Comparison of SARS-CoV-2 detection in Saliva by real-time RT-PCR and RT-PCR/MALDI-TOF Methods

The coronavirus disease 2019 (COVID-19) pandemic has accelerated the need for rapid implementation of diagnostic assays for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in respiratory specimens. While multiple molecular methods utilize nasopharyngeal specimens, supply chain constraints and need for easier and safer specimen collection warrant alternative specimen types, particularly saliva. Although saliva has been found to be a comparable clinical matrix for detection of SARS-CoV-2, evaluations of diagnostic and analytic performance across platforms for this specimen type are limited. Here, we compared two methods for SARS-CoV-2 detection in saliva: the Roche cobas(R) 6800/8800 SARS-CoV-2 real-time RT-PCR Test and the Agena Biosciences MassARRAY(R) SARS-CoV-2 Panel/MassARRAY(R) System. Overall, both systems had high agreement with one another, and both demonstrated high diagnostic sensitivity and specificity when compared to matched patient upper respiratory specimens. We also evaluated the analytical sensitivity of each platform and determined the limit of detection of the Roche assay was four times lower than that of Agena for saliva specimens (390.6 v. 1,562.5 copies/mL). Furthermore, across individual target components of each assay, T2 and N2 targets had the lowest limits of detection for each platform, respectively. Together, we demonstrate that saliva represents an appropriate specimen for SARS-CoV-2 detection in two technologies that have high agreement and differ in analytical sensitivities overall and across individual component targets. The addition of saliva as an acceptable specimen and understanding the sensitivity for testing on these platforms can further inform public health measures for screening and detection to combat the COVID-19 pandemic.

Use of pharmaceutical salts and cocrystals to address the issue of poor solubility.

Salt and cocrystal formation are the most commonly used method of increasing solubility and dissolution rate of pharmaceutical compounds, and are of particular interest for compounds with an intermediate to low aqueous solubility. However, selection of the most appropriate form does not necessarily equate to selection of the salt/cocrystal with the optimal aqueous solubility, but rather a balance between the best solubility and the best physicochemical properties. This review provides a presentation of salt and cocrystal selection, from a high throughput screening perspective and then an assessment of counter ion properties, common ion effects and the potential impact on the biopharmaceutical performance of the compound. In addition, there is a brief discussion of the impact on polymorphism, the potential use of salts and stoichiometric amorphous mixtures to stabilise amorphous forms and other potential issues for consideration from a pharmaceutical development perspective.

Formation of solid solutions between racemic and enantiomeric citalopram oxalate.

The X-ray powder diffractograms of racemic citalopram oxalate and (S)-citalopram oxalate are very similar, but the melting point of the racemate is higher than that of the pure enantiomer. The higher melting point indicates that the racemate is a racemic compound, rather than a conglomerate. The crystal structure of the enantiomer contains two molecules of (S)-citalopram in the asymmetric unit. The conformation of the two molecules is different but they approximate mirror images of each other if the aromatic groups are interchanged. The crystal structure of the racemate is essentially isostructural with that of the enantiomer, having almost the same cell parameters but containing a crystallographic inversion centre that is not retained in the enantiomer structure. The closely-comparable crystal structures permit solid solutions to be formed between racemic and enantiomeric citalopram oxalate. Phase diagrams of the (R)-citalopram and (S)-citalopram oxalate system are constructed, and they show that solid solutions are formed at all ratios of the two enantiomers.

Phase transformations of amlodipine besylate solid forms.

Hydrate formation and dehydration phenomena are frequently encountered phase transformations during manufacturing and storage of the drug products. It is essential to understand, monitor, and control these transformations to ensure that the quality attributes of the drug product are not affected. In this work, phase transformations of the solid forms of amlodipine besylate (AMB) were studied using Raman and near-infrared (NIR) spectroscopy. AMB exists as anhydrate (AH), monohydrate (MH), dihydrate (DH), and amorphous (AM) form. Solid form quantification models based on multivariate data analysis of the Raman and NIR spectra were developed. The AH, MH, and AM form were transformed to the DH during solubility measurements. The AH to DH transformation also occurred during wet granulation. The transformation kinetics were faster during wet granulation than during the solubility experiments. This was due to the shear forces involved in granulation that can facilitate nucleation and can enhance the overall transformation. The DH form present in the wet granules persisted after drying, and final granules contained a mixture of the AH and DH. The relative importance of the dissolution, nucleation, and growth steps for the transformation was elucidated using optical microscopy experiments. The transformation kinetics were found to be limited by nucleation and growth.

Integrated approach to study the dehydration kinetics of nitrofurantoin monohydrate.

There is a need for thorough knowledge of solid-state transformations in order to implement quality by design (QbD) methodology in drug development. The present study was aimed at gaining a mechanistic understanding of the dehydration of nitrofurantoin monohydrate II (NF-MH). The dehydration was studied using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), hot-stage microscopy (HSM), and variable temperature X-ray powder diffraction (VT-XRPD). Isothermal TGA data were used to study dehydration kinetics using model-fitting and model-free approaches. Model-fitting analysis indicated a good fit for several models derived from nucleation-growth and/or geometric contraction mechanisms. However, based on visual observations during HSM, Avrami-Erofeyev equations A3 and A4, indicating nucleation-growth phenomenon, were found to be the most suitable kinetic models. HSM showed initiation of dehydration with random nucleation, and nuclei coalesced with the progress of dehydration reaction. VT-XRPD revealed formation of anhydrate beta form on dehydration of NF-MH. The phenomenon of random nucleation is justified based on the crystal structure of NF-MH, which showed presence of water molecules in an isolated manner, prohibiting directional dehydration. It was found that supplementary information from HSM and VT-XRPD can be valuable to gain a better understanding of dehydration from formal solid-state kinetics analysis.

Structural characterisation and dehydration behaviour of siramesine hydrochloride.

In this study the crystal structures of siramesine hydrochloride anhydrate alpha-form and siramesine hydrochloride monohydrate were determined, and this structural information was used to explain the physicochemical properties of the two solid forms. In the crystal structure of the monohydrate, each water molecule is hydrogen bonded to two chloride ions, and thus the water is relatively strongly bound in the crystal. No apparent channels for dehydration were observed in the monohydrate structure, which could allow transmission of structural information during dehydration. Instead destructive dehydration occurred, where the elimination of water from the monohydrate resulted in the formation of an oily phase, which subsequently recrystallised into one or more crystalline forms. Solubility and intrinsic dissolution rate of the anhydrate alpha-form and the monohydrate in aqueous media were investigated and both were found to be lower for the monohydrate compared to the anhydrate alpha-form. Finally, the interactions between water molecules and chloride ions in the monohydrate as well as changes in packing induced by water incorporation could be detected by spectroscopic techniques.

Near-infrared spectroscopy for cocrystal screening. A comparative study with Raman spectroscopy.

Near-infrared (NIR) spectroscopy is a well-established technique for solid-state analysis, providing fast, noninvasive measurements. The use of NIR spectroscopy for polymorph screening and the associated advantages have recently been demonstrated. The objective of this work was to evaluate the analytical potential of NIR spectroscopy for cocrystal screening using Raman spectroscopy as a comparative method. Indomethacin was used as the parent molecule, while saccharin and l-aspartic acid were chosen as guest molecules. Molar ratios of 1:1 for each system were subjected to two types of preparative methods. In the case of saccharin, liquid-assisted cogrinding as well as cocrystallization from solution resulted in a stable 1:1 cocrystalline phase termed IND-SAC cocrystal. For l-aspartic acid, the solution-based method resulted in a polymorphic transition of indomethacin into the metastable alpha form retained in a physical mixture with the guest molecule, while liquid-assisted cogrinding did not induce any changes in the crystal lattice. The good chemical peak selectivity of Raman spectroscopy allowed a straightforward interpretation of sample data by analyzing peak positions and comparing to those of pure references. In addition, Raman spectroscopy provided additional information on the crystal structure of the IND-SAC cocrystal. The broad spectral line shapes of NIR spectra make visual interpretation of the spectra difficult, and consequently, multivariate modeling by principal component analysis (PCA) was applied. Successful use of NIR/PCA was possible only through the inclusion of a set of reference mixtures of parent and guest molecules representing possible solid-state outcomes from the cocrystal screening. The practical hurdle related to the need for reference mixtures seems to restrict the applicability of NIR spectroscopy in cocrystal screening.

Solvent diversity in polymorph screening.

Selecting a diverse set of solvents to be included in polymorph screening assignments can be a challenging task. As an aid to decision making, a database of 218 organic solvents with 24 property descriptors was explored and visualized using multivariate tools. The descriptors included, among others, log P, vapor pressure, hydrogen bond formation capabilities, polarity, number of pi-bonds and descriptors derived from molecular interaction field calculations (e.g., size/shape parameters and hydrophilic/hydrophobic regions). The data matrix was initially analyzed using principal component analysis (PCA). Results from the PCA showed 57% cumulative variance being explained in the first two principal components (PCs), although relevant information was also found in the third, fourth and fifth component, revealing distinct clusters of solvents. Since five dimensions were not suitable for visual presentation, a nonlinear method, self-organizing maps (SOMs), was applied to the dataset. The constructed SOM displayed features of clusters observed in the first three PCs, however in a more compelling way. Thus, the SOM was chosen as the visually most convenient way to display the diversity of the 218 solvents. In addition, it was demonstrated how safety aspects can be considered by labeling a large fraction of the solvents in the SOM with toxicological information.