Amount Of Degradation Products Research Articles

Degradation of oil and oil products by microorganisms isolated from the Azerbaijani coast of the Caspian Sea at low temperatures

The object of the study is microorganisms isolated from the Azerbaijani territory of the Caspian Sea, capable of decomposing oil and oil products at low temperatures of 4–6 °C. The purpose of this work was to study bacteria and fungi capable of actively assimilating oil, gasoline, kerosene, diesel fuel at a temperature of 4–6 °C in order to solve the problem of environmental pollution in this region. The studied most effective biodestructors are combined into associations that can be used to create an active biological product based on native microorganisms with its further use for water bioremediation, as well as treatment of tanks and other containers used for long-term storage of petroleum products at a temperature of 4–6 °C. The process of degradation of samples of used oils at the molecular level of the selected compounds was studied by reverse-phase high-performance liquid chromatography. As a result of chromatographic and spectroscopic studies, it was found that almost all microorganisms decompose the tested oil products. Only weak peaks remained on the chromatogram, which is a clear indicator of the deep biodegradation carried out by these microorganisms. Thanks to the chromatographic and spectral analysis of oil and products of its decomposition, an oxidation sequence has been established, indicating the decomposition of the last aromatic fraction. As a result, a general picture was obtained on the amount of degradation products containing carboxyl, keto-, hydroxyl groups, which is not enough to determine the processes of transformation of initial products into final ones. All this testifies to the multidirectionality of biodegradation processes

Comparison of Chemical and Electrochemical Approaches to Abacavir Oxidative Stability Testing.

A novel electrochemical approach using two different electrode materials, platinum and boron-doped diamond (BDD), was employed to study the oxidative stability of the drug abacavir. Abacavir samples were subjected to oxidation and subsequently analysed using chromatography with mass detection. The type and amount of degradation products were evaluated, and results were compared with traditional chemical oxidation using 3% hydrogen peroxide. The effect of pH on the rate of degradation and the formation of degradation products were also investigated. In general, both approaches led to the same two degradation products, identified using mass spectrometry, and characterised by 319.20 and m/z 247.19. Similar results were obtained on a large-surface platinum electrode at a potential of +1.15 V and a BDD disc electrode at +4.0 V. Degradation of 20% of abacavir, the rate required for pharmaceutical stability studies, took only a few minutes compared to hours required for oxidation with hydrogen peroxide. Measurements further showed that electrochemical oxidation in ammonium acetate on both types of electrodes is strongly pHdependent. The fastest oxidation was achieved at pH 9. The pH also affects the composition of the products, which are formed in different proportions depending on the pH of the electrolyte.

Degradation mechanism of cement-based materials under the effects of stray current, chloride and sulfate

When infrastructure is exposed to a complicated environment containing stray current, chloride and sulfate, it may be subjected to severe degradation. In order to analyze the interaction of different factors and the degradation mechanism of cement-based materials in a complicated environment, the blended solution with four molar concentration ratios of Cl-/SO42- (0, 0.5, 1, and 2) were prepared to simulate a range of aggressive medium. Meanwhile, 40 V DC (direct current) electrical field was applied to cement-based materials to simulate stray current. After exposure, the transport properties, macro-properties, and microstructures of specimens were observed and measured. Additionally, the types and amounts of degradation products were tested to further analyze the degradation mechanism of cement-based materials. Experimental results show that chloride in the blended solution could inhibit SO42- transport into cement-based materials, causing the SO42- concentration in a specimen to decrease with the increase of Cl-/SO42- ratio. Therefore, the specimens appeared less degraded in terms of macro-properties. But the results of microstructural analysis showed that degradation products and cracks were generated in the interior of specimens. Friedel’s salt was detected in the specimens exposed to the solutions containing chloride. The formation of Friedel’s salt consumed calcium aluminate hydrate, which caused the amount of Al-phase involved in the formation of ettringite to reduce. Consequently, the amount of ettringite was reduced, and the degradation of compressive strength and appearance was also reduced. Additionally, an electrical field could induce the ion concentration in pore solution to change and even break the ion chemical equilibrium, which resulted in the decomposition of ettringite and Friedel’s salt. However, Friedel’s salt can also transform into ettringite at a specific condition. Therefore, the amounts of ettringite and Friedel’s salt in specimens were changed over the exposure time.

A study of variables that affect the process of sulfate attack of cement‐based materials subjected to stray current

AbstractDurability is one of the most important considerations for subway structures. However, the cement‐based materials used in subway engineering can be jeopardized by sulfate and the stray current and therefore can be subjected to severe damage during service. This study aims to investigate the degradation process of cement‐based materials exposed to stray current and external sulfate. In particular, the effects of three key variables were evaluated: concentration of external sulfate solution, level of stray current and cement type. After exposure, the visual appearance, compressive strength, SO42− profiles and microstructure of cement‐based materials were analyzed. Moreover, the amounts of degradation products were measured to evaluate the influencing degree of different variables and further explain the degradation mechanisms. Experimental results of this study reveal that (a) sulfate attack was related to the external sulfate concentration, but the relation was changed due to the presence of stray current. One explanation is that sulfate ions can be quickly accumulated in the pores of cement‐based materials under the effect of stray current, which makes the chemical reaction of sulfate attack exchange from diffusion control to reaction control. (b) Stray current could accelerate external sulfate attack of cement‐based materials, and the greater the stray current, the more severe sulfate attack was induced. (c) Compared with P·I type cement, high sulfate resistant (HSR) cement showed a better resistance to external sulfate attack. (iv) And, the main degradation products, ettringite and gypsum, were observed in specimens after exposure. Additionally, stray current might induce the decomposition of ettringite. It should be paid more attention on the durability of cement‐based materials exposed to the coupled conditions of stray current and sulfate.

The Influence of Cycling, Temperature, and Electrode Gapping on the Safety of Prismatic Lithium-Ion Batteries

Lithium-ion batteries are prevalent in every aspect of modern life (cell phones, laptops, electric vehicles, and energy storage systems for the electric grid). For all applications, the battery safety is an important consideration. Compared to numerous studies on the safety behavior of 18650-type cells, limited research has been conducted to characterize prismatic cells with their unique challenges including how electrode gapping, cycling history, electrolyte degradation, or lithium plating affect the safety. In this paper, a systematic study is reported on prismatic cells cycled at 0 °C, 23 °C, and 45 °C. The safety aspect of the cells with electrode gapping is evaluated using accelerating rate calorimetry (ARC). The evolution of gaps was monitored using X-ray computed tomography. Our study demonstrates that gaps are intrinsic even in fresh cells. The gaps can evolve during cycling and are closely related to the localized lithium plating and electrolyte degradation, which can be more severe for cells cycled at 45 °C. However, the safety behavior is not simply correlated to cell internal structure (e.g. gapping), or the amount of degradation products after cycling; the aging mechanism at different temperatures also plays an important role. Understanding the thermal stability of prismatic cells during their lifecycle is necessary for risk mitigation in numerous applications.

The effect of concentration, reconstitution solution and pH on the stability of a remifentanil hydrochloride and propofol admixture for simultaneous co-infusion

BackgroundThere are scenarios where pre-mixing and infusing analgesic and anaesthetic agents as a single intravenous (IV) solution is highly desirable; however, it is important to ensure the agents are compatible when mixed. As such, the long-term stability of a remifentanil-propofol mixture, and means of improving this, were assessed across a range of remifentanil concentrations, diluents, and time points.MethodsRemifentanil was reconstituted with ultrapure water, 0.9% saline, 20% saline, or 8.4% sodium bicarbonate solution (the latter two chosen for their pH characteristics, rather than their use in pharmaceutical reconstitution) and then mixed with propofol (1%) or further diluted with water to derive concentrations of 10–50 μg mL− 1. Remifentanil and propofol concentrations were determined initially and then periodically for up to 24 h using high performance liquid chromatography (HPLC). Mass spectrometry (MS) was used to detect degradation products in solutions containing 30 μg mL− 1 of remifentanil. Statistical analysis was performed using ANOVA and Student’s t-test, with a significance value of 0.05.ResultsIsolated remifentanil (pH < 4) and propofol (pH 7.35) did not degrade significantly when reconstituted with water or saline solution over 24 h, while remifentanil reconstituted with sodium bicarbonate degraded significantly (P < 0.001, pH 8.65). Mixing with propofol substantially increased the pH of the mixture and resulted in significant remifentanil degradation for all reconstitution solutions used, while propofol remained stable (pH 6.50). The amount of degradation product detected in samples containing isolated remifentanil and a mixture of the drugs was proportional to the remifentanil degradation observed.ConclusionsRemifentanil stability is affected by both the reconstitution solution used and when mixed with propofol, with pH appearing to be a contributing factor to degradation. If the pH of the solution and concentration of remifentanil are correctly controlled, e.g. through the use of a more acidic diluent, an admixture of remifentanil and propofol may be useful clinically.

Abstract P115: Troponin I and Dystrophin Proteolysis by Matrix Metalloproteinase 2 Contribute to Chronic Cardiac Remodeling and Dysfunction in Renovascular Hypertension

Aim: Increased MMP-2 activity contributes to hypertension-induced chronic cardiac morphological and functional changes by proteolyzing troponin I and dystrophin. Methods: Male Wistar rats were sham or two kidney-one clip (2K1C) operated and treated with water or doxycycline (15 mg/kg/day) by gavage for six weeks, starting from the tenth week. To establish this aim tail-cuff plethysmography, echocardiogram, in situ zymography, troponin I western blotting and dystrophin immunofluorescence were performed. Results were analyzed by Two-way ANOVA (Ethics Committee approval number: 023/2015-1). Results: Systolic blood pressure (SBP) of 2K1C rats was higher than sham after sixteen weeks of hypertension (215.7 ± 4.8 vs. 120.9 ± 4.4, p&lt;0.05) and doxycycline did not decrease it (216.1 ± 6.1, p&gt;0.05). After four months of hypertension, 25% (6 of 24) of 2K1C rats had left ventricular eccentric hypertrophic remodeling (LVEHR (2K1C-D)) while others remained at left ventricular concentric hypertrophic remodeling (LVCHR (2K1C-H)). Interestingly, 17.6% (3 of 17) of 2K1C rats treated with doxycycline had LVEHR while others 2K1C had LVCHR. Furthermore, 2K1C-D rats had reduced ejection fraction (EF: 39.8 ± 2.9 vs. 66.7 ± 1.7 and 66.2 ± 1.6, p&lt;0.05) and shortening fraction (SF: 20.4 ± 1.6 vs. 38.7 ± 1.4 and 38.4 ± 1.3, p&lt;0.05) when compared to 2K1C-H and Sham. Doxycycline preserved both EF and SF (66.4 ± 2.3 and 38.6 ± 1.8, p&lt;0.05). MMP-2 activity was increased in both 2K1C rats (2K1C-H: 7.2 ± 0.5 and 2K1C-D: 6.5 ± 0.4 vs. 5.3 ± 0.3 Sham, p&lt;0.05) and treatment with doxycycline decreased it (5.5 ± 0.3, p&lt;0.05). Higher amounts of degradation products of troponin I were observed in 2K1C-D when compared to 2K1C-H and Sham (3.6x10 -8 ± 7.9x10 -9 vs. 2.0x10 -8 ± 2.3x10 -9 and 2.0x10 -8 ± 3.4x10 -9 , p=0.06) and doxycycline prevented it (1.9x10 -8 ± 3.0x10 -9 , p&lt;0.05). The levels of dystrophin also decreased in 2K1C-H and -D when compared to Sham (13.5 ± 1.4 and 10.7 ± 2.1 vs. 15.6 ± 1.7) and doxycycline reversed it (16.9 ± 0.6, p&lt;0.05). Conclusion: Increased MMP-2 activity contributes to diminish troponin I and dystrophin levels in 2K1C and this effect may lead to chronic cardiac remodeling and dysfunction.

The influence of degree of fragmentation of Pinus sibirica on flammability, thermal and thermomechanical behavior of the epoxy-composites

Abstract In this study, lignocellulosic flour obtained through grinding of Pinus siberica wood (S) was used as a filler for developing epoxy-based (EP) composites characterized by reduced flammability. The influence of filler particle size on flammability, thermal and thermomechanical behavior of the natural composites was discussed in reference to the morphological analysis of final products as well as filler geometrical changes induced by processing. The structural analysis was realized with using 3D tomography. It was found, that introduction of 20 wt% of Pinus siberica wood caused a sharp drop in the heat release rate values, and the lowest results (2.5 times lower compared to EP) was determined for composite with the highest degree of fragmentation. The residue investigation confirmed the influence of particles size on the formation of the swelling structure of char, which effectively inhibits the combustion processes. Moreover, the analysis of gaseous products emitted during the thermal decomposition of composites was performed. The addition of investigated filler caused a decrease in the amount of degradation products, while the quantity of polycyclic aromatic hydrocarbons (PAHs) increased.

CO2 Product Quality: Assessment of the Range and Level of Impurities in the CO2 product Stream from MEA Testing at Technology Centre Mongstad (TCM)

During the recent MEA campaign at the Technology Centre Mongstad (TCM), a broad range of operational conditions have been explored for the post-combustion amine-based CO2 capture demonstration plant. This paper presents CO2 product composition data from online gas analyzers, originating from CO2 capture of two different flue gas sources available at TCM. Detailed composition data obtained by manual sampling and laboratory analysis, both internally at TCM and by Airborne Labs International Inc. is presented. Among the impurities identified and analyzed for, ammonia, formaldehyde and acetaldehyde are the compounds not commonly reported in the literature. The solvent quality, in terms of metal content and amount of degradation products, seemed to be the most influential parameter affecting the concentration of acetaldehyde and ammonia in the CO2 product gas. In addition, ammonia slip was found to be correlated with operating temperature of the overhead stripper system.

Macrophage phagocytosis of biomedical Mg alloy degradation products prepared by electrochemical method

Biomedical Mg alloy is promising for its widespread use clinically. In vitro and in vivo studies showed that the degradation products of biomedical Mg alloy were composed of O, P, Ca, Mg and other alloying elements. However, little is known about the metabolism of the degradation products. In this study, the in vitro macrophage phagocytosis of the degradation products of a biomedical Mg-Nd-Zn-Zr alloy was directly observed. This result affirms the necessity to investigate the long-term fate of Mg alloy degradation products in physiological environments. Besides, an electrochemical method was proposed to prepare enough amount of degradation products in vitro efficiently.

Degradation study of different brands of paracetamol by UV spectroscopy

Objective: To investgate the forced degradation study for the determination of degradation of the drug substance. Methods: Paracetamol was exposed to different conditions according to International Conference on Harmonization guideline. The amount of degradation product can be calculated with the help of UV spectrophotometer. The official test limits according to British Pharmacopoeia/United States Pharmacopoeia should not less than and should not more than lapelled amount. Forced degradation of drug substance was exposed to acidic and basic medium of panadol. Forced degradation of drug substance of panadol, disprol and calpol were also observed negligible difference in availability on exposure to UV and heat. This method can be used successfully for studying the stress degradation factors. Because this method is less time consuming and simple and cost effective also. Results: The brands i.e. calpol, panadol and disprol, when they come in contact with different degradation parameters (before, acid, base, heat and UV treatments) according to statistical analysis, the result showed significant values (P < 0.05) which indicated that there was no degradation in any of the brand. Conclusions: The result indicated there is no degradation found in these brands.

Influence of Different Hop Products on the cis/trans Ratio of Iso-α-Acids in Beer and Changes in Key Aroma and Bitter Taste Molecules during Beer Ageing

Isomerization of α-Acids from hops can result in different ratios of cis-to trans-iso-α-acids. In contrast to oxidative degradation processes, the conversion of iso-α-acids to tri- and tetracyclic compounds during beer ageing is only possible in the trans form. Therefore, it might be an advantage to use pre-isomerized hop products in order to maximize the cis form in beer. For this study, beers conventionally hopped with regular pellets were compared with beers produced with isomerized pellets and isomerized extracts. In addition, inline pre-isomerization was investigated as another alternative. The four variants were produced in a pilot brewery. Bottled beers were stored at 5 and 28°C. Analysis of fresh and aged samples after 4, 8, and 12 months was focused on bitter compounds (HPLC-MS/MS) and on volatile aroma compounds (GC-MS/MS). Beers produced with isomerized hop products showed higher ratios of cis- to trans-iso-α-acids, and their amounts of degradation products of trans-iso-α-acids were accordingly lower after ageing. However, all of the four types of beers hopped in different ways showed very similar profiles of volatile aroma compounds even during ageing. Moreover, sensory evaluation of aged beers gave no preference for the variations with pre-isomerized hop products.

Plasma Fibrin Degradation Products (Fdp ) In Patients With Pre Eclamptic Toxaemia (Pre-eclampsia)

Plasma fibrin degradation products (F.D.P.) assay measures the amount of fibrin split products in the blood. The high levels of F.D.P. in pre-eclampsia suggests that abnormal amount of degradation products are most likely due to localized lysis of fibrin in the uterine vascular compartment. The present case control study was designed to determine the relationship between plasma fibrin degradation products and pre-eclampsia. The study was carried out in the department of Biochemistry, Chittagong Medical College during the period of September 2011 to August 2012. The samples were collected from the department of Obstetrics and Gynecology, Chittagong Medical College Hospital, age from 18- 40 years. The data were collected by a structured questionnaire including age, socioeconomic condition, gravida, para, edema, blood pressure, proteinuria, history of hypertension, family history of pre-eclampsia and diabetes mellitus. Considering the cost of experiment and length of time, total 60 patients were included in this study. Among them 40 were considered as case (Blood Pressure &gt; or = 140/90 mm of Hg and proteinuria &gt; 0.3 gram/day) and 20 were considered as control (Blood Pressure &lt; 140/90 mm of Hg and no proteinuria). Plasma fibrin degradation products were measured in all samples. Study showed that plasma FDP were more increased in pre-eclamptic patients than that of normal pregnancy (21.52 + 16.47 ugm/ml Vs 10.63 + 7.12 ugm/ml), P = &lt;0.01. Result showed that percentage of raised plasma FDP is more in preeclamptic patients (100%) than that of normal pregnancy (75%), P= &lt;0.01. Pearson's Correlation Coefficient (r) showed that there were positive correlation between blood pressure and plasma FDP (systolic blood plessure and plasma FDP, r=0.221,P=&gt;.0.05 and diastolic blood pressure and plasma FDP, r=0.285,P=&lt;0.05). Significantly raised level of plasma F.D.P. in pre-eclampsia help to formulate a management plan and thereby reduce the complications of this disease. JCMCTA 2013; 24 (1):9-13

Analysis of broad leaf lignin of Japanese angiospermous trees by DFRC (derivatization followed by reductive cleavage) method

The DFRC (Derivatization Followed by Reductive Cleavage) analysis of lignin gives phenylpropanoid (C6-C3 type) degradation products from arylether linkages and can be used to detect lignin specifically from plant tissues, excluding other (poly) phenolic substances. DFRC analysis was employed to investigate the lignin composition in broad-leaves of typical Japanese angiosperms. Many leaves gave both guaiacyl- (G, 3-methoxyphenyl-) and syringyl- (S, 3,5-dimethoxyphenyl-) type degradation products, which indicates the existence of lignin in angiospermous leaves, and the leaf lignin has common structural elements with xylem lignin of the same tree. However, the results sometimes differed among species. Persimmon and cherry leaves revealed only trace amounts of degradation products. These results show the heterogeneous distribution of leaf lignin in angiospermous tree species. The syringyl/guaiacyl ratio of leaf lignin DFRC products was apparently lower than that of xylem lignin, which suggests the role of leaf lignin as a material to reinforce leaf vascular tissue for water conduction, like the low S/G ratio in woody vessel element cells.

Fermentation of the Straw Material Paja Brava by the Yeast &amp;lt;i&amp;gt;Pichia stipitis&amp;lt;/i&amp;gt; in a Simultaneous Saccharification and Fermentation Process

Paja Brava is a native South American grass with a high carbohydrate content. In the current work, the potential of using this feedstock for ethanol production using a simultaneous saccharification and fermentation (SSF) process with the xylose-fermenting yeast Pichia stipitis (Scheffersomyces stipitis) CBS6054 was investigated. The straw material was subjected to SO2 catalyzed steam pretreatment at 200°C and 5 min residence time, which resulted in a solubilization of pentose sugars (mainly xylose) of 64% with only minor amounts of degradation products. The obtained material, including the pretreatment liquid, was subsequently hydrolyzed and fermented in an SSF process at microaerobic conditions using either a batch or a fed-batch process at a total water-insoluble solids loading of 10%. Overall yields of ethanol based on all available sugars of 0.24 g/g and 0.27 g/g were obtained for batch and fed-batch mode of operation, respectively. The higher yield in the fed-batch process coincided with a higher degree of conversion of the sugars in the liquid medium, in particular of arabinose, for which the conversion was doubled (from 48% to 97%).

Preparing for Chemical Terrorism: A Study of the Stability of Expired Pralidoxime (2-PAM)

Oximes such as pralidoxime (2-PAM) are essential antidotes for life-threatening organophosphate poisoning. Unfortunately, oximes are expensive, have limited use, and have short shelf lives. As such, maintaining large stockpiles in preparation for terrorist activity is not always possible. We have demonstrated that atropine is stable well beyond its labeled shelf life and that recently expired 2-PAM was clinically efficacious in a series of poisoned patients. Because 2-PAM is often dosed empirically, clinical improvement does not guarantee pharmacological stability. We therefore chose to analyze the chemical stability of expired 2-PAM. Samples of lyophylized 2-PAM were maintained according to the manufacturer's recommendations for 20 years beyond the published shelf life. We studied 2-PAM contained in a MARK I autoinjector that was stored properly for 3 years beyond its expiration date. An Agilent LC/MSD 1100 with diode-array detector and an Agilent Sorbax SB-C-18, 4.6 × 150-mm, 5-μm column were used with the following solvent systems: water with 0.01% trifluoroacetic acid and methanol with 0.01% trifluoroacetic acid. Fresh reagent grade 2-PAM was used as a standard. Results were repeated for consistency. Lyophylized 2-PAM was a white powder that was clear and colorless in solution. Liquid chromatography was identical to the standard and resulted in 2 isolated peaks with identical mass spectra, suggesting that they are stereoisomers. The autoinjector discharged a clear, yellowish solution. In addition to the 2 peaks identified for lyophylized 2-PAM, a small third peak was identified with a mass spectra corresponding to the reported N -methyl pyridinium carboxaldehyde degradation product. When properly stored, lyophylized 2-PAM appears to be chemically stable well beyond its expiration date. Although the relative amount of degradation product found in solubilized (autoinjector) 2-PAM was small, it is unclear whether this may be toxic and therefore is of concern. Further studies performed with lots of drug stored under varied conditions would be required to fully determine the stability of expired 2-PAM.

Cross-Linking and Depolymerisation of γ-Irradiated Fish Gelatin and Porcine Gelatin Studied by SEC-MALLS and SDS-PAGE: A Comparative Study

γ-Irradiation of gelatin and collagen hydrogels can be used for sterilization and mechanical stabilization, providing biomaterials suitable for both tissue engineering and drug-delivery systems. Controversial results have been reported regarding the extent of irradiation-induced cross-linking and degradation, which depend on both protein concentration and irradiation dose. In this work the relative contributions of these processes were studied for irradiation doses between 0 and 1.0 kGy and concentrations between 0.3% and 5% using size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) and viscosity detection, as well as SDS-PAGE. It was demonstrated that chain degradation and cross-linking occur simultaneously in fish gelatin (FG), porcine gelatin (PG) and porcine collagen (PC), by the gradual appearance of protein fragments (10–80) × 103 concomitant with the formation of structures of high molecular weight. FG and PG behaved rather similarly, despite the fact they were irradiated and analyzed above and below their denaturation temperatures, respectively, suggesting little or no influence of molecular ordering under the conditions used. PC showed an increasing amount of degradation products following heat treatment prior to SEC-MALLS, suggesting that chain cleavage may occur within ordered collagen structures without complete release of the protein fragments.

Predicting drug hydrolysis based on moisture uptake in various packaging designs

An attempt was made to predict the stability of a moisture sensitive drug product based on the knowledge of the dependence of the degradation rate on tablet moisture. The moisture increase inside a HDPE bottle with the drug formulation was simulated with the sorption–desorption moisture transfer model, which, in turn, allowed an accurate prediction of the drug degradation kinetics. The stability prediction, obtained by computer simulation, was made in a considerably shorter time frame and required little resources compared to a conventional stability study. The prediction was finally upgraded to a stochastic Monte Carlo simulation, which allowed quantitative incorporation of uncertainty, stemming from various sources. The resulting distribution of the outcome of interest (amount of degradation product at expiry) is a comprehensive way of communicating the result along with its uncertainty, superior to single-value results or confidence intervals.

Activation time-course and in vivo pharmacological analysis of native BoNT/E

In the present study, a detailed activation time-course was examined for single chain native BoNT/E, enzymatically nicked in solution with trypsin. Through SDS-PAGE and Western blot analysis, the optimal nicking time was determined to be approximately 30 min. At 30 min, maximal di-chain BoNT/E and minimal secondary nicking (degradation) products were observed. The identity and integrity of the resulting light chain and heavy chain products were confirmed by N-terminal sequence analysis. Trypsin-activated BoNT/E samples covering a broad time-course were subsequently evaluated for potency and efficacy in pharmacological (DAS) and toxicological (im-LD50, ip-LD50) mouse models. In all three models, sample potencies varied with the degree of BoNT/E activation. In the DAS model, compared to single-chain BoNT/E, all samples displayed improved maximal efficacy and potency, except for 96 h-treated samples, which displayed lower potency. Both im- and ip-LD50 studies displayed similar dose–response profiles across the time-course. All three models yielded maximal potencies with samples activated for 30–100 min. These data demonstrate that purified, native BoNT/E can be activated in a controlled and reproducible manner with trypsin to near completion prior to the formation of any significant amounts of degradation products, with concomitant maximal in vivo potency.

Automated semipreparative purification with mass spectrometric fraction collection trigger: Modeling and experimental evaluation of a setup employing passive splitting

A semipreparative HPLC setup was evaluated for automated fractionation with both photometric- and mass spectrometric trigger. The goals of the work were to systematically study and optimize the flow-splitting setup for mass-directed purifications by mathematical modeling and experimental verification. The system comprised a passive splitting device with make-up flow capability, which directed a small fraction of the column effluent to the mass spectrometer and the remainder to the fraction collector. Tubing lengths and diameters of the splitter as well as make-up flow rates were varied in order to address and optimize peak dispersion, delay times between mass detector and fraction collector, and mass spectrometric signal quality. A paraben standard mixture was analyzed and purified on both microparticulate and monolithic columns with 10 mm inner diameter and at typical flow rates of 5-10 mL/min. Fraction purities and recoveries close to 100% were achieved. The system allowed mass-triggered fractionations on a 1 mg scale at flow rates of 10 mL/min in combination with monolithic columns in less than 2 min. Finally, the system was successfully applied to the fully automated isolation of milligram quantities of degradation products in a pharmaceutical preparation to successfully allow for structure elucidation with NMR spectroscopy.