1H qNMR Method Research Articles

Absolute purity determination of an organic fluorine pharmaceutical voriconazole via quantitative 19F NMR and method validation

The 19F NMR spectrum has a smaller number of signals than 1H NMR, therefore, quantitative 19F NMR (19F-qNMR) can replace quantitative 1H NMR (1H-qNMR) for the absolute quantitation of organic fluorine-bearing compounds. In this study, we determined the purity of voriconazole (VCZ), an organic fluorine pharmaceutical, using 19F-qNMR, validated the results in multiple laboratories, and compared them with those obtained using an established 1H-qNMR method. 3,5-Bis(trifluoromethyl)benzoic acid (3,5-BTFMBA) was selected as the reference standard for both 19F-qNMR and 1H-qNMR owing to its solubility characteristics and 19F-qNMR and 1H-qNMR chemical shifts.Since VCZ contains three fluorines, the 19F-qNMR spectrum of VCZ showed three major fluorine signals (FA, FB, and FC). 19F-qNMR measurements were performed under three conditions with each 19F signal of VCZ (FA, FB, and FC) and the 19F signal of RS as the center (offset) of each observation. The quantitative values of the three target signals FA, FB, and FC were calculated and were comparable; 99.40 %, 99.66 %, and 99.49 %, respectively.Owing to a difference of up to 8.8 % between the quantitative values of the two signals other than the targeted quantitative signal, the average FA, FB, and FC quantitative values were considered for purity (%). The purity of VCZ determined by 1H- and 19F-qNMR was comparable (99.65±0.29 % and 99.52±0.44 %, respectively), exhibiting variations within an acceptable range. Compared to conventional 1H-qNMR methods, the developed 19F-qNMR method has been proven efficient and highly promising for determining the accurate purity of organic fluorine pharmaceuticals.

Quantitative 31P-NMR for Purity Determination of Organophosphorus Compounds (Pharmaceuticals)

Quantitative NMR (qNMR), particularly 1H-qNMR, is useful for determining the absolute purity of organic molecules. However, identifying the target signal(s) for quantification is difficult, because of the overlap and complexity of organic molecules. Therefore, we focused on the 31P nucleus, owing to the simplicity of its signals, and investigated the 31P-qNMR absolute determination method by using organophosphorus drugs, water-soluble cyclophosphamide hydrate (CP), and water-insoluble sofosbuvir (SOF). The optimized and reproducible 31P-qNMR conditions, such as qNMR sample preparation [i.e., selecting suitable deuterated solvents and a reference standard (RS) for 31P-qNMR], hygroscopicity and solution stability of the analyte and RS, and qNMR measurements-such as acquisition time, relaxation delay time, and spectral width-were examined. The CP purities determined using 31P-qNMR agreed well with those for the established 1H-qNMR method in D2O. In contrast, the SOF purity determined using 31P-qNMR was 1.6% higher than that for 1H-qNMR in the protic solvent CD3OD. Therefore, using a protic solvent, such as CD3OD, was not suitable for 31P-qNMR; the deuterium exchange with the RS for 31P-qNMR (i.e., phosphonoacetic acid) resulted in a small integrated intensity. Consequently, the aprotic solvent DMSO-d6 was employed to determine the SOF purity. The data revealed that the SOF purities determined using 31P-qNMR agreed well with the established 1H-qNMR values, indicating that the absolute quantification of SOF using both 31P-qNMR and 1H-qNMR is possible in DMSO-d6. Thus, we established an optimized and reproducible 31P-qNMR method in validation study across multiple laboratories.

Characterization of defective coffee beans and blends differentiation based on 1H qNMR technique

Defective coffee beans (DCB) are one of the main reasons for poor coffee quality. In the current research, chemical difference of three common DCB including sour beans (SCB), black beans (BCB), and mold beans (MCB) were clarified using 1H qNMR method and compared with that of non-defective beans (NDCB). The results indicated that DCB has lower sugar and lipid content compared to NDCB, yet it boasts a higher acetate concentration. The 1H NMR from water-soluble content was shown to be more effective than that of oil fraction for qualitative of DCB blends, regardless of whether partial least squares discriminant analysis (PLS-DA) or machine learning (ML) algorithms were used. Support vector machine (SVM) was proved to be excellent for distinguishing DCB blends. Finally, a partial least squares regression (PLS) model was built for quantitative analysis of DCB blends. In summary, current research will not only help to reveal the material basis of DCB and their impact on coffee flavor, but also provide feasible strategies for the identification of DCB.

Utility and greenness appraisal of nuclear magnetic resonance for sustainable simultaneous determination of three 1,4-benzodiazepines and their main impurity 2-amino-5-chlorobenzophenone

A robust, stability-indicating, and eco-friendly proton nuclear magnetic resonance (1H-qNMR) method was developed for the concurrent determination of three 1,4-benzodiazepines (BDZs), namely diazepam (DZP), alprazolam (ALP), and chlordiazepoxide (CDP) and their common impurity, synthesis precursor, and degradation product; 2-amino-5-chlorobenzophenone (ACB). In the present method, a novel approach was developed for composing a green and cost-efficient solvent system as an alternative to the common NMR organic solvents utilizing 0.3 M sodium dodecyl sulfate prepared in deuterated water. The conducted method is characterized by simplicity with no need for sample pretreatment or labeling. Phloroglucinol was used as an internal standard. The chosen signals for the determinations of ALP, CDP, DZP and ACB were at 2.35 ppm (singlet), 2.84 ppm (singlet), 3.11 ppm (singlet), and 6.90 ppm (doublet of doublet), respectively. The proposed method possessed linearity over the concentration range of 0.25–15.0 mg ml−1 for DZP, ALP, CDP and of 0.5–25.0 mg ml−1 for ACB with LOD values of 0.06, 0.03, 0.07 and 0.16 mg ml−1 respectively, and LOQ values of 0.18, 0.09, 0.21 and 0.49 mg ml−1, respectively. Accuracy of the method was evidenced by excellent recovery% (99.57–99.90%) and small standard deviation (≥ 1.10) for the three analyzed drugs. Intra- and inter-day precision were determined with coefficient of variation ranging from 0.12 to 1.14 and from 0.72 to 1.67, respectively. For the studied compounds, appraisal of the method greenness was achieved via four approaches: Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), Analytical greenness metric (AGREE), and RGB Additive Color Model. The results proved that the proposed method has the privilege of being a green analytical method.

Implementation of analytical quality by design concepts for the optimization of quantitative 1H nuclear magnetic resonance (1H-qNMR) method for quantitation of novel anti-covid drugs (molnupiravir and favipiravir) in their pharmaceutical dosage forms

For the first time in the literature, the Analytical Quality by Design (AQbD) principles were applied to the development of a specific and simple quantitative proton nuclear magnetic resonance (1H-qNMR) method for quantitation of two antiviral drugs, molnupiravir (MOL) and favipiravir (FAV). This 1H-qNMR method was developed within the QbD framework, the new paradigm of quality recently outlined in the ICH Q14 guidelines. Basic principles of AQbD have been adapted starting with early risk assessment and identification of the analytical target profile (ATP), the analytical procedure attributes (APA) and the analytical procedure parameters (APPs). During the scouting phase, dimethyl sulfoxide-d6 (DMSO‑d6) was selected as the deuterated solvent and maleic acid as the internal standard. The quantitative determination was based on the selective proton signals from MOL's pyrimidine ring at 6.82 ppm (doublet) and FAV's hydroxyl group at 8.71 ppm (singlet), as well as a singlet signal from the IS at 6.24 ppm. A Box-Behnken Design was employed in response surface methodology to develop mathematical predictive models relating the APPs to the APAs followed by multicriteria- decision making with the aid of Derringer's desirability function. Finally, the method operable design regions (MODRs) were computed with the aid of Monte Carlo simulations and identified as the multidimensional combination of the APPs where the risk of failure to fulfill the requirements for the APAs was lower than 5 %. The most robust NMR conditions for MOL, with MODR intervals in brackets, were as follows: no. of scans of 72 (16–124.4), a relaxation delay of 19.8 s (1.2–40 s) and an acquisition time of 3.98 s (2.1–6 s). Similarly, for FAV NMR quantitation, the most robust conditions were found to be 72 scans (16–117.3), a relaxation delay of 19.8 s (1.4–40 s) and an acquisition time of 3.98 s (2.2–6 s). The developed method was validated in accordance with the recently endorsed ICH Q2R2 guidelines, and it exhibited detection limits of 1.04 and 0.88 mg mL−1, as well as quantitation limits of 3.45 and 2.95 mg mL−1 for MOL and FAV, respectively. The method was successfully applied to the analysis of the studied analytes in their pharmaceutical products, proving to be simple, fast, non-destructive, and accurate. Additionally, it can be considered an environmentally friendly alternative for quantitation and quality control analysis of pharmaceutical products.

Pharmaceutical process-omics for quality control of traditional Chinese medicine preparations: A 1H-qNMR assisted case study of Guanxinning injection

Guanxinning injection (GXNI) is a traditional Chinese medicine preparation derived from Salviae Miltiorrhizae Radix et Rhizoma and Chuanxiong Rhizoma. It is produced through a series of processes involving water extraction, ethanol precipitation, and other techniques. GXNI is primarily used for treating angina pectoris in coronary heart disease, and it has shown remarkable efficacy in clinical practice. One of the key components responsible for its pharmacological effects is salvianolic acids. However, these components are known for their poor stability and susceptibility to pH and temperature variations. Therefore, it is crucial to focus on ensuring the quality and stability of salvianolic acids in GXNI. In this study, we employed Proton nuclear magnetic resonance (1H NMR) to analyze the intermediate products formed during the manufacturing of GXNI. We thoroughly examined the spectra of these intermediates and developed a precise 1H-qNMR method for the accurate quantification of various classes of chemical components present in GXNI. Additionally, we applied this method to investigate how the composition of GXNI evolves and transfers throughout the entire production process. To further enhance our understanding, we employed Principal Component Analysis (PCA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) to identify critical steps in the production process and to identify potential quality markers (Q-markers) associated with these processes. Our study sheds light on the dynamic changes in composition that occur during the production of GXNI. This research serves as a foundation for establishing a comprehensive quality evaluation system for GXNI, ensuring its efficacy and safety in clinical applications.

Application of quantitative NMR in pharmaceutical analysis: Method qualification and potency determination for two new chemical entities in early-stage clinical studies

Quantitative NMR (qNMR), being a well-established analytical tool featuring efficiency, simplicity as well as versatility, has been extensively employed in pharmaceutical and medicinal testing. In this study, two 1H qNMR methods were developed to determine the %wt/wt potency of two new chemical entities (compound A and compound B) used in early clinical phase process chemistry and formulation development. The qNMR methods were demonstrated to be significantly more sustainable and efficient than the LC-based approach by substantially reducing the cost, hands-on-time, and materials consumed for testing. The qNMR methods were achieved on a 400 MHz NMR spectrometer equipped with 5 mm BBO S1 broad band room temperature probe. The methods with CDCl3 (for compound A) and DMSO-d6 (compound B) as solvent as well as commercially certified standards for quantitation were phase-appropriately qualified in terms of specificity, accuracy, repeatability/precision, linearity, and range. Both qNMR methods were demonstrated to be linear over the range of 0.8–1.2 mg/mL (80% to 120% of the nominal sample concentration of 1.0 mg/mL) with a correlation coefficient greater than 0.995. The methods were also demonstrated to be accurate with average recoveries ranging from 98.8% to 98.9% and 99.4–99.9% for compound A and compound B respectively as well as precise with %RSD of 0.46% and 0.33% for compound A and compound B respectively. The potency results of compound A and compound B determined by qNMR were compared with those determined by the conventional LC-based method and the qNMR results were demonstrated to be consistent with the LC-based method with absolute difference of 0.4% and 0.5% for compound A and B respectively.

New trends of new psychoactive substances (NPS)-infused chocolate: Identification and quantification of trace level of NPS in complex matrix by GC-MS and NMR

For the first time, the identification and quantification of trace level of new psychoactive substances (NPS) in a complex chocolate matrix have been reported. Since the beginning of 2022, suspected NPS-infused chocolate samples confiscated in inbound packages have been continuously sent to our laboratory for analysis. The qualitative gas chromatography-mass spectrometry (GC-MS) results were verified by 1H nuclear magnetic resonance (1H NMR) and 19F NMR to distinguish between potential aromatic isomers. A total of 11 NPS including deoxymethoxetamine, 3-OH-PCP, 6-APB, 4-APB, 4-OH-MiPT, 3-FEA, 2-FEA, 3-MMC, bromazolam, 2-FDCK, and ADB-BUTINACA were detected in 65 seized chocolate samples. A general 1H quantitative NMR (1H qNMR) method for quantification of 297 types of NPS in complex chocolate matrixes was devised for the first time after rigorous analysis of various critical features of merit, including suitable deuterated solvent, internal standard, quantitative peaks, and instrument acquisition parameters. Validation of the method using six different types of NPS afforded limits of detection of 0.05–0.1 mg/mL, limits of quantification of 0.01–0.03 mg/mL, repeatability and reproducibility lower than 0.5% and 3.6%, recoveries of 91.7%∼104.4%, and absence of matrix effect. The quantitative analysis of 65 seized chocolate samples by 1H qNMR and 19F qNMR showed that the content of NPS was in the range of 0.5 mg/g∼44.1 mg/g. Generally, the developed qNMR method was simple, fast, precise, and can be performed without reference materials of NPS. Since the type and content of NPS are relatively random, chocolate consumers will face huge health risks. Therefore, this new trend of NPS-infused chocolate deserves and requires more attention from national NPS monitoring departments as well as forensic laboratories.

NMR method for simultaneous determination of 21 chemical components in Danhong Injection

Danhong Injection, a compound Chinese medicine injection prepared from Salviae Miltiorrhizae Radix et Rhizoma and Carthami Flos, is used in the clinical treatment of coronary heart disease, cerebral thrombosis, myocardial infarction, angina pectoris and other cardiovascular and cerebrovascular diseases. In this study, a quantitative method for simultaneous determination of multiple components in Danhong Injection was developed based on ~1H-qNMR technology and then methodological verification was carried out. The results showed that the established method had good methodological indexes. This method can simultaneously determine the content of 21 chemical components including 6 amino acids, 4 small molecular organic acids, 5 sugars and their derivatives, 1 nucleoside, and 5 aromatic compounds in Danhong Injection. The total content accounted for about 85% of the total solid mass, which reflected the great advantage of ~1H-qNMR method in the analysis of Chinese medicine injections. The ~1H-qNMR method for simultaneous determination of multiple components in Danhong Injection developed in this study has simple operation, short analysis time, and wide application range, which has practical significance for the quality evaluation of Danhong Injection and provides reference for the development of quality control methods for Chinese medicine injections.

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures.

The widespread use of phytocannabinoids or cannabis extracts as ingredients in numerous types of products, in combination with the legal restrictions on THC content, has created a need for the development of new, rapid, and universal analytical methods for their quantitation that ideally could be applied without separation and standards. Based on previously described qNMR studies, we developed an expanded 1H qNMR method and a novel 2D-COSY qNMR method for the rapid quantitation of ten major phytocannabinoids in cannabis plant extracts and cannabis-based products. The 1H qNMR method was successfully developed for the quantitation of cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabielsoin (CBE), and cannabidivarin (CBDV). Moreover, cannabidivarinic acid (CBDVA) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) can be distinguished from CBDA and Δ9-THCA respectively, while cannabigerovarin (CBGV) and Δ8-tetrahydrocannabivarin (Δ8-THCV) present the same 1H-spectra as CBG and Δ8-THC, respectively. The COSY qNMR method was applied for the quantitation of CBD, CBDA, CBN, CBG/CBGA, and THC/THCA. The two methods were applied for the analysis of hemp plants; cannabis extracts; edible cannabis medium-chain triglycerides (MCT); and hemp seed oils and cosmetic products with cannabinoids. The 1H-NMR method does not require the use of reference compounds, and it requires only a short time for analysis. However, complex extracts in 1H-NMR may have a lot of signals, and quantitation with this method is often hampered by peak overlap, with 2D NMR providing a solution to this obstacle. The most important advantage of the COSY NMR quantitation method was the determination of the legality of cannabis plants, extracts, and edible oils based on their THC/THCA content, particularly in the cases of some samples for which the determination of THC/THCA content by 1H qNMR was not feasible.

1H NMR spectrometry for methanol quantification in apple wines and ciders as optimised by comparison to SIDA-HS-GC-MS

Methanol, a hepato- and neurotoxic compound, is present in apple-based beverages as a by-product of the enzymatic degradation of pectin. Stable isotope dilution assay (SIDA) employing gas chromatography-mass spectrometry (GC–MS) is the state-of-the-art method for methanol determination in beverages. Despite higher initial investment costs, quantitative 1H NMR spectroscopy (qNMR) is a simpler and faster analytical technique to quantify numerous analytes in liquid foods. Beyond targeted analyses, qNMR spectral fingerprints in the product might be used for non-targeted analytical goals, such as adulteration and contamination detections. Here, an existing 1H-qNMR method used for wine profiling was optimised for methanol quantification in apple-based products, including cross-validation against a SIDA-HS-GC–MS method and reference values from interlaboratory trials. The optimisation involved a pivotally important estimation of a correction factor by an external calibration approach, making qNMR results comparable to SIDA-HS-GC–MS. The optimised qNMR method is suggested to be an alternative for methanol quantification in beverages.

QNMR characterization of potential peptide calibrators for isotope dilution LC–MS quantification of a SARS-CoV-2 IgG monoclonal antibody

The year 2020 will be remembered for the most pernicious epidemic of modern history. The SARS-CoV-2 coronavirus has affected millions of people worldwide and has triggered an unprecedented race for the development of diagnostic tests and therapeutical vaccines. These technologies require befitting measurements that underpin reliability and patient safety. The level of blood-borne antibodies against SARS-CoV-2 is correlated initially with the extent of infection and subsequently with the degree of immunity attained. This renders their accurate measurement an important target for immunometric and liquid chromatography–mass spectrometry methods. Suitable reference materials and methods are being developed to ensure the reliability and consistency of such measurements. Solutions of purified monoclonal IgG may prove useful as primary calibrator materials for the development of calibration hierarchies for the measurement of blood-borne antibodies against SARS-CoV-2. Bottom-up approaches can be applied to the quantification of such structurally complex large molecules. These consist of trypsin and Lys C digestion and quantification of the resulting proteotypic peptides as surrogate analytes for an IgG monoclonal antibody. Establishing the metrological traceability of this procedure requires peptides whose purity has been determined accurately, for example with quantitative nuclear magnetic resonance (qNMR). The 1H qNMR method allows for the mass fraction assignment of peptides by accurate quantification of 1H resonance signals specific for both the peptide and for a certified reference material used as internal standard in the peptide solution. The current paper describes the qNMR characterization of five peptides that could be used in a double isotope dilution method for the quantification of SARS-CoV-2 IgG monoclonal antibodies in solution.

Quantitative proton nuclear magnetic resonance method for simultaneous analysis of fluticasone propionate and azelastine hydrochloride in nasal spray formulation.

A facile, rapid, accurate and selective quantitative proton nuclear magnetic resonance (1H-qNMR) method was developed for the simultaneous determination of fluticasone propionate (FLP) and azelastine hydrochloride (AZH) in pharmaceutical nasal spray for the first time. The 1H-qNMR analysis of the studied analytes was performed using inositol as the internal standard and dimethyl sulfoxide-d6 (DMSO-d6) as the solvent. The quantitative selective proton signal of FLP was doublet of doublet at 6.290, 6.294, 6.316 and 6.319 ppm, while that of AZH was doublet at 8.292 and 8.310 ppm. The internal standard (inositol) produced a doublet signal at 3.70 and 3.71 ppm. The method was rectilinear over the concentration ranges of 0.25–20.0 and 0.2–15.0 mg ml−1 for FLP and AZH, respectively. No labelling or pretreatment steps were required for NMR analysis of the studied analytes. The proposed 1H-qNMR method was validated efficiently according to the International Council on Harmonisation guidelines in terms of linearity, limit of detection, limit of quantification, accuracy, precision, specificity and stability. Moreover, the method was applied to assay the analytes in their combined nasal spray formulation. The results ensured the linearity (r2 > 0.999), precision (% RSD < 1.5), stability, specificity and selectivity of the developed method.

Purity Determination of Cyclophosphamide Hydrate by Quantitative &lt;sup&gt;31&lt;/sup&gt;P-NMR and Method Validation

Recently, quantitative NMR (qNMR), especially 1H-qNMR, has been widely used to determine the absolute quantitative value of organic molecules. We previously reported an optimal and reproducible sample preparation method for 1H-qNMR. In the present study, we focused on a 31P-qNMR absolute determination method. An organophosphorus compound, cyclophosphamide hydrate (CP), listed in the Japanese Pharmacopeia 17th edition was selected as the target compound, and the 31P-qNMR and 1H-qNMR results were compared under three conditions with potassium dihydrogen phosphate (KH2PO4) or O-phosphorylethanolamine (PEA) as the reference standard for 31P-qNMR and DSS-d6 as the standard for 1H-qNMR. Condition 1: separate sample containing CP and KH2PO4 for 31P-qNMR or CP and DSS-d6 for 1H-qNMR. Condition 2: mixed sample containing CP, DSS-d6, and KH2PO4. Condition 3: mixed sample containing CP, DSS-d6, and PEA. As conditions 1 and 3 provided good results, validation studies at multiple laboratories were further conducted. The purities of CP determined under condition 1 by 1H-qNMR at 11 laboratories and 31P-qNMR at 10 laboratories were 99.76±0.43% and 99.75±0.53%, respectively, and those determined under condition 3 at five laboratories were 99.66±0.08% and 99.61±0.53%, respectively. These data suggested that the CP purities determined by 31P-qNMR are in good agreement with those determined by the established 1H-qNMR method. Since the 31P-qNMR signals are less complicated than the 1H-qNMR signals, 31P-qNMR would be useful for the absolute quantification of compounds that do not have a simple and separate 1H-qNMR signal, such as a singlet or doublet, although further investigation with other compounds is needed.

Review on Quantification of Mineral Oil Aromatic Hydrocarbon (MOAH) from Cosmetics by Analytical Method

Mineral oils which consists mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH), which are largely applied in various consumer products like medicines cosmetics etc. MOAH which is potential public health hazard because it include carcinogenic polycyclic compounds. There is a rapid method for quantifying MOAH by proton nuclear magnetic resonance spectroscopy (1H qNMR) in anhydrous cosmetics. The 1H qNMR method is a good complement to the LC-GC-FID method. Another method is a simple and fast developed that uses columns packed with silver-modified silica in supercritical fluid chromatography with flame ionization and UV detection (SFC-FID/UV) for the determination of mineral oil saturated hydrocarbons (MOSH) and also mineral oil aromatic hydrocarbons (MOAH) in purified mineral oil samples. Another method which is based on gas chromatography with vacuum ultraviolet detection (GC-VUV) and relies on the spectral differences between the aliphatic and aromatic compounds in the sample. The detector provides a good selectivity for aromatics, direct quantification of the MOAH content is possible without the need for a laborious pre-separation of the mineral oil. GC-VUV method good sensitive for the analysis of all but gives highest purity mineral oils.

Quantification of Cathinone Analogues without Reference Standard Using 1H Quantitative NMR.

Synthetic cathinones are a type of new psychoactive substances (NPS) that have been seriously abused. Owing to the rapid variation in their structures, the absence of reference standards poses a challenge in quantitative investigations. In this study, a 1H quantitative nuclear magnetic resonance (1H qNMR) method was established using maleic acid as the internal standard and the shared signal (i.e., the methylidyne hydrogen) on the parent synthetic cathinones structure as the quantitative peak. Taking 3-methoxy-2-(methylamino)-1-(4-methylphenyl)propan-1-one (mexedrone) as an example, this study optimized the acquisition parameters and conducted method validation, including an evaluation of the specificity, linearity, accuracy, precision, and robustness. Using this 1H qNMR method, the contents of mexedrone and its analogues, including 1-(3-chlorophenyl)-2-(ethylamino)-propan-1-one (3-CEC), 4-chloro-α-pyrroli-dinopropiophenone (4-Cl-α-PVP), 1-(3,4-methylenedioxy-phenyl)-2-propylamino-propan-1-one (propylone), and methcathinone, were obtained. The obtained results showed that the method was accurate, rapid, versatile, and can be used to address the qualitative and quantitative issues related to similar substances.

1H qNMR-based quantitative analysis of total macamides in five maca (Lepidium meyenii Walp.) dried naturally

Macamides are considered bioactive markers of maca, and they have been used as food and medicine for centuries. In the present study, a 1H qNMR method was developed, and the analyses of the specificity, linearity, sensitivity, accuracy, and precision further indicate the validity of this method. Furthermore, the 1H qNMR method was applied for the quantification of total macamides in five naturally air-dried maca samples with different origins and colors. The results showed that the content of total macamides was correlated with the origins and colors: China purple maca (1845.75 ± 3.45 μg g−1) > China yellow maca (1090.20 ± 2.07 μg g−1) > Peru yellow maca (393.30 ± 2.42 μg g−1) > China and Peru black maca (each 269.10 ± 1.03 μg g−1). In addition, a new macamide, N-benzyl-(9-carboxymethyl)-octanamide (2), which is the first example of a macamide with an azelaxyl motif, was identified from Chinese purple maca. Our research not only provides a valid method to determine the content of total macamides in different maca samples but also offers a reference for quality control of maca.

Compositional variation of atranorin-related components of lichen Myelochroa leucotyliza dependent on extraction solvent and their quantitative analysis by qHNMR.

Quantitative nuclear magnetic resonance (qNMR) is one of the effective and reliable quantification tools for natural product research. Myelochroa leucotyliza belongs to the genus Myelochroa, a common foliose lichen genus found in the Korean Peninsula, and has not been quantitatively analysed using NMR. Previous chemical studies on M. leucotyliza have been limited to the main components by traditional thin-layer chromatography (TLC) experiments. We explored the stability of atranorin, a major component of M. leucotyliza, in methanol and acetone using NMR and characterised the changes in the chemical profiles of the lichen extracts in methanol and acetone using qNMR. Atranorin transformation in the presence of methanol was analysed using time-dependent proton (1 H)-NMR analysis (600 MHz NMR spectrometer). A 1 H qNMR (qHNMR) method was established using dimethyl sulfone as the internal standard for quantifying the selected components isolated from M. leucotyliza. Homogenous mixtures of the samples were dissolved in deuterated chloroform. Time-dependent 1 H-NMR experiments revealed that atranorin (5) from lichen M. leucotyliza decomposed into atraric acid (1) and methyl haemmatommate (2) in methanol. Four components were identified from M. leucotyliza: 1, 2, usnic acid (4), and 5, and their respective contents were determined using qHNMR. The percentages (w/w) of 1, 2, and 4 in the methanol extract were calculated as 5.66%, 0.69%, and 0.90%, while those of 1, 4, and 5 in the acetone extract were 1.70%, 1.68%, and 19.11%, respectively. We used qHNMR to effectively analyse quantitative compositional variations in two different M. leucotyliza extracts and reliably determined the chemical conversion of the unstable compound atranorin.

Development and Validation of 2-Azaspiro [4,5] Decan-3-One (Impurity A) in Gabapentin Determination Method Using qNMR Spectroscopy.

The authors developed a 1H qNMR test procedure for identification and quantification of impurity A present in gabapentin active pharmaceutical ingredient (API) and gabapentin products. The validation studies helped to determine the limit of quantitation and assess linearity, accuracy, repeatability, intermediate precision, specificity, and robustness of the procedure. Spike-and-recovery assays were used to calculate standard deviations, coefficients of variation, confidence intervals, bias, Fisher’s F test, and Student’s t-test for assay results. The obtained statistical values satisfy the acceptance criteria for the validation parameters. The authors compared the results of impurity A quantification in gabapentin APIs and capsules by using the 1H qNMR and HPLC test methods.

Development of a Quantitative NMR Method for Direct and Simultaneous Determination of Four Flavonoids in Scutellaria baicalensis georgi Extracts

Introduction: Scutellaria Baicalensis Georgi (SBG), a Traditional Chinese Medicine (TCM) used for the treatment of antiviral therapy, contains many flavonoids. Materials and Methods: A simple, fast and accurate 1H quantitative nuclear magnetic resonance (1HqNMR) method was established for simultaneously determination of four flavonoids (Baicalin, Baicalein, Wogonin and Wogonoside) in SBG extract by using 3,4,5-trichloropyridine as an internal standard (IS). All the NMR determination work was performed at 308 K on an NMR sample tube inserted with a co-axial tube. The NMR sample tube was added with the sample (SBG extract and IS), the co-axial insert tube was added with D2O. Quantification of four flavonoids was carried out by calculating the relative peak area ratio of the selected proton signals from the target compounds and the IS. Results: The validated 1H-qNMR method was successfully used to determine the four flavonoids in 9 batches of SBG from different origins and the results were in good accordance with those obtained from HPLC. Conclusion: The established 1H-qNMR method could be used as a powerful tool for the quality control of SBG from different sources and had potential in the quantification of main components from other TCMs.