A GC-MS/MS Method for Trace Level Quantification of Six Nitrosamine Impurities (NDMA, NDEA, NEIPA, NDIPA, NDPA, and NDBA) in Commercially Used Organic Solvents: Dichloromethane, Ethyl Acetate, Toluene, and O-xylene.

Nitrosamines, the probable carcinogens have been reported with Angiotensin II Receptor Blocker (ARB) drugs, Ranitidine, and other medicines. Solvents play a vital role in the pharmaceutical industry in the separation, purification, and cleaning process for manufacturing APIs and drug products. According to the FDA and EMA, solvents used in the drug manufacturing process are potential root causes of Nitrosamine impurities. Hence, monitoring nitrosamines in solvents is an essential step for manufacturers. A sensitive direct injection GC-MS/MS, an essential analytical tool for low-level nitrosamine quantification in solvents, was developed by utilizing multiple reactions monitoring mode (MRM) for the simultaneous determination of six nitrosamines, namely, NDMA, NDEA, NEIPA, NDIPA, NDPA and NDBA in common solvents such as dichloromethane, ethyl acetate, toluene, and o-xylene. NDMA-d6 was used as an internal standard. The FDA reported a combined direct injection method for nitrosamine impurity assay by GC-MS/MS, which had several challenges for commercial-grade solvents in terms of interferences and resolution of unknown impurities and nitrosamine peaks. A novel method was developed to optimize the critical parameters of GC-MS/MS according to the solvent samples. The method validation was performed through the following parameters, sensitivity, linearity, accuracy, precision, specificity, and stability. The quantification of nitrosamines in commercial-grade solvents ranged from 100 ppb to 8000 ppb with respect to the sample concentration of 25 mg/mL with good sensitivity in LOQ level. The quantification ranged from 5 ppb (for NDMA, NDEA, NEIPA, NDIPA, NDPA) and 13 ppb (NDBA) to 2000 ppb with respect to the sample concentration of 100 mg/mL for analytical grade solvents with good sensitivity in the proposed method. Hence, it will be useful to quantify the low-level nitrosamines in commercial-grade solvents as well as analytical-grade solvents.

Metabolism and biological production of resolvins derived from docosapentaenoic acid (DPAn-6).

17S-HDPAn-6 (17S-hydroxydocosa-4Z,7Z,10Z,13Z,15E-pentaenoic acid) and 10S,17S-HDPAn-6 (10S,17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E-pentaenoic acid) are potent anti-inflammatory resolvins derived from DPAn-6 (docosapentaenoic acid n-6) and are analogous in structure and action to DHA (docosahexaenoic acid)-derived resolvins. These resolvins have proven to be potential drug candidates, albeit with therapeutic profiles that need optimization. The main objectives of this study were to evaluate key features of DPAn-6 derived resolvins that are important for therapeutic efficacy, demonstrate that these DPAn-6 resolvins could be produced naturally, and could therefore have physiological significance. Here we demonstrate biological production, examine pharmacokinetic profiles and identify key routes of metabolic inactivation of DPAn-6 derived resolvins. We compare their metabolic stability to a known resolvin, 17S-HDHA (17S-hydroxydocosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid) and show that order of their stabilities is 10S,17S-HDPAn-6>17S-HDPAn-6>17S-HDHA. We show that both these compounds are not strong inhibitors of cytochrome-P450 enzymes. We evaluate activity of compounds in the delayed-type hypersensitivity model, results of which show that compounds need optimization for enhanced duration and magnitude of action. Analysis of the metabolic stability and identification of metabolites of these compounds could play an important role in the design of better analogs with longer durations of action and hence better efficacy.

Biogenic synthesis, purification, and chemical characterization of anti-inflammatory resolvins derived from docosapentaenoic acid (DPAn-6).

Enzymatically oxygenated derivatives of the omega-3 fatty acids cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and cis-5,8,11,14,17-eicosapentaenoic acid, known as resolvins, have potent inflammation resolution activity (Serhan, C. N., Clish, C. B., Brannon, J., Colgan, S. P., Chiang, N., and Gronert, K. (2000) J. Exp. Med. 192, 1197-1204; Hong, S., Gronert, K., Devchand, P. R., Moussignac, R., and Serhan, C. N. (2003) J. Biol. Chem. 278, 14677-14687). Our objective was to determine whether similar derivatives are enzymatically synthesized from other C-22 fatty acids and whether these molecules possess inflammation resolution properties. The reaction of DHA, DPAn-3, and DPAn-6 with 5-, 12-, and 15-lipoxygenases produced oxylipins, which were identified and characterized by liquid chromatography coupled with tandem mass-spectrometry. DPAn-6 and DPAn-3 proved to be good substrates for 15-lipoxygenase. 15-Lipoxygenase proved to be the most efficient enzyme of the three tested for conversion of long chain polyunsaturated fatty acids to corresponding oxylipins. Since DPAn-6 is a major component of Martek DHA-S oil, we focused our attention on reaction products obtained from the DPAn-6 and 15-lipoxygenase reaction. (17S)-hydroxy-DPAn-6 and (10,17S)-dihydroxy-DPAn-6 were the main products of this reaction. These compounds were purified by preparatory high performance liquid chromatography techniques and further characterized by NMR, UV spectrophotometry, and tandem mass spectrometry. We tested both compounds in two animal models of acute inflammation and demonstrated that both compounds are potent anti-inflammatory agents that are active on local intravenous as well as oral administration. These oxygenated DPAn-6 compounds can thus be categorized as a new class of DPAn-6-derived resolvins.

Thiophenes as traps for benzyne. 3. Diaryl sulfides and the role of dipolar intermediates.

The reactions of seven thiophenes with benzyne generated from diphenyliodonium-2-carboxylate (DPIC) under a standard set of conditions led among other products to the formation of alpha- and beta-naphthyl phenyl sulfides 2a and 2b from thiophene (1a) and of 2c and 2d from 2-methylthiophene (1b). Dithienyl sulfides 4a-f were produced from the halothiophenes 1c-g. The structures of the naphthyl sulfides were proven by comparison with authentic samples of 2a-f, thus eliminating one of two possible mechanisms of formation. The remaining mechanism involves [4+2]-cycloaddition of benzyne to thiophene or to an S-phenylthiophenium ylide 10 to give the dipolar 2:1 benzyne/thiophene adduct 8 followed by ring-opening. Stevens-like rearrangements of 11, formed from 10 by proton transfer, may also explain the origin of arylated thiophenes such as 12 and 3 found in some reactions of benzynes with thiophene.