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Background: QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) is a methodology previously developed to extract pesticides from vegetables and fruits and has been fully applied for different analytical approaches. Objective: In the present study, a rapid and less laborious modified QuEChERS extraction method for the quantification of 13 opioids [codeine, morphine, heroin, 6-acetylmorphine (6-AM), desomorphine, ethylmorphine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 2-ethyl-5-methyl-3,3diphenyl-1-pyrroline (EMDP), papaverine, tramadol, O-desmetyltramadol (M1) and, tapentadol], cocaine and cocaethylene in whole blood was developed and validated by Gas Chromatography-Mass Spectrometry. Method: The modification of QuEChERS method consisted in the pretreatment of the whole blood samples using ultrasonication, the use of ethyl acetate as extraction solvent and a previous step of sample alcalinization. The use of dispersive separation steps such as Dispersive Solid-Phase Extraction (dSPE) or sorbents such as Primary Secondary Amine (PSA) was suppressed to minimize the errors and, to improve the velocity of the analysis. Results: The method proved to be selective and the regression analysis for the analytes was linear in the range of 31.2-2000 ng/mL with correlation coefficients > 0.98. The coefficients of variation did not exceed 15%. The lowest limit of detection and quantification for all the analytes were below the therapeutic range of the drugs. The recoveries of the analytes ranged from 52.4 to 95.0%. Conclusion: The developed method can provide a rapid, effective and "greener" process for the analysis of a wide range of opioids drugs in whole blood samples and can be applied to clinical and forensic antemortem and postmortem cases.

The use of in vitro human skin permeation tests is of value when addressing the quality and equivalence of topical drug products in Europe and the US. Human skin is the membrane of choice for these studies. The use of human skin as a membrane is hindered by limited access, high variability of results, and limited applicability for drugs with low skin permeability. Reconstructed human epidermis (RhE) models are validated as skin surrogates for safety tests and have been explored for percutaneous absorption testing. Clotrimazole poorly permeates human skin and is widely available for topical treatments. In this study, clotrimazole creams were used to test the ability of RhE to be used as biological membrane for bioequivalence testing, based on the Draft Guideline on Quality and Equivalence of Topical Products (CHMP/QWP/708282/2018) using a discriminative and modified in vitro permeation test (IVPT). To fulfill the validation of a discriminatory method, Canesten® 10 mg/g cream was compared with a test product with the same drug strength, along with two “negative controls” dosed at a 50% and 200% drug strength. Products were compared in finite dose conditions, regarding maximal flux (Jmax) and the total amount of drug permeated (Atotal). The results showed the discriminatory power of the method among the three drug strengths with no interference of the placebo formulation. The study design and validation complied with the requirements established in the guideline for a valid IVPT. This new test system allowed for the equivalence comparison between test and comparator product. Higher permeability of the RhE compared to human skin could be observed. This arose as a strength of the model for this modified IVPT bioequivalence testing, since comparing permeation profiles among products is envisaged instead of drawing absolute conclusions on skin permeation extent. These results may support the acceptance of RhE as biological membranes for modified IVPT in bioequivalence testing of topical products.

Methylisothiazolinone (MI) is one of the most used preservatives in shampoos and also one of the most effective. A preservative mixture known as Kathon™ CG is commercially available. It contains 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) (3:1) and stabilizers. The aim of this study is to evaluate the influence of formulation factors in the quantification and stability of isothiazolinones in shampoos. Two shampoo bases containing Kathon™ CG as a preservative were prepared. Some ingredients that are at risk of interfering with the preservative stability were added to these formulations. The preservative was quantified by HPLC-DAD (High-performance liquid chromatography with a diode-array detector) after preparation of the formulation and after storage at room temperature and at 40 °C. The addition of magnesium silicate proved to be essential for the breakdown of the interaction between the matrix and the analytes in the extraction procedure. The content of CMI/MI decreased right after preparation indicating that immediate interactions between CMI/MI and the ingredients may have occurred after preparation resulting in a decrease in the preservative concentration. Detrimental interactions between the ingredients, regarding the stability of the isothiazolinones were detected immediately after preparation and over time resulting in the reduction of CMI/MI concentration in these cosmetic shampoos.

Cutaneous wound healing consists of a complex series of events involving inflammation, cellular migration/proliferation, vasculogenesis, and tissue remodeling. Chemical exposure to toxic agents