Greenness Assessment and Validation of HPLC Method for Simultaneous Determination of Resveratrol and Vitamin E in Dietary Supplements.

BACKGROUND: There is an increasing interest in the use of a combination of trans-resveratrol and vitamin E in dietary supplements. Determination of the content of both components is essential for confirmation of the quality of the product. OBJECTIVE: To establish the applicability and ensure the greenness of the previously developed high-throughput HPLC/UV method for the simultaneous determination of trans-resveratrol and alpha-tocopherol acetate (vitamin E) in dietary supplements. METHOD: Separation was performed on RP C8 Select B chromatographic column, using acetonitrile and water in the mobile phase, with gradient elution. Full method validation was performed in accordance with ICH Q2(R1). The greenness of the method was assessed using the analytical eco-scale (AES) methodology and the analytical greenness metric (AGREE). RESULTS: The method is selective, linear, precise, and accurate over defined concentration ranges (185-369 µg/mL of trans-resveratrol and 37-75 µg/mL of alpha-tocopherol acetate), and it has a suitable sensitivity (limits of detection and quantification are 7.7 and 23.3 µg/mL for resveratrol and 2.6 and 7.8 µg/mL for tocopherol acetate, respectively). The obtained analytical eco-scale score of 77 and the pale green AGREE pictogram with an overall score of 0.61 confirm the method's greenness. CONCLUSIONS: The sensitivity and selectivity of the method, its short analysis time (7 min), the low negative environmental impact, and the simple sample preparation make the method readily applicable to inline quality control procedures. HIGHLIGHTS: A method for simultaneously analyzing vitamin E and resveratrol in dietary supplements is presented. The method is rapid, includes a simple sample preparation procedure, and has a low environmental impact.

Comprehensive Assessment of Degradation Behavior of Simvastatin by UHPLC/MS Method, Employing Experimental Design Methodology.

This manuscript describes comprehensive approach for assessment of degradation behavior of simvastatin employing experimental design methodology as scientific multifactorial strategy. Experimental design methodology was used for sample preparation and UHPLC method development and optimization. Simvastatin was subjected to stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Using 2n full factorial design degradation conditions were optimized to obtain targeted level of degradation. Screening for optimal chromatographic condition was made by Plackett-Burman design and optimization chromatographic experiments were conducted according to Box-Behnken design. Successful separation of simvastatin from the impurities and degradation products was achieved on Poroshell 120 EC C18 50 × 3.0 mm 2.7 µm, using solutions of 20 mM ammonium formate pH 4.0 and acetonitrile as the mobile phase in gradient mode. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines. Validation results have shown that the proposed method is selective, linear, sensitive, accurate, and robust and it is suitable for quantitative determination of simvastatin and its impurities. Afterwards, the degradation products were confirmed by a direct hyphenation of liquid chromatograph to ion-trap mass spectrometer with heated electrospray ionization interface. This study highlights the multiple benefits of implementing experimental design, which provides a better understanding of significant factors responsible for degradation and ensures successful way to achieve degradation and can replace the trial and error approach used in conventional forced degradation studies.

High-Throughput HPLC-MS/MS Method for Quantification of Ibuprofen Enantiomers in Human Plasma: Focus on Investigation of Metabolite Interference.

In this research, as a part of the development of fast and reliable HPLC-MS/MS method for quantification of ibuprofen (IBP) enantiomers in human plasma, the possibility of IBP acylglucoronide (IBP-Glu) back-conversion was assessed. This involved investigation of in source and in vitro back-conversion. The separation of IBP enantiomers, its metabolite and rac-IBP-d3 (internal standard), was achieved within 6 min using Chiracel OJ-RH chromatographic column (150 × 2.1 mm, 5 µm). The followed selected reaction monitoring transitions for IBP-Glu (m/z 381.4 → 205.4, m/z 381.4 → 161.4 and m/z 205.4 → 161.4) implied that under the optimized electrospray ionization parameters, in source back-conversion of IBP-Glu was insignificant. The results obtained after liquid-liquid extraction of plasma samples spiked with IBP-Glu revealed that the amount of IBP enantiomers generated by IBP-Glu back-conversion was far <20% of lower limit of quantification sample. These results indicate that the presence of IBP-Glu in real samples will not affect the quantification of the IBP enantiomers; thereby reliability of the method was improved. Additional advantage of the method is the short analysis time making it suitable for the large number of samples. The method was fully validated according to the EMA guideline and was shown to meet all requirements to be applied in a pharmacokinetic study.

Efficacy assessment of local doxycycline treatment in periodontal patients using multivariate chemometric approach.

The aim of our study was application of chemometric algorithms for multivariate data analysis in efficacy assessment of the local periodontal treatment with doxycycline (DOX). Treatment efficacy was evaluated by monitoring inflammatory biomarkers in gingival crevicular fluid (GCF) samples and clinical indices before and after the local treatment as well as by determination of DOX concentration in GCF after the local treatment. The experimental values from these determinations were submitted to several chemometric algorithms: principal component analysis (PCA), partial least square discriminant analysis (PLS-DA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA). The data structure and the mutual relations of the selected variables were thoroughly investigated by PCA. The PLS-DA model identified variables responsible for discrimination of classes of data, before and after DOX treatment. The OPLS-DA model compared the efficacy of the two commonly used medications in periodontal treatment, chlorhexidine (CHX) and DOX, at the same time providing insight in their mechanism of action. The obtained results indicate that application of multivariate chemometric algorithms can be used as a valuable approach for assessment of treatment efficacy.

Critical development by design of a rugged HPLC-MS/MS method for direct determination of ibuprofen enantiomers in human plasma.

Development and validation of a HPLC-MS/MS method for direct determination of R- and S-ibuprofen (Ibu) in human plasma without a need of derivatization or other complexities such as postcolumn infusion of solvents or reagents was performed. Critical steps were investigated during method development using experimental design to achieve a reliable and rugged assay. The LC-MS/MS separation of R-Ibu and S-Ibu was obtained on Lux Cellulose chiral column utilizing 0.1% (v/v) acetic acid in mixture of methanol and water (90:10%, v/v) as a mobile phase. Two types of extraction procedure for Ibu and Ketoprofen (internal standard, IS) were optimized using Full factorial 3(2) design (LLE) and D-Optimal Experimental Design (SPE). Excellent recovery values, 80% (mean) and 95% (mean) for LLE and SPE respectively, were obtained using 50µL plasma. The matrix effect was assessed for both of the extraction procedures, including hyperlipidaemic and haemolyzed plasma. The extensive investigation of matrix effect showed that LLE yields cleaner extracts than the SPE. The result of the investigation of in vitro interconversion of R-Ibu and S-Ibu showed that it does not occur under the influence of pH, temperature, and in the overall analytical procedure. The validation data, adhered to EMA guideline for validation of bioanalytical methods, showed that the proposed method provides accurate and reproducible results in range of 0.1-50mg/L with a lower limit of detection of 0.02mg/L. The applicability of the method was demonstrated through determination of R-Ibu and S-Ibu in human plasma after oral administration of 400mg rac-Ibu.