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The commercially available Polysorbate 80 (PS-80) is a highly heterogeneous product. It is a complex and structurally diverse mixture consisting of polymeric species containing polyoxyethylenes (POEs), fatty acid esters, with/or without a carbohydrate core. The core is primarily sorbitan, with some isosorbide and sorbitol. Depending on the sources of fatty acids and the degrees of esterification, multiple combinations of fatty acid esters are commonly observed. A number of POE intermediates, such as polyoxyethylene glycols, POE-sorbitans, POE-isosorbides, and an array of fatty acid esters from these intermediates remain in the raw material as well. The complex composition of PS-80 is difficult to control and poses a significant characterization challenge for its use in the pharmaceutical industry. Here, we present a novel solution for PS-80 characterization using ultra high-performance liquid chromatography coupled with charge-reduction high resolution mass spectrometry. Post column co-infusion of triethylamine focused the signal into mainly singly charged molecular ions and reduced the extent of in-source fragmentation, resulting in a simpler ion map and enhanced measurement of PS-80 species. The data processing workflow is designed to programmatically identify PS-80 component classes and reduce the burden of manually analyzing complex MS data. The 2-dimensional graphical representation of the data helps visualize these features. Together, these innovative methodologies enabled us to analyze components in PS-80 with unprecedented detail and shall be a useful tool to study formulation and stability of pharmaceutical preparations. The power of this approach was demonstrated by comparing the composition of PS-80 obtained from different vendors.

High-resolution capillary zone electrophoresis - mass spectrometry (CZE-MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle-down and intact CZE-MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post-translational modifications (PTMs) and glycosylation structures. Middle-down and intact CZE separations were performed in an acidified methanol-water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle-down analysis of the IdeS-digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X-deamidated, 1X-deamidated, and non-deamidated forms at baseline resolution. In the course of the middle-down CZE-MS analysis, separation of glycoforms of the FC /2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE-MS2 . Incorporation of TCEP-HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE-MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X-glycosylated, 1X-glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE-MS represents a complementary approach to the more conventional liquid-chromatography - mass spectrometry-based approaches.

In addition, Arg, 18 O2-Arg, 15 N2-Arg and 15 N2- 18 O2-Arg were also analyzed to elucidate some of the minor fragments in greater detail. Mosquito excreta from individual females were collected before and at different times after feeding a blood meal, mixed with 15 N2-Arg, an internal standard, and then derivatized as isobutyl esters. Based on the fragmentation mechanisms of Arg standards, studied by MS 2 and MS 3 , Arg in the mosquito excreta was successfully analyzed by ESI-multiple reaction monitoring in a triple-quadrupole mass spectrometer. Arg excretion was monitored over a 120 h window before and after feeding female mosquitoes with a blood meal, with the maximum level of Arg excretion observed at 36–48 h post blood feeding. This method provides an efficient and rapid tool to quantify Arg in individual blood-fed mosquitoes, and can be applied to other organisms, whose small size severally limits the use of conventional biochemical analysis. Copyright © 2012 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this article.

New and improved methods are required for the enhanced characterization of complex biopharmaceuticals, especially those with charge and glycan heterogeneity. High resolution separation and mass spectrometry (MS) analysis of intact proteoforms can contribute significantly to the characterization of such proteins, many of which are glycoproteins. Here, we report on capillary zone electrophoresis (CZE) coupled via a commercial CESI sheathless interface to an Orbitrap ELITE MS for the intact analysis of recombinant human interferon-β1 (Avonex, rhIFN-β1), a biopharmaceutical with complex glycosylation at a single N-linked site. Using a cross-linked polyethylenimine coating, column efficiencies between 350,000 and 450,000 plates were produced, allowing separation based on charge and subtle hydrodynamic volume differences. A total of 138 proteoforms were found, and 55 were quantitated. Charge species due to deamidation and sialylation were separated by CZE. Given the high column efficiency, isobaric positional isomers of a single sialic acid on biantennary glycan antennae were resolved. Further, triantennary isomers (antenna on α(1-3) or α(1-6) arms) were separated and confirmed by exoglycosidase digestion. Proteoforms of the N-terminal cleavage of methionine were detected by precursor molecular weight and top-down ETD and HCD analysis of the reduced protein. Quantitative analysis suggested potential correlations between the methionine loss with the relative amount of the deamidation, as well as the level of deamidation with glycan structure. We demonstrate that high resolution CZE separation of intact glycoprotein species coupled to MS has significant potential for the in-depth characterization and quantitative analysis of biopharmaceutical proteoforms.

Investigations of the prison compound latrines from the Johnson’s Island Civil War Military Prison site provide a unique opportunity to explore a multitude of questions related to military life and POW treatment during the American Civil War. Johnson’s Island (Ohio), a Union prison for Confederate officers, contained special use areas within the compound and their associated latrines which can be evaluated chronologically. Coupled with the extensive primary documentation existing for this prison, investigations to date have centered around how these unique latrines exhibit both the POW experience as well as changing Union policy towards prisoners. These latrines captured lost items, were the repository for primary and secondary deposits reflecting the material culture available to the prisoner at the time, and served as the staging areas for escape attempts.

Sodium sulfate, Na2SO4, is principally used as a filler in soaps and detergents and in the manufacturing process of kraft paper. These uses consume about 80% of the world’s production. It is also used in glassmaking, in textiles, glass, and carpet fresheners. Both sodium sulfate and sodium bisulfate, NaHSO4, are used to adjust the pH of various solutions and to dilute certain dyes. Sodium bisulfate is a mild acid safe for household cleaning and where a mild acid is needed. About half of the sodium sulfate production is derived from naturally occurring salts and brines. The other half is produced as a by-product in the manufacture of rayon, sodium dichromate, resorcinol, and silica pigments. Sodium hydrosulfide, sodium sulfide, and sodium tetrasulfide are somewhat interchangeable in many applications. Uses range from ore flotation to leather tanning and from pulp processing in kraft pulping to lubricant manufacture. The processes used to manufacture these compounds are described. Sulfide solubilities are discussed. Keywords: sodium sulfate; soap; rayon; sodium sulfite; sodium sulfate; sodium sulfate decahydrate; sodium bisulfate; minerals; glauber’s salt; paper; detergents; sodium hydrosulfide; sodium sulfide; sodium tetrasulfide; pulp processing; ore flotation

Throughout the United States’ Civil War, the treatment of prisoners of war varied. Confederate and Union soldiers captured early in the war had a preconceived notion of how they were to be treated. As the war progressed, the Union changed their policies of treatment toward POWs. From Lincoln on down, those in charge struggled with this newly defined class of formerly recognized the US citizen. Johnson’s Island prison, as the only stand-alone facility constructed by the Union, encapsulated their early commitment to the “humane treatment” of prisoners. As the Union’s treatment policies changed, so did the physical landscape of Johnson’s Island. The long-term archaeological exploration of Johnson’s Island addresses changing prisoner treatment through this altered landscape.

Midazolam is a potent benzodiazepine derivative with sedative, hypnotic, anticonvulsant, muscle-relaxant, and anxiolytic activities. It undergoes oxidative metabolism catalyzed almost exclusively by the CYP3A subfamily to a major metabolite, 1'-hydroxymidazolam, which is equipotent to midazolam. 1'-Hydroxymidazolam is subject to glucuronidation followed by renal excretion. To date, the glucuronidation of 1'-hydroxymidazolam has not been evaluated in detail. In the current study, we identified an unreported quaternary N-glucuronide, as well as the known O-glucuronide, from incubations of 1'-hydroxymidazolam in human liver microsomes enriched with uridine 5'-diphosphoglucuronic acid (UDPGA). The structure of the N-glucuronide was confirmed by nuclear magnetic resonance analysis, which showed that glucuronidation had occurred at N-2 (the imidazole nitrogen that is not a part of the benzodiazepine ring). In a separate study, in which midazolam was used as the substrate, an analogous N-glucuronide also was detected from incubations with human liver microsomes in the presence of UDPGA. Investigation of the kinetics of 1'-hydroxymidazolam glucuronidation in human liver microsomes indicated autoactivation kinetics (Hill coefficient, n = 1.2-1.5). The apparent S(50) values for the formation of O- and N-glucuronides were 43 and 18 microM, respectively, and the corresponding apparent V(max) values were 363 and 21 pmol/mg of microsomal protein/min. Incubations with recombinant human uridine diphosphate glucuronosyltransferases (UGTs) indicated that the O-glucuronidation was catalyzed by UGT2B4 and UGT2B7, whereas the N-glucuronidation was catalyzed by UGT1A4. Consistent with these observations, hecogenin, a selective inhibitor of UGT1A4, selectively inhibited the N-glucuronidation, whereas diclofenac, a potent inhibitor of UGT2B7, had a greater inhibitory effect on the O-glucuronidation than on the N-glucuronidation. In summary, our study provides the first demonstration of N-glucuronidation of 1'-hydroxymidazolam in human liver microsomes.