Quantitative Nuclear Magnetic Resonance Spectroscopy Research Articles

Quantitative estimation and validation of Alectinib hydrochloride drug substance using quantitative nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectroscopy has been recently used for quantitative estimation of drug substance which has numerous merits in pharmaceutical applications when compared to traditional methods of chromatography. Present study focuses on method development and validation of quantitative proton nuclear magnetic spectroscopy method for estimation of alectinib hydrochloride drug substance. This method involves use of internal standard in presence of alectinib hydrochloride. For quantitation, 1H NMR signals at 8.4 ppm and 6.7 ppm corresponding to analyte proton of alectinib and ethyl 4-(dimethyl amino) benzoate internal standard respectively were used. Moreover, this method was validated as per ICH guidelines for accuracy, precision, linearity, limit of detection, limit of quantitation, range and robustness. Assay estimation by Q-NMR is facile and time-efficient which does not require reference standard of analyte and yet accurate and precise alternative as compared to estimation by other chromatographic techniques.

Quantitative Blood Serum IVDr NMR Spectroscopy in Clinical Metabolomics of Cancer, Neurodegeneration, and Internal Medicine.

Despite more than two decades of metabolomics having joined the "omics" scenery, to date only a few novel blood metabolite biomarkers have found their way into the clinic. This is changing now by massive large-scale population metabolic phenotyping for both healthy and disease cohorts. Here, nuclear magnetic resonance (NMR) spectroscopy is a method of choice, as typical blood serum markers can be easily quantified and by knowledge of precise reference concentrations, more and more NMR-amenable biomarkers are established, moving NMR from research to clinical application. Besides customized approaches, to date two major commercial platforms have evolved based on either 600MHz (14.1 Tesla) or 500MHz (11.7 Tesla) high-field NMR systems. This chapter provides an introduction into the field of quantitative in vitro diagnostics research (IVDr) NMR at 600 MHzand its application within clinical research of cancer, neurodegeneration, and internal medicine.

Unlocking the potential of NMR spectroscopy for precise and efficient quantification of microplastics

Precise, fast, and reliable identification and quantification of microplastic contamination are essential for determining their environmental concentrations for risk assessments. This study investigates the use of nuclear magnetic resonance (NMR) spectroscopy to quantify microplastics by analysing dilution series of polystyrene (PS), polyisoprene-cis (PI), polybutadiene-cis (PB), polylactic acid (PLA), polyvinyl chloride (PVC) and polyurethane (PU). Each polymer type was dissolved in a suitable solvent and an internal standard was utilized for quantification. Detection and quantification limits for each polymer type were established in two ways: (1) by using an equation based on proton signals and an internal standard with known concentration and (2) by using the LOQ based on the signal-to-noise ratio. Both data sets were compared and showed that using the internal standard (method 1) results in more accurate and lower concentration limits in the range of 0.2–8 µg mL−1 for all six polymer types, while the LOQ based on the SNR (method 2) gives consistently higher concentration limits (1–10 µg mL−1). The research shows the accuracy, efficacy, and reliability of quantitative NMR spectroscopy for polymer analysis in these concentration ranges compared to established quantifying methods, such as, PyGC/MS, FTIR, or Raman spectroscopy.

Analysis of rebaudioside A in probiotic-fermented milk via quantitative nuclear magnetic resonance spectroscopy

Rebaudioside A is used in the food and beverage industries owing to its sweetness, even in extremely small amounts, as well as its natural origin. Liquid chromatography is commonly used to quantify rebaudioside A; however, it suffers from complicated sample pretreatment and time consumption. External-calibration quantitative nuclear magnetic resonance (qNMR) analysis of probiotic-fermented milk has thus far not been reported. Moreover, it involves more factors contributing to errors than internal standard approaches used in the official methods, such as those recommended by the International Organization for Standardization. In this study, we developed a method for quantifying rebaudioside A in probiotic-fermented milk using internal-calibration qNMR. This method entails a simple pretreatment step with dilution and solid-phase extraction, enabling rebaudioside A determination in five types of beverages including probiotic-fermented milk. The results revealed a recovery rate and relative standard deviation of 85.4–104 % and 1.57–4.09 %, respectively, satisfying the international guidelines. The working range of qNMR was 0.80–26.7 mg/100 g of product, and the analysis time was 10–20 min. Therefore, the proposed internal-calibration qNMR method facilitates easy, rapid, and accurate quantification of rebaudioside A in probiotic-fermented milk and is applicable for analyzing various other beverages and foods.

Quantitative analysis of selected alkaloids of Mitragyna speciosa using 1H quantitative nuclear magnetic resonance spectroscopy.

Mitragyna speciosa is a perennial plant native to Asia, well known for its psychoactive properties. Its major alkaloid mitragynine is known to have sedative and euphoric effects. Hence, the plant has been a subject of abuse, leading to addiction, necessitating efficient analytical methods to detect its psychoactive constituents. However, current chromatography-based methods for detecting the alkaloids are time consuming and costly. Quantitative nuclear magnetic resonance (qNMR) spectroscopy emerges as a promising alternative due to its nondestructive nature, structural insights, and short analysis time. Hence, a rapid and precise qNMR method was developed to quantify selected major psychoactive alkaloids in various parts of M.speciosa. Mitragynine, specioliatine, and speciogynine were quantified in relation to the integral value of the -OCH3 groups of the alkaloids and the internal standard 1,4-dinitrobenzene. The precision and reproducibility of the method gave a relative standard deviation (RSD) of 2%, demonstrating the reliability of the method. In addition, the method showed excellent specificity, sensitivity, high linearity range (R2 = 0.999), and limits of detection (LOD) and quantification (LOQ) values. The analysis revealed that the red-veined M.speciosa leaves contained higher levels of mitragynine (32.34 mg/g), specioliatine (16.84 mg/g) and speciogynine (7.69 mg/g) compared to the green-veined leaves, stem bark, or fruits.

Trace element detection in anhydrous minerals by micro-scale quantitative nuclear magnetic resonance spectroscopy

Nominally anhydrous minerals (NAMs) composing Earth’s and planetary rocks incorporate microscopic amounts of volatiles. However, volatile distribution in NAMs and their effect on physical properties of rocks remain controversial. Thus, constraining trace volatile concentrations in NAMs is tantamount to our understanding of the evolution of rocky planets and planetesimals. Here, we present an approach of trace-element quantification using micro-scale Nuclear Magnetic Resonance (NMR) spectroscopy. This approach employs the principle of enhanced mass-sensitivity in NMR microcoils. We were able to demonstrate that this method is in excellent agreement with standard methods across their respective detection capabilities. We show that by simultaneous detection of internal reference nuclei, the quantification sensitivity can be substantially increased, leading to quantifiable trace volatile element amounts of about 50 ng/g measured in a micro-meter sized single anorthitic mineral grain, greatly enhancing detection capabilities of volatiles in geologically important systems.

Characterisation of Tenebrio molitor Reared on Substrates Supplemented with Chestnut Shell.

Tenebrio molitor larvae represent a sustainable protein source for food and feed. The aim of this study was to evaluate the supplementation of chestnut shell, a by-product of the agro-industrial chain, in growth substrates for T. molitor larvae rearing. Seven-week-old larvae were reared on three different growth substrates: the control group (CTRL) was fed wheat bran, treatment group one was fed wheat bran supplemented with 12.5% w/w chestnut shell (TRT1), and treatment group two was fed wheat bran supplemented with 25% w/w chestnut shell (TRT2). Larval weight, substrate consumption, and mortality were recorded weekly. After 14 days, insect meals were produced for bromatological and colorimetric analysis, and bacterial inhibition activity assay using a microdilution method. The amino acid profile of insects was determined using quantitative nuclear magnetic resonance spectroscopy. Our results showed a lower feed conversion ratio and higher larval survival rate % in TRT2 compared to CTRL (p < 0.05). Proteins and lipids of TRT2 were higher than other groups (p < 0.05). Important differences were observed in the amino acid profile of TRT1 and TRT2 compared to CTRL (p < 0.05). TRT1 and TRT2 showed higher E. coli inhibitory activity than CTRL (p < 0.05). In conclusion, chestnut shell supplementation improved the survival and functional characteristics of larvae and likely impacted the insects' metabolism.

Quality Control of L-Ascorbic Acid 2-Phosphate Magnesium Using Reversed-Phase Liquid Chromatography.

L-Ascorbic acid 2-phosphate magnesium (AP-Mg) salt is a Vitamin C derivative frequently used as a raw material in cell culture media for research purposes as well as for Good Manufacturing Practice (GMP)-manufacturing of cell and tissue advanced therapy medicinal products. A selective reversed-phase HPLC (RP-LC) method was developed and validated. Commercially available AP-Mg products from different suppliers were analyzed. Various new impurities were found using this newly developed RP-LC method. Using quantitative nuclear magnetic resonance spectroscopy, a mass balance of roughly 99.9% was obtained; the total numbers of impurities detected in both methods are also identical. The values of the relative ultraviolet (UV) response factors at λ = 210nm of the impurities in this RP-LC method were discussed. When equaling the overall mean relative response factor of the impurities to 0.6 (estimated central value), the mass balance in the RP-LC method was nearly 100%. The structures of the new impurities are proposed as ethylation derivatives of open-ring AP-Mg products as well as phosphorylated derivatives of ascorbic acid.

Purity Assessment of Tripropyl Phosphate through Mass Balance and 1H and 31P Quantitative Nuclear Magnetic Resonance.

Tripropyl phosphate (TnPP) is a commonly used organic phosphate flame retardant in the textiles, plastics, and coating industries. Residues are commonly detected in samples from the environment and food. The availability of certified reference materials (CRMs) is essential to ensure the accuracy and traceability of detection results. In this study, a comprehensive characterization of a CRM for TnPP was carried out, and its purity was evaluated using two distinct methodologies: mass balance (MB) and quantitative nuclear magnetic resonance spectroscopy (qNMR). In the MB method, the levels of structurally related organic impurities are 1.37 mg/g. The water content was determined to be 3.16 mg/g, while inorganic impurities were found to be 0.87 mg/g, and no residual organic solvents were detected. Benzoic acid and monocrotophos were chosen as internal standards for 1H-qNMR and 31P-qNMR, respectively. The purity of the TnPP CRM was assessed as 994.6 mg/g, 994.1 mg/g, and 993.5 mg/g using MB, 1H-qNMR, and 31P-qNMR techniques, respectively. The verified purity of the TnPP CRM was ultimately determined to be 994.1 mg/g, with an expanded uncertainty of 3.4 mg/g (k = 2), ensuring traceability to the International System of Units (SI). This CRM can be effectively utilized for preparing calibration solutions suitable for the routine monitoring of TnPP residues in plastics and food samples.

The co-promoting mechanism of EMEA/DEEA/PZ ternary water-lean solvent on CO2 capture- a study based on 13C-qNMR and DFT calculations

Amine based CO2 absorption is currently the most promising means of capturing CO2 in industry, and the water-lean amine solvent has received widespread attentions. The reaction mechanism of the EMEA/DEEA/PZ ternary water-lean amine solvent (WLS) during the process of CO2 capture was investigated theoretically to understand the synergistic effect between the different amine components. The concentrations of intermediate products were analyzed by 13C-qNMR (quantitative Nuclear Magnetic Resonance) spectroscopy, while the reaction mechanisms were calculated using gaussian 16. The NMR results showed that carbamate (EMEACOO-), HCO3–/CO32– were the main products in the carbon capture process of EMEA WLS. The protonated amine concentration varies according to the change of carbon dioxide concentration. The balance between deprotonation (forming carbamates) and CO2 removal (regenerating EMEA) depends largely on how ampholytes interact with nearby water molecules. The DFT result showed that EMEA is the key component of CO2 capture, while DEEA and PZ primarily assist in transferring protons, catalyzing the conversion of H2CO3 into HCO3– to accelerate the process of CO2 capture. Furthermore, they assist in the redaction of EMEAH+ to EMEA, allowing it continue to participate in capture reactions. DEEA, PZ and water could promote activating the protonated EMEA and enhanced its capacity of CO2 capture. Furthermore, the desorption process confirmed the proton transfer from DEEAH+, ultimately leading to the regeneration of EMEA, thereby demonstrating the effective performance of regeneration.

Scent Detection Threshold of Trained Dogs to Eucalyptus Hydrolat.

Dogs' (Canis lupus familiaris) sense of smell is based on a unique anatomy and physiology that enables them to find and differentiate low concentrations of odor molecules. This ability is exploited when dogs are trained as search, rescue, or medical detection dogs. We performed a three-part study to explore the scent detection threshold of 15 dogs to an in-house-made Eucalyptus hydrolat. Here, decreasing concentrations of the hydrolat were tested using a three-alternative forced-choice method until the first incorrect response, which defined the limit of scent detection for each tested dog. Quantitative proton nuclear magnetic resonance spectroscopy was used to identify and measure the contents of ten commercial Eucalyptus hydrolats, which are used in a dog scent training sport called "nose work". In this study, the dogs' limit of detection initially ranged from 1:104 to 1:1023 but narrowed down to 1:1017-1:1021 after a training period. The results show that, with training, dogs learn to discriminate decreasing concentrations of a target scent, and that dogs can discriminate Eucalyptus hydrolat at very low concentrations. We also detected different concentrations of eucalyptol and lower alcohols in the hydrolat products and highlight the importance of using an identical source of a scent in training a dog for participation in canine scent sport competitions and in olfactory research.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine-10,11-epoxide in human serum and plasma.

A reference measurement procedure (RMP) using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated with the aim of accurately measuring carbamazepine-10,11-epoxide concentrations in human serum and plasma. To establish traceability to SI units, the absolute content of the reference material was determined using quantitative nuclear magnetic resonance (qNMR) spectroscopy. As sample preparation a protein precipitation protocol followed by a high dilution step was established. Chromatographic separation from carbamazepine and potential metabolites was achieved using a C18 stationary phase. Selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement were evaluated based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. The RMP demonstrated very good selectivity and specificity, showing no evidence of a matrix effect. This enabled accurate quantification of carbamazepine-epoxide in the concentration range of 0.0400-12.0 μg/mL. The intermediate precision was found to be less than 2.1 %, and the repeatability coefficient of variation (CV) ranged from 1.2 to 1.8 % across all concentration levels. Regarding accuracy, the relative mean bias varied from 1.4 to 2.5 % for native serum levels and from 1.4 to 3.5 % for Li-heparin plasma levels. The measurement uncertainty for single measurements ranged from 1.6 to 2.1 %. In this study, we introduce a new LC-MS/MS-based candidate RMP for accurately measuring carbamazepine-10,11-epoxide in human serum and plasma. This novel method offers a traceable and dependable platform, making itsuitable for standardizing routine assays and assessing clinically relevant samples.

Analytical tools for monitoring glycol degradation

This paper aims to develop new methods for monitoring oxidative and thermal glycol degradation through solvent analysis. Methods for quantifying TEG, and for quantifying some degradation products (small glycols and acids), were successfully developed. The results were validated by applying two independent analytical methods for each compound monitored. The glycols were successfully quantified with gas chromatography coupled with flame ionization detection (GC-FID) and with quantitative carbon-13 nuclear magnetic resonance (13C NMR) spectroscopy. The acidic degradation compounds were successfully quantified with high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) and heat-stable salt (HSS) analysis. The thermal stability of TEG decreased with the addition of impurities, especially formic acid. Some color changes were observed during the degradation, but they were not linked with the amount of TEG degraded. Finally, it was found that TEG samples have limited stability, even if stored cold.

Fully automatic quantitation of eight different metabolites in coffee using 1H‐NMR spectroscopy and the PULCON methodology

AbstractBackgroundCoffee contains a plethora of constituents with some of them being especially important either due to their physiological effects or as quality markers. As quantitative proton nuclear magnetic resonance spectroscopy (1H‐NMR) has been established as a fast and reliable analytical tool its application was evaluated for the simultaneous quantitation of lactic acid, acetic acid, formic acid, caffeine, caffeoylquinic acid (CQA) isomers, N‐methylpyridinium, trigonelline, and 5‐hydroxymethylfurfural (HMF) in aqueous extracts of roasted Coffea arabica samples.ResultsSimultaneous quantitative determination was achieved by an automated analysis based on the PULCON methodology (pulse length‐based concentration determination). The method was validated regarding linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). Recovery rates were between 76% (CQA) and 116% (HMF), and precision was between 1.7% (caffeine) and 10.3% (HMF). The LOD varied between 0.06 g/kg (HMF) and 1.35 g/kg (caffeine and CQA), with the LOQ being between 0.22 g/kg (HMF) and 4.87 g/kg (CQA). To verify the results of the 1H‐NMR method, caffeine, trigonelline, HMF, 3‐CQA, 4‐CQA, and 5‐CQA were additionally quantitated by HPLC‐DAD and the results were compared. The described 1H‐NMR method was additionally applied to coffee samples that contained different coffee defects. Results showed only slight changes in the concentrations of the analytes by adding defective beans to defect‐free coffee.DiscussionThe developed 1H‐NMR approach was proven to be fast (30 min), reliable, and precise. Thus, it is well suited to analyze several coffee constituents of interest in a large number of samples in, for example, quality control.

Quantitative Metabolomics and Lipoprotein Analysis of PDAC Patients Suggests Serum Marker Categories for Pancreatic Function, Pancreatectomy, Cancer Metabolism, and Systemic Disturbances.

Pancreatic ductal adenocarcinoma (PDAC) is difficult to diagnose in the early stages and lacks reliable biomarkers. The scope of this project was to establish quantitative nuclear magnetic resonance (NMR) spectroscopy to comprehensively study blood serum alterations in PDAC patients. Serum samples from 34 PDAC patients obtained before and after pancreatectomy as well as 83 age- and sex-matched control samples from healthy donors were analyzed with in vitro diagnostics research (IVDr) proton NMR spectroscopy at 600 MHz. Uni- and multivariate statistics were applied to identify significant biofluid alterations. We identified 29 significantly changed metabolites and 98 lipoproteins when comparing serum from healthy controls with those of PDAC patients. The most prominent features were assigned to (i) markers of pancreatic function (e.g., glucose and blood triglycerides), (ii) markers related to surgery (e.g., ketone bodies and blood cholesterols), (iii) PDAC-associated markers (e.g., amino acids and creatine), and (iv) markers for systemic disturbances in PDAC (e.g., gut metabolites DMG, TMAO, DMSO2, and liver lipoproteins). Quantitative serum NMR spectroscopy is suited as a diagnostic tool to investigate PDAC. Remarkably, 2-hydroxybutyrate (2-HB) as a previously suggested marker for insulin resistance was found in extraordinarily high levels only after pancreatectomy, suggesting this metabolite is the strongest marker for pancreatic loss of function.

Semi-biological photosystem: harnessing carbon dots and Geobacter sulfurreducens for solar-driven hydrogenation

Abstract Semi-biological photosynthesis utilizes the unique ability of microbial catalysts together with synthetic photosensitizers (semiconductors) to produce high-value chemicals from sustainable feedstocks. In this work, we devise a semi-biological hybrid system consisting of sustainable photosensitizers, carbon dots in the size range of 5–35 nm (CDs) interfaced with bacteria, Geobacter sulfurreducens, to reduce fumarate to succinate as a model hydrogenation reaction. After 7 days of solar irradiation, using quantitative proton nuclear magnetic resonance spectroscopy (qNMR), the CD−G. sulfurreducens photosystem produced ∼18 mM of succinate without the need for a redox mediator. Moreover, in reusing the CDs, ∼70% of the succinate (compared to the previous cycle) was recovered. The proposed photobiohybrid system paves a new avenue for sustainable solar-to-chemical conversion in high-value chemical production.

Preparation and characterization of the aflatoxin B1 purity certified reference material (GBW (E) 100599)

Aflatoxin B1 (AFB1), the most harmful mycotoxin, is widely distributed in many important foodstuffs, especially in peanuts, maize, and oil products. AFB1 certified reference material (CRM), essential for an accurate and reliable detection, is not easily obtained due to the difficulty of preparing high purity candidate. In this study, the high-purity AFB1 candidate was prepared by the preparative high performance liquid chromatography (Pre-HPLC) after high-yields of AFB1 on the maize medium by Aspergillus flavus. The structure of AFB1 was successively characterized by UV, IR, LC-TOF-MS/MS, 1H NMR, and 13C NMR. According to the principle of ISO 17034: 2016 and ISO guide 35: 2017, the purity of the candidate was accurately determined by the mass balance method (MB) and the quantitative nuclear magnetic resonance spectroscopy method (qNMR), while the homogeneity, long-term stability, short-term stability, and uncertainty were systematically studied by the MB method. Under the optimum conditions, the certified purity value for the AFB1 CRM was 99.53 % with an extended uncertainty of 0.56 % (k = 2, 95 % confidence interval), which was also sufficiently homogeneous and stable for 6 months at 4 °C and −20 °C. After approval by State Administration for Market Regulation, a new AFB1 purity CRM (GBW (E) 100599) was finally prepared. This is the first report for the preparation of a reliable AFB1 purity CRM, benefit for enhancing the accuracy, traceability, and comparability of measurements of AFB1 in various foodstuffs, and finally ensure the food quality and safety.

Highly selective solid-liquid extraction of microplastic mixtures as a pre-preparation tool for quantitative nuclear magnetic resonance spectroscopy studies.

Despite various developments in the application of quantitative nuclear magnetic resonance (qNMR) spectroscopy toward microplastics in recent years, this method still lacks suitable sample preparation and fractionation procedures. As this poses a crucial obstacle for its utilisation on environmental samples, which contain various mixtures of polymers along with other matrix substances, this research aims to address this missing link by presenting an easy-to-apply procedure based on common laboratory equipment. The process selectively separates microplastics from inorganic constituents while performing the necessary fractionation of different types of microplastics prior to qNMR analysis. It allows subsequent quantification of polystyrene (PS), polybutadiene rubber (BR), polymethylmethacrylate (PMMA), polyvinylchloride (PVC), polyethylene terephthalate (PET) and polyamide (PA) from a single sample, establishing recovery rates greater than 88% for all tested polymer types. Additionally, we extended our previous qNMR protocol to include two common polymer types: polymethylmethacrylate (PMMA) and polyacrylonitrile (PAN), achieving limits of detection down to 1.76 μg ml-1 and 12.53 μg ml-1 as well as limits of quantification down to 5.88 μg ml-1 and 41.78 μg ml-1, respectively. Thus, the qNMR method presented herein is now applicable to eight abundant polymer types, allowing the quantification of up to three different types simultaneously.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of zonisamide in human serum and plasma.

To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations. Quantitative nuclear magnetic resonance (qNMR) spectroscopy was employed to determine the absolute content of the reference material used in order to establish traceability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of zonisamide within the range of 1.50-60.0 μg/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8-1.0 %, respectively. We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Assessment of the Purity of IMM-H014 and Its Related Substances for the Treatment of Metabolic-Associated Fatty Liver Disease Using Quantitative Nuclear Magnetic Resonance Spectroscopy.

An accurate, rapid, and selective quantitative nuclear magnetic resonance method was developed and validated to assess the purity of IMM-H014, a novel drug for the treatment of metabolic-associated fatty liver disease (MAFLD), and four related substances (impurities I, II, III, and IV). In this study, we obtained spectra of IMM--H014 and related substances in deuterated chloroform using dimethyl terephthalate (DMT) as the internal standard reference. Quantification was performed using the 1H resonance signals at δ 8.13 ppm for DMT and δ 6.5-7.5 ppm for IMM-H014 and its related substances. Several key experimental parameters were investigated and optimized, such as pulse angle and relaxation delay. Methodology validation was conducted based on the International Council for Harmonization guidelines and verified with satisfactory specificity, precision, linearity, accuracy, robustness, and stability. In addition, the calibration results of the samples were consistent with those obtained from the mass balance method. Thus, this research provides a reliable and practical protocol for purity analysis of IMM-H014 and its critical impurities and contributes to subsequent clinical quality control research.