Absolute quantitation of stevioside and rebaudioside A in commercial standards by quantitative NMR.

The extract prepared from the leaves of Stevia rebaudiana BERTONI (Asteraceae) contains sweet steviol glycosides, mainly stevioside and rebaudioside A. Highly purified stevia extracts have become popular worldwide as a natural, low-calorie sweetener. They contain various types of steviol glycosides, and their main components are stevioside and rebaudioside A. The content of each steviol glycoside is quantified by comparing the ratios of the molecular weights and the chromatographic peak areas of the samples to those of stevioside or rebaudioside A standards of the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Joint Expert Committee on Food Additives (JECFA) and other specifications. However, various commercial standard reagents of stevioside and rebaudioside A are available. Their purities are different and their exact purities are not indicated. Therefore, the measured values of stevioside and rebaudioside A contained in a sample vary according to the standard used for the quantification. In this study, we utilized an accurate method, quantitative NMR (qNMR), for determining the contents of stevioside and rebaudioside A in standards, with traceability to the International System of Units (SI units). The purities of several commercial standards were determined to confirm their actual values.

Organic tailored batteries materials using stable open-shell molecules with degenerate frontier orbitals.

Secondary batteries using organic electrode-active materials promise to surpass present Li-ion batteries in terms of safety and resource price. The use of organic polymers for cathode-active materials has already achieved a high voltage and cycle performance comparable to those of Li-ion batteries. It is therefore timely to develop approaches for high-capacity organic materials-based battery applications. Here we demonstrate organic tailored batteries with high capacity by using organic molecules with degenerate molecular orbitals (MOs) as electrode-active materials. Trioxotriangulene (TOT), an organic open-shell molecule, with a singly occupied MO (SOMO) and two degenerate lowest-unoccupied MOs (LUMOs) was investigated. A tri-tert-butylated derivative ((t-Bu)(3)TOT)exhibited a high discharge capacity of more than 300 A h kg(-1), exceeding those delivered by Li-ion batteries. A tribrominated derivative (Br(3)TOT) was also shown to increase the output voltage and cycle performance up to 85% after 100 cycles of the charge-discharge processes.

NMR metabolic profiling combined with two-step principal component analysis for toxin-induced diabetes model rat using urine.

Nuclear magnetic resonance-based metabolic profiling (NMR-MP) was applied to evaluate disorder model animals using urine. Diabetic nephropathy was established in this experiment by administering streptozotocin to Wistar rats, which immediately developed diabetes after toxin-treatment and then gradually produced albumin-containing urine (albuminuria). Urine samples were collected for the first 4 weeks after toxin treatment. Predominant urinary sugar signals were seen in (1)H-NMR spectra of diabetes rat urine, and spectra were processed and subjected to multivariate analysis. Principal component analysis (PCA) identified 3 outliers among 20 individuals. Outlier rats did not develop urinary sugar and were later found to be rats insufficient to establish diabetes models. A second PCA was performed excluding the additional glucose-signal region (3.2-6.3 ppm), as glucose signals had a predominant effect that may mask details of other metabolic profiles. Consequently another outlier was revealed. This exceptional rat did not develop albuminuria even after producing glucosuria for 14 weeks. NMR metabolic profiling provides good guidance to evaluate biophysical conditions of animals, enabling detection of abnormalities in the early stage of toxicological experiments.

Multivariate analysis for 1H-NMR spectra of two hundred kinds of tea in the world.

NMR measurements coupled with pattern-recognition analysis offer a powerful mixture-analysis tool for latent-feature extraction and sample classification. As fundamental applications of this analysis for mixtures, the 1H spectra of 176 kinds of green, black, oolong and other tea infusions were acquired by a 500 MHz NMR spectrometer. Each spectrum pattern was analyzed by a multivariate statistical pattern-recognition method where Principal Component Analysis (PCA) was used in combination with Soft Independent Modeling of Class Analogy (SIMCA). SIMCA effectively selected variables that contribute to tea categorization. The final PCA resulted in clear classification reflecting the fermentation and processing of each tea, and revealed marker variables that include catechin and theanine peaks.

Pattern recognition analysis for classification of hypertensive model rats and diurnal variation using 1H-NMR spectroscopy of urine.

Urine samples were collected during the daytime and nighttime from spontaneously hypertensive model rats and normal rats without dosing. The 1H NMR spectra were measured for their urine samples, and analyzed by a pattern recognition method, known as Principal Component Analysis (PCA) and Soft Independent Modeling of Class Analogy (SIMCA). The separation of urinary data due to the diurnal variation (daytime and nighttime) and also to the difference between the two strains of rat was achieved in the PCA score plot. Differences of the urinary profiles in the respective separation were effectively extracted as marker variables by the SIMCA method. NMR measurements coupled with pattern recognition methods provide a straightforward approach to inspect the disease metabolic status and the preliminary screening tool of marker candidates for further development.

[Determination of the Plant Origin of Licorice Oil Extract, a Natural Food Additive, by Principal Component Analysis Based on Chemical Components].

"Licorice oil extract" (LOE) (antioxidant agent) is described in the notice of Japanese food additive regulations as a material obtained from the roots and/or rhizomes of Glycyrrhiza uralensis, G. inflata or G. glabra. In this study, we aimed to identify the original Glycyrrhiza species of eight food additive products using LC/MS. Glabridin, a characteristic compound in G. glabra, was specifically detected in seven products, and licochalcone A, a characteristic compound in G. inflata, was detected in one product. In addition, Principal Component Analysis (PCA) (a kind of multivariate analysis) using the data of LC/MS or (1)H-NMR analysis was performed. The data of thirty-one samples, including LOE products used as food additives, ethanol extracts of various Glycyrrhiza species and commercially available Glycyrrhiza species-derived products were assessed. Based on the PCA results, the majority of LOE products was confirmed to be derived from G. glabra. This study suggests that PCA using (1)H-NMR analysis data is a simple and useful method to identify the plant species of origin of natural food additive products.

Application of mixture analysis to crude materials from natural resources (III): NMR spectral studies to analyze chalcones from Angelica keiskei.

This study presents the application of the NMR-based analyses, DOSY and ROSY, to the chalcones, xanthoangelol (1) and 4-hydroxyderricin (2) from Angelica keiskei. We investigated whether virtual separation and structural information from each compound can be obtained. DOSY displays spectra of (1) and (2) in one dimension and diffusion spectra in the other. And the 1H slice spectra were analyzed in detail by comparison with authentic samples previously isolated from the same material. The resulting ROSY spectrum clearly showed two distinct peaks in the 1H T1 dimension. Each slice of the ROSY spectrum along the 13C dimension contains over-lapped signals, which are difficult to assign at this time.

Application to classification of mulberry leaves using multivariate analysis of proton NMR metabolomic data.

Recently, NMR-based metabolomic analysis has been used to acquire information based on differentiation among biological samples. In the present study, we examined whether multivariate analysis was able to be applied to natural products and/or material field. Each extraction of 24 leaf samples, divided into six locations from the tip of the stem in each of four strains, was analyzed by pattern recognition methods, known as Principal Component Analysis (PCA) and Soft Independent Modeling of Class Analogy (SIMCA). Twenty-four extracts from mulberry leaf showed independent spectra by 1H NMR. The separation of leaf extraction data due to the difference at six locations was achieved in the PCA score plot as correlation PC1 (86.1%) and PC3 (4.6%) and showed two loading plots, suggesting classification by leaf position as an independent variable in the loading plot. Moreover, the difference among six locations clarified the seven highest discrimination powers by the SIMCA method. Meanwhile, the PCA score plot obtained classification by the variety of mulberry strains with three loading plots, but the SIMCA method did not give a peak by classification.

Identification of Glycyrrhiza species by direct analysis in real time mass spectrometry.

DART (Direct Analysis in Real Time)-MS is a novel mass spectrometric ion source, and allows the analysis of most compounds at ambient pressure and ground potential by producing [M+H]+ molecular ion species. Using this method, we examined the compounds characteristic of several kinds of licorices. For the analysis of Glycyrrhiza inflata Batalin, the peak at m/z 339 originates mainly from [M+H]+ of licochalcone A (LA), a species-specific compound. This peak was hardly detected in G. glabra Linné and G. uralensis Fischer. These results indicate that G. inflata can be differentiated from the other two species by detection of LA peaks using DART-MS analysis.

[Absolute quantitation of quercetin and the glycosides in natural food additives by quantitative NMR].

We are developing a simple absolute quantitation method for organic compounds, by means of quantitative nuclear magnetic resonance (qNMR), with traceability to the International System of Units (SI units). The qNMR method was applied to the absolute quantitation of rutin, isoquercitrin and quercetin in natural food additives, rutin (extract), enzymatically decomposed rutin extract and quercetin, and those compounds as commercial reagents. In this study, 1,4-bis-(trimethylsilyl)benzene-d(4) (1,4-BTMSB-d(4)) whose purity was precisely evaluated on the basis of metrology, was newly used as a qNMR reference material, to be added to the sample solution as an internal standard. The contents of quercetin and quercetin glycosides were calculated from the ratio of the signal intensities of each aromatic proton at the 2' position of the three compounds (these are observed at different chemical shifts) to the eighteen protons of the six methyl groups on 1,4-BTMSB-d(4) used as a qNMR reference material. Rapid and simple qNMR method with only one step process was carried by using 1,4-BTMSB-d(4). It was demonstrated that the purities of rutin, isoquercitrin and quercetin can be separately determined by qNMR without the need for a separation process or reference materials for all the target compounds.

[Absolute quantification of carminic acid in cochineal extract by quantitative NMR].

A quantitative NMR (qNMR) method was applied for the determination of carminic acid. Carminic acid is the main component in cochineal dye that is widely used as a natural food colorant. Since several manufacturers only provide reagent-grade carminic acid, there is no reference material of established purity. To improve the reliability of analytical data, we are developing quantitative nuclear magnetic resonance (qNMR), based on the fact that the intensity of a given NMR resonance is directly proportional to the molar amount of that nucleus in the sample. The purities and contents of carminic acid were calculated from the ratio of the signal intensities of an aromatic proton on carminic acid to nine protons of three methyl groups on DSS-d6 used as the internal standard. The concentration of DSS-d6 itself was corrected using potassium hydrogen phthalate, which is a certified reference material (CRM). The purities of the reagents and the contents of carminic acid in cochineal dye products were determined with SI-traceability as 25.3-92.9% and 4.6-30.5% based on the crystalline formula, carminic acid potassium salt trihydrate, which has been confirmed by X-ray analysis. The qNMR method does not require a reference compound, and is rapid and simple, with an overall analysis time of only 10 min. Our approach thus represents an absolute quantitation method with SI-traceability that should be readily applicable to analysis and quality control of any natural product.