Rapid Structure Determination of Ranitidine Hydrochloride API in Two Crystal Forms Using Microcrystal Electron Diffraction.

The solid-state properties of drug candidates play a crucial role in their selection. Quality control of active pharmaceutical ingredients (APIs) based on their structural information involves ensuring a consistent crystal form and controlling water and residual solvent contents. However, traditional crystallographic techniques have limitations and require high-quality single crystals for structural analysis. Microcrystal electron diffraction (microED) overcomes these challenges by analyzing difficult-to-crystallize or small-quantity samples, making it valuable for efficient drug development. In this study, microED analysis was able to rapidly determine the configuration of two crystal forms (Forms 1, 2) of the API ranitidine hydrochloride. The structures obtained with microED are consistent with previous structures determined by X-ray diffraction, indicating microED is a useful tool for rapidly analyzing molecular structures in drug development and materials science research.

In Silico Prediction of N-Nitrosamine Formation Pathways of Pharmaceutical Products.

The recent discovery of N-nitrosodimethylamine (NDMA), a mutagenic N-nitrosamine, in pharmaceuticals has adversely impacted the global supply of relevant pharmaceutical products. Contamination by N-nitrosamines diverts resources and time from research and development or pharmaceutical production, representing a bottleneck in drug development. Therefore, predicting the risk of N-nitrosamine contamination is an important step in preventing pharmaceutical contamination by DNA-reactive impurities for the production of high-quality pharmaceuticals. In this study, we first predicted the degradation pathways and impurities of model pharmaceuticals, namely gliclazide and indapamide, in silico using an expert-knowledge software. Second, we verified the prediction results with a demonstration test, which confirmed that N-nitrosamines formed from the degradation of gliclazide and indapamide in the presence of hydrogen peroxide, especially under alkaline conditions. Furthermore, the pathways by which degradation products formed were determined using ranitidine, a compound previously demonstrated to generate NDMA. The prediction indicated that a ranitidine-related compound served as a potential source of nitroso groups for NDMA formation. In silico software is expected to be useful for developing methods to assess the risk of N-nitrosamine formation from pharmaceuticals.

Quantitative 31P-NMR for the Purity Determination of the Organophosphorus Compound Brigatinib and Its Method Validation.

The spectrum of 31P-NMR is fundamentally simpler than that of 1H-NMR; consequently identifying the target signal(s) for quantitation is simpler using quantitative 31P-NMR (31P-qNMR) than using quantitative 1H-NMR (1H-qNMR), which has been already established as an absolute determination method. We have previously reported a 31P-qNMR method for the absolute determination of cyclophosphamide hydrate and sofosbuvir as water-soluble and water-insoluble organophosphorus compounds, respectively. This study introduces the purity determination of brigatinib (BR), an organophosphorus compound with limited water solubility, using 31P-qNMR at multiple laboratories. Phosphonoacetic acid (PAA) and 1,4-BTMSB-d4 were selected as the reference standards (RSs) for 31P-qNMR and 1H-qNMR, respectively. The qNMR solvents were chosen based on the solubilities of BR and the RSs for qNMR. CD3OH was selected as the solvent for 31P-qNMR measurements to prevent the influence of deuterium exchange caused by the presence of exchangeable intramolecular protons of BR and PAA on the quantitative values, while CD3OD was the solvent of choice for the 1H-qNMR measurements to prevent the influence of water signals and the exchangeable intramolecular protons of BR and PAA. The mean purity of BR determined by 31P-qNMR was 97.94 ± 0.69%, which was in agreement with that determined by 1H-qNMR (97.26 ± 0.71%), thus indicating the feasibility of purity determination of BR by 31P-qNMR. Therefore, the findings of this study may provide an effective method that is simpler than conventional 1H-qNMR for the determination of organophosphorus compounds.

Significant association between HLA-B*35:01 and onset of drug-induced liver injury caused by Kampo medicines in Japanese patients.

AIM: Drug-induced liver injury (DILI) is a severe and life-threatening immune-mediated adverse effect, occurring rarely among treated patients. We examined genomic biomarkers in the Japanese population that predict the onset of DILI after using a certain class of drugs, such as Kampo products (Japanese traditional medicines). METHODS: A total of 287 patients diagnosed as DILI by hepatology specialists were recruited after written informed consent was obtained. A genome-wide association analysis and human leukocyte antigen (HLA) typing in four digits were performed. RESULTS: We found a significant association (p = 9.41 × 10-10 ) of rs146644517 (G > A) with Kampo product-related DILI. As this polymorphism is located in the HLA region, we evaluated the association of HLA types and found that 12 (63.2%) of 19 Kampo-DILI patients contained HLA-B*35:01, whereas only 15.2% were positive for this HLA among healthy volunteers. The odds ratio was 9.56 (95% confidence interval 3.75-24.46; p = 2.98 × 10-6 , corrected p = 4.17 × 10-5 ), and it increased to 13.55 compared with the DILI patients not exposed to Kampo products. The individual crude drug components in the Kampo products, including Scutellaria root (ougon in Japanese), rhubarb (daiou), Gardenia fruit (sanshishi), and Glycyrrhiza (kanzou), were significantly associated with HLA-B*35:01. CONCLUSIONS: HLA-B*35:01 is a genetic risk factor and a potential predictive biomarker for Kampo-induced DILI in the Japanese population.

Folded, undulating, and fibrous doxorubicin sulfate crystals in liposomes.

High-resolution cryogenic transmission electron microscopy (cryo-TEM) evidenced that doxorubicin sulfate crystals in liposomes (prepared by remote loading with ammonium sulfate) form folded, undulating, and fibrous crystals with a diameter of approximately 2.4 nm. An undulating, fibrous crystal considered to be undergrowth, in addition to bundles of fibrous crystals, was also observed in doxorubicin-loaded liposomes. This explains the validity of the formation of doxorubicin sulfate crystals of various shapes, e.g., curved, U-shaped, or circular, in addition to cylinder and/or rod-like crystals reported in the literature. Liposomes that do not contain crystals have inner aqueous phases with high electron density, suggesting that the doxorubicin is remotely loaded and remains as a solute without precipitation.

Advanced Solid-State NMR Analysis of Two Crystal Forms of Ranitidine Hydrochloride: Detection of 1H-14N Intra-/Intermolecular Correlations.

Understanding the characteristics of crystal polymorphism of active pharmaceutical ingredients and analyzing them with high sensitivity is important for quality of drug products, appropriate characterization strategies, and appropriate screening and selection processes. However, there are few methods to measure intra- and intermolecular correlations in crystals other than X-ray crystallography, for which it is sometimes difficult to obtain suitable single crystals. Recently, solid-state NMR has been recognized as a straightforward method for measuring molecular correlations. In this study, we selected ranitidine hydrochloride, which is known to exist in two forms, 1 and 2, as the model drug and investigated each form using solid-state NMR. In conducting the analysis, rotating the sample tube, which had a 1-mm inner diameter, increased the solid-state NMR resolution at 70 kHz. The 1H-14N dipolar-based heteronuclear multiple quantum coherence (D-HMQC) analysis revealed the intermolecular correlation of Form 1 between the N atom of the nitro group and a proton of the furan moiety, which were closer than those of the intramolecular correlation reported using single X-ray crystal analysis. Thus, 1H-14N D-HMQC analysis could be useful for characterizing intermolecular interaction in ranitidine hydrochloride crystals. In addition, we reassigned the 13C solid-state NMR signals of ranitidine hydrochloride according to the liquid-state and multiple solid-state NMR experiments.

A New Method to Visualize the Internal Morphology of Crude Drugs Using High-Resolution X-Ray Computed Tomography.

The microscopic test method (microscopic examination) used to identify crude drugs is a common method in the identification of the original plant source by determining the characteristics from a small sample quantity. However, in recent years, the number of examiners who are familiar with the microscopic examination technique has decreased. In recent years, high-resolution X-ray computed tomography (HRXCT) has been used to visualize the internal structure of plants. HRXCT scans an object using X-rays and enables visualization of the internal structure of the crude drug using a computer. Therefore, in this report, HRXCT was used to easily observe the internal morphology of crude drugs using the Ephedra Herb as an example. The same internal morphological characteristics were observed using both, microscopic examination and HRXCT methods. Image analysis using HRXCT did not require specific techniques, such as sectioning, and the same tissue could be observed from any orientation using a single scan. It afforded remarkable technical simplification and reduction in time to inspect the organization's characteristics. Therefore, image analysis using HRXCT is a useful method for crude drug identifications.

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.

Flavonoids from Sedum japonicum subsp. oryzifolium (Crassulaceae).

Twenty-two flavonoids were isolated from the leaves and stems of Sedum japonicum subsp. oryzifolium (Crassulaceae). Of these compounds, five flavonoids were reported in nature for the first time, and identified as herbacetin 3-O-xyloside-8-O-glucoside, herbacetin 3-O-glucoside-8-O-(2'''-acetylxyloside), gossypetin 3-O-glucoside-8-O-arabinoside, gossypetin 3-O-glucoside-8-O-(2'''-acetylxyloside) and hibiscetin 3-O-glucoside-8-O-arabinoside via UV, HR-MS, LC-MS, acid hydrolysis and NMR. Other seventeen known flavonoids were identified as herbacetin 3-O-glucoside-8-O-arabinoside, herbacetin 3-O-glucoside-8-O-xyloside, gossypetin 3-O-glucoside-8-O-xyloside, quercetin, quercetin 3-O-glucoside, quercetin 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, quercetin 3-O-rhamnoside-7-O-glucoside, kaempferol, kaempferol 3-O-glucoside, kaempferol 7-O-rhamnoside, kaempferol 3,7-di-O-rhamnoside, kaempferol 3-O-glucoside-7-O-rhamnoside, kaempferol 3-O-glucosyl-(1→2)-rhamnoside-7-O-rhamnoside, kaempferol 3-O-xylosyl-(1→2)-rhamnoside, kaempferol 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, myricetin 3-O-glucoside and cyanidin 3-O-glucoside. Some flavonol 3,8-di-O-glycosides were found in Sedum japonicum subsp. oryzifolium as major flavonoids in this survey. They were presumed to be the diagnostic flavonoids in the species. Flavonoids were reported from S. japonicum for the first time.

Isolation of New Colibactin Metabolites from Wild-Type Escherichia coli and In Situ Trapping of a Mature Colibactin Derivative.

Colibactin is a polyketide-nonribosomal peptide hybrid secondary metabolite that can form interstrand cross-links in double-stranded DNA. Colibactin-producing Escherichia coli has also been linked to colorectal oncogenesis. Thus, there is a strong interest in understanding the role colibactin may play in oncogenesis. Here, using the high-colibactin-producing wild-type E. coli strain we isolated from a clinical sample with the activity-based fluorescent probe we developed earlier, we were able to identify colibactin 770, which was recently identified and proposed as the complete form of colibactin, along with colibactin 788, 406, 416, 420, and 430 derived from colibactin 770 through structural rearrangements and solvolysis. Furthermore, we were able to trap the degrading mature colibactin species by converting the diketone moiety into quinoxaline in situ in the crude culture extract to form colibactin 860 at milligram scale. This allowed us to determine the stereochemically complex structure of the rearranged form of an intact colibactin, colibactin 788, in detail. Furthermore, our study suggested that we were capturing only a few percent of the actual colibactin produced by the microbe, providing a crude quantitative insight into the inherent instability of this compound. Through the structural assignment of colibactins and their degradative products by the combination of LC-HRMS and NMR spectroscopies, we were able to elucidate further the fate of inherently unstable colibactin, which could help acquire a more complete picture of colibactin metabolism and identify key DNA adducts and biomarkers for diagnosing colorectal cancer.

Synthesis of Norgestomet and its 17ß-isomer and evaluation of their agonistic activities against progesterone receptor.

Norgestomet is a synthetic progesterone derivative applied in veterinary medicine to control estrus and ovulation in cattle. Norgestomet has been widely used in the livestock industry to promote the synchronization of estrus in cattle and increase pregnancy rates. However, highly reproducible synthetic methods for Norgestomet have been rarely reported. Here, we described a method for the synthesis of Norgestomet and performed quantitative NMR analysis to determine the purity of the products. Moreover, the agonistic activity of the synthesized compounds against progesterone receptors (PRs) was evaluated using an alkaline phosphatase assay. We synthesized Norgestomet with 97.9% purity that exhibited agonistic activity against PR with EC50 values of 4.5 nM. We also synthesized the 17ß-isomer of Norgestomet with 92.7% purity that did not exhibit any PR agonistic activity. The proposed synthetic route of Norgestomet can facilitate the assessment of residual Norgestomet in foods.

Miroestrol Quantification in Pueraria mirifica Crude Drugs and Products by Single-Reference UPLC/PDA/MS Using Relative Molar Sensitivities to Kwakhurin.

Owing to occasional health damages caused by health food products derived from Pueraria mirifica (PM), the Japanese government has designated PM as an "ingredient calling for special attention." Miroestrol is a specific isoflavone isolated from PM and possesses very strong estrogenic activity enough to induce side effects in small amount. Therefore, routine analyses for miroestrol quantification is recommended to control the safety and quality of PM products. However, miroestrol content in PM is quite low, and commercial reagent for its detection is rarely available. In this study, we developed a quantitative analysis method for miroestrol in PM without using its analytical standard by using the relative molar sensitivity (RMS) of miroestrol to kwakhurin, another PM-specific isoflavone, as a reference standard. The RMS value was obtained by an offline combination of 1H-quantitative NMR spectroscopy and a LC/photo diode array (PDA) and miroestrol content was determined by single-reference LC/PDA using RMS. Furthermore, we investigated miroestrol content in commercially available PM crude drugs and products, and the RMS method was compared with the conventional calibration curve method in terms of performance. The rate of concordance of miroestrol contents determined by two method was 89-101%. The results revealed that our developed LC/PDA/MS method with RMS using kwakhurin as a reference standard was accurate for routine monitoring of miroestrol content in PM crude drugs and products to control their quality.

UHPLC/MS and NMR-Based Metabolomic Analysis of Dried Water Extract of Citrus-Type Crude Drugs.

Citrus-type crude drugs (CCDs) are commonly used to formulate decoctions in Kampo formula (traditional Japanese medicine). Our previous study reported metabolomic analyses for differentiation of the methanol extracts of Citrus-type crude drugs (CCDs) using ultra-HPLC (UHPLC)/MS, and 13C- and 1H-NMR. The present study expanded the scope of its application by analyzing four CCD water extracts (Kijitsu, Tohi, Chimpi, and Kippi); these CCDs are usually used as decoction ingredients in the Kampo formula. A principal component analysis score plot of processed UPLC/MS and NMR analysis data indicated that the CCD water extracts could be classified into three groups. The loading plots showed that naringin and neohesperidin were the distinguishing components. Three primary metabolites, α-glucose, ß-glucose, and sucrose were identified as distinguishing compounds by NMR spectroscopy. During the preparation of CCD dry extracts, some compounds volatilized or decomposed. Consequently, fewer compounds were detected than in our previous studies using methanol extract. However, these results suggested that the combined NMR- and LC/MS-based metabolomics can discriminate crude drugs in dried water extracts of CCDs.

N-Nitrosodimethylamine (NDMA) Formation from Ranitidine Impurities: Possible Root Causes of the Presence of NDMA in Ranitidine Hydrochloride.

N-Nitrosodimethylamine (NDMA) is a probable human carcinogen. This study investigated the root cause of the presence of NDMA in ranitidine hydrochloride. Forced thermal degradation studies of ranitidine hydrochloride and its inherent impurities (Imps. A, B, C, D, E, F, G, H, I, J, and K) listed in the European and United States Pharmacopeias revealed that in addition to ranitidine, Imps. A, C, D, E, H, and I produce NDMA at different rates in a solid or an oily liquid state. The rate of NDMA formation from amorphous Imps. A, C, and E was 100 times higher than that from crystalline ranitidine hydrochloride under forced degradation at 110 °C for 1 h. Surprisingly, crystalline Imp. H, bearing neither the N,N-dialkyl-2-nitroethene-1,1-diamine moiety nor a dimethylamino group, also generated NDMA in the solid state, while Imp. I, as an oily liquid, favorably produced NDMA at moderate temperatures (e.g., 50 °C). Therefore, strict control of the aforementioned specific impurities in ranitidine hydrochloride during manufacturing and storage allows appropriate control of NDMA in ranitidine and its pharmaceutical products. Understanding the pathways of the stability related NDMA formation enables improved control of the pharmaceuticals to mitigate this risk.

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.

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.

Facile Synthesis of Kwakhurin, a Marker Compound of Pueraria mirifica and Its Quantitative NMR Analysis for Standardization as a Reagent.

The side effects of kwao keur dietary supplements (obtained from the tuberous root of Pueraria mirifica) have recently been reported by the Ministry of Health, Labour and Welfare, Japan. To control the quality of kwao keur products, its ingredients need to be maintained by characteristic marker compounds, such as miroestrol, deoxymiroestrol, and kwakhurin (KWA). In this study, we described the facile synthesis of KWA, a marker compound of P. mirifica. Our revised synthetic method produced KWA with shorter steps and higher yield than the reported method. Furthermore, the absolute purity of KWA was determined by quantitative NMR analysis for standardization as a reagent, and its purity was 92.62 ± 0.12%.

Temperature-Dependent Formation of N-Nitrosodimethylamine during the Storage of Ranitidine Reagent Powders and Tablets.

The purpose of this study was to elucidate the effect of high-temperature storage on the stability of ranitidine, specifically with respect to the potential formation of N-nitrosodimethylamine (NDMA), which is classified as a probable human carcinogen. Commercially available ranitidine reagent powders and formulations were stored under various conditions, and subjected to LC-MS/MS analysis. When ranitidine tablets from two different brands (designated as tablet A and tablet B) were stored under accelerated condition (40 °C with 75% relative humidity), following the drug stability guidelines issued by the International Conference on Harmonisation (ICH-Q1A), for up to 8 weeks, the amount of NDMA in them substantially increased from 0.19 to 116 ppm and from 2.89 to 18 ppm, respectively. The formation of NDMA that exceeded the acceptable daily intake limit (0.32 ppm) at the temperature used under accelerated storage conditions clearly highlights the risk of NDMA formation in ranitidine formulations when extrapolated to storage under ambient conditions. A forced-degradation study under the stress condition (60 °C for 1 week) strongly suggested that environmental factors such as moisture and oxygen are involved in the formation of NDMA in ranitidine formulations. Storage of ranitidine tablets and reagent powders at the high temperatures also increased the amount of nitrite, which is considered one of the factors influencing NDMA formation. These data indicate the necessity of controlling/monitoring stability-related factors, in addition to controlling impurities during the manufacturing process, in order to mitigate nitrosamine-related health risks of certain pharmaceuticals.

Quality Evaluation and Characterization of Fractions with Biological Activity from Ephedra Herb Extract and Ephedrine Alkaloids-Free Ephedra Herb Extract.

Previously, we reported that the c-Met inhibitory effect of Ephedra Herb extract (EHE) is derived from ingredients besides ephedrine alkaloids. Moreover, analgesic and anti-influenza activities of EHE and ephedrine alkaloids-free Ephedra Herb extract (EFE) have been reported recently. In this study, we examined the fractions containing c-Met kinase inhibitory activity from EHE and the fractions with analgesic and anti-influenza activities from EFE, and elucidated the structural characteristics of the active fractions. Significant c-Met kinase activity was observed in 30, 40, and 50% methanol (MeOH) eluate fractions obtained from water extract of EHE using Diaion HP-20 column chromatography. Similarly, 20 and 40% MeOH, and MeOH eluate fractions obtained from water extract of EFE were found to display analgesic and anti-influenza activities. Reversed phase-HPLC analysis of the active fractions commonly showed broad peaks characteristic of high-molecular mass condensed tannin. The active fractions were analyzed using 13C-NMR and decomposition reactions; the deduced structures of active components were high-molecular mass condensed tannins, which were mainly procyanidin B-type and partly procyanidin A-type, including pyrogallol- and catechol-type flavan 3-ols as extension and terminal units. HPLC and gel permeation chromatography (GPC) analyses estimated that the ratio of pyrogallol- and catechol-type was approximately 9 : 2, and the weight-average molecular weight based on the polystyrene standard was >45000. Furthermore, GPC-based analysis was proposed as the quality evaluation method for high-molecular mass condensed tannin in EHE and EFE.