Optimization of feather degradation by a Bacillus thuringiensis isolate using response surface methodology and investigation of the feather protein hydrolysate structure.

Valorization of chicken feather is a long-sought approach for its sustainable disposal. Being protein rich, hydrolyzed chicken feather has a wide range of applications, not limited to formulation of microbiological culture media, animal feed, and biofertilizers, but extends to synthesis of bioplastic films, cosmetics, and biomedicals. In this study, a potent keratinolytic isolate was recovered from soil and identified by 16S rRNA as Bacillus thuringiensis. Feather degradation by the isolate was optimized through response surface methodology. First, one-variable-at-a-time technique to assign the factors that affect feather degradation, then Box-Behnken central composite design model were employed. The model, involving three independent variables (initial pH, inoculum size, and concentration of supplementary glucose), was significant (R2  = 0.9716). According to the model, complete feather degradation is obtained at an inoculum size of B. thuringiensis B4 equal to 1 × 1010  CFU/ml, when feather meal broth is supplemented with 1.5% (w/v) glucose and pH adjusted to 8.5. Protein content of the lysate was 327.8 ± 25 µg/ml, and no carbohydrates were detected. SEM/EDX analysis has shown that the hydrolysate consisted mainly of O, P, S, and Se in addition to carbon, while FTIR images assured the presence of carboxyl and amino groups characteristic of peptides and amino acids.

Smart Eco-Friendly Spectrophotometric Methods Resolving Highly Overlapping Spectra: Application to Veterinary Antibiotic Injections.

BACKGROUND: Over the last few years, mathematical manipulation has proved to be a very powerful means of successfully resolving severely overlapped spectra for various multicomponent mixtures. OBJECTIVE: Smart and environmentally friendly spectrophotometric determination approaches were used for two binary mixtures of fixed dose veterinary injections containing flunixin meglumine (FLU) combined with either florfenicol (FLR), or oxytetracycline HCl (OXY). METHODS: Regarding the first mixture, both FLU and FLR were determined by three successive resolution techniques, which were; constant multiplication coupled with spectrum subtraction (CM-SS), derivative ratio (DD1), and ratio difference (RD), and two progressive resolution techniques which were absorbance subtraction (AS) and amplitude modulation (AM). Also, graphical representation of concentration of the two drugs through concentration value (CNV) method was also applied. Concerning the second mixture, both FLU and OXY showed severely overlapped spectra and a comparative study was conducted for the determination of each drug by constant center (CC), ratio subtraction via amplitude difference coupled with spectrum subtraction (RS/AD-SS), constant value via amplitude difference (CV-AD), and advanced concentration value (ACV) methods. RESULTS: Calibration graphs of the first mixture were linear over the range 5-40 µg/mL for FLU, and 3-40 µg/mL for FLR. The proposed methods overcame the problem of the overlapped spectra and the presence of a minor component in the mixture. Regarding the second mixture, calibration graphs were linear over the range 2.5-24 µg/mL for FLU and 4-28 µg/mL for OXY. CONCLUSION: The proposed methods were successfully validated as per International Council for Harmonization (ICH) guidelines. The obtained results were statistically compared with the official or reported methods, showing no significant difference concerning accuracy and precision. The methods were evaluated for greenness by three different assessment tools: NEMI, analytical ecoscale, and GAPI. HIGHLIGHTS: The methods were successfully applied for the simultaneous determination of the two combinations in synthetic mixtures and their marketed antibiotic veterinary injections: Megluflor® and Floxon®.

Fourier Transform Infrared Spectroscopic, Spectrofluorimetric Assays of Canagliflozin, and Stability-Indicating UV-Spectrophotometric Method for the Simultaneous Determination of Canagliflozin and Metformin.

BACKGROUND: Green Fourier transform infrared (FTIR) spectroscopy, using potassium bromide (KBr) disc technique, eliminates the consumption of hazardous chemicals. Spectrofluorimetry for drugs that have native fluorescence. Mean centering of ratio spectra (MCRS) analysing overlapped spectra without preliminary separation. OBJECTIVE: Three simple, accurate, and sensitive methods have been developed and validated for the determination of canagliflozin (CANA); one is a stability-indicating method for CANA and metformin (MET) determination. METHODS: Method A is FTIR using a KBr disc for CANA determination measuring alkyl halide C-F peak area centered on 1230/cm. Method B is spectrofluorimetry using Δ λ = 50 nm synchronous mode at a peak maximum of 291.8 nm for CANA determination using methanol as solvent. Method C is a stability-indicating MCRS method measuring the peak amplitude of CANA and MET at 306.2 and 246.6 nm, respectively, in their mixture with complete CANA oxidation degradation. RESULTS: The linear ranges were: FTIR spectroscopy, 1.50-24.70 µg/mg CANA; spectrofluorimetry, 100.00-600.00 ng/mL CANA; and MCRS, 1.00-25.00 µg/mL CANA and 1-30 µg/mL MET. CONCLUSION: All results were statistically compared with a reported method: no significant difference was observed. HIGHLIGHTS: The proposed methods can be used efficiently for routine analysis in QC laboratories. A green FTIR method utilizes only one reagent, KBr. Spectrofluorimetry using a constant wavelength synchronous scan of CANA native fluorescence in nanogram concentrations overcomes conventional excitation/emission spectra drawbacks and proves that the solvent effect in the fluorescence intensity differs according to concentration used. Stability-indicating MCRS determines the studied drugs in bulk, pharmaceutical formulations, accelerated and long-term stability study samples.

Investigation of a QuEChERS-Based Method for Determination of Polycyclic Aromatic Hydrocarbons in Rat Plasma by GC-MS.

Owing to the toxic effects of polycyclic aromatic hydrocarbons (PAHs) on humans and the environment, their sensitive biomonitoring is essential and significant. In this work, a sensitive, simple and rapid bioanalytical method was established for the simultaneous determination of 13 PAHs in rat plasma depending on QuEChERS (Quick, easy, cheap, effective and rugged method) as a preliminary step and gas chromatography-mass spectrometry (GC-MS) for identification. QuEChERS procedure was optimized where acetonitrile was employed for plasma samples extraction, which was further cleaned using primary secondary amine as the sorbent material. Optimization of GC-MS conditions was performed to produce optimum selectivity of the proposed method. The method was fully validated for rat plasma samples where recoveries, matrix effects, limit of quantitation (LOQ), linearity and precision were evaluated. Linearity range was 5-100 ng/mL for most of the 13 analytes. Average recoveries of the 13 PAHs ranged between 85.57% and 109.64% in fortified rat plasma with standard deviations <8.91 except for anthracene that showed 19.24. The limits of detection and LOQs for the 13 compounds ranged from 0.045 to 0.372 ppb and from 0.137 to 1.128 ppb, respectively. The established method was successfully implemented to perform a minor toxicokinetic study in intraperitoneally dosed rats (0.25 and 2 mg/kg in vegetable oil). The 13 PAHs were tracked in rat plasma samples for 6 h after administration, and most of the target compounds were recognized in plasma samples only at the higher dose.

Spectrophotometric Determination of Polyvinyl Pyrrolidone in Pure and Pharmaceutical Dosage Form.

BACKGROUND: Recently, functional polymers have attracted significant attention in the areas of pharmaceuticals and biomedical applications, so it is important to develop simple techniques to analyze functional polymers in their pharmaceutical dosage forms. OBJECTIVE: Three simple, accurate, and sensitive UV spectrophotometric methods have been developed and validated for determination of polyvinyl pyrrolidone (PVP) in the presence of benzalkonium chloride (BZ) and sodium lactate in ternary mixtures. METHOD: Method A is a derivative ratio spectra zero-crossing (DRSZ) method which measures PVP peak amplitude at 303.1 nm. Method B is a double divisor ratio derivative (DDRD), used for determination of both PVP and BZ in the presence of sodium lactate at 272.6 and 271.5 nm, respectively. Method C is a double divisor ratio derivative-ratio difference spectrophotometric method (DDRD-RDSM), a new and hybrid method of double divisor and ratio difference that hasn't been applied before. It measures peak amplitude difference of the ratio spectra at ΔP278 - 252.4 and ΔP260.9 - 213 for PVP and BZ, respectively. RESULTS: Linear ranges for PVP (5.00-35.00, 10.00-40.00, and 10.00-40.00 µg/mL) was obtained by using DRSZ, DDRD, and DDRD-RDSM, respectively. While the linear range for BZ (5.00-60 µg/mL) was obtained by using both DDRD and DDRD-RDSM. CONCLUSIONS: All results were statistically compared with reported methods. No significant differences were observed. The developed methods were applied to the analysis of the investigated drugs in pure and pharmaceutical dosage forms. HIGHLIGHTS: The proposed methods are of great value, improving the efficiency of routine analysis of PVP and BZ in their pharmaceutical dosage forms.

Analytical comparability study of anti-CD20 monoclonal antibodies rituximab and obinutuzumab using a stability-indicating orthogonal testing protocol: Effect of structural optimization and glycoengineering.

Glycoengineering and biosimilarity are the key factors for growing, promising and progressive approaches in monoclonal antibodies development. In this study, the physicochemical stability of anti-CD20 rituximab (RTX); originator and biosimilar was compared to its glycoengineered humanized version; obinutuzumab (OBZ). An orthogonal stability-indicating protocol using a set of validated bioanalytical techniques; size exclusion high performance liquid chromatography (SE-HPLC), reversed phase liquid chromatography (RP-HPLC), quantitative gel electrophoresis by TapeStation, receptor binding assay and dynamic light scattering (DLS) was used to investigate the effect of different stress factors on the pattern and kinetics of degradation. SE-HPLC results supported with spectral purity showed similar degradation extent with a different pattern of degradation between RTX and OBZ. A lower tendency to form degraded fragments and a relatively higher favorability for degradation through aggregate formation has been revealed in case of OBZ. Results were in agreement with those of DLS and receptor binding assay which showed specificity to the intact antibodies in the presence of their degradation products. Furthermore, results were additionally confirmed through denaturing quantitative gel electrophoresis which suggested reducible covalent bonds as the mechanism for aggregates formation. RP-HPLC results showed two oxidized forms via excessive oxidation of RTX and OBZ with nearly the same degradation percent. Comparability data of RTX and OBZ using the applied methodologies showed that although glycoengineering; carried out to enhance the therapeutic and biological activity of OBZ altered the pattern of degradation but did not significantly affect the overall stability. Results showed also consistent stability profile between the biosimilar and its originator RTX products.