RESUMEN
The goal of the qNMR Summit is to take stock of the status quo and the recent developments in qNMR research and applications in a timely and accurate manner. It provides a platform for both advanced and novice qNMR practitioners to receive a well-rounded update and discuss potential qNMR-related applications and collaborations. For over a decade, scientists from academia, industry, nonprofit institutions, and governmental bodies have focused on the standardization of qNMR methodology, as well as its metrological and pharmacopeial utility. This paper reviews key content of qNMR Summits 1.0 to 4.0 and puts into perspective the outcomes and available transcripts of the October 2019 Summit 5.0, with attendees from the United States, Canada, Japan, Korea, and several European countries. Summit presentations focused on qNMR methodology in the pharmaceutical industry, advanced quantitation algorithms, and promising developments.
Asunto(s)
Tecnología , Canadá , Japón , Estándares de Referencia , Estados UnidosRESUMEN
The present study demonstrates the relationship between conventional and quantum mechanical (QM) NMR spectroscopic analyses, shown here to assist in building a convincingly orthogonal platform for the solution and documentation of demanding structures. Kaempferol-3-O-robinoside-7-O-glucoside, a bisdesmosidic flavonol triglycoside and botanical marker for the aerial parts of Withania somnifera, served as an exemplary case. As demonstrated, QM-based 1H iterative full spin analysis (HiFSA) advances the understanding of both individual nuclear resonance spin patterns and the entire 1H NMR spectrum of a molecule and establishes structurally determinant, numerical HiFSA profiles. The combination of HiFSA with regular 1D 1H NMR spectra allows for simplified yet specific identification tests via comparison of high-quality experimental with QM-calculated spectra. HiFSA accounts for all features encountered in 1H NMR spectra: nonlinear high-order effects, complex multiplets, and their usually overlapped signals. As HiFSA replicates spectrum patterns from field-independent parameters with high accuracy, this methodology can be ported to low-field NMR instruments (40-100 MHz). With its reliance on experimental NMR evidence, the QM approach builds up confidence in structural characterization and potentially reduces identity analyses to simple 1D 1H NMR experiments. This approach may lead to efficient implementation of conclusive identification tests in pharmacopeial and regulatory analyses: from simple organics to complex natural products.
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Glicósidos/análisis , Espectroscopía de Resonancia Magnética/normas , Withania/química , Flavonoles/análisis , Quempferoles/análisis , Monosacáridos/análisis , Componentes Aéreos de las Plantas/químicaRESUMEN
In the last two decades, quantitative NMR (qNMR) has become increasingly important for the analysis of pharmaceuticals, chemicals, and natural products including dietary supplements. For the purpose of quality control and chemical standardization of a large variety of pharmaceutical, chemical, and medicinal products, qNMR has proven to be a valuable orthogonal quantification method and a compelling alternative to chromatographic techniques. This work reviews a fundamental component of the early development of qNMR, reflected in the pioneering work of the late George M. Hanna during the years between 1984 and 2006 at the US Food and Drug Administration (FDA). Because Hanna performed the majority of his groundbreaking work on a 90-MHz instrument, his legacy output connects with recent progress in low-field benchtop NMR instrumentation. Hanna gradually established the utility of qNMR for the routine quality control analyses practiced in pharmaceutical and related operations well ahead of his peers. His work has the potential to inspire new developments in qNMR applied to small molecules of biomedical importance.
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Espectroscopía de Resonancia Magnética/historia , Preparaciones Farmacéuticas/análisis , Historia del Siglo XX , Humanos , Espectroscopía de Resonancia Magnética/instrumentación , Espectroscopía de Resonancia Magnética/métodos , Preparaciones Farmacéuticas/química , Control de Calidad , Estados Unidos , United States Food and Drug AdministrationRESUMEN
NMR spectroscopy has recently been utilized to determine the absolute amounts of organic molecules with metrological traceability since signal intensity is directly proportional to the number of each nucleus in a molecule. The NMR methodology that uses hydrogen nucleus (1H) to quantify chemicals is called quantitative 1H-NMR (1H qNMR). The quantitative method using 1H qNMR for determining the purity or content of chemicals has been adopted into some compendial guidelines and official standards. However, there are still few reports in the literature regarding validation of 1H qNMR methodology. Here, we coordinated an international collaborative study to validate a 1H qNMR based on the use of an internal calibration methodology. Thirteen laboratories participated in this study, and the purities of three samples were individually measured using 1H qNMR method. The three samples were all certified via conventional primary methods of measurement, such as butyl p-hydroxybenzoate Japanese Pharmacopeia (JP) reference standard certified by mass balance; benzoic acid certified reference material (CRM) certified by coulometric titration; fludioxonil CRM certified by a combination of freezing point depression method and 1H qNMR. For each sample, 1H qNMR experiments were optimized before quantitative analysis. The results showed that the measured values of each sample were equivalent to the corresponding reference labeled value. Furthermore, assessment of these 1H qNMR data using the normalized error, En-value, concluded that statistically 1H qNMR has the competence to obtain the same quantification performance and accuracy as the conventional primary methods of measurement.
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Espectroscopía de Resonancia Magnética/normas , Ácido Benzoico/química , Calibración , Dioxoles/química , Hidroxibenzoatos/química , Cooperación Internacional , Espectroscopía de Resonancia Magnética/métodos , Pirroles/química , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
The ICH guidelines recommend reporting thresholds for regular impurities in drug substances at the level of 0.05% or 0.03% (w/w) depending on the maximum daily intake. Therefore, any instrumental method of analysis applicable to the impurity analysis should be able to detect and quantify the analytes at those levels. This investigation was designed to verify the suitability of 1H NMR spectroscopy for the detection of impurities, as a first step in the process before attempting quantification. In order to minimize demand on equipment, this study employed a 400 MHz instrument for structural confirmation and signal assignments of choline (1) and O-(2-hydroxyethyl)choline (2), a known impurity. The limit of detection (LOD) of 2 in 10 mg of 1 was established as 0.01% on a 400 MHz instrument and 2% on a 60 MHz (benchtop) NMR spectrometer. Thus, impurities for which quantification is required are readily detected at 400 MHz or above. These results are in contrast to the widespread belief that 1H NMR sensitivity is insufficient for pharmaceutical impurity analysis. The choice of solvent was recognized as a critical parameter for 1H NMR LOD analysis. Furthermore, publicly available NMR raw data (HMDB) proved to be valuable for unveiling the otherwise cryptic information hidden in complex signal patterns via 1H NMR iterative Full Spin Analysis. Finally, the study uncovered the less noticed, yet characteristic, 14N-1H coupling in the -N+(CH3)3 groups, adding strong arguments for the Raw NMR Data Initiative. Collectively, the data prove that the analytical capabilities of high-field NMR easily fulfill the ICH requirements for detection of impurity in the presence of an actual substance of interest which makes it a step closer to achieving regulatory standards.
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Colina , Contaminación de Medicamentos , Cromatografía Líquida de Alta Presión/métodos , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/métodos , Preparaciones FarmacéuticasRESUMEN
Whereas generic, LC-based pharmaceutical control quality procedures depend largely on the detection mode and can be particularly 'blind' to certain impurities, NMR is a more versatile and, thus, often more judicious detector. While adulteration presents ever-evolving challenges for the analysis of active pharmaceutical ingredients (APIs) and finished products sold in the worldwide (online) marketplace, research chemicals are usually trusted rather than being considered flawed or even adulterated. This report shows how NMR analysis uncovered the unanticipated presence of substantial amounts of mannitol (20 and 43% w/w) as undeclared constituent in two custom synthetic peptides, DR and DRVYI, that were sourced commercially. Quantitative 1H NMR (qHNMR) readily detected the contaminant, even on a 60â¯MHz benchtop instrument, and quantified the highly polar and UV-transparent adulterant. Quantum-mechanical 1H iterative Full Spin Analysis (HiFSA) not only achieved unambiguous identification of both the mannitol and the peptides, but also confirmed the quantitative results. The cases show that experimental verification supersedes trust in both pharmaceutical and research QC. They also highlight the promising utility of both established high-field and recently re-evolving low-field benchtop qHNMR. The unanticipated findings remind manufacturers and researchers alike about the advantages of including/performing NMR and qNMR with routine CofA documentation and/or verification of research grade chemicals. Especially when done jointly, this can greatly improve confidence in research and help streamline the pharmaceutical QC toolbox.
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Péptidos/química , Preparaciones Farmacéuticas/química , Contaminación de Medicamentos , Imagen por Resonancia Magnética/métodos , Control de CalidadRESUMEN
BACKGROUND: Levels of marinobufagenin (MBG), an endogenous bufadienolide Na/K-ATPase (NKA) inhibitor, increase in preeclampsia and in NaCl-sensitive hypertension. METHODS: We tested a 3E9 monoclonal anti-MBG antibody (mAb) for the ability to lower blood pressure (BP) in NaCl-sensitive hypertension and to reverse the preeclampsia-induced inhibition of erythrocyte NKA. Measurements of MBG were performed via immunoassay based on 4G4 anti-MBG mAb. RESULTS: In hypertensive Dahl-S rats, intraperitoneal administration of 50 microg/kg 3E9 mAb lowered BP by 32 mmHg and activated the Na/K-pump in the thoracic aorta by 51%. NaCl supplementation of pregnant rats (n = 16) produced a 37 mmHg increase in BP, a 3.5-fold rise in MBG excretion, and a 25% inhibition of the Na/K-pump in the thoracic aorta, compared with pregnant rats on a normal NaCl intake. In eight pregnant hypertensive rats, 3E9 mAb reduced the BP (21 mmHg) and restored the vascular Na/K-pump. In 14 patients with preeclampsia (mean BP, 126 +/- 3 mmHg; 26.9 +/- 1.4 years; gestational age, 37 +/- 0.8 weeks), plasma MBG was increased three-fold and erythrocyte NKA was inhibited compared with that of 12 normotensive pregnant women (mean BP, 71 +/- 3 mmHg) (1.5 +/- 0.1 vs. 3.1 +/- 0.2 micromol Pi/ml/h, respectively; P < 0.01). Ex-vivo 3E9 mAb restored NKA activity in erythrocytes from patients with preeclampsia. As compared with 3E9 mAb, Digibind, an affinity-purified antidigoxin antibody, was less active with respect to lowering BP in both hypertensive models and to restoration of NKA from erythrocytes from patients with preeclampsia. CONCLUSION: Anti-MBG mAbs may be a useful tool in studies of MBG in vitro and in vivo and may offer treatment of preeclampsia.
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Anticuerpos Monoclonales/uso terapéutico , Bufanólidos/inmunología , Hipertensión/tratamiento farmacológico , Preeclampsia/tratamiento farmacológico , Preñez/fisiología , Embarazo/fisiología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Digoxina/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Hipertensión/fisiopatología , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Preeclampsia/fisiopatología , Tercer Trimestre del Embarazo , Ratas , Ratas Endogámicas Dahl , Sensibilidad y Especificidad , Cloruro de Sodio Dietético , ATPasa Intercambiadora de Sodio-Potasio/fisiologíaRESUMEN
BACKGROUND: In order to define appropriate quality of botanical dietary supplements, botanical drugs, and herbal medicines, the United States Pharmacopeia (USP) and the Herbal Medicines Compendium (HMC) contain science-based quality standards that include multiple interrelated tests to provide a full quality characterization for each article in terms of its identity, purity, and content. PURPOSE: To provide a comprehensive description of the pharmacopeial tests and requirements for articles of botanical origin in the aforementioned compendia. Selective chromatographic procedures, such as high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC), are used as Identification tests in pharmacopeial monographs to detect species substitution or other confounders. HPLC quantitative tests are typically used to determine the content of key constituents, i.e., the total or individual amount of plant secondary metabolites that are considered bioactive constituents or analytical marker compounds. Purity specifications are typically set to limit the content of contaminants such as toxic elements, pesticides, and fungal toxins. Additional requirements highlight the importance of naming, definition, use of reference materials, and packaging/storage conditions. METHODS: Technical requirements for each section of the monographs were illustrated with specific examples. Tests were performed on authentic samples using pharmacopeial reference standards. The chromatographic analytical procedures were validated to provide characteristic profiles for the identity and/or accurate determination of the content of quality markers. RESULTS: The multiple tests included in each monograph complement each other to provide an appropriate pharmacopeial quality characterization for the botanicals used as herbal medicines and dietary supplements. The monographs provide detailed specifications for identity, content of bioactive constituents or quality markers, and limits of contaminants, adulterants, and potentially toxic substances. Additional requirements such as labeling and packaging further contribute to preserve the quality of these products. CONCLUSION: Compliance with pharmacopeial specifications should be required to ensure the reliability of botanical articles used for health care purposes.
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Suplementos Dietéticos/normas , Preparaciones de Plantas/normas , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Plantas Medicinales/química , Estándares de Referencia , Reproducibilidad de los Resultados , Estados UnidosRESUMEN
Ganoderma lucidum is a well-known medicinal mushroom. At present, numerous G. lucidum products have emerged in the form of dietary supplements in the United States due to its various benefits. However, the quality consistency of these products based on their label ingredients has seldom been evaluated due to the lack of a suitable toolkit. In this study, 19 batches of products of G. lucidum (Red Reishi, Reishi), herbal/mushroom supplements purchased in the United States, were evaluated based on their bioactive components including triterpenes and polysaccharides by using chromatographic methods and saccharide mapping. The results showed that the measured ingredients of only 5 tested samples (26.3%) were in accordance with their labels, which suggested the quality consistency of G. lucidum dietary supplements in the U.S. market was poor, which should be carefully investigated.
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Productos Biológicos/normas , Suplementos Dietéticos/normas , Reishi/química , Productos Biológicos/química , Polisacáridos Fúngicos/análisis , Triterpenos/análisis , Estados UnidosRESUMEN
Hypericum perforatum L. is the most commonly used herb for treating depression. Due to the popularity of this botanical, there is a potential for economically driven adulteration of St. John's wort (SJW) products. The goal of this study was to investigate SJW ingredients suspected to be adulterated based on simple preliminary HPTLC tests. Commercial samples were analyzed by HPTLC following the United States Pharmacopeia (USP) monograph methodology, with additional visualization under white light. A number of these samples presented odd methanolic solution colors and unconventional HPTLC fingerprints, suggesting the presence of other species and/or extraneous polar additives. To achieve identification and separation of the polar additives, a new reversed-phase HPTLC method was developed. The adulterants were identified as synthetic dyes in the amounts of 0.51 to 1.36% by weight. Identities of the dyes were confirmed by scanning densitometry and HPTLC-MS. A modified USP method with additional detection mode permitted the identification of eight SJW samples adulterated with dyes and six others with flavonoid fingerprints different from those specified by USP from a total of 37 samples of dry extracts, finished products, and bulk raw herb. A decision flowchart is proposed to guide the detection of adulteration of SJW in a systematic fashion.
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Medicamentos Falsificados/química , Hypericum/química , Cromatografía Líquida de Alta Presión , Extractos Vegetales/químicaRESUMEN
OBJECTIVE: The pathogenesis of pre-eclampsia (PE), a major cause of maternal and fetal mortality, is not fully understood. Digitalis-like sodium pump ligands (SPLs) are believed to be implicated in PE, as illustrated by clinical observations that DIGIBIND, a digoxin antibody that binds SPLs, lowers blood pressure (BP) in PE. We recently reported that plasma levels of marinobufagenin (MBG), a vasoconstrictor SPL, are increased four-fold in patients with PE. In the present study, we tested whether a polyclonal antibody to MBG can lower BP in rats with pregnancy-associated hypertension. METHODS: Systolic BP (SBP), 24-h renal excretion of MBG and endogenous ouabain (EO), and sodium pump activity in the thoracic aortae were measured in virgin and pregnant Sprague-Dawley rats without and with NaCl supplementation (drinking 1.8% NaCl solution). RESULTS: NaCl supplementation of virgin rats stimulated renal excretion of MBG by 60%, but not that of EO, and did not change the BP. Compared with virgin rats, the last week of pregnancy in non-NaCl-loaded rats was associated with a decrease in SBP (106 +/- 2 versus 117 +/- 2 mmHg); a moderate increase in renal excretion of MBG (97.6 +/- 4.9 versus 57.4 +/- 7.0 pmoles/24 h) and EO (36.2 +/- 4.3 versus 24.1 +/- 3.2 pmoles/24 h). NaCl-loaded pregnant rats exhibited elevation in SBP (139 +/- 3 mmHg; P < 0.01 versus non-NaCl-loaded pregnant rats), in renal excretion of MBG (160.0 +/- 17.5 pmoles/24 h; P < 0.01 versus non-NaCl-loaded pregnant rats), but not in EO, and showed fetal growth retardation. Administration of the anti-MBG antibody to NaCl-loaded pregnant rats lowered SBP (111 +/- 2 mmHg; P < 0.01) and increased aortic sodium pump activity (144 +/- 3 versus 113 +/- 5 nmol Rb/g per min; P < 0.01 versus non-NaCl-loaded pregnant rats). CONCLUSIONS: These observations provide evidence that MBG contributes to BP elevation in pregnant rats rendered hypertensive by NaCl supplementation.
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Presión Sanguínea , Bufanólidos/inmunología , Hipertensión Inducida en el Embarazo/terapia , Inmunoterapia/métodos , Animales , Anticuerpos/farmacología , Peso Corporal , Bufanólidos/orina , Ingestión de Líquidos , Femenino , Embarazo , Conejos , Ratas , Ratas Sprague-Dawley , Sodio , Cloruro de Sodio Dietético/farmacología , Cloruro de Sodio Dietético/orinaRESUMEN
An evaluation system including colorimetric assay with iodine and potassium iodide, HPSEC-MALLS-RID analysis, GC-MS analysis, and saccharide mapping based on PACE analysis was proposed for the identification and discrimination of commercial product of Hericium erinaceus based on the chemical characters of polysaccharides in H. erinaceus fruiting body collected from different regions of China. The results showed that the molecular weights, the compositional monosaccharides and the glycosidic linkages of polysaccharides in H. erinaceus collected from different regions of China were similar, respectively. However, polysaccharides in the widely consumed product of H. erinaceus in China were significantly different from those of H. erinaceus fruiting body. The implications from these results were found to be beneficial to improve the quality control of polysaccharides from the H. erinaceus fruiting body, and suggest that the proposed evaluation system could be used as a routine approach for the quality control of polysaccharides in other edible and medicinal mushrooms.
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Basidiomycota/química , Polisacáridos/química , Basidiomycota/metabolismo , Cromatografía Líquida de Alta Presión , Colorimetría , Cuerpos Fructíferos de los Hongos/química , Cuerpos Fructíferos de los Hongos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Peso Molecular , Monosacáridos/análisis , Monosacáridos/química , Monosacáridos/aislamiento & purificación , Polisacáridos/análisis , Polisacáridos/aislamiento & purificación , Análisis de Componente PrincipalRESUMEN
Elevated levels of endogenous Na/K-ATPase (NKA) inhibitors, cardiotonic steroids (CTSs) including marinobufagenin (MBG), contribute to pathogenesis of preeclampsia (PE) and represent a target for immunoneutralization by Digibind (Ovine Digoxin Immune Antibody, Glaxo-Smith Kline). Because Digibind is no longer commercially available, we studied whether DigiFab (BTG International Ltd, UK) can substitute Digibind for immunoneutralization of CTS in patients with PE. We compared DigiFab, Digibind, and anti-MBG monoclonal antibody (mAb) with respect to their ability to interact with CTS in PE plasma and to restore NKA activity in erythrocytes from patients with PE. Using immunoassays based on DigiFab, Digibind, and anti-MBG mAb, we studied the elution profile of CTS following high-performance liquid chromatography (HPLC) fractionation of PE plasma. Totally, 7 patients with mild PE (28 ± 2 years; gestational age, 39 ± 0.5 weeks; blood pressure 156 ± 5/94 ± 2 mm Hg) and 6 normotensive pregnant participants (28 ± 1 years; gestational age, 39 ± 0.4 weeks; blood pressure 111 ± 2/73 ± 2 mm Hg) were enrolled. Preeclampsia was associated with a substantial inhibition of erythrocyte NKA (1.47 ± 0.17 vs 2.65 ± 0.16 µmol Pi/mL per h in control group, P < .001). Ex vivo, at 10 µg/mL concentration, which is consistent with the clinical dosing of Digibind administered previously in PE, DigiFab and Digibind as well as anti-MBG mAb (0.5 µg/mL) restored erythrocyte NKA activity. Following HPLC fractionation of pooled PE and control plasma, PE-associated increase in CTS material was detected by Digibind (176 vs 75 pmoles), DigiFab (221 vs 70 pmoles), and anti-MBG mAb (1056 vs 421 pmoles). Therefore, because DigiFab interacts with CTS from PE plasma and reverses PE-induced NKA inhibition, it can substitute Digibind for immunoneutralization of CTS in patients with PE.