Nannostelium ampullaceum gen. et sp. nov., a tiny new member of the protosteloid amoeba of the Cavosteliida (Variosea, Amoebozoa).

Protosteloid amoebae are slime molds characterized by simple sporocarp comprised only of a stalk and typically single spore. Recent molecular phylogenetic studies showed that protosteloid amoebae are scattered among the Amoebozoa, but most of them belongs to the Variosea. The Cavosteliida is one of the largest protosteloid group of the Variosea. We have established novel protosteloid amoeba strain YIP-63 from the fruiting body of jelly fugus Auricularia sp. The strain YIP-63 is unique to have a tiny sporocarp and amoeba comparing to the other protosteloid species, and trophic amoebae forming both round and branched shape. The molecular phylogenetic analyses based on 18S rRNA gene suggest that YIP-63 represents a novel lineage in the Cavosteliida. Therefore, we propose the new genus and new species for the strain YIP-63 as Nannostelium ampullaceum gen. et sp. nov. We provide morphological and molecular data on this novel protosteloid amoeba.

Investigation of the Fracture Behaviour of Al-CFRP Cross-Lap Joint Fabricated by Coaxial One-Side Resistance Spot Welding.

In the present research, coaxial one-side resistance spot welding was performed to join Al5052 and CFRP sheets with different welding currents. The mechanical performance of the cross-lap joint was clarified experimentally. The cross-section of the welded joint and the fracture surfaces was subjected to multi-scale characterization. The fracture behaviours and mechanisms of cross-lap joints are discussed in detail. The results showed that the thermal degradation of CFRP was detected on the cross-section under a 6000 A welding current and the O element was enriched in the decomposed area. The joining zone could be divided into four subregions according to their morphology, which were defined, from outside to inside, as the squeezed zone, the adhesion zone, the cohesion zone and the decomposed zone. After welding, the O-C=O bond disappeared on the CFRP surface while the O=C-N bond was detected on the Al5052 surface. The cross-lap joints demonstrated brittle and ductile fracture behaviours in a cross-tension test, which included two sub-modes: brittle-transition mode and ductile-degradation mode. The transformation of failure modes had a relationship with the heat input and corresponding joining zone composition. The maximum cross-tension load was about 1279 ± 40 N with a welding current of 5600 A.

[High Purity Solvents for qNMR Measurement].

Quantitative NMR (qNMR) has been adopted by documentary standards, including the Japanese Pharmacopoeia (JP), United States Pharmacopoeia (USP), and International Organization for Standardization (ISO), owing to its reliability and efficiency. Note that qNMR can be used for quantifying target components using the signal integration ratio of an analyte to a reference. In qNMR, a modern NMR instrument with high resolution and sensitivity is used to record reliable spectra. This instrument can detect small signals from impurities in a solvent, which may result in inaccurate signal integration in the spectrum. In this study, we investigated the influence of solvent quality on qNMR accuracy focusing on organic impurities, water content, and deuteration ratio. If signals from organic impurities and signals from the analyte overlap, the duplication of signal integration will directly affect the qNMR analytical result. To examine overlapping, we performed blank solvent tests. Additionally, a high water content and low deuteration ratio affect the detection sensitivity, thus reducing the signal-to-noise (S/N) ratio of the target. Thus, these factors must be considered to obtain accurate qNMR results.

Re-examination of a rare protosteloid amoeba Schizoplasmodiopsis micropunctata, and the revision of Tychosporium (Cavosteliida, Variosea, Amoebozoa).

The genus Schizoplasmodiopsis is one of the most morphologically diverse groups among the class Variosea. Recent phylogenetic studies suggest that Schizoplasmodiopsis is polyphyletic, but there are few taxonomic studies of this genus. We established S. micropunctata strain YIP-40, observed in detail its of morphology and lifecycle, and conducted a phylogenetic analysis. The phylogenetic analysis revealed that S. micropunctata was sister to Tychosporium acutostipes. Scanning electron microscopy showed S. micropunctata had a non-deciduous hilum structure that is unique to Tychosporium. The morphology of amoebae, mitotic behavior, and prespore cells of S. micropunctata also supported the close relationship to Tychosporium. We propose to transfer S. micropunctata to Tychosporium and emend the generic concept of Tychosporium to include this species.

Quantitative 31P-NMR for Purity Determination of Sofosbuvir and Method Validation.

Quantitative 1H-NMR (1H-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for quantitation because of their overlap and complexity. Therefore, we focused on the 31P nucleus because of the simplicity of its signals and previously reported 31P-qNMR in D2O. Here we report 31P-qNMR of an organophosphorus compound, sofosbuvir (SOF), which is soluble in organic solvents. Phosphonoacetic acid (PAA) and 1,4-bis(trimethylsilyl)benzene-d4 (1,4-BTMSB-d4) were used as reference standards for 31P-qNMR and 1H-qNMR, respectively, in methanol-d4. The purity of SOF determined by 31P-qNMR was 100.63 ± 0.95%, whereas that determined by 1H-qNMR was 99.07 ± 0.50%. The average half bandwidths of the 31P signal of PAA and SOF were 3.38 ± 2.39 and 2.22 ± 0.19 Hz, respectively, suggesting that the T2 relaxation time of the PAA signal was shorter than that of SOF and varied among test laboratories. This difference most likely arose from the instability in the chemical shift due to the deuterium exchange of the acidic protons of PAA, which decreased the integrated intensity of the PAA signal. Next, an aprotic solvent, dimethyl sulfoxide-d6 (DMSO-d6), was used as the dissolving solvent with PAA and sodium 4,4-dimethyl-4-silapentanesulfonate-d6 (DSS-d6) as reference standards for 31P-qNMR and 1H-qNMR, respectively. SOF purities determined by 31P-qNMR and 1H-qNMR were 99.10 ± 0.30 and 99.44 ± 0.29%, respectively. SOF purities determined by 31P-qNMR agreed with the established 1H-qNMR values, suggesting that an aprotic solvent is preferable for 31P-qNMR because it is unnecessary to consider the effect of deuterium exchange.

Purity Determination of Cyclophosphamide Hydrate by Quantitative 31P-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 sodium 4,4-dimethyl-4-silapentanesulfonate-d6 (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.

Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 2).

As a new absolute quantitation method for low-molecular compounds, quantitative NMR (qNMR) has emerged. In the Japanese Pharmacopoeia (JP), 15 compounds evaluated by qNMR are listed as reagents used as the HPLC reference standards in the assay of crude drug section of the JP. In a previous study, we revealed that humidity affects purity values of hygroscopic reagents and that (i) humidity control before and during weighing is important for a reproducible preparation and (ii) indication of the absolute amount (not purity value), which is not affected by water content, is important for hygroscopic products determined by qNMR. In this study, typical and optimal conditions that affect the determination of the purity of ginsenoside Rb1 (GRB1), saikosaponin a (SSA), and barbaloin (BB) (i.e., hygroscopic reagents) by qNMR were examined. First, the effect of humidity before and during weighing on the purity of commercial GRB1, with a purity value determined by qNMR, was examined. The results showed the importance afore-mentioned. The results of SSA, which is relatively unstable in the dissolved state, suggested that the standardization of humidity control before and during weighing for a specific time provides a practical approach for hygroscopic products. In regard to BB, its humidity control for a specific time, only before weighing, is enough for a reproducible purity determination.

Absolute Purity Determination of a Hygroscopic Substance, Indocyanine Green, Using Quantitative NMR (qNMR).

Quantitative NMR (qNMR) is applied to determine the absolute quantitative value of analytical standards for HPLC-based quantification. We have previously reported the optimal and reproducible sample preparation method for qNMR of hygroscopic reagents, such as saikosaponin a, which is used as an analytical standard in the assay of crude drug section of Japanese Pharmacopoeia (JP). In this study, we examined the absolute purity determination of a hygroscopic substance, indocyanine green (ICG), listed in the Japanese Pharmaceutical Codex 2002, using qNMR for standardization by focusing on the adaptation of ICG to JP. The purity of ICG, as an official non-Pharmacopoeial reference standard (non-PRS), had high variation (86.12 ± 2.70%) when preparing qNMR samples under non-controlled humidity (a conventional method). Additionally, residual ethanol (0.26 ± 0.11%) was observed in the non-PRS ICG. Next, the purity of non-PRS ICG was determined via qNMR when preparing samples under controlled humidity using a saturated sodium bromide solution. The purity was 84.19 ± 0.47% with a lower variation than that under non-controlled humidity. Moreover, ethanol signal almost disappeared. We estimated that residual ethanol in non-PRS ICG was replaced with water under controlled humidity. Subsequently, qNMR analysis was performed when preparing samples under controlled humidity in a constant temperature and humidity box. It showed excellent results with the lowest variation (82.26 ± 0.19%). As the use of a constant temperature and humidity box resulted in the lowest variability, it is recommended to use the control box if the reference ICG standard is needed for JP assays.

[Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 1)].

Quantitative NMR (qNMR) has been developed as an absolute quantitation method to determine the purity or content of organic compounds including marker compounds in crude drugs. The "qNMR test" has been introduced into the crude-drug section of the Japanese Pharmacopoeia (JP) for determining the purity of reagents used for the assay in the JP. In Supplement II to the JP 17th edition published in June 2019, fifteen compounds adopted qNMR test were listed as the reagents for the assay. To establish the "qNMR test" in the crude drug section of the JP, there were several problems to be solved. Previously, we reported that the handling impurity signals from reference substances and targeted marker compounds, chemical shifts of reference substances, and peak unity of signals of targeted marker compounds are important factors to conduct qNMR measurements with intended accuracy. In this study, we investigated that the hygroscopicity of reagents could cause the changes in the compounds' purity depending on increasing their water content. Twenty-one standard products used for the crude-drug test in JP were examined by water sorption-desorption analysis, and ginsenosides and saikosaponins were found to be hygroscopic. To prepare a sample solution of saikosaponin b2 for qNMR analysis, samples need to be maintained for 18 h at 25°C and 76% relative humidity; further, samples need to be weighed at the same humidity for the qNMR analysis.

Collaborative Study to Validate Purity Determination by 1H Quantitative NMR Spectroscopy by Using Internal Calibration Methodology.

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.