Development of Analytical Method for the Quantitation of Monoclonal Antibodies Solutions via Raman Spectroscopy: The Case of Bevacizumab.

Personalized dosages of monoclonal antibodies are being used more regularly to treat various diseases, rendering their quantitation more essential than ever for the right dose administration to the patients. A promising alternative, which overcomes the obstacles of the well-established chromatographic techniques regarding the quantification of biopharmaceuticals, is Raman spectroscopy. This study aimed to develop and validate a novel analytical method for the quantitation of bevacizumab in solutions via Raman spectroscopy. For this purpose, a droplet of the solution was left to dry on a highly reflective carrier and a home-made apparatus was employed for rotation of the sample. Hence, each recorded Raman spectrum was the average of the signal acquired simultaneously from multiple points on a circular circumference. The method was validated, and the detection limit of the antibody was found to be 1.06 mg/mL. Bevacizumab was found to be highly distributed at the formed coffee ring of the dried droplet, though this was a function of solution concentration. Finally, Raman spectra at different distances on the coffee ring were obtained from the four quarters. The lowest bevacizumab detection limit was found at a distance of 75 µm from the external side of the coffee ring and it was determined to be equal to 0.53 mg/mL.

Quantitative determination of alcohols in human biological fluids through Raman spectroscopy: An alternative alcohol test.

The severe effects of alcohols on humans trigger the continuous research on the alcohols level measurement in biological fluids. The officially established technique is Headspace Gas Chromatography (HS-GC), while breathalyzers are commonly used by police on the road. However, they all exhibit drawbacks; HS-GC is expensive and labor-intensive, while the precision of breathalyzers is controversial. In the present study, a novel method was developed, for ethanol and methanol detection and quantification in human urine, saliva and blood serum, based on Raman spectroscopy. Biological fluids from healthy adult volunteers were collected, standard solutions of the alcohols in a concentration range from 0.00 µL/mL to 5.00 µL/mL were prepared and analysed using an air-tight and small volume sample carrier. Calibration curves for each binary system (alcohol - biological fluid) were created. Ethanol calculated detectable concentrations were below permissible limits for all biological fluids. In the case of methanol, the limits were not as satisfactory, but lower than intoxication level, due to the difficult spectral discrimination. For both alcohols, the lowest detection limits were recorded for saliva. All detection limits were verified by visual inspection of the spectra. The proposed quantitative method was validated in all cases regarding their specificity, working range, accuracy, precision and sensitivity.

Comparative Study of Sample Carriers for the Identification of Volatile Compounds in Biological Fluids Using Raman Spectroscopy.

Vibrational spectroscopic techniques and especially Raman spectroscopy are gaining ground in substituting the officially established chromatographic methods in the identification of ethanol and other volatile substances in body fluids, such as blood, urine, saliva, semen, and vaginal fluids. Although a couple of different carriers and substrates have been employed for the biochemical analysis of these samples, most of them are suffering from important weaknesses as far as the analysis of volatile compounds is concerned. For this reason, in this study three carriers are proposed, and the respective sample preparation methods are described for the determination of ethanol in human urine samples. More specifically, a droplet of the sample on a highly reflective carrier of gold layer, a commercially available cuvette with a mirror to enhance backscattered radiation sealed with a lid, and a home designed microscope slide with a cavity coated with gold layer and covered with transparent cling film have been evaluated. Among the three proposed carriers, the last one achieved a quick, simple, and inexpensive identification of ethanol, which was used as a case study for the volatile compound, in the biological samples. The limit of detection (LoD) was found to be 1.00 µL/mL, while at the same time evaporation of ethanol was prevented.

Formation and Characterisation of Posaconazole Hydrate Form.

Posaconazole is an API added as Form I for the production of oral suspensions, but it is found as Form-S in the final formulation. In this study, it was found that this polymorphic conversion, which may affect the bioavailability, is due to an interaction with water. However, the relatively poor wettability of posaconazole Form I renders the complete wetting of its particles and production of pure Form-S challenging. Consequently, for its isolation, Form I should be dispersed in water followed by application of sonication for at least 10 min. Pure posaconazole Form-S was characterised using X-ray powder diffraction (XRPD), Raman spectroscopy, attenuated total reflection (ATR) spectroscopy, thermogravimetric analysis (TGA) and optical microscopy. From these techniques, posaconazole Form-S was characterised as a hydrate form, which includes three molecules of water per API molecule.

Warfarin Sodium Stability in Oral Formulations.

Warfarin sodium is a low-dose pharmaceutical blood thinner that exists in two forms: the clathrate form and the amorphous form. In commercially available warfarin sodium oral suspension, the active pharmaceutical ingredient (API) is added in the amorphous state. This study investigates the apparent instability of the commercially available warfarin liquid oral formulation using Raman and IR spectroscopy, X-ray diffraction, differential scanning calorimetry, UV spectroscopy, and optical microscopy. Warfarin, not its sodium salt, was identified as the undissolved solid existing in the suspension. This was found to be due to the dissociation of sodium salt and the protonation of the warfarin ion in the liquid phase, which triggered the crystallization of the sparingly soluble unsalted form. The coexistence of protonated and unprotonated warfarin ions in the supernatant, as detected by Raman and UV spectroscopy, confirmed this assumption. Study of the dissolution of warfarin sodium amorphous salt and crystalline sodium clathrate in the placebo and pure water verified the results. The effect of pH and temperature on warfarin precipitation was also explored.

Sample Preparation of Posaconazole Oral Suspensions for Identification of the Crystal Form of the Active Pharmaceutical Ingredient.

Determination of the polymorphic form of an active pharmaceutical ingredient (API) in a suspension could be really challenging because of the water phase and the low concentration of the API in this formulation. Posaconazole is an antifungal drug available also as an oral suspension. The aim of this study was to develop a sample-preparation method for polymorphic identification of the dispersed API by increasing the concentration of the API but with no compromise of polymorph stability. For this purpose, filtration, drying and centrifugation were tested for separating the API from the suspending medium. Centrifugation was selected because it succeeded in separating Posaconazole API with no polymorph transformation during the process. During this study, it was found that Posaconazole in oral suspensions is Form-S. However, when slower scanning rates were used for acquiring an XRPD pattern with better signal/noise ratio, Posaconazole was converted to Form I due to water loss. In order to protect the sample from conversion, different approaches were tested to secure an airtight sample including a commercially available XRPD sample holder with a dome-like transparent cap, standard polymethylmethacrylate (PMMA) sample holders covered with Mylar film, transparent pressure-sensitive tape and a transparent food membrane. Only usage of the transparent food membrane was found to protect the API from conversion for a period of at least two weeks and resulted in a Posaconazole Form-S XRPD pattern with no artificial peaks.

Effect of Plant Extracts on the Characteristics of Silver Nanoparticles for Topical Application.

Silver nanoparticles (AgNPs) were synthesized using hydroalcoholic extracts of dittany (Origanum dictamnus), sage (Salvia officinalis), sea buckthorn (Elaeagnus rhamnoides, syn. Hippophae rhamnoides), and calendula (Calendula officinalis) as reducing agents. AgNPs synthesized using NaBH4 and citric acid were used as control. The impact of the origin of the extract and preparation conditions (light, temperature, reaction time) on the properties of the synthesized AgNPs was investigated. The structure, morphology, composition, physicochemical characteristics, and colloidal stability were characterized using dynamic laser scattering (DLS), ultraviolet-visible spectrophotometry (UV-/Vis), XRD, X-ray fluorescence (XRF), TEM, and FTΙR. The reduction of total phenolic and flavonoid content of the extracts after the reaction of AgNPs synthesis was also determined. Low IC50 values for all types of AgNPs revealed good antioxidant activity, attributable to the phenolic and flavonoid content of their surface. The results suggest that plant extract selection is important to the green synthesis of AgNPs because it affects the kinetics of their synthesis as well as their morphology, physicochemical characteristics, and colloidal stability. In vitro permeation studies on porcine skin revealed that AgNPs remained at the upper layers of stratum corneum and did not penetrate the skin barrier after 4 h of cutaneous application suggesting the safety of their application on intact skin for a relatively short time.

NMR study of non-structural proteins-part III: 1H, 13C, 15N backbone and side-chain resonance assignment of macro domain from Chikungunya virus (CHIKV).

Macro domains are conserved protein domains found in eukaryotic organisms, bacteria, and archaea as well as in certain viruses. They consist of 130-190 amino acids and can bind ADP-ribose. Although the exact role of these domains is not fully understood, the conserved binding affinity for ADP-ribose indicates that this ligand is important for the function of the domain. Such a macro domain is also present in the non-structural protein 3 (nsP3) of Chikungunya Alphavirus (CHIKV) and consists of 160 amino acids. In this study we describe the high yield expression of the macro domain from CHIKV and its preliminary structural analysis via solution NMR spectroscopy. The macro domain seems to be folded in solution and an almost complete backbone assignment was achieved. In addition, the α/ß/α sandwich topology with 4 α-helices and 6 ß-strands was predicted by TALOS+.