eremophilane lactones (parasalbolides A-J), including three pairs of C-10 epimers (parasalbolides A and G, B and H, and F and I, respectively), were isolated and identified from the whole plant of Parasenecio albus. Their structures were established on the basis of the HRESIMS and NMR spectroscopic analyses, combined with the comparison of the ECD spectra. The absolute configuration of parasalbolide A was confirmed by single-crystal X-ray diffraction using Cu Kα radiation. Parasalbolides A-J represent the first examples of 1,2,10-trioxygenated eremophila-7(11),8-dien-12,8-olides. The cytotoxic and immunosuppressive activities of selected isolates were evaluated and the (10S)-eremophilane lactones (parasalbolides A, B, and F) showed more potent activities than the (10R)-ones (parasalbolides G, H, and I).

Amygdalin epimers exert discrepant anti-pulmonary fibrosis activity via inhibiting TGF-ß1/Smad2/3 pathway.

Idiopathic pulmonary fibrosis (IPF) represents a chronic and progressive tissue repair response that leads to irreversible scarring and lung remodeling. The decoction of bitter almond usually contains amygdalin epimers in traditional clinical application for lung disease. To reveal the differences of cytotoxicity and antifibrotic effect between amygdalin epimers, and potential mechanism is also explored. The cytotoxicity of amygdalin epimers were evaluated with MRC-5 cells in vitro. Their antifibrotic activities were evaluated in bleomycin-induced C57BL/6 mice and TGF-ß1-induced MRC-5 cells. Here we demonstrated that L-amygdalin is more toxic of the amygdalin epimers in MRC-5 cells, and D-amygdalin is more effective in anti-pulmonary fibrosis among the amygdalin epimers in bleomycin-induced C57BL/6 mice. Herein, it was observed that D-amygdalin had a stronger inhibitory effect on inflammation than L-amygdalin, and had similar results in inhibiting the mRNA and protein expression levels of fibrosis-related biomarkers. The mechanism of anti-pulmonary fibrosis showed that amygdalin epimers suppressing expression of phosphorylation of Smads2/3, which implying deactivation of the TGF-ß1induced Smads2/3 signal pathway. This study evaluates the amygdalin epimers cytotoxicity and antifibrotic effect, and its mechanisms were related to the TGF-ß1/Smads2/3 signal pathway. It provides a reference for clinical safety and effectiveness of amygdalin epimers.

Comparing derivatization reagents for quantitative LC-MS/MS analysis of a variety of vitamin D metabolites.

The present study systematically compares the sensitivity and selectivity of the analysis of multiple vitamin D metabolites after chemical derivatization using different reagents for liquid chromatography-tandem mass spectrometry (LC-MS/MS). Generally, chemical derivatization is applied to vitamin D metabolites to increase the ionization efficiency, which is particularly important for very low abundant metabolites. Derivatization can also improve the selectivity of the LC separation. A wide variety of derivatization reagents has been reported in recent years, but information on their relative performance and applicability to different vitamin D metabolites is, unfortunately, not available in the literature. To fill this gap, we investigated vitamin D3, 3ß-25-hydroxyvitamin D3 (3ß-25(OH)D3), 3α-25-hydroxyvitamin D3 (3α-25(OH)D3), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and compared response factors and selectivity after derivatizing with several important reagents, including four dienophile reagents (4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD), Amplifex, 2-nitrosopyridine (PyrNO)) as well as two reagents targeting hydroxyl groups: isonicotinoyl chloride (INC) and 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). In addition, a combination of dienophiles and hydroxyl group reagents was examined. For LC separations, reversed-phase C-18 and mixed-mode pentafluorophenyl HPLC columns using different compositions of the mobile phase were compared. With respect to detection sensitivity, the optimum derivatization reagent for the profiling of multiple metabolites was Amplifex. Nevertheless, FMP-TS, INC, PTAD, or PTAD combined with an acetylation reaction showed very good performance for selected metabolites. These reagent combinations provided signal enhancements on the order of 3- to 295-fold depending on the compound. Chromatographic separation of the dihydroxylated vitamin D3 species was readily achieved using any of the derivatization reactions, while for 25(OH)D3 epimers, only PyrNO, FMP, INC, and PTAD combined with acetylation enabled complete separation. In conclusion, we believe this study can serve as a useful reference for vitamin D laboratories, to help analytical and clinical scientists decide which derivatization reagent to choose for their application.

Accurate determination of four tetracycline residues in chicken meat by isotope dilution-liquid chromatography/tandem mass spectrometry.

An analytical method based on isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC‒MS/MS) was developed to accurately determine four representative tetracyclines (tetracycline, chlortetracycline, doxycycline, and oxytetracycline) in chicken meat. Tetracyclines are known to have a great tendency for epimerization and keto-enol tautomerism, which often provoke major challenges in their determination. Since this isomerization was found to be unavoidable during the whole chain of the current analysis, the total content (µg kg‒1) of individual tetracycline was quantified as a sum of each parent compound and its respective isomeric forms. Using this approach in combination with IDMS analysis, more consistent, accurate, and reproducible measurement results for the four tetracyclines in chicken meat were acquired. LC-MS/MS conditions and sample preparation processes were comprehensively optimized to minimize the chelating effect of tetracyclines and possible co-extracted interferences. Details of the sample preparation scheme, LC‒MS/MS detection, calculation equation, and method validation are described in this article. The method provided very good accuracy (97.7-102.6%) for all analytes across the concentration range of 10-200 µg kg‒1, with relative standard deviations for intra-day and inter-day precision of less than 4%. The limits of quantification were below 0.2 µg kg‒1, demonstrating the high sensitivity of the method. Furthermore, the measurement uncertainty was generally below 5.5%. Hence, the established method exhibits high-order metrological quality with superior performance over various existing methodologies. Moreover, this method can provide references for general food testing laboratories close to and far below the established maximum residue limits (100 µg kg‒1) for animal muscle tissues.

Analysis of vitamin D metabolic markers by mass spectrometry: Recent progress regarding the "gold standard" method and integration into clinical practice.

Liquid chromatography/tandem mass spectrometry is firmly established today as the gold standard technique for analysis of vitamin D, both for vitamin D status assessments as well as for measuring complex and intricate vitamin D metabolic fingerprints. While the actual mass spectrometry technology has seen only incremental performance increases in recent years, there have been major, very impactful changes in the front- and back-end of MS-based vitamin D assays; for example, the extension to new types of biological sample matrices analyzed for an increasing number of different vitamin D metabolites, novel sample preparation techniques, new powerful chemical derivatization reagents, as well the continued integration of high resolution mass spectrometers into clinical laboratories, replacing established triple-quadrupole instruments. At the same time, the sustainability of mass spectrometry operation in the vitamin D field is now firmly established through proven analytical harmonization and standardization programs. The present review summarizes the most important of these recent developments.

COX-2 and iNOS inhibitory epimeric meroterpenoids from Ganoderma cochlear and structure revision of cochlearol Q.

Four novel epimeric meroterpenoids, ganadone A (1), 3',10'-di-epi-ganadone A (2), 10'-epi-ganadone A (3), and 3'-epi-ganadone A (4) as well as another pairs of epimers, ganadone B (5) and 10'-epi-ganadone B (6), with a same basic skeleton compound ganadone C (7), together with two lactonized meroterpenoids, ganadones D and E (8 and 9) were isolated from the fruiting bodies of Ganoderma cochlear. Compounds 1-7 were constructed with fascinating adjacent 6',7'-bifuran ring system. Fortunately, we have revised our previously reported structure cochlearol Q, which was proposed pyrano[6',7'-b]pyran ring system into 6',7'-bifuran motif. All the isolates were characterized by analysis of HRESIMS, NMR spectroscopy and 1 was supported by X-ray crystallography analysis. The absolute stereochemistry of 1-9 were assigned by quantum chemical calculations. Biological evaluation of 1-9 showed that 5, 6, and 9 have significant anti-inflammatory potentials.

Jatrolignans C and D: New Neolignan Epimers from Jatropha curcas.

Two new neolignans jatrolignans, C (1) and D (2), a pair of epimers, were isolated from the whole plants of Jatropha curcas L. (Euphorbiaceae). Their structures were determined with HRESIMS, IR, and NMR data analysis, and electronic circular dichroism (ECD) experiments via a comparison of the experimental and the calculated ECD spectra. Their antichlamydial activity was evaluated in Chlamydia abortus. They both showed dose-dependent antichlamydial effects. Significant growth inhibitory effects were observed at a minimum concentration of 40 µM.

A pair of novel phenylethanol glycosides from Cistanche tubulosa (Schenk) Wight.

A pair of differential epimers with opposite C-7 configurations, crenatosides A and B (1 and 2), and 10 known phenylethanoid glycosides (PhGs) (3-12) were obtained from the succulent stem of Cistanche tubulosa. The structures were elucidated based on extensive spectral data (UV, IR, 1D and 2D NMR, HR-ESIMS), which are first reported natural products with unique glycoside structures. After acid hydrolysis, the configuration of the sugar was determined by comparing it with the normative sugar by HPLC. The absolute configurations of both compounds were determined by ECD spectrum analysis. All the obtained compounds were examined for their inhibitory effect on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse microglial cells (BV-2 cells), and compounds 1 and 2 showed potent inhibition on NO production with IC50 values of 5.62 µM and 6.30 µM, respectively.

Continuous flow process for preparing budesonide.

Budesonide, a glucocorticosteroid, is used as anti-asthmatic drug that became generic in 2019. Existing preparation methods of budesonide require utilization of corrosive acids and involve expensive purification process. Thus, a new cost-effective continuous flow process for the synthesis of budesonide which belongs to the class of 16,17 acetals of pregnane core, is discussed in the present research findings. Flow reactor parameters such as flow rate, temperature, residence time, solution volumes, anti-solvents and reactor frequency are subjected to investigation on the preparation of molar ratio of budesonide epimers. Further, the suitable parameters entail for obtaining the desired molar ratio of epimers. In another aspect, particle size optimization studies are also performed to get the desired budesonide solid product. A continuous flow process for preparation of budesonide is identified from the present research investigation which can be readily transferred to industrial scale up.

Identification and Quantification by NMR Spectroscopy of the 22R and 22S Epimers in Budesonide Pharmaceutical Forms.

The authors developed four variants of the qNMR technique (1H or 13C nucleus, DMSO-d6 or CDCl3 solvent) for identification and quantification by NMR of 22R and 22S epimers in budesonide active pharmaceutical ingredient and budesonide drugs (sprays, capsules, tablets). The choice of the qNMR technique version depends on the drug excipients. The correlation of 1H and 13C spectra signals to molecules of different budesonide epimers was carried out on the basis of a comprehensive analysis of experimental spectral NMR data (1H-1H gCOSY, 1H-13C gHSQC, 1H-13C gHMBC, 1H-1H ROESY). This technique makes it possible to identify budesonide epimers and determine their weight ratio directly, without constructing a calibration curve and using any standards. The results of measuring the 22S epimer content by qNMR are comparable with the results of measurements using the reference HPLC method.