Surface-enhanced Raman spectroscopy for comparison of biochemical profile of bacteriophage sensitive and resistant methicillin-resistant Staphylococcus aureus (MRSA) strains.

Methicillin-resistant Staphylococcus aureus (MRSA) is gram positive bacteria and leading cause of a wide variety of diseases. It is a common cause of hospitalized and community-acquired infections. Development of increasing antibiotic-resistance by methicillin-resistant S. aureus (MRSA) strains demand to develop alternate novel therapies. Bacteriophages are now widely used as antibacterial therapies against antibiotic-resistant gram-positive pathogens. So, there is an urgent need to find fast detection techniques to point out phage susceptible and resistant strains of methicillin-resistant S. aureus (MRSA) bacteria. Samples of two separate strains of bacteria, S. aureus, in form of pellets and supernatant, were used for this purpose. Strain-I was resistant to phage, while the other (strain-II) was sensitive. Surface Enhanced Raman Spectroscopy (SERS) has detected significant biochemical changes in these bacterial strains of pellets and supernatants in the form of SERS spectral features. The protein portion of these two types of strains of methicillin-resistant S. aureus (MRSA) in their relevant pellets and supernatants is major distinguishing biomolecule as shown by their representative SERS spectral features. In addition, multivariate data analysis techniques such as principal component analysis (PCA) and a partial least squares-discriminant analysis (PLS-DA) were found to be helpful in identifying and characterizing various strains of S. aureus which are sensitive and resistant to bacteriophage with 100% specificity, 100% accuracy, and 99.8% sensitivity in case of SERS spectral data sets of bacterial cell pellets. Moreover, in case of supernatant samples, the results of PLS-DA model including 95.5% specificity, 96% sensitivity, and 96.5% accuracy are obtained.

Qualitative and Quantitative Analysis of Azithromycin as Solid Dosage by Raman Spectroscopy.

Active pharmaceutical ingredients (APIs) and excipients are main drug constituents that ought to be identified qualitatively and quantitatively. Raman spectroscopy is aimed to be an efficient technique for pharmaceutical analysis in solid dosage forms. This technique can successfully be used in terms of qualitative and quantitative analysis of pharmaceutical drugs, their APIs, and excipients. In the proposed research, Raman spectroscopy has been employed to quantify Azithromycin based on its distinctive Raman spectral features by using commercially prepared formulations with altered API concentrations and excipients as well. Along with Raman spectroscopy, principal component analysis and partial least squares regression (PLSR), two multivariate data analysis techniques have been used for the identification and quantification of the API. For PLSR, goodness of fit of the model (R2) was found to be 0.99, whereas root mean square error of calibration was 0.46 and root mean square error of prediction was 2.42, which represent the performance of the model. This study highlights the efficiency of Raman spectroscopy in the field of pharmaceutics by preparing pharmaceutical formulations of any drug to quantify their API and excipients to compensate for the commercially prepared concentrations.

Characterization of structural changes occurring in insulin at different time intervals at room temperature by surface-enhanced Raman spectroscopy.

BACKGROUND: Insulin storage above the temperature recommended by food and drug administration (FDA) causes decrease in its functional efficacy due to degradation and aggregation of its protein based active pharmaceutical ingredient (API) that results poor glycemic control in diabetic patients. The aggregation of protein causes serious neurodegenerative diseases such as type-2 diabetes, Huntington disease, Parkinson's disease, and Alzheimer's disease. Surface-enhanced Raman spectroscopy (SERS) has been employed for the denaturation study of many proteins at the temperature above the recommendations of food and drug administration (FDA) (above 30 °C) which indicates potential of technique for such studies. OBJECTIVE: SERS along with multivariate discriminating analysis techniques-based analysis of degradation of liquid pharmaceutical insulin protein after regular intervals of time at room temperature to analyze the structural changes in this protein during the storage of insulin pharmaceutical at room temperature. METHODS: Silver nanoparticles (Ag-NPs) prepared by chemical reduction method are used as SERS active substrate for the surface enhancement of the insulin spectral signal. SERS spectral measurements of insulin were collected from eight different samples of insulin in the time range of 7 pm to 7 am first at fridge temperature (5 °C), second after half hour and next six with the time difference of 2 h each time at room temperature. The acquired SERS spectral data was preprocessed and analyzed. SERS structural transformations detection and discrimination potential in insulin was further confirmed by applying multivariate discriminating analysis techniques including principal component analysis (PCA) and Partial least square regression analysis (PLSR). RESULTS: SERS significantly detects the structural changes produced in insulin even after 2 h of insulin placement at room temperature. PCA successfully differentiates the insulin spectral data obtained after regular intervals of time according to PC-1 (77 %) explained variance. Application of PLSR model provides quantitative confirmation of SERS efficiency, by providing insulin data regression coefficients plot, efficient prediction of time with calibration data set having 0.77 mean square absolute error of calibration (RMSAEC), validation data set with 0.80 mean square absolute error of prediction (RMSAEP) and 0.98 coefficient of determination (R2) for both calibration and validation data set. CONCLUSION: SERS is proved as a highly sensitive and discriminating technique to detect and discriminate insulin structural changes after regular intervals of time at room temperature.