3D characterisation of dry powder inhaler formulations: Developing X-ray micro computed tomography approaches.

Carrier-based dry powder inhaler (DPI) formulations need to be accurately characterised for their particle size distributions, surface roughnesses, fines contents and flow properties. Understanding the micro-structure of the powder formulation is crucial, yet current characterisation methods give incomplete information. Commonly used techniques like laser diffraction (LD) and optical microscopy (OM) are limited due to the assumption of sphericity and can give variable results depending on particle orientation and dispersion. The aim of this work was to develop new three dimensional (3D) powder analytical techniques using X-ray computed tomography (XCT) that could be employed for non-destructive metrology of inhaled formulations. α-lactose monohydrate powders with different characteristics have been analysed, and their size and shape (sphericity/aspect ratio) distributions compared with results from LD and OM. The three techniques were shown to produce comparable size distributions, while the different shape distributions from XCT and OM highlight the difference between 2D and 3D imaging. The effect of micro-structure on flowability was also analysed through 3D measurements of void volume and tap density. This study has demonstrated for the first time that XCT provides an invaluable, non-destructive and analytical approach to obtain number- and volume-based particle size distributions of DPI formulations in 3D space, and for unique 3D characterisation of powder micro-structure.

Examination of inequivalent wetting on the crystal habit surfaces of RS-ibuprofen using grid-based molecular modelling.

Synthonic engineering tools, including grid-based searching molecular modelling, are applied to investigate the wetting interactions of the solute and four crystallisation solvents (ethanol, ethyl acetate, acetonitrile and toluene) with the {100}, {001} and {011} forms of RS-ibuprofen. The grid-based methods, in particular the construction of a crystal slab parallel to a given plane in a coordinate system with one axis perpendicular to the surface, are defined in detail. The interaction strengths and nature (dispersive, hydrogen bonding (H-bonding) or coulombic forces) are related to the crystal growth rates and morphologies. The solute is found to interact strongest with the capping {011}, then the side {001} and weakest with the top {100} habit surfaces. The solute interactions with the {100} and {001} surfaces are found to be almost solely dominated by dispersive force contributions, whilst the same with the {011} surfaces are found to have a greater contribution from H-bonding and coulombic forces. The increased surface rugosity, at the molecular level of the {011} surfaces, results in a favourable docking site in a surface 'valley', not present in the {100} and {001} surfaces. The H-bonding solvents ethanol, acetonitrile and ethyl acetate are found to strongly interact with the {011} surfaces and weakly with the {001} surfaces, with the {011} interactions having a much greater contribution from H-bonding and coulombic forces. The interaction energies of the apolar and aprotic solvent toluene, with the {011} and {001} surfaces, are found to be very close. Toluene is found having slightly stronger interactions with the {001} than the {011} surfaces, which are all dominated by dispersive interactions. The ratio of the average energy of the top 100 solvent interactions with the {001} surface divided by the average energy of the top 100 interactions with the {011} surface is compared to the ratio of the experimentally measured growth rates of the same forms. In general, the interaction energy ratio is found to have an inverse ratio with the growth rates, implying that the solvents which are calculated to interact strongly with a particular surface are impeding the growth of that surface and reducing the growth rate, in turn impacting upon the final morphology of the material.

The influence of solution environment on the nucleation kinetics and crystallisability of para-aminobenzoic acid.

The influence of solvent type on the solution thermodynamics, nucleation-kinetics and crystal growth of alpha para-aminobenzoic acid (PABA) crystallising from supersaturated ethanol, acetonitrile and water solutions, is examined using poly-thermal analysis of the metastable zone width. Application of a recently proposed model for analysis of crystallisation kinetics (J. Cryst. Growth, 2010, 312, 698-704) indicates a solvent and concentration dependence of the nucleation mechanism and key nucleation parameters for the alpha form of PABA. The mechanism of nucleation is found to change from instantaneous to progressive with decreasing concentration and also when changing the solvent from ethanol to acetonitrile to water. The dependence of the nucleation mechanism is correlated to the kinetic component of the nucleation rate through calculated values of instantaneously nucleated crystallites, which increase from 1.40 × 109 m-3 in ethanol to 1.08 × 1010 m-3 in acetonitrile to 2.58 × 1010 m-3 in water. This in combination with low calculated number concentrations of interfacial tension between 1.13 and 2.71 mJ m-2, supports the conclusion that the kinetic component of the nucleation rate is more limiting when crystallising PABA from ethanol solutions in comparison to water solutions. This finding is further supported by molecular dynamics simulations of the solvation free energy of PABA, which is found to be greatest in water, -42.4 kJ mol-1 and lowest in ethanol, -58.5 kJ mol-1.

Towards an understanding of the nucleation of alpha-para amino benzoic acid from ethanolic solutions: a multi-scale approach.

The molecular assembly and subsequent nucleation of para-amino benzoic acid (PABA) from ethanolic solutions is probed using a multi-scale and multi-technique approach. This is applied by examining and interrelating information regarding the molecular, solution-state, cluster, solid-state and surface structures to understand why the alpha form of PABA is crystallised in preference to its low temperature beta form. Calculations suggest that conformational changes within the solute molecule play little or no role in directing the nucleation of either the alpha or beta crystal forms. Combined ab initio and molecular dynamics calculations of the stability of small clusters in solution suggests that the hydrogen-bonded carboxylic acid dimers, present in the alpha structure, are the most stable in solution and play a major role in the self-assembly and polymorphic expression of the alpha form in ethanol in preference to the beta form. These calculations are in good agreement with X-ray small-angle scattering analysis which reveals the presence of PABA clusters in ethanol which are consistent with the size and shape of a carboxylic acid dimer. SAXS studies also reveal the presence of larger cluster structures in a size range 10-40 nm which appear to grow, perhaps reflecting a change in the balance between monomers and dimers within the solution during the nucleation process. The results of crystallisation-kinetics experiments indicate an instantaneous nucleation mechanism where the number of instantaneously nucleated crystallites is calculated to be 1360-660 nuclei per ml and the subsequent growth is found to be only rate limited by diffusion of the growth unit to the crystallite surface. A linear dependence of growth rate with respect to supersaturation is observed for the (0 1 -1) capping face, which is associated with strong π-π stacking interactions. This is consistent with a solid-on-solid mechanism associated with surface roughened growth and concomitant poor lattice-perfection. Conversely, the side (1 0 -1) surface has a growth mechanism consistent with a 2D nucleation birth and spread mechanism. Hence, these mechanisms result in very fast growth along the b-axis and the needle-like morphology that is observed for alpha-PABA.

An examination of binding motifs associated with inter-particle interactions between facetted nano-crystals of acetylsalicylic acid and ascorbic acid through the application of molecular grid-based search methods.

Grid-based intermolecular search methods using atom-atom force fields are used to assess the structural nature of potential crystal-crystal interfacial binding associated with the examination of representative pharmaceutical formulation components, viz acetylsalicylic acid (aspirin) and ascorbic acid (vitamin C). Molecular models of nano-sized molecular clusters for these two compounds, shaped in accordance with an attachment energy model of the respective particle morphologies, are constructed and used together with a grid-based search method to model the likely inter-particle interactions. The most-stable, mutual alignments of the respective nano-clusters based on their interaction energies are identified in the expectation that these are indicative of the most likely inter-particle binding configurations. The stable inter-particle binding configurations identified reveal that the number of interfacial hydrogen bonds formed between the binding particles is, potentially, an important factor in terms of the stability of inter-particle cohesion. All preferred inter-particle alignments are found to involve either the (1 0 0) or the (1 1 0) face of aspirin crystals interacting with a number of the growth forms of ascorbic acid. Four main types of interfacial hydrogen bonds are found to be associated with inter-particle binding and involve acceptor-donor interactions between hydroxyl, carbonyl, ester and lactone acceptor groups and hydroxyl donor groups. This hydrogen bonding network is found to be consistent with the surface chemistry of the interacting habit faces with, in general, the number of hydrogen bonds increasing for the more stable alignments. The likely usefulness of this approach for predicting solid-state formulation properties is reviewed.

Simulation of energetic stability of facetted l-glutamic acid nanocrystalline clusters in relation to their polymorphic phase stability as a function of crystal size.

A molecular modeling approach is used to study the stability of different polymorphic forms of l-glutamic acid through building and optimizing molecular clusters of different sizes and shapes with the latter corresponding to the predicted crystal growth morphologies. The results reveal that the initially nucleating (according to Oswald rule) metastable (alpha) form is the more energetically stable form at small cluster sizes of ca. 200 molecular units, whereas the stable (beta) form is more stable when the cluster size is larger.

Field resistance of two soybean germplasm lines, HC95-15MB and HC95-24MB, against bean leaf beetle (Coleoptera: Chrysomelidae), western corn rootworm (Coleoptera: Chrysomelidae), and Japanese beetles (Coleoptera: Scarabaidae).

Two recently released, Mexican bean beetle, Epilachna varivestis, Mulsant, resistant soybean, Glycine max (L.) Merrill, germplasm lines, HC95-15MB and HC95-24MB, were examined for foliar and pod feeding resistance to adult bean leaf beetles, Cerotoma trifurcata (Forster), western corn rootworms, Diabrotica virgifera virgifera LeConte, and Japanese beetles, Popillia japonica Newman. Both lines were planted along with a susceptible control cultivar in 18 by 30-m plots and separate 0.8-ha size fields. Insects were sampled on a weekly basis with a sweep net. In late summer, defoliation ratings were recorded along with data on percentage pod feeding. Although a few significant differences in insect densities were obtained among the soybean lines on some sampling dates, no specific trends were observed in the ability of the resistant germplasm to reduce insect numbers. Insect population densities were similarly on all lines. However, both resistant lines were able to reduce defoliation during the growing season. Conversely, percentage pod feeding was similar among all the soybean lines, with no differences observed. The resistant germplasm lines appear able to lower levels of defoliation, and thus, offer a potential management tactic where leaf feeding, i.e., defoliation, is of concern. However, their ability to greatly reduce beetle population densities, and for the bean leaf beetle, to reduce pod feeding, appears limited.

Simulated insect defoliation on soybean: influence of row width.

An ongoing change in soybean production gaining popularity in the United States is a reduction in row spacing. Plant canopy closure is quicker and leaf area index is greater, thus yield is usually higher. Because yield response to insect defoliation is primarily a function of how defoliation causes changes in light interception, the possibility exists that the insect-injury-yield-loss relationship might differ among row widths. Soybean was grown in four states using similar methodologies. Insect defoliation was simulated by picking leaflets based on an insect defoliation model. Plant growth measurements were taken immediately following the end of defoliation. Numerous independent variables were measured or calculated, including percentage light interception, leaf area index, percentage defoliation, and leaf area per plot. Analyses of covariance were conducted on the resultant data to determine whether insect-injury-yield-loss relationships interact with row width. A significant interaction would indicate that the impact of the variables on yield was dependent on the row width, whereas a nonsignificant interaction would suggest that the relationship between the variables and yield is similar at all row widths. Few significant interactions were obtained, indicating that the impact of the variables on yield is similar across row widths. Because of the lack of significant interactions, the insect-injury-yield-loss relationships previously developed should be usable across varying row widths. Thus, treatment decisions based on light interception and leaf area indices, both considered more appropriated measures of insect injury, should be applicable for all row spacings.

Collisionally activated dissociations of aminocyclitol-aminoglycoside antibiotics and their application in the identification of a new compound in tobramycin samples.

Several aminocyclitol-aminoglycoside antibiotics have been studied by tandem mass spectrometry. Glycosidic bond cleavages were the major reactions in the low energy collisionally activated decomposition (CAD) of the protonated antibiotics. Only the glycoside residing on the C6-O of the 2-deoxystreptamine was observed to undergo significant decomposition at the C2-C3 and O-C1 bonds. The comprehension of the CAD of known aminoglycosides aided in the identification of an unknown impurity in tobramycin. The unknown compound was initially detected by reverse phase high-performance liquid chromatography following dinitrofluorobenzene derivatization of the amino groups. The molecular weight of the dinitrobenzene derivative measured by LC mass spectrometry (MS) led to the detection of two isomeric impurities in tobramycin by LC-MS using an amino column. Their CAD spectra were subsequently acquired by LC-MS/MS. One of the two compounds was determined to be a known compound, 6"-O-carbamyltobramycin with the carbamyl group substituted on the glycoside residing on the C6-O of 2-deoxystreptamine. The fragmentation pattern of the other compound was interpreted as that the unknown was also a carbamyltobramycin. The carbamyl group was, however, substituted on 2-deoxystreptamine. It was speculated that the carbamyl group was substituted at the C1 amino group. This compound, to our knowledge, has not been reported before.

Eliminating interferences in a compendial test for oxidizable substances in water.

The United States Pharmacopoeia (USP) uses acidified potassium permanganate to test for dissolved organics in pharmaceutical-grade water. In the test, a standard permanganate concentration is added to a boiling, acidified water sample. Visually inspecting the sample for residual permanganate determines whether the sample passes (pink color remains) or fails (pink color disappears) the test. The permanganate redox chemistry is complex, however, and test samples are prone to developing suspended particulate and colors other than pink. Forming hazy or off-colored solutions interferes with the subjective end point determination according to the USP test. We report two alternative end point determinations that essentially eliminate interferences from the compendial test method. The first alternative involves recording a uv-visible spectrum of the reduced permanganate test solution. Residual permanganate shows three distinct absorbance maxima at 510, 526, and 545 nm. It is straightforward to differentiate the characteristic permanganate fingerprint from the broad, lower-wavelength extinction that results from interfering substances. The second alternative involves filtering the reduced permanganate test solution through sintered glass. This filtration step removes manganese oxides and other colloidal particles that contribute to haze and off color formation in test samples. Visually inspecting the filtrate for residual pink color remains the end point determination for the test method. A third alternative method, namely spectrophotometric determination of permanganate loss rate constants is not a suitable alternative owing to a strong dependence of permanganate reduction rate on organic substrate structure.