Development of an LC-MS/MS method for simultaneous quantitative analysis of macromolecular pharmaceutical adjuvant 2-hydroxypropyl-ß-cyclodextrin and active pharmaceutical ingredients butylphthalide in rat plasma.

Hydroxypropyl-ß-cyclodextrin, which possesses a high water solubility and low hemolycity, is widely used as a solubilizer and an excipient. It had also been reported that hydroxypropyl-ß-cyclodextrin has the activity of regulating lipid homeostasis. In order to further understand the metabolism, the primary focus was to establish a quantitative method for hydroxypropyl-ß-cyclodextrin. The analytes were extracted from plasma by protein precipitation with methanol and then carried out on a Waters CORTECS T3 column in the gradient elution of pure water and methanol. Finally, liquid chromatography-tandem mass spectrometry was applied in multiple reaction monitoring mode to complete the quantitative analysis of hydroxypropyl-ß-cyclodextrin. This validated method had been successfully applied to investigate the interaction between hydroxypropyl-ß-cyclodextrin and butylphthalide in vivo by optimizing the extraction reagent, simplifying the experimental procedure, and improving the sensitivity while considering the difference of drug chemical properties. Results showed that the inclusion of hydroxypropyl-ß-cyclodextrin with butylphthalide significantly improved the pharmacokinetic behavior of free body hydroxypropyl-ß-cyclodextrin and 3-n-butylphthalide in vivo. It had been implied that the metabolism of hydroxypropyl-ß-cyclodextrin and the drug active ingredients could impact each other. It will help better application of hydroxypropyl-ß-cyclodextrin and the developed method might lay the foundation for development of hydroxypropyl-ß-cyclodextrin as a treatment drug for brain diseases.

Recombinant PBP2a of methicillin-resistant S. aureus formulation in Alum and Montanide ISA266 adjuvants induced cellular and humoral immune responses with protection in Balb/C mice.

Staphylococcus aureus is an important cause of both hospital and community acquired infections worldwide. S.aureus can develop multidrug resistance; thus, immunotherapy can be a rational alternative. High level ß-lactam resistance of S. aureus has been attributed to the penicillin binding protein 2a (PBP2a). In this study, we assessed the immunogenicity and protectivity of PBP2a formulated in Montanide ISA266 and Alum adjuvants. Recombinant PBP2a with a molecular weight of approximately 13 kDa was expressed and purified by nickel-nitrilotriacetic acid (NI-NTA) affinity chromatography and characterized by SDS-PAGE and Western blot. To investigate the immunogenicity and protective effects of recombinant protein, 20 µg of r-PBP2a in various formulations were subcutaneously injected in different groups. Two booster vaccinations were carried out in two-week intervals and blood samples were collected two weeks after each injection. To determine the type of induced immune response, sera and splenocytes were analyzed by ELISA for total IgG and isotypes (IgG1 and IgG2a) and cytokine secretion (IFN-γ, IL-4, IL-17 and TNF-α), respectively. Three weeks following the last immunization, experimental mice were challenged with 5 × 108 CFU of bacteria intraperitoneally and mortality rate and bacterial load were assessed. Interestingly, analysis of humoral immune responses revealed that administration of r-PBP2a with Montanide ISA266 significantly increased specific IgG responses and also IgG1 isotype compared to alum-adjuvanted vaccine group. Also, r-PBP2a formulation with alum and MontanideISA266 adjuvants raised IFN-γ, IL-4, IL-17 cytokines secretion, and protectivity following experimental challenge. The results of the present study provide evidences for immunogenicity and protectivity of PBP2a protein as a vaccine candidate.

ß2-adrenoceptor signaling reduction is involved in the inflammatory response of fibroblast-like synoviocytes from adjuvant-induced arthritic rats.

OBJECTIVE: To investigate the effects of ß-AR signaling on fibroblast-like synoviocytes (FLS) from adjuvant-induced arthritis (AA) rats and the partial mechanisms focused on ß-AR desensitization mediated by GRK2 and ß-arrestin2. METHODS: Animals were divided into a control group and an AA model group, and FLSs were cultured. Arthritis index, histopathology of joints, epinephrine (Epi) and norepinephrine (NE) were detected in vivo. The effect of the ß-AR agonist isoprenaline (ISO) and the ß2-AR agonist salbutamol on FLS cell viability were detected by CCK8. Cytokines TNF-α, IL-1ß, OPG and RANKL were examined by ELISA. The expression of ß2-AR was detected by immunofluorescence and flow cytometry. The cytomembrane expression and desensitization of ß2-AR, GRK2, and ß-arrestin2 were measured by flow cytometry and western blot. RESULTS: The concentration of NE increased to a peak on day 21, which was consistent with the arthritis index. The levels of Epi and NE in synovial tissues were decreased. ISO inhibited FLS cell viability and TNF-α, IL-1ß, and RANKL secretion, and promoted OPG secretion. ß2-AR mediated the effects of ISO on FLS cell viability. ß2-AR signaling was weaker in AA rats compared to the controls. Elevated GRK2 and ß-arrestin2 in cytomembranes promoted ß2-AR desensitization and may decrease the anti-inflammatory effect of ß2-AR signaling. CONCLUSION: The activation of ß2-AR signaling exerts its anti-inflammatory activities on FLS. ß2-AR signaling decreased in the AA model, which might be related to the increased membrane expression of GRK2 and ß-arrestin2, and promoted the excessive desensitization of ß2-AR. Decreased ß2-AR signaling may be relevant to the exacerbation of arthritis inflammation.

Simultaneous Determination of Cefalexin, Cefazolin, Flucloxacillin, and Probenecid by Liquid Chromatography-Tandem Mass Spectrometry for Total and Unbound Concentrations in Human Plasma.

BACKGROUND: Pharmacokinetic studies and therapeutic drug monitoring of antibiotics require a simple, rapid, and reliable analytical method for monitoring the concentrations in plasma, including unbound concentrations for highly protein-bound drugs. The aim of the current work was to develop and validate a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of total and unbound concentrations of 3 widely used ß-lactam antibiotics (cefalexin, cefazolin, and flucloxacillin) and the often coadministered drug probenecid in human plasma, suitable for pharmacokinetic studies and for routine use in ordinary, busy hospital laboratories. METHODS: Unbound drug was separated from bound drug by ultrafiltration. A simple 1-step protein precipitation was used for sample preparation. Cefalexin, cefazolin, flucloxacillin, probenecid, and their corresponding isotopically labeled internal standards were then resolved on a C18 (2) column. All the compounds were detected using electrospray ionization in the positive mode. RESULTS: Standard curves were linear for all compounds over the concentration range of 0.2-100 mg/L (r > 0.99) for total drug in plasma and 0.01-10 mg/L (r > 0.99) for unbound drug in plasma ultrafiltrate. For both total and unbound drugs, bias was <±10%, and intra- and interday coefficients of variation (imprecision) were <10%. The limit of quantification was 0.2 mg/L for total plasma concentrations and 0.01 mg/L for plasma ultrafiltrate concentrations of all drugs. CONCLUSIONS: The method has proven to be simple, rapid, robust, and reliable and is currently being used in clinical pharmacokinetic studies and in the routine clinical service to enhance the effective use of the ß-lactam antibiotics.

Effect of 2 Emulsion-Based Adjuvants on the Structure and Thermal Stability of Staphylococcus aureus Alpha-Toxin.

The effects of 2 squalene-based emulsion adjuvant systems (MedImmune emulsion 0 [ME.0] and Stable Emulsion [SE]) on the structure and stability of the recombinant protein antigen alpha-toxin (AT), a potential vaccine candidate for Staphylococcus aureus infection, were investigated using Fourier-transform infrared spectroscopy and both steady-state and time-resolved intrinsic fluorescence spectroscopy as well as differential scanning calorimetry (DSC). A component study, performed to identify the effects of the individual emulsion's components, showed negligible interactions between AT and ME.0. DSC analysis showed the ME.0 emulsion thermally destabilized AT, probably because of changes in the buffer composition of AT upon mixing. The SE emulsion caused increased alpha-helix and decreased beta-sheet content in AT, and a significant blue shift in the fluorescence spectra relative to that of AT in solution. DSC analysis showed SE exerted a dramatic thermal stabilization effect on AT, probably attributable to an interaction between AT and SE. Size exclusion chromatography showed a complete loss in the recovery of AT when mixed with SE, but not ME.0, indicating a high degree of interaction with SE. This work successfully characterized the biophysical properties of AT in the presence of 2 emulsion adjuvants including a component study to rationalize how emulsion components affect protein antigen stability.

Adulteration Practices of Psychoactive Illicit Drugs: An Updated Review.

BACKGROUND: Powdery drugs such as cocaine and heroin are frequently adulterated or diluted predominantly to obtain more doses and to increase the drug dealer's profits, but also to enhance, to modify or to oppose drug effects. The aim of this report is to provide an overview of the recent scientific literature on medicines as well as on new psychoactive substances, used as cutting agents (i.e. pharmacologically active adulterants) and on the related adverse health effects on consumers, possibly due to the synergistic effect of the adulterants laced with substances of abuse. METHOD: A literature search up to January 2017 was performed on MEDLINE, Scopus and Web of Science and reports and documents of international agencies or institutions were also searched. RESULTS: Pharmacologically active substances such as: paracetamol, caffeine, dextromethorphan, clenbuterol for heroin; levamisole, phenacetine, lidocaine, hydroxyzine and diltiazem for cocaine; caffeine and phentermine for amphetamine, have been identified over the years. Furthermore, since cocaine and morphine (this latter as a precursor of heroin) are both extracted from natural products, some impurities and minor alkaloids can be present in the final preparation. In this context, it is worth considering that new psychoactive substances are also used as cutting agents. CONCLUSION: The wide availability of illicit psychotropic drugs is the most serious hazard threatening consumers. Indeed emergency departments are often responsible in evaluating damages caused not only by the base substance, but also by other eventual compounds added to mimic or antagonize drug effects or simply dilute the drug amount, with a possible harmful synergic toxic action.

Development of a high-throughput screening platform to study the adsorption of antigens onto aluminum-containing adjuvants.

Aluminum-containing salts are important adjuvants in the formulations of many licensed human vaccines. However, in the early stage of the design of a new vaccine, a thorough understanding of the adsorption mechanisms of an antigen onto an aluminum salt is required. Therefore, we have developed a robust, rapid, and reproducible high-throughput screening (HTS) platform to study the adsorption capacity of aluminum-containing vaccines. The adsorption isotherms on aluminum hydroxide and aluminum phosphate of two model proteins, ß-casein, and bovine serum albumin, were evaluated using a liquid handling system, which permitted rapid sample preparation in a small volume without nonspecific adsorption. Highly reproducible adsorption capacities and adsorptive coefficients were estimated based on the Langmuir model. To demonstrate the potential of this HTS platform, we evaluated the adsorption isotherms for two antigens, hepatitis B surface antigen and a pneumococcal serotype polysaccharide conjugated to a protein-D carrier, onto aluminum-containing vaccines at either a constant protein or a constant aluminum concentration. The automated assay enabled the rapid quantification of antigen adsorption with a significant reduction in operator workload and reagent use. This platform should accelerate data acquisition during the development of a new vaccine.

Toxicity of formulated glyphosate (glyphos) and cosmo-flux to larval and juvenile colombian frogs 2. Field and laboratory microcosm acute toxicity.

The spraying of coca (Erythroxylum coca) with glyphosate (coca mixture, a combination of formulated glyphosate, Glyphos, and an adjuvant, Cosmo-Flux) in Colombia has raised concerns about possible impacts on amphibians. Although acute LC50 for 8 species of Colombian frogs ranged from 1.2 to 2.78 mg acid equivalents (a.e.)/L, these exposures were conducted in the laboratory in the absence of sediments and organic matter such as would occur under realistic field conditions. In order to assess the effects of overspray of frog habitat under field conditions, Gosner stage 25 tadpoles of Rhinella granulosa, R. marina, Hypsiboas crepitans, and Scinax ruber were placed in outdoor microcosms made from polyethylene plastic fish ponds (2.07 m in diameter, 37 cm high) in an experimental area in Tolima, Colombia. The bottoms of the microcosms were covered with a 3-cm layer of local soil and they were filled to a depth of 15 cm (above the sediment) with local spring water. After up to 100 tadpoles of each frog species were placed in the microcosms, they were sprayed with the coca mixture at concentrations greater and less than the normal application rate (3.69 kg glyphosate a.e./ha). Mortality at 96 h in the control microcosms was between 0 and 16% and LC50 values were between 8.9 and 10.9 kg glyphosate a.e./ha (equivalent to initial concentrations of 5963 to 7303 microg glyphosate a.e./L). Mortality >LC50 was only observed in the tested species when the application rate was >2-fold the normal application rate. In other experiments, juvenile and adult terrestrial stages of frogs were exposed by direct spraying to a range of concentrations of coca mixture. Juveniles and adults were exposed in plastic food containers (19 x 19 cm). The bottom of the container was filled with moistened soil and leaf litter to a depth of 1 cm and 0.5 cm, respectively. Mortality in the controls was low, from 0 to 10%, and from 0 to 35% at the normal application rate. LC50 values ranged between 4.5 kg a.e./ha and 22.8 kg a.e./ha, 1.5- to 6-fold greater than the normal application rate. Data indicate that, under realistic worst-case exposure conditions, the mixture of Glyphos and Cosmo-Flux as used for control of coca in Colombia exerts a low toxicity to aquatic and terrestrial stages of anurans and that risks to these organisms under field conditions are small.

Risks to Colombian amphibian fauna from cultivation of coca (Erythroxylum coca): a geographical analysis.

The Colombian amphibian fauna is among the richest known in the world, with about 20 species of salamanders (order Caudata), 35 of the limbless caecilians (order Gymnophiona), and more than 700 species of frogs and toads (order Anura) recorded from localities within the country. The potential effects of exposure to glyphosate on amphibians arising from production of illegal crops (coca) were examined. The analysis was based on (1) behavior and ecology of species and (2) proximities of actual museum records to localities in which illegal crops are being grown and the subset of those that have been sprayed with glyphosate. Based on data on the location of amphibians collected in Colombia, records were obtained for 193 species (28% of the national diversity) of frogs and toads found in localities within 10 km of areas where coca is grown. Further analyses with ARC MAP software allowed for measurement of the direct distance separating collection locations for frogs, known coca fields, and areas where aerial spraying was being conducted. Records in or near coca fields included data for 11 of 13 families of frogs and toads known to be present in Colombia. Only Ceratophryidae and Pipidae were not reported from these locations and appear not to be at risk. For eight species (Dendrobates truncatus, Craugastor raniformis, Pristimantis gaigeae, Smilisca phaeota, Elachistocleis ovale, Hypsiboas crepitans, Trachycephalus venulosus, and Pseudis paradoxa) selected to represent several habitat preferences and life-cycle strategies, large areas of their distributions lie outside coca production regions and their populations as a whole are at low risk. For a limited number of species that barely enter Colombian territory, the consequences of coca production may be more serious and may have placed several species of frogs at risk. These include Ameerega bilingua, Dendropsophus bifurcus, Pristimantis colomai, P. degener, P. diadematus, P. quaquaversus, P. variabilis, and Trachycephalus jordani. Other species may also be at risk but exact numbers are unknown since few investigations were undertaken in these areas during the past 30 yr. The main ranges for these species were assumed to be in Ecuador.

A mathematical feasibility argument for the use of aptamers in chemotherapy and imaging.

A challenge for drug design is to create molecules with optimal functions that also partition efficiently into the appropriate in vivo compartment(s). This is particularly true in cancer treatments because cancer cells upregulate their expression of multidrug resistance transporters, which necessitates a higher concentration of extracellular drug to promote sufficiently high intracellular concentrations for cell killing. Pharmacokinetics can be improved by ancillary molecules, such as cyclodextrins, that increase the effective concentrations of hydrophobic drugs in the blood by providing hydrophobic binding pockets. However, the extent to which the extracellular concentration of drug can be increased is limited. A second approach, different from the 'push' mechanism just discussed, is a 'pull' mechanism by which the effective intracellular concentrations of a drug is increased by a molecule with an affinity for the drug that is located inside the cell. Here we propose and give a proof in principle that intracellular RNA aptamers might perform this function. The mathematical model considers the following: Suppose I denotes a drug (inhibitor) that must be distributed spatially throughout a cell, but that tends to remain outside the cell due the transport properties of the cell membrane. Suppose that E, an enzyme that binds to I, is expressed by the cell and remains in the cell. It may be that the equilibrium E+I[right arrow over left arrow]{k(-1)k(1)}P is not sufficiently far enough to the right to drive enough free inhibitor into the cell to completely inhibit the enzyme. Here we evaluate the use of an intracellular aptamer with affinity for the inhibitor (I) to increase the efficiency of inhibitor transport across the cell membrane and thus drive the above equilibrium further to the right than would ordinarily be the case. We show that this outcome will occur if: (1) the aptamer neither binds too tightly nor too weakly to the inhibitor than the enzyme and (2) the aptamer is much more diffusible in the cell cytoplasm than the enzyme. Thus, we propose and show by simulation that an intracellular aptamer can be enlisted for an integrated approach to increasing inhibitor effectiveness and imaging aptamer-expressing cells.

Determination of metalaxyl and identification of adjuvants in wettable powder pesticide technical formulas.

Foliar runoff is one of the most important processes affecting off-target movement of fungicides. In this way, Ridomil Gold Plus and Ridomil Gold MZ are two types of wettable powder technical formulations which contain metalaxyl and they are used for such a purpose. A method for quantitative determination of metalaxyl in pesticide formulas has been developed, validated, and subsequently applied to Ridomil Gold Plus and Ridomil Gold MZ. The method employs liquid-liquid extraction followed by liquid chromatography coupled with UV detection (LC-UV), using gas chromatography coupled with mass spectrometry as confirmation technique and to carry out a screening of organic adjuvants of these two selected pesticide formulas. Metalaxyl of 26.5 and 41 g/kg was detected in Ridomil Gold Plus and Ridomil Gold MZ, close to the manufacture specified level of 25 and 40 g/kg, respectively. Activator and utility adjuvants were detected in these two wettable powder technical formulations. Only methyl-ester-based surfactants were found within the group of nonionic surfactants, but the long-term fates of most adjuvants in soils and elsewhere in the environment are largely unknown, partially because of the lack of long-term monitoring data.

Unlocking interpretation in near infrared multivariate calibrations by orthogonal partial least squares.

Near infrared spectroscopy (NIR) was developed primarily for applications such as the quantitative determination of nutrients in the agricultural and food industries. Examples include the determination of water, protein, and fat within complex samples such as grain and milk. Because of its useful properties, NIR analysis has spread to other areas such as chemistry and pharmaceutical production. NIR spectra consist of infrared overtones and combinations thereof, making interpretation of the results complicated. It can be very difficult to assign peaks to known constituents in the sample. Thus, multivariate analysis (MVA) has been crucial in translating spectral data into information, mainly for predictive purposes. Orthogonal partial least squares (OPLS), a new MVA method, has prediction and modeling properties similar to those of other MVA techniques, e.g., partial least squares (PLS), a method with a long history of use for the analysis of NIR data. OPLS provides an intrinsic algorithmic improvement for the interpretation of NIR data. In this report, four sets of NIR data were analyzed to demonstrate the improved interpretation provided by OPLS. The first two sets included simulated data to demonstrate the overall principles; the third set comprised a statistically replicated design of experiments (DoE), to demonstrate how instrumental difference could be accurately visualized and correctly attributed to Wood's anomaly phenomena; the fourth set was chosen to challenge the MVA by using data relating to powder mixing, a crucial step in the pharmaceutical industry prior to tabletting. Improved interpretation by OPLS was demonstrated for all four examples, as compared to alternative MVA approaches. It is expected that OPLS will be used mostly in applications where improved interpretation is crucial; one such area is process analytical technology (PAT). PAT involves fewer independent samples, i.e., batches, than would be associated with agricultural applications; in addition, the Food and Drug Administration (FDA) demands "process understanding" in PAT. Both these issues make OPLS the ideal tool for a multitude of NIR calibrations. In conclusion, OPLS leads to better interpretation of spectrometry data (e.g., NIR) and improved understanding facilitates cross-scientific communication. Such improved knowledge will decrease risk, with respect to both accuracy and precision, when using NIR for PAT applications.

Aluminum granuloma after administration of the quadrivalent human papillomavirus vaccine. Report of a case.

We report the case of a young woman who developed a subcutaneous granulomatous response after administration of the quadrivalent human papillomavirus vaccine. The inciting agent was most likely an aluminum adjuvant, which previously has been reported to be associated with a granulomatous response after administration of other vaccines. Histologically, the lesion consisted of a necrotic/necrobiotic center surrounded by palisading epithelioid histiocytes closely resembling deep granuloma annulare or rheumatoid nodule. The histiocytes contained abundant intracytoplasmic violaceous/gray granular material. An ammonium aurintricarboxylate (Aluminon) stain demonstrated the presence of aluminum in the granular material. Aluminum granulomas should be included in the differential diagnosis of deep granulomatous reaction in young women, due to the high frequency of vaccination in this population.

[Comparative study of drug efficacy and drug additives between generic drugs and original drugs].

In the present study, we tested three kinds of sleeping drugs, consisting mainly of triazolam, brotizolam, and flunitrazepam, to compare the drug efficacy of generic drugs with that of original drugs. After these drugs were administered orally to mice, drug efficacy was evaluated in terms of ambulation, onset time of sleep, and duration of sleep in the open field test. For all kinds of sleep-inducing drugs, the drug efficacy of most generic drugs is not necessarily equal to that of the original drug. The main reason for the difference appears to be due to differences in the rate of absorption of the main drug. Any other differences between an original drug and a generic drug are caused by drug additives, the crystal form of the main drug, the formulation, and so on. In this study, the formulation was not the reason for the differences because all of the drugs were pulverized in a mortar and had no special coating. The drug additives for all the drugs are listed and the drug efficacy compared. Unfortunately, the information was not sufficient to shed any light on the differences in drug efficacy. For effective drug therapy, more information on drug additives should be provided.

Screening of active ingredients of herbal medicine for interaction with CYP450 3A4.

A two-step algorithm is adopted in the screening of herbal species which possess significant inhibitory effects on cytochrome P450 3A4 (CYP450 3A4). The algorithm comprises an initial stage of high throughput screening with Herbochip for the identification of herbal fractions that exhibit interactions with CYP450 3A4. Fifty commonly used TCM species were screened with seven showing a positive signal reflecting interaction. In the inhibition assays that followed, six of the seven species gave a signal. Sophora flavescens stood out as it gave the highest number of wells with a response, the highest maximum index was 0.96, and the median index was 0.55. The selection of TCM species with inhibitory effects on CYP450 carries the potential role of its use to boost the effects of known therapeutic agents, a mechanism that has been exploited in the design of regimens for the treatment of HIV infection.

Assessment of priority pesticides, degradation products, and pesticide adjuvants in groundwaters and top soils from agricultural areas of the Ebro river basin.

Gas chromatography-mass spectrometry (GC/MS) was employed for the determination of 30 widely used pesticides including various transformation products and alkylphenols in water and agricultural soils with the aim of assessing the impact of these compounds in agricultural soils and the underlying aquifer. The extraction, clean-up, and analytical procedures were optimized for both water and soil samples to provide a highly robust method capable of determining target analytes at the ppb-ppt level with high precision. For water samples, different solid-phase extraction cartridges and conditions were optimized; similarly, pressurized liquid extraction conditions were tested to provide interference-free extracts and high sensitivity. Instrumental LODs of 3-4 pg were obtained. The multi-residue extraction procedures were applied to the analysis of groundwaters and agricultural soils from the Ebro river basin (NE Spain). Most ubiquitous herbicides detected were triazines but some acetanilides and organophosphorus pesticides were also found; the pesticide additive tributylphosphate was found in all water samples. Levels varied between 0.57 and 5.37 microg/L in groundwater, whereas nonylphenol was the sole compound detected in soil. Alkylphenols are used as adjuvants in pesticide formulations and are present in sludges employed as soil fertilizers. Occurrence was found to be similar to other environmental studies.

Aluminum hydroxide adjuvant produced under constant reactant concentration.

Aluminum hydroxide adjuvant, AlO(OH), is used to potentiate the immune response to vaccines by adsorbing the antigen. The structure of aluminum hydroxide adjuvant is unusual as it is crystalline but has a high surface area due to its very small primary particles. The purpose of this study was to investigate the chemical and thermal conditions required to synthesize aluminum hydroxide adjuvant that is stable and exhibits a high protein adsorptive capacity. Aluminum hydroxide adjuvant was precipitated using a procedure in which the concentration of reactants was maintained constant throughout the precipitation. The precipitation variables were: 2.50, 2.75, and 3.00 OH/Al molar ratio; 0.5, 4.0, and 5.0 M NaCl; and 25, 60, and 65 degrees C. High sodium chloride concentration and high temperature facilitated the formation of AlO(OH) rather than crystalline forms of aluminum hydroxide, Al(OH)(3). The AlO(OH) produced was not stable because crystalline forms of aluminum hydroxide formed during aging at room temperature. Aluminum hydroxide adjuvant was stabilized for the study period of 12 weeks at room temperature by either the addition of 3.0 M NaCl after precipitation and washing or hydrothermal treatment at 110 degrees C for 4 h. Stabilization by the addition of sodium chloride required a hypertonic concentration of sodium chloride and was not practical as vaccines for parenteral administration are desired to be isotonic (equivalent to 0.15 M NaCl). Stabilization by hydrothermal treatment produced aluminum hydroxide adjuvant, which exhibited a high protein adsorptive capacity that did not change during the 12-week study period.

Concentration determination of a recombinant vaccine antigen adsorbed onto an alum adjuvant by chemiluminescent nitrogen detection.

PURPOSE: A chemiluminescent nitrogen detector (CLND) has been evaluated for determining the concentration of an aluminum-adsorbed recombinant vaccine antigen. METHODS: Quantification of the antigen was based upon several nitrogen-containing compounds used to calibrate the CLND. All calibrants (6.75-400 microg/ml) generated linear standard curves, with slopes being directly proportional to the % nitrogen. The limit of quantification (LOQ) was determined to be 6.75 microg/ml based on the performance of the antigen standard curve, and the limit of detection (LOD) was defined by setting the CLND minimum peak area to 40,000 U. The CLND was capable of analyzing antigen-adjuvant suspensions (adsorbed + unbound antigen) without any sample pretreatment. To measure unbound antigen, the suspension was centrifuged and an aliquot of supernatant removed for analysis; the difference between these two measurements was the amount of adsorbed antigen. RESULTS: The adjuvant exhibited no significant matrix effect. Samples were analyzed in triplicate with observed relative standard deviation values ranging from 0.065% to 10.0%. The most accurate concentrations of the antigen were recovered relative to the antigen itself and to glycine as standards. CONCLUSION: This methodology provides a direct measurement of the concentration of a vaccine antigen adsorbed onto an aluminum adjuvant.

[The problem of the presence of dangerous adjuvants in pesticide preparations used in agriculture or households]. / La problematica relativa alla presenza di coformulanti pericolosi nei preparati antiparassitari di uso agricolo o domestico.

Problems related to the presence of dangerous co-formulating agents and adjuvants in plant protection products and biocides are described. Usually, these kind of preparations are made including big quantities of different inerts (solvents, adhesives, wetting agents, surfactants, etc.), often more dangerous than the active ingredients. The obligatory declaration on the label about identity and concentration of some of these substances is not provided by the actual legislation. Sometimes, as in the case of accidental poisoning, the real nature of the toxicological damage is difficult to recognize. Also for the environmental compartment, the persistence of some preparation can be hugely increased by the presence of inerts added on purpose for lasting the efficacy.

Quantitative analysis quantitation of 2-n-nonyl-1,3-dioxolane by stable-isotope dilution gas chromatography-mass spectrometry.

We report here a quantitative methodology developed for determination of SEPA (2-n-nonyl-1,3-dioxolane) in human serum. The method employed solid-phase extraction of SEPA and internal standard, [13C2]SEPA, from serum followed by gas chromatography-mass spectrometry analysis using EI monitoring m/z 73 and 75. We have investigated the utility of stable isotope dilution gas chromatography-mass spectrometry (GC-MS) for the determination of SEPA concentrations in serum using chemical ionization (positive ion, CI) or electron ionization (EI). The comparison of the specificity and sensitivity between EI and CI indicated that monitoring the m/z 73 ion in EI was superior to monitoring either MH+ or m/z 73 using CI. The method was simple, sensitive and accurate, demonstrating a lower limit of quantitation (LLOQ) of 0.25 ng/ml and intra- and inter-assay accuracy and precision of < or = 7.5%.