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Quantitative

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.

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.

Recently, quantitative NMR (qNMR), especially

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.

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 (

The azaspiracids (AZAs) are a group of marine toxins implicated in several intoxications whose mechanism of action is unknown. These phycotoxins include the five compounds shown in : AZA-1 (1), AZA-2 (2), AZA-3 (3), AZA-4 (4), and AZA-5 (5). The aim of this work was to study the effects of the five naturally occurring azaspiracids (AZA-1 to -5, Fig. 1) and four synthetic analogues (6-9, Fig. 2) on intracellular pH, and the influence of Ca2+ upon this effect. The AZAs (1-5) were found to modulate cytosolic Ca2+ levels in human lymphocytes, while some of them, but not all, had effects on the intracellular pH. AZA-1 (1) and AZA-2 (2) did not modify intracellular pH in a Ca2+-containing or a Ca2+-free medium. AZA-3 (3) increased intracellular pH by 0.16 units in the presence of extracellular Ca2+, an effect that was blocked when a 1 mM solution of Ni2+ was added. In a Ca2+-free medium, the increase in pH induced by AZA-3 (3) was reduced to 0.08 pH units. AZA-4 (4) inhibited the basal pH increase even in the presence of a 1 mM solution of Ni2+. In a Ca2+-free medium, the inhibition caused by AZA-4 (4) was small, but when Ca2+ was added back to the medium, the pH basal increase was again significantly inhibited. The alkalinization was also inhibited when AZA-4 (4) was added simultaneously, 10 min before or 10 min after thapsigargin (Tg), and also when the Ca2+-influx induced by Tg was inhibited by Ni2+. AZA-5 (5), on the other hand, did not modulate the intracellular pH profile in either a Ca2+-containing or a Ca2+-free medium. Finally, we investigated four synthetic analogues (6-9, Fig. 2) whose structures were based on the four originally proposed structures of azaspiracid-1, with an opened E-ring. Compound 6 induced a small cytosolic Ca2+ increase, but did not modify intracellular pH in saline solution. In a Ca2+-free medium, compound 6 blocked the pH fall when Ca2+ was added back to the medium. Compound 7 also did not modify intracellular pH in saline solutions, however it significantly blocked basal pH increases in a Ca2+-free medium. Compound 8 did not alter intracellular pH, however compound 9 induced a small acidification when Ca2+ was present in the extracellular medium. These results point to a structure-activity relationship in AZAs pH effect that affects the modulation and the coupling of intracellular pH and Ca2+.

Henrycinols A (1) and B (2), two novel indole alkaloids, together with three known compounds, (+)-Δ14-vincamine (3), (+)-16-epi-Δ14-vincamine (4), and (+)-isoeburnamine (5), were isolated from the roots of Melodinus henryiCraib. Their structures were established on the basis of 1D- and 2D-NMR spectroscopic analysis. The relative configuration of henrycinols A and B was determined by NOESY analysis.

Toxicological effects of orally administered azaspiracid (AZA), a new toxin isolated from mussels, were investigated. First, a total of 25 mice were administered AZA twice at 300-450 microg/kg doses and observed for recovery processes from severe injuries. Slow recoveries from injuries were revealed: erosion and shortened villi persisted in the stomach and small intestine for more than 3 months: edema, bleeding, and infiltration of cells in the alveolar wall of the lung for 56 days; fatty changes in the liver for 20 days; and necrosis of lymphocytes in the thymus and spleen for 10 days. Secondly, low doses of AZA (50, 20, 5 and 1 microg/kg) were administered twice a week up to 40 times to four groups of mice. Many mice, nine out of ten at 50 microg/kg and three out of ten at 20 microg/kg, became so weak that they were sacrificed before completion of 40 injections. All these mice showed interstitial pneumonia and shortened small intestinal villi. Most importantly, lung tumor were observed in four mice, one out of ten (10%) at 50 microg/kg and three out of ten (30%) at 20 microg/kg. Tumors were not observed in 11 mice treated at lower doses and in 19 control mice. Hyperplasia of epithelial cells was also observed in the stomach of six mice out of ten administered at 20 microg/kg.

A new type of food poisoning resulting from ingestion of mussels produced in Ireland occurred in the Netherlands in 1995 and then reoccurred in Ireland in 1997. As the causative agent, azaspiracid, was isolated in pure form and revealed to have a structure entirely unlike other known algal toxins, in vivo studies with mice were carried out to elucidate the pathological injuries caused by the toxin. By per os administration, the toxin caused necrosis in the lamina propria of the small intestine and in lymphoid tissues such as thymus, spleen and the Peyer's patches. Both T and B lymphocytes were injured. Additionally a fatty change was observed in the liver. These injuries distinctly differed from those caused by the representative diarrhetic shellfish toxin, okadaic acid.

Two new analogs of azaspiracid, azaspiracid-2 and azaspiracid-3, were isolated from mussels collected at Arranmore Island, Ireland in 1997 as additional causes of human intoxication. Their structures were determined to be 8-methylazaspiracid and 22-demethylazaspiracid, respectively by NMR and negative ion FAB CID MS/MS experiments. Copyright © 1999 John Wiley & Sons, Ltd.

Two new analogs of azaspiracid, azaspiracid-4 and azaspiracid-5, isolated from the mussel Mytilus edulis, involved in a newly emerged shellfish poisoning in Europe were determined to be 3-hydroxy-22-demethylazaspiracid and 23-hydroxy-22-demethylazaspiracid, respectively.

Azaspiracids (AZs) are a new group of phycotoxins discovered in the Ireland coast that includes the isolated analogues: AZ-1, AZ-2, AZ-3, AZ-4 and AZ-5 and the recently described AZ-6–11. Azaspiracid toxic episodes show gastrointestinal illness, but neurotoxic symptoms are also observed in mouse bioassay. Despite their great importance in human health, so far its mechanism of action is largely unknown. In this report, we present the first data about the effect of AZ-4 on cytosolic calcium concentration [Ca2+]i in freshly human lymphocytes. Cytoslic Ca2+ variations were determined by fluorescence digital imaging microscopy using Fura2 acetoxymethyl ester (Fura2-AM). AZ-4 did not modify cytosolic Ca2+ in resting cells. However, the toxin dose-dependent inhibited the increase in cytosolic Ca2+ levels induced by thapsigargin (Tg). AZ-4 decreased Ca2+-influx induced by Tg but did not affect the Ca2+-release from internal stores induced by this drug. The effects of AZ-4 on Ca2+-influx induced by Tg were reversible and not regulated by adenosine 3′,5′-cyclic monophosphate (cAMP) pathway. When AZ-4 was added before, after or together with nickel, an unspecific blocker of Ca2+ channels, the effects were indistinguishable and additive. AZ-4 also inhibited maitotoxin (MTX)-stimulated Ca2+-influx by 5–10%. Thus, AZ-4 appeared to be a novel inhibitor of plasma membrane Ca2+ channels, affecting at least to store operated channels, showing an effect clearly different from other azaspiracid analogues.

Azaspiracids (AZs) are a new group of phycotoxins discovered in the Ireland coast that includes the isolated analogues: AZ-1, AZ-2, AZ-3, AZ-4, and AZ-5 and the recently described AZ-6-11. Toxic episodes of AZs show gastrointestinal illness as in diarrhetic shellfish poisoning, but neurotoxic symptoms are also observed in a mouse bioassay. Despite their great importance in human health, so far, its mechanism of action is largely unknown. In this report, we present the first data of AZ-2 and AZ-3 effects on intracellular cyclic adenosine monophosphate (cAMP), intracellular calcium ([Ca 2 + ] i ), and cytosolic pH levels (pH;) in freshly human lymphocytes. The variations of cAMP, calcium, and pH were determined by fluorescence digital imaging microscopy using recombinant fluorescein- and rhodamine-labeled protein kinase A, Fura2-AM, and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, respectively. Our experiments show that both analogues, AZ-2 and AZ-3, clearly increase cytosolic cAMP levels of human lymphocytes. In calcium studies, we found that only if cells are initially in a calcium-free medium, AZ-2 increases the intracellular calcium concentration with two components: Ca 2 + release from internal stores and Ca 2 + influx from extracellular medium. AZ-2 sensitive Ca 2 + stores seem to be different from the thapsigargin sensitive one. AZ-2-induced Ca 2 - influx is mediated through Ni 2 + and SKF96365 blockable channels, and it is additive with Tg-induced Ca 2 + influx. Surprisingly, AZ-3 does not empty intracellular stores but also increases cytosolic calcium levels. This AZ-3-induced Ca 2 + influx is mediated through Ni 2 + blockable channels, and it is not additive with Tg-induced Ca 2 + influx. In addition, AZ-3 slightly alkalinizes cytosol. In accordance with cAMP studies, we found that adenylyl cyclase (AC) modulation inhibits AZ-2- and AZ-3-evoked Ca 2 + increase and AZ-3-induced pH i rise. Thus, both analogues seem to involve an AC pathway, although its effects on [Ca 2 + ] i and pH; are quite different.

This paper reports on potential cellular targets of azaspiracid-1 (AZ-1), a new phycotoxin that causes diarrhoeic and neurotoxic symptoms and whose mechanism of action is unknown. In excitable neuroblastoma cells, the systems studied were membrane potential, F-actin levels and mitochondrial membrane potential. AZ-1 does not modify mitochondrial activity but decreases F-actin concentration. These results indicate that the toxin does not have an apoptotic effect but uses actin for some of its effects. Therefore, cytoskeleton seems to be an important cellular target for AZ-1 effect. AZ-1 does not induce any modification in membrane potential, which does not support for neurotoxic effects. In human lymphocytes, cAMP, cytosolic calcium and cytosolic pH (pHi) levels were also studied. AZ-1 increases cytosolic calcium and cAMP levels and does not affect pHi (alkalinization). Cytosolic calcium increase seems to be dependent on both the release of calcium from intracellular Ca2+ pools and the influx from extracellular media through Ni2+-blockable channels. AZ-1-induced Ca2+ increase is negatively modulated by protein kinase C (PKC) activation, protein phosphatases 1 and 2A (PP1 and PP2A) inhibition and cAMP increasing agents. The effect of AZ-1 in cAMP is not extracellularly Ca2+ dependent and unsensitive to okadaic acid (OA).

A liquid chromatography/mass spectrometry (LC/MS) method was developed for the sensitive and specific determination of azaspiracid and its two analogs, the causative toxins of azaspiracid poisoning that occurred in the Netherlands and Ireland. The LC/MS method provided a detection limit of 50 pg for azaspiracid. The sensitivity was approximately 8 x 10(4) times greater than the mouse bioassay. The method was used to confirm the presence of azaspiracids in toxic mussels collected at Arranmore Island, Ireland in 1997.