Factorial design applied to LC-ESI-QTOF mass spectrometer parameters for untargeted metabolomics.

Investigations of untargeted metabolomics are based on high-quality data acquisition usually from multiplatform systems that include high-resolution mass spectrometry equipment. The comprehensive set of results is used as data entry for bioinformatics and machine learning sciences to access reliable metabolic and biochemical information for clinical, forensic, environmental, and endless applications. In this context, design of experiments is a powerful tool for optimizing data acquisition procedures, using a multivariate approach, which enables the maximization of a high-quality amount of information with reduced number of tests. In this study, we applied a 33 Box-Behnken factorial design with central point triplicate for optimizing the ionization of an HPLC-ESI-QTOF method used for screening urine samples. Nozzle voltage (V), fragmentor voltage (V) and nebulizer pressure (psig) were the factors selected for variation. The response surface methodology was applied in the molecular features extracted at each level, resulting in a statistical model that helps evaluating the synergic interaction between these factors. Together with the qualitative analysis of the resulting total ion chromatograms, we came across a reproducible (6.14% RSD) and highly efficient method for untargeted metabolomics of human urine samples. The proposed method can be useful for applications in several urine-based metabolomics-driven studies, as the factorial design can be applied in the development of any analytical protocol considering different LC-MS setups.

Determination of hydroquinone and benzoquinone in pharmaceutical formulations: critical considerations on quantitative analysis of easily oxidized compounds.

Hydroquinone is a skin-lightening agent used as an active ingredient in topical dermatological formulations prescribed for treating cutaneous diseases caused by hyperpigmentation. Despite being widely used, some toxicological aspects have been associated with these products, mainly due to overdosage and long-term use combined with the easy oxidation of hydroquinone. In this work, an investigative study has been done to gather enough data for selecting a quantitative analytical method for quality control purposes that considers the ease of oxidation not only within the product but also during the experimental procedures. After studying the influence of pH, reversibility, sampling, and standard solution preparation on the redox reaction between hydroquinone and benzoquinone by using spectroscopic, electrophoretic, and electroanalytical measurements, a reliable, fast, and selective chronoamperometric method was achieved. The optimized method was used for the analysis of samples, previously diluted in Britton-Robinson (BR) buffer (pH 5.5) and methanol (1 : 9, v/v), by applying a potential fixed at 0.4 V. A glassy-carbon working electrode, lab-made Ag/AgCl(sat) and platinum wire as a reference electrode and auxiliary electrodes, respectively, and BR buffer (pH 5.5) as supporting electrolyte were the additional experimental conditions used. Analytical performance parameters were verified to confirm the applicability of the new method (LOD 4.22 µmol L-1 and LOQ 14.1 µmol L-1; recovery mean value of 100% with 0.22% RSD). A gel topical formulation containing 4% (w/w) hydroquinone was analyzed through the developed method for determination of dosage and oxidation traces, and a content of 3.53 ± 0.095% (w/w) was found with no indications of degradation.