Application of design space and quality by design methodologies combined with ultra high-performance liquid chromatography for the optimization of the sample preparation of complex pharmaceutical dosage forms.

Accurate and precise analytical measurements play a significant role in assessments and decisions that are made throughout the drug development process. Developing a robust and reliable sample preparation is essential for drug product formulations to generate consistent results guaranteeing the product quality. However, due to the complex nature of the different pharmaceutical formulations with diverse excipients, developing robust sample preparation methods can be challenging and time consuming. Ensuring sample extraction robustness of pharmaceutical dosage forms becomes increasingly important with the potential impact to patient safety, product efficacy, and business efficiency. In this work we demonstrate and evaluate potential application of Quality by Design (QbD) principles to develop and optimize a robust sample preparation method in combination with the chromatographic analytical technique for a solid pharmaceutical dosage form. Practicability and utility of a QbD approach in optimization of sample preparation of this drug product are demonstrated as the active pharmaceutical ingredient (API) used in the drug product is proven to be highly sensitive for hydrolysis during analysis. Finally, the ultra-high-performance liquid chromatography method with UV detection that was applied during the design of experiments (DoE) was validated as per regulatory requirements. This systematic approach in analytics could provide guidance for the pharmaceutical industry in the development of robust sample preparation methods for different pharmaceutical dosage forms thus significantly reduce risks associated with the method transfers at clinical and commercial manufacturing sites.

Parental frequencies and spatial configuration shape bumblebee behavior and floral isolation in hybridizing Rhinanthus.

To shed light on the role played by pollinators in the diversification of angiosperms, focus is needed on how floral isolation varies locally in the early stages of plant divergence. The few studies performed so far have often used species pairs with distinct pollination syndromes and contrasting floral displays. Here, we focus on a hybridizing pair (Rhinanthus minor and Rhinanthus angustifolius) with strong similarities in flower morphology and pollinators (bumblebees). We examined how ethological isolation changes locally in relation to relative Rhinanthus frequencies, spatial configurations, and pollinator assemblages. Interestingly, floral divergence based on adaptation to different pollinators is unlikely in Rhinanthus: no relationship was found between floral isolation and the local pollinator assemblage. In contrast, species frequency and spatial arrangement strongly influenced bumblebee behavior, ethological isolation, and thus potentially hybrid formation. When both Rhinanthus were present in equal proportions, bees generally preferred the more rewarding and conspicuous species. However, when the Rhinanthus frequencies were unbalanced, the more abundant species was preferred, although this was less pronounced when the less rewarding R. minor predominated. Ethological isolation is highly sensitive to site characteristics, and can be as high as in species with contrasting floral displays and pollinator suites, even though flowers are similar.

Post-pollination barriers and their role in asymmetric hybridization in Rhinanthus (Orobanchaceae).

PREMISE OF THE STUDY: Several barriers against hybrid formation exist, and their combined action can affect the evolutionary outcomes of hybridization. To explain the asymmetrical introgression observed between two bumblebee-pollinated plant species (Rhinanthus angustifolius and R. minor), we focused on post-pollination barriers and ethological isolation of hybrids. METHODS: We studied pollen competition in conspecific and heterospecific crosses on both species and analyzed germination rates of hybrid and pure seeds. We also measured bumblebee visitation rates to hybrids relative to their parents using potted Rhinanthus placed in populations of each parental species. KEY RESULTS: In mixed pollinations, there was a conspecific siring advantage in both species, but no difference in pollen tube growth rates in either cross type. F(1) seeds with a R. angustifolius maternal plant germinated poorly, while those with R. minor as the maternal parent germinated better than pure seeds. Interestingly, bumblebees treated hybrids almost as equal to the background species and more often rejected the nonresident Rhinanthus. In a R. angustifolius background, bumblebees preferred R. angustifolius, but visited hybrids more often than R. minor. In contrast, visitation rates were similar on a R. minor background. CONCLUSIONS: Our results suggest that hybridization rates in Rhinanthus remain low because of several leaky barriers that make R. minor the maternal parent of most F(1) offspring. Preference for R. angustifolius and the equal treatment of F(1) and background species by bumblebees induce a visitation pattern that directs gene flow toward R. angustifolius when this species predominates.

Shared pollinators and pollen transfer dynamics in two hybridizing species, Rhinanthus minor and R. angustifolius.

Gene flow between hybridizing plant species depends strongly on pollinator behaviour, which affects pollen transfer among floral types and reproductive isolation. We examined bumblebee behaviour and pollen transfer between two hybridizing Rhinanthus species that are very similar in ecology and floral traits. The two species, Rhinanthus minor and R. angustifolius, shared similar pollinator guilds and assemblages, but pollinator recruitment and flower visitation rates were higher in R. angustifolius sites, probably because of its higher reward levels and better visibility. When presented with Rhinanthus flowers, bumblebees that previously foraged on R. angustifolius were less prone to visit R. minor inflorescences, while R. minor foragers accepted both species in similar proportions. Although Rhinanthus has been cited as a case of mechanical isolation resulting from interactions between bee behaviour and differences in stigma and anther placement, we found no support for efficient mechanical reproductive isolation. Bumblebees that foraged on R. minor flowers carried more pollen, but pollen placement on their bodies was similar to that of bees that visited R. angustifolius, and cross-specific stigmatic pollen deposition was similar in both directions. However, the asymmetry in pollinator handling time between the two species, due to dissimilar pollen rewards, may have lowered relative heterospecific pollen receipt on R. angustifolius, suggesting that net gene flow resulting from pollen transfer dynamics is more likely towards R. minor, although this effect remains weak and will be most likely counterbalanced by context-based labile pollinator preference.