H-NMR Method Research Articles

A multivariate curve resolution-alternating least squares (MCR-ALS) technology assisted 1 H-NMR methodology for multi-component quantitation of Trichosanthis Pericarpium injection.

Trichosanthis Pericarpium injection (TPI) is a traditional Chinese medicine preparation obtained from Trichosanthis Pericarpium by extraction, purification and sterilisation. It contains amino acids, alkaloids, nucleotides and other components. Existing quantitative methods only analyse a few components in injections, so this study intends to develop a method for comprehensive analysis of TPI components. To develop a method for quantification of components in TPI by multivariate curve resolution-alternating least squares (MCR-ALS) assisted proton nuclear magnetic resonance (1 H-NMR). A 1 H-NMR method was developed for the quantification of components in TPI. For components with independent signals, 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TSP) was used as an internal standard to calculate the component contents. For components with overlapping signals, the method of MCR-ALS was used. A total of 36 components were identified in TPI, of which 33 were quantified. Methodological validation results showed that the developed 1 H-NMR method has good linearity, accuracy, precision, robustness and specificity. The use of 1 H-NMR provides a reliable and universal method for the TPI components identification and quantification. Also, it can be used as a powerful tool for analysing the contents in a complex mixture as a quality control measure.

1 H-NMR Based Metabolomics Profiling of Citrus Juices Produced in Veracruz, México.

This study describes the 1 H-NMR-based metabolomics profiling of juices from citrus fruits harvested in the state of Veracruz, México. The hydrophilic profile of commercial lemons (Agrio and Persian), tangerines (Fremont and Mónica), oranges (Valencia and Washington Navel), and grapefruits (Red Ruby and Rio Red) was determined. According to our results, 35 metabolites were identified in the 1 H-NMR profiling. The statistical differences obtained by PCA and OPLS-DA revealed that specific amino acids, sugars, and organic acids were differential metabolites in the species and cultivars studied. High endogenous levels of sucrose (10-190 mM), α-glucose, β-glucose (α- and β-isomers, 40-205 mm), and fructose (36-170 mm) were detected in the juices of grapefruits, oranges, and tangerines, whereas citric acid (40-530 mm) was the principal organic acid in the juices of lemons. To calculate the specific amounts of metabolites from these species and their cultivars, the results were finely analyzed using the qNMR method. According to these calculations, Valencia oranges had the highest concentration of ascorbic acid (>2 mm). The described 1 H-NMR method is highly reproducible, inexpensive, and highly robust in comparison to other analytical methods used to determine the hydrophilic profile of citrus juices.

Study on Selectivity of Tungstate-based catalyst in Epoxidation Reaction of modified and non-modified Rubber Seed Oil

The selectivity of tungstate-based catalyst in the epoxidation reaction of rubber seed oil and modified rubber seed oil was studied in two ways. The titration method was performed according to the ASTM standard and the H-NMR method was based on the peaks of the three different standard groups. The catalyst selectivity of the epoxidation reaction of modified rubber seed oil was much higher than that of non-modified rubber seed oil for both methods. The average catalyst selectivity obtained by using H-NMR method of the modified rubber seed oil epoxidation reaction was equal, while of the rubber seed oil epoxidation reaction was 11% lower than that of the titrimetric method. For both types of oils, the difference between the catalytic selectivity obtained by using titration method and by using protons in group α-CH2-(C=O)-O- as the standard peak was the smallest and was the biggest when the protons in group -CH3 was used as the standard peak.

Combined Application of UHPLC-QTOF/MS, HPLC-ELSD and 1 H-NMR Spectroscopy for Quality Assessment of DA-9801, A Standardised Dioscorea Extract.

DA-9801, a standardised 50% aqueous ethanolic extract of a mixture of Dioscorea japonica and D. nipponica, is a botanical drug candidate for the treatment of diabetic neuropathy, which finished its US phase II clinical trials recently. An advanced quality control method is needed for further development of DA-9801, considering its high contents of both primary and secondary metabolites. Development of a quality assessment strategy for DA-9801, based on the combination of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H-NMR spectroscopy. The method was developed and tested with 15 batch products of DA-9801. The steroidal saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS and were quantified with the validated HPLC-ELSD method. Primary metabolites of DA-9801 were identified and profiled using 1 H-NMR spectrometry. The batch-to-batch equivalence of DA-9801 was tested with the 1 H-NMR spectra using spectral binning, correlation analysis, and principal component analysis. Six major saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS. Among them, protodioscin and dioscin were quantified by the validated HPLC-ELSD method. Twenty-six metabolites were identified in 1 H-NMR spectra. The similarity between DA-9801 batches could be evaluated with the NMR spectra of DA-9801. The 1 H-NMR method also revealed that two Dioscorea species contributed distinct amino acids to the contents of DA-9801. This study validates the effectiveness of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H NMR-combined method for quality control of DA-9801 and its crude materials. Copyright © 2016 John Wiley & Sons, Ltd.

(1)H NMR Metabolic Fingerprinting to Probe Temporal Postharvest Changes on Qualitative Attributes and Phytochemical Profile of Sweet Cherry Fruit.

Sweet cherry fruits (Prunus avium cvs. ‘Canada Giant’, ‘Ferrovia’) were harvested at commercial maturity stage and analyzed at harvest and after maintenance at room temperature (storage at ∼20°C, shelf life) for 1, 2, 4, 6, and 8 days, respectively. Fruit were initially analyzed for respiration rate, qualitative attributes and textural properties: ‘Canada Giant’ fruit were characterized by higher weight losses and stem browning index, being more intense over the late stages of shelf life period; meanwhile ‘Ferrovia’ possessed appreciably better performance even after extended shelf life period. A gradual decrease of respiration rate was monitored in both cultivars, culminated after 8 days at 20°C. The sweet cherry fruit nutraceutical profile was monitored using an array of instrumental techniques (spectrophotometric assays, HPLC, 1H-NMR). Fruit antioxidant capacity was enhanced with the progress of shelf life period, concomitant with the increased levels of total anthocyanin and of phenolic compounds. ‘Ferrovia’ fruit presented higher contents of neochlorogenic acid and p-coumaroylquinic acid throughout the shelf life period. We further developed an 1H-NMR method that allows the study of primary and secondary metabolites in a single running, without previous separation and isolation procedures. Diagnostic peaks were located in the aliphatic region for sugars and organic acids, in the aromatic region for phenolic compounds and at 8.2–8.6 ppm for anthocyanins. This NMR-based methodology provides a unifying tool for quantitative and qualitative characterization of metabolite changes of sweet cherry fruits; it is also expected to be further exploited for monitoring temporal changes in other fleshy fruits.

Application of quantitative 1H-NMR method to determination of gentiopicroside in Gentianae radix and Gentianae scabrae radix

A quantitative (1)H-NMR method (qHNMR) was used to measure gentiopicroside content in Gentianae radix and Gentianae scabrae radix. Gentiopicroside is a major component of Gentianae radix and Gentianae scabrae radix. The purity of gentiopicroside was calculated from the ratio of the intensity of the H-3 signal at δ 7.44ppm or the H-8 signal at δ 5.78ppm in methanol-d 4 of gentiopicroside to that of a hexamethyldisilane (HMD) signal at 0ppm. The concentration of HMD was corrected with SI traceability by using potassium hydrogen phthalate of certified reference material (CRM) grade. As a result, the gentiopicroside content in two lots of Gentianae radix as determined by qHNMR was found to be 1.76 and 2.17%, respectively. The gentiopicroside content in two lots of Gentianae scabrae radix was 2.73 and 3.99%, respectively. We demonstrated that this method is useful for the quantitative analysis of crude drugs.

Application of a quantitative 1H-NMR method for the determination of amygdalin in Persicae semen, Armeniacae semen, and Mume fructus

A quantitative (1)H-NMR method (qHNMR) was used to measure the amygdalin content of Persicae semen, Armeniacae semen, and Mume fructus, in each of which amygdalin constitutes a major component. The purity of amygdalin was calculated from the ratio of the intensity of the amygdalin H-2 signal at δ 6.50 ppm in pyridine-d 5 to that of the hexamethyldisilane (HMD) signal at 0 ppm. The HMD concentration was corrected by the International System of Units (SI) traceability with certified reference material (CRM)-grade bisphenol A. qHNMR revealed the amygdalin contents to be 2.72 and 3.13% in 2 lots of Persicae semen, 3.62 and 5.19% in 2 lots of Armeniacae semen, and 0.23% in Mume fructus. Thus, we demonstrated the utility of this method for the quantitative analysis of crude drugs.

Developmental Changes in the Composition of Five Anthraquinones from Rheum palmatum as Quantified by 1H‐NMR

Rheum palmatum is an important traditional Chinese medicine featuring anthraquinones with several activities. Generally, rhein, emodin, aloe-emodin, physcion and chrysophanol are used as chemical markers for the quality control of rhubarb products. To develop a simple protocol for the quantification of rhein, emodin, aloe-emodin, physcion and chrysophanol in R. palmatum collected at different developmental stages. (1) H-NMR spectra were measured on samples dissolved in acetone-d6 , quantification was carried out using the signals of H-4 of rhein (δH 8.36), H-7 of emodin (δH 6.68), CH2 OH of aloe-emodin (δH 4.81), OCH3 of physcion (δH 4.02) and CH3 of chrysophanol (δH 2.50), which were well separated from other signals. Quantitative analysis was based on the relative ratio of the intensity of each compound to the known amount of internal standard maleic acid. The quantitative (1) H-NMR (qHNMR) method developed showed good precision, trueness, linearity, repeatability and stability for the quantification of rhein, emodin, aloe-emodin, physcion and chrysophanol. This method was applied successfully to explore the seasonal variations of the five major anthraquinones in R. palmatum, and provided quantitative results in reasonable agreement with those obtained by the HPLC-UV method. Compared with the conventional HPLC-based methods, the qHNMR analysis is rapid, reference-free and convenient with less sample pre-treatment. This technique should be a feasible choice for the quality control of R. palmatum.

Application of quantitative 1H-NMR method to determination of paeoniflorin in Paeoniae radix

A quantitative (1)H-NMR method (qHNMR) was used to measure paeoniflorin content in Paeoniae radix (dried root of Paeonia lactiflora), of which paeoniflorin is a major component. The purity of paeoniflorin was calculated from the ratio of the intensity of the H-9 signal at δ 5.78 ppm of paeoniflorin to that of a hexamethyldisilane (HMD) signal at 0 ppm. The concentration of HMD was corrected with SI traceability by using bisphenol A of certified reference material (CRM) grade. The paeoniflorin content in 2 separate samples of Paeoniae radix was determined by qHNMR and was found to be 2.15 and 2.45%. We demonstrated that this method is useful for quantitative analysis of crude drugs.

The Measurement of the β/α Anomer Composition Within Amorphous Lactose Prepared by Spray and Freeze Drying Using a Simple 1H-NMR Method

Reports of the anomeric composition of amorphous lactose are rare and state a highly variable range of composition (between 0% and 60% w/w β content). We aimed to develop a quantitative measurement by (1)H-NMR of α and β anomer content in amorphous lactose produced by different production methods. Amorphous lactose was prepared by spray and freeze drying 10% w/v aqueous solutions of lactose. NMR analysis was performed in DMSO; peak areas of partially resolved doublets at 6.3 and 6.6ppm were used to calculate % of α and β lactose present. Polarimetery was used to determine optical rotation of lactose solutions. Observed specific rotation for supplied crystalline alpha lactose monohydrate of 88° recorded in DMSO was constant for the length of a typical NMR experiment (max. 10min). β/α anomer contents of amorphous lactose measured by (1)H-NMR had standard deviations as low as 0.1% w/w (n = 6). Drying a lactose solution 4h after its preparation led to almost 35% w/w difference in anomer composition within solid amorphous material compared to samples dried after only 30min, e.g. for freeze dried samples, β content was 60 ± 0.1% w/w (4h) and 25 ± 1.0% w/w (30min). Mutarotation leads to this increase in β anomer concentration in aqueous solution and within the solid amorphous lactose stored at 25°C. e.g. after 56 d storage the β content of freeze dried lactose (30 min solution) increased from 25±1.0% to 50±0.5% w/w. A simple solution-based (1)H-NMR method for measurement of anomeric composition of lactose has been established. The solution β/α ratio at the time of drying is mirrored in the composition of the resulting solid amorphous material. In order to produce a consistent anomer composition within spray and freeze dried amorphous lactose, the standing time for the feed solution should be greater than 4 h, such that the most dynamic region of the mutarotation profile has been exceeded. If the amorphous material has been formed from a solution that has not been allowed to equilibrate for 4 h, the resulting solid will continue to undergo mutarotation if trace amounts of moisture are present, until the anomeric β/α ratio slowly approaches 1.7.

Isovaline in naturally occurring peptides: A nondestructive methodology for configurational assignment

The nonproteinogenic, C(α)-tetrasubstituted, helicogenic, chiral α-amino acid isovaline (Iva) is remarkably spread in the biosphere. Together with its achiral, lower homolog α-aminoisobutyric acid (Aib), it represents a characteristic marker of a class of naturally occurring peptide antibiotics, for which the acronym "peptaibiotics" became established. In these peptides, Iva occurs as the (S)-(= L) or the (R)-(= D) enantiomer, but peptide sequences containing both Iva enantiomers are also common. Here, we applied our recently developed (1)H-NMR method, which enables the nondestructive assignment of the configuration of each Iva residue in a peptide of known helical screw sense, to natural and synthetic peptaibiotics. Our method proved to be generally applicable and provided evidence that, in the peptaibiotic bergofungin A, the Iva(12) configuration is (R) and not (S) as reported previously. Moreover, we extended our NMR method by including a (13)C-NMR parameter. A statistical analysis of the preferred main- and side-chain conformations of the Iva residues in peptides, performed based on their published X-ray diffraction structures, allowed us to provide a sound rationale to the NMR criteria exploited to establish the configuration of this amino acid.

Application of 1H-NMR spectroscopy to validation of berberine alkaloid reagents and to chemical evaluation of Coptidis Rhizoma

Berberine, palmatine, and coptisine are major pharmacologically active protoberberine alkaloids in Coptidis Rhizoma, and have been used as indices for chemical evaluation of the crude drug. (1)H-NMR spectroscopy was applied to determination of purities of commercial reagents of protoberberine alkaloids. The purities of the alkaloids were calculated from the ratios of the intensities of the H-13 singlet signal at about δ 8.7 ppm of target protoberberine alkaloids to integration of a hexamethyldisilane (HMD) signal at 0 ppm. The concentration of HMD was corrected with SI traceability using potassium hydrogen phthalate of certified reference material (CRM) grade. The purity of the reagent estimated by the (1)H-NMR was, in general, lower than that claimed by the manufacturer, leading to over-estimation of the alkaloid contents of Coptidis Rhizoma when determined by HPLC. The present quantitative (1)H-NMR method was also applicable to direct determination of protoberberine alkaloid contents in Coptidis Rhizoma.

1H-NMR Studies of Metabolic Effects of Endo- and Exogeneous Compounds on Astroglia Cells Cultivated In Vitro

A 1 H-NMR method is described for the study of metabolic reactions in-vitro of neonatal cerebellar rat astroglia cells cultivated on micro carrier beads. In the NMR tube, the formation of metabolic effects on astroglia cells caused by addition of nutrients (pyruvate and fumarate) or neurotoxins can be monitored. Metabolic changes of both endogenous metabolites and toxins, 1,2-epoxybutane and ethyl acrylate are investigated. 1,2,4-Benzenetricarboxylate (BTC) served as an extracellular reference. Dy(ATP) -1 was used to depress signals from extracellular compounds. 6-Diazo-5-oxo-L- norleucin was added to cause accumulation of conjugated intracellular metabolites to an extent that permitted detection by 1 H-NMR spectroscopy. 1,2-Epoxybutane is shown to be detoxified due to a transferase reaction involving conjugation of reduced glutathione. The sulphur is coupled to carbon-1 or carbon-2 of the epoxide. Addition of ethyl acrylate leads in a similar way to a C1-GSH derivative. The metabolic status of the astroglia cells were monitored during different exposure conditions.

Quantification of artemisinin in Artemisia annua extracts by 1H‐NMR

Artemisinin is a polycyclic sesquiterpene lactone that is highly effective against multidrug-resistant strains of Plasmodium falciparum, the etiological agent of the most severe form of malaria. Determination of artemisinin in the source plant, Artemisia annua, is a challenging problem since the compound is present in very low concentrations, is thermolabile and unstable, and lacks chromophoric or fluorophoric groups. The ain of this study was to develop a simple protocol for the quantification of artemisinin in a plant extract using an (1)H-NMR method. Samples were prepared by extraction of leaf material with acetone, treatment with activated charcoal to remove chlorophylls and removal of solvent. (1)H-NMR spectra were measured on samples dissolved in deuterochloroform with tert-butanol as internal standard. Quantification was carried out using the using the delta 5.864 signal of artemisinin and the delta 1.276 signal of tert-butanol. The method was optimised and fully validated against a reference standard of artemisinin. The results were compared with those obtained from the same samples quantified using an HPLC-refractive index (RI) method. The (1)H-NMR method gave a linear response for artemisinin within the range 9.85-97.99 mm (r(2) = 0.9968). Using the described method, yields of artemisinin in the range 0.77-1.06% were obtained from leaves of the A. annua hybrid CPQBA x POP, and these values were in agreement with those obtained using an HPLC-RI.

Functional group analysis of high-molecular weight compounds in the water-soluble fraction of organic aerosols

In this study, we present the analysis of functional groups of humic-like substances (HULIS) isolated from atmospheric aerosols (<1 μm). Aerosol samples were collected in an urban area (Dübendorf, Switzerland) both in summer and winter. Quantification of carboxylic, arylic, phenolic and aliphatic functional groups of HULIS was performed by a specially adapted and optimized H-NMR method. The concentrations of carboxylic, arylic, phenolic and aliphatic functional groups were between 9×10 −11 and 6×10 −8 mol/m 3 for all samples, corresponding up to 14% of the total HULIS mass. A good correlation between the H-NMR results and the potentiometric titration of carboxylic groups was observed for all winter and summer samples. The p K distributions of carboxylic groups of HULIS were calculated from potentiometric titration data. p K spectra showed that p K values of most carboxylic groups is between 3 and 5. The H-NMR data show that the content of aromatic groups is higher in winter than in summer. This may either be due to emission of aromatic compounds by wood burning or to slower degradation reaction of aromatics in winter.

The first preparation of enantiopure 1‐methyl‐7‐oxabicyclo[2.2.1]heptan‐2‐one, a versatile chiral building block for terpenoids

Methods for the resolution of (+/-)-1-methyl-7-oxabicyclo[2.2.1]heptan-2-one 1, a versatile chiral building block for terpenoids, have been investigated. While no efficient result was obtained with kinetic resolution methods, both enantiomers of 1 were prepared optically pure for the first time via esterification of the reductive products of 1 with (+)-mandelic acid and oxidation of the saponified products of diastereomer esters, in an overall yield of 70%. The absolute configurations of (-)-1 and (+)-1 were determined as (1S,4R)-(-)-1 and (1R,4S)-(+)-1 by the CD exciton chirality method and confirmed by Moshers (1)H-NMR method.

Quantitative analysis of cannabinoids from Cannabis sativa using 1H-NMR.

A (1)H-NMR method has been developed for the quantitative analysis of pure cannabinoids and for cannabinoids present in Cannabis sativa plant material without any chromatographic purification. The experiment was performed by the analysis of singlets in the range of delta 4.0-7.0 in the (1)H-NMR spectrum, in which distinguishable signals of each cannabinoid are shown. Quantitation was performed by calculating the relative ratio of the peak area of selected proton signals of the target compounds to the known amount of the internal standard, anthracene. For this method no reference compounds are needed. It allows rapid and simple quantitation of cannabinoids with a final analysis time of only 5 min without the need for a pre-purification step.

Development of Super Water-absorbent from Cellulosic Materials (Part 2)

Water absorbents were developed from a bleached hardwood kraft pulp by carboxymethylation (CM-LBKP) and cross-linking with ethylene glycol diglycidyl ether under different conditions. For the determination of degree of substitution of carboxymethyl group in the cross-linked materials, three different methods, atomic absorption method, copper precipitation method, 'H-NMR method, were evaluated. It was found that atomic absorption method is most applicable to these materials. Number of cross -linking agent bound to CM-LBKP was calculated and the cross-linking density of products was evalu-ated in terms of the cross-linking reaction conditions and its affect on the water absorbency.

Study of non-isothermal crystallization kinetics and sequence distribution in poly(ethylene terephthalate-co-isophthalate)

The non-isothermal crystallization kinetics of poly(ethylene terephthalate-co-isophthalate) (PEIT) with different isophthalate contents (4, 8 and 12%) were studied by differential scanning calorimetry. Avrami exponent n was obtained. It was found that the n values of PEIT4 and PEIT8 are same as that of poly(ethylene terephthalate) (PET). The n value of PEIT12 is larger than that of PET. PEIT crystallization mechanism appears different with varied isophthalate composition. The sequence distribution in macromolecular chain of PEIT was also investigated by 1 H-NMR method. It revealed that PEIT4 and PEIT8 are block-copolymers with isophthalate sequence length >5, and PEIT12 is a random copolymer in which most isophthalate sequence length is 2 or 1. The relationship between the crystallization mechanism and sequence distribution characteristic of PEIT has been discussed.