In-house standards derived from doping peptides: Enzymatic and serum stability and degradation profile of GHRP and GHRH-related peptides.

Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP-4, GHRP-5, GHRP-6, Sermorelin (1-11), Sermorelin (13-20) and Sermorelin (22-29) were synthesized using solid-phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high-performance liquid chromatography-diode array detector (HPLC-DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP-4, GHRP-6 and Sermorelin (22-29) can be considered as in-house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP-6 and Sermorelin (22-29) were used to analyse a dimeric peptide (26 [F] LfcinB (20-30)2 ) in four different matrices to test these peptides as in-house IS.

Gradient Retention Factor Concept Applied to Method Development for Peptide Analysis by Means of RP-HPLC.

Using the van Deemter model, the efficiency of three stationary phase systems in the analysis of a mixture of synthetic peptides was evaluated: (i) monolithic, (ii) packed, and (iii) core-shell columns, and it was shown that the efficiency of the monolithic column is superior to the others, specifically using it, the lowest values of H min (0.03 and 0.1 mm) were obtained, and additionally its efficiency was not significantly affected by increasing the flow. Using the concept of the gradient retention factor (k*), a method for chromatographic separation of a peptide complex mixture was designed, implemented, and optimized and then transferred from a packed column to a monolithic one. The results showed that it was possible to separate all components of the mixture using both evaluated columns; moreover, the analysis time was reduced from 70 to 10 min, conserving the critical pair resolution (1.4), by the transfer method using the k* concept. The method developed was tested against a mixture of doping peptides, showing that this method is efficient for separating peptides of various natures. This investigation is very useful for the development of methods for the analysis of complex peptide mixtures since it provides a systematic approach that can be extrapolated to different types of columns and instrumentation.

Synthetic Peptides in Doping Control: A Powerful Tool for an Analytical Challenge.

Peptides are very diverse molecules that can participate in a wide variety of biological processes. In this way, peptides are attractive for doping, since these molecules can activate or trigger biological processes that can improve the sports performance of athletes. Peptide molecules are found in the official World Anti-Doping Agency lists, mainly in sections S2, S4, and S5. In most cases, these molecules have a very short half-life in the body and/or are identical to natural molecules in the body, making it difficult to analyze them as performance-enhancing drugs. This article reviews the role of peptides in doping, with special emphasis on the peptides used as reference materials, the pretreatment of samples in biological matrices, the instrumentation, and the validation of analytical methodologies for the analysis of peptides used in doping. The growing need to characterize and quantify these molecules, especially in complex biological matrices, has generated the need to search for robust strategies that allow for obtaining sensitive and conclusive results. In this sense, strategies such as solid phase peptide synthesis (SPPS), seeking to obtain specific peptides, metabolites, or isotopically labeled analogs, is a key tool for adequate quantification of different peptide molecules in biological matrices. This, together with the use of optimal methodologies for sample pretreatment (e.g., SPE or protein precipitation), and for subsequent analysis by high-resolution techniques (mainly hyphenated LC-HRMS techniques), have become the preferred instrumentation to meet the analytical challenge involved in the analysis of peptides in complex matrices.

Omics in the detection and identification of biosynthetic pathways related to mycotoxin synthesis.

Mycotoxins are secondary metabolites that are known to be toxic to humans and animals. On the other hand, some mycotoxins and their analogues possess antioxidant as well as antitumor properties, which could be relevant in the fields of pharmaceutical analysis and food research. Omics techniques are a group of analytical tools applied in the biological sciences in order to study genes (genomics), mRNA (transcriptomics), proteins (proteomics), and metabolites (metabolomics). Omics have become a vital tool in the field of mycotoxins, especially contributing to the identification of biomarkers with potential use for the detection of mycotoxigenic species and the gathering of information about the biosynthetic pathways of mycotoxins in different environments. This approach has provided tools for the development of prevention strategies and control measures for different mycotoxins. Additionally, research has revealed important information about the impact of global warming and climate change on the prevalence of mycotoxin issues in society. In the context of foodomics, the aim is to apply omics techniques in order to ensure food safety. The objective of the present review is to determine the state of the art regarding the development of analytical techniques based on omics in the identification of biosynthetic pathways related to mycotoxin synthesis.