Calcium/Phosphate Solubility Curves for Premasol and Trophamine Pediatric Parenteral Nutrition Formulations.

OBJECTIVES: Calcium and phosphate incompatibility in parenteral nutrition formulations remains a critical concern for patient safety. This study examined calcium phosphate solubility for 2-in-1 admixtures prepared using 2 commercially available pediatric amino acid solutions (Premasol, Baxter Healthcare Corp; or Trophamine, B. Braun Medical Inc), applying identical test methods, storage conditions, and acceptance criteria. METHODS: Parenteral 2-in-1 admixtures included amino acid; dextrose; static concentrations of sodium, potassium, and magnesium, and varying concentrations of calcium (0-60 mEq/L), phosphate (15-50 mmol/L), and cysteine. Three replicate samples were stored for 48 hours at 40°C ± 2°C and then visually inspected for particulate matter, evaluated for subvisible particulate matter, when particulate matter was noted, microscopic examination was performed to confirm the presence of calcium phosphate crystals. Pass criteria were: all replicates free of visible particulate matter related to calcium phosphate crystals and particle counts below US Pharmacopeia <788> limits. RESULTS: Premasol and Trophamine generated identical calcium phosphate curves for 2% amino acid formulations containing 20% dextrose with/without cysteine, and similar curves for the 1% or 3% amino acid formulations containing 10% or 20% dextrose with/without cysteine. Calcium phosphate particles were identified in failed samples by scanning electron microscopy/energy dispersive X-ray spectroscopy. Calcium phosphate solubility was higher in formulations containing cysteine 40 mg/g amino acid vs. cysteine 20 mg/g amino acid and in cysteine 20 mg/g amino acid vs. no cysteine. CONCLUSIONS: Admixtures made with 1%, 2%, or 3% Premasol or Trophamine have essentially equivalent calcium phosphate solubility curves when tested with identical methods, storage conditions, and acceptance criteria.

Automated system for kinetic analysis of particle size distributions for pharmaceutically relevant systems.

Detailing the kinetics of particle formation for pharmaceutically relevant solutions is challenging, especially when considering the combination of formulations, containers, and timescales of clinical importance. This paper describes a method for using commercial software Automate with a stream-selector valve capable of sampling container solutions from within an environmental chamber. The tool was built to monitor changes in particle size distributions via instrumental particle counters but can be adapted to other solution-based sensors. The tool and methodology were demonstrated to be highly effective for measuring dynamic changes in emulsion globule distributions as a function of storage and mixing conditions important for parenteral nutrition. Higher levels of agitation induced the fastest growth of large globules (≥5 µm) while the gentler conditions actually showed a decrease in the number of these large globules. The same methodology recorded calcium phosphate precipitation kinetics as a function of [Ca(2+)] and pH. This automated system is readily adaptable to a wide range of pharmaceutically relevant systems where the particle size is expected to vary with time. This instrumentation can dramatically reduce the time and resources needed to probe complex formulation issues while providing new insights for monitoring the kinetics as a function of key variables.

Probability-based compatibility curves for calcium and phosphates in parenteral nutrition formulations.

BACKGROUND: The information content of the calcium phosphate compatibility curves for adult parenteral nutrition (PN) solutions may benefit from a more sophisticated statistical treatment. Binary logistic regression analyses were evaluated as part of an alternate method for generating formulation compatibility curves. MATERIALS AND METHODS: A commercial PN solution was challenged with a systematic array of calcium and phosphate concentrations. These formulations were then characterized for particulates by visual inspection, light obscuration, and filtration followed by optical microscopy. Logistic regression analyses of the data were compared with traditional treatments for generating compatibility curves. RESULTS: Assay-dependent differences were observed in the compatibility curves and associated probability contours; the microscopic method of precipitate detection generated the most robust results. Calcium and phosphate compatibility data generated from small-volume glass containers reasonably predicted the observed compatibility of clinically relevant flexible containers. CONCLUSIONS: The published methods for creating calcium and phosphate compatibility curves via connecting the highest passing or lowest failing calcium concentrations should be augmented or replaced by probability contours of the entire experimental design to determine zones of formulation incompatibilities. We recommend researchers evaluate their data with logistic regression analysis to help build a more comprehensive probabilistic database of compatibility information.

Interactions between Parenteral Lipid Emulsions and Container Surfaces.

OBJECTIVE: To evaluate the relationship between changes in emulsion globule size distributions and container uptake of lipid emulsions in total nutrient admixtures. METHODS: A total nutrient admixture was prepared from a commercial lipid emulsion, 20% ClinOleic®, separated into glass (borosilicate) and ethylene vinyl acetate (EVA) plastic containers, and then stored at ambient conditions for approximately 24 h. The large globule size distribution was monitored continuously for both containers, and the quantity of triglycerides associated with both containers was measured by liquid chromatography. The changes in mass of the EVA containers were also measured gravimetrically. RESULTS: The volume percent of globules greater than 5 microns in diameter (PFAT5) levels for an emulsion admixture in EVA containers showed a 75% reduction compared to a marginal decrease of PFAT5 when in the glass container. Extraction of the containers showed that the quantity of triglycerides associated with the EVA surfaces steadily increased with emulsion exposure time, while the glass showed a significantly lower triglyceride content compared to the EVA. Gravimetric measurements confirmed that the EVA containers gained significant mass during exposure to the emulsion admixture. CONCLUSION: A time-dependent decrease in PFAT5 values for an emulsion admixture was associated with container triglyceride absorption where EVA containers had a greater uptake than glass containers. The larger globules appear to absorb preferentially, and the admixture globule size distribution fraction represented by PFAT5 accounts for 15-20% of the total triglyceride adsorption to the container. LAY ABSTRACT: The goal of this work is to evaluate how emulsions in total nutrition admixtures are affected by the containers within which they are stored. Specifically, the study examines how the emulsion globule size distribution in different containers is related to adsorption or absorption of the lipids onto or into the container. The admixtures were prepared from a commercial lipid emulsion, 20% ClinOleic®, and the containers were either glass (borosilicate) or plastic (ethylene vinyl acetate, EVA). The large globule size distribution was monitored continuously for both containers over the course of 24 h, and the quantity of triglycerides taken up by both containers was measured by liquid chromatography. The lipid uptake by the EVA containers was also monitored by gravimetric methods. Briefly, the percent of fat globules greater than 5 micrometers (PFAT5) in EVA containers showed a 75% reduction compared to a marginal decrease of PFAT5 when in the glass container. Extraction of the lipids from the containers showed that the quantity of triglycerides associated with the EVA surfaces steadily increased with admixture exposure time, while the glass showed a significantly lower triglyceride content. Gravimetric measurements confirmed that the EVA containers gained measurable mass during exposure to the emulsion admixture.

Antimicrobial testing for surface-immobilized agents with a surface-separated live-dead staining method.

Modification of a traditional live-dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface-immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3-(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si-QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si-QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si-QAC surfaces showed no kill after removing all Si-QAC elutables via rinsing procedures. The semi-quantitative surface-separated live-dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents.

Review of immobilized antimicrobial agents and methods for testing.

Antimicrobial surfaces for food and medical applications have historically involved antimicrobial coatings that elute biocides for effective kill in solution or at surfaces. However, recent efforts have focused on immobilized antimicrobial agents in order to avoid toxicity and the compatibility and reservoir limitations common to elutable agents. This review critically examines the assorted antimicrobial agents reported to have been immobilized, with an emphasis on the interpretation of antimicrobial testing as it pertains to discriminating between eluting and immobilized agents. Immobilization techniques and modes of antimicrobial action are also discussed.

Forces between insulin microspheres and polymers surfaces for a dry powder inhaler.

Here we demonstrate the use of a colloidal probe atomic force microscope (AFM) to compare the interactions between a model protein microsphere (insulin) and a set of common device polymers (polytetrafluoroethylene, polyethylene and polypropylene) with and without antistatic additive. For inhalation-based delivery devices the solid protein microspheres will interact with the device surfaces under ambient atmospheric conditions, and as such we studied the particle device interaction at a range of relative humidities. The results clearly discriminate between the five different polymer choices, and the impact of the antistatic additive. Although the mechanistic understanding is incomplete, it is evident that the polypropylene with antistatic additive gives consistent and relatively small interaction forces over the entire humidity range. The other polymer surfaces have humidity ranges where the pull-off forces are substantially greater. At 80% relative humidity, the insulin-polymer adhesion forces were similar for all the polymers probably due to the dominance of static charge mitigation and surface hydration effects. Overall, direct measurement of adhesion forces between pharmaceutical microspheres and container substrates can help direct rational choice of plastics/coatings for medical devices.

Patterning of cantilevers with functionalized nanoparticles for combinatorial atomic force microscopy.

In this letter, we demonstrate a substantial improvement in the ease of attaching and recognizing chemical libraries on cantilevers for use with combinatorial atomic force microscopy. These experiments exploit chemically modified nanoparticles as a means of encoding various chemistries onto the cantilevers with structures that can be readily distinguished. For the first time, 16 combinations of interactions were measured in a single experiment and in the same solution. Furthermore, these experiments have not begun to push the limits of the technique; it is plausible that in the very near future hundreds of interactions could be rapidly measured, helping to transform AFM into a truly high throughput technique.

Patterning of cantilevers with inverted dip-pen nanolithography: efforts toward combinatorial AFM.

We report patterning of AFM cantilevers by inverted dip-pen nanolithography, thereby markedly enhancing the development of combinatorial AFM as a high-throughput force-measuring instrument capable of determining interactions between opposing libraries of biomolecules.

Tapping mode imaging and measurements with an inverted atomic force microscope.

This report demonstrates the successful use of the inverted atomic force microscope (i-AFM) for tapping mode AFM imaging of cantilever-supported samples. i-AFM is a mode of AFM operation in which a sample supported on a tipless cantilever is imaged by one of many tips in a microfabricated tip array. Tapping mode is an intermittent contact mode whereby the cantilever is oscillated at or near its resonance frequency, and the amplitude and/or phase are used to image the sample. In the process of demonstrating that tapping mode images could be obtained in the i-AFM design, it was observed that the amplitude of the cantilever oscillation decreased markedly as the cantilever and tip array were approached. The source of this damping of the cantilever oscillations was identified to be the well-known "squeeze film damping", and the extent of damping was a direct consequence of the relatively shorter tip heights for the tip arrays, as compared to those of commercially available tapping mode cantilevers with integrated tips. The functional form for the distance dependence of the damping coefficient is in excellent agreement with previously published models for squeeze film damping, and the values for the fitting parameters make physical sense. Although the severe damping reduces the cantilever free amplitude substantially, we found that we were still able to access the low-amplitude regime of oscillation necessary for attractive tapping mode imaging of fragile molecules.

Developments for inverted atomic force microscopy.

Atomic force microscopy (AFM) has been used to study a wide range of systems. Chemically and biologically modified probes have extended AFM by coupling chemical and biological information with the physical measurements. In an effort to further expand the capabilities of modified AFM probes, previous studies investigated the use of an inverted AFM design (i-AFM), wherein a microfabricated tip array is used to image a cantilever-supported sample. This report details developments in cantilever and tip array fabrication which are aimed at improving the applicability and performance of this i-AFM design. Using an epoxy-based procedure, commercial cantilevers were modified with a series of standard substrates, including template-stripped gold, highly oriented pyrolytic graphite, and mica. The samples on these cantilevers were imaged with i-AFM, and lateral force images are obtained. This paper demonstrates the first use of i-AFM for measuring friction.