Isolation of Caribbean Ciguatoxin-5 (C-CTX5) and confirmation of its structure by NMR spectroscopy.

Ciguatera poisoning occurs throughout subtropical and tropical regions globally. The Virgin Islands in the Caribbean Sea is a known hyperendemic region for ciguatera and has been associated with Caribbean ciguatoxin (C-CTX) contamination in fish. An algal C-CTX (C-CTX5) was identified in Gambierdiscus silvae and G. caribeaus isolated from benthic algal samples collected in waters south St. Thomas, US Virgin Islands. The highest CTX-producing isolate, G. silvae 1602 SH-6, was grown at large-scale to isolate sufficient C-CTX5 for structural confirmation by NMR spectroscopy. A series of orthogonal extraction and fractionation procedures resulted in purification of approximately 40 µg of C-CTX5, as estimated by quantitative NMR. A suite of 1D and 2D NMR experiments were acquired that verified the structure originally proposed for C-CTX5. The structural confirmation and successful isolation of C-CTX5 opens the way for work on the stability, toxicology and biotransformation of C-CTXs, as well as for the production of quantitative reference materials for analytical method development and validation. The strategies developed for purification of C-CTX5 may also apply to isolation and purification of CTXs from the Pacific Ocean and other regions.

Identification, synthesis and structural confirmation of process-related impurities in proparacaine hydrochloride.

Proparacaine hydrochloride is an ester-type local anesthetic agent that is extensively used in ophthalmic operations. The process-related impurities of proparacaine hydrochloride were investigated, and seven impurities were detected in the reaction solution of the last step at the level of 0.03-1.08 % by a newly developed HPLC method. Based on the synthetic process and the results of LC-HRMS, the structures of five impurities were proposed as 3-amino-4-propoxybenzoic acid (Imp-A), ethyl 3-amino-4-propoxybenzoate (Imp-B), 2-(ethylamino)ethyl 3-amino-4-propoxybenzoate hydrochloride (Imp-C), 2-(diethylamino)ethyl 3-nitro-4-propoxybenzoate hydrochloride (Imp-F), and 3-nitro-4-propoxybenzoic acid (Imp-G). And the structures were confirmed by synthesis, followed by varieties of spectral and chromatographic analyses. The structures of two impurities with almost same molecular weight in LC-HRMS were elucidated as 2-(diethylamino)ethyl 3-(ethylamino)-4-propoxy-benzoate hydrochloride (Imp-D) and 2-(diethylamino)ethyl 3-formamido-4-propoxybenzoate hydrochloride (Imp-E) by NMR and IR. An HPLC-based method was developed, and validation study demonstrated that the approach was precise, accurate, and sensitive. The impurities information of proparacaine hydrochloride can be used for the quality control of intermediate, raw material drug and its commercial products.

Preparation process and structure confirmation of impurity C of rupatifen fumarate / 中国药科大学学报

@#The high-purity impurity C was isolated and purified from a new anti-allergic drug, rupatifen, by nucleophilic substitution reaction with bromobutaric acid, and its structure was confirmed by IR, UV, MS, 1H NMR, 13C NMR, DEPT135°,HSQC, HMBC, and 1H-1HCOSY. The preparation process of impurity C in this study was simple and easy to obtain under mild conditions, with the purity of 99.0% and the yield of 25%-30%;and the sample met the target compound by structural confirmation. The preparation process and structure confirmation provided sufficient impurity C reference substance for impurity research of raw materials and preparations of rupatifen fumarate, which laid a solid foundation for quality research of new drugs.

Establishment of National Reference Standards of Sulfadimidine Impurities / 中国药学杂志

OBJECTIVE: To establish the national reference standards of sulfadimidine impurities thus to provide guarantee for improving the standard of quality control of sulfadiazine in China. METHODS: First, the structures of sulfadimidine impurities A and E were validated by infrared spectrocopy, mass spectrum and nuclear magnetic resonance method. Then, the purities of impurities A and E were determined using the method of related substance test for sulphadiazine in the European Pharmacopoeia (version 9.0),their water content and residue on ignition were determined as well. The contents of sulfadimidine impurities A and E were determined by using mass balance method. Meanwhile, external standard method and nuclear magnetic quantitative method were used to calculate the content, which were mutually verified with the mass balance method. Finally, the correction factors of sulfadiazine impurities A and E at 241 nm were determined using standard curve method. RESULTS: The structures of sulfadimidine impurities A and E were confirmed, and the contents of impurities A and E were 99.1% and 98.7%, respectively, which were calculated by mass balance method. The results were consistent with those obtained from external standard method and nuclear magnetic quantification method. The correction factors of impurities A and E to sulfadimidine were 0.97 and 0.63, respectively. CONCLUSION: The first batch of national standard substances of sulfadimidine impurities A and E were established successfully.

Structure Elucidation, Relative LC-MS Response and In Vitro Toxicity of Azaspiracids 7-10 Isolated from Mussels (Mytilus edulis).

Azaspiracids (AZAs) are marine biotoxins produced by dinoflagellates that can accumulate in shellfish, which if consumed can lead to poisoning events. AZA7-10, 7-10, were isolated from shellfish and their structures, previously proposed on the basis of only LC-MS/MS data, were confirmed by NMR spectroscopy. Purified AZA4-6, 4-6, and 7-10 were accurately quantitated by qNMR and used to assay cytotoxicity with Jurkat T lymphocyte cells for the first time. LC-MS(MS) molar response studies performed using isocratic and gradient elution in both selected ion monitoring and selected reaction monitoring modes showed that responses for the analogues ranged from 0.3 to 1.2 relative to AZA1, 1. All AZA analogues tested were cytotoxic to Jurkat T lymphocyte cells in a time- and concentration-dependent manner; however, there were distinct differences in their EC50 values, with the potencies for each analogue being: AZA6 > AZA8 > AZA1 > AZA4 ≈ AZA9 > AZA5 ≈ AZA10. This data contributes to the understanding of the structure-activity relationships of AZAs.

Research on impurity of pentoxifylline and glucose injection / 中国药学杂志

OBJECTIVE: To study the impurity of pentoxifylline and glucose injection and confirm its structure and source. METHODS: LC-MS was used to speculate the structure of the impurity. The impurity was synthesized by using theobromine and 1, 3-di-bromopropane as the starting materials and the structure was identified by IR, LC-MS, 1H-NMR, and 13C-NMR. RESULTS: The impurity was identified to be 1, 1′-(propane-1, 3-diyl)bis(3, 7-dimethyl-3, 7-dihydro-1H-purine-2, 6-dione). CONCLUSION: The study provides theoretical basis for the control of impurity in the production process and quality assessment of pentoxifylline and glucose injection.

Research of the main impurities in Compound Tilidine Hydrochloride Oral Solution / 中国药学杂志

OBJECTIVE: To explore the sources and structures of the main impurities in compound tilidine hydrochloride oral solution.

Structure confirm of impurities of cefatrizine propylene glycolate / 中国药学杂志

OBJECTIVE: To confirm the structures of the impurities of cefatrizine propylene glycolate.

Preparation of impurity C in benzathine benzylpenicillin products / 中国药学杂志

OBJECTIVE: To prepare impurity C in benzathine benzylpenicillin products. METHODS: Using the mother liquor of benzathine benzylpenicillin, impurity C was prepared by means of vacuum distillation, column chromatography, and recrystallization. Its molecular structure was confirmed with UV, IR, MS, NMR and elemental analysis. Its purity and correction factor were determined by HPLC method. The contents of impurity C in the samples were determined by external standard method. RESULTS: The prepared product was proved to be impurity C by structure confirmation. CONCLUSION: The preparation of impurity C in benzathine benzylpenicillin products has important significance for the control of related substances of benzathine benzylpenicillin products.