Identification of selected substances with a hormonal effect (peptides) using LC-MS and MS/MS methods

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00007064%3AK01__%2F24%3AN0000063" target="_blank" >RIV/00007064:K01__/24:N0000063 - isvavai.cz</a>

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Identification of selected substances with a hormonal effect (peptides) using LC-MS and MS/MS methods

Popis výsledku v původním jazyce

The poster, which focused on the qualitative analysis of peptide hormones, was presented at the international conference called TIAFT (September 2024). Due to the increasing number of abuses of these substances in the Czech Republic, there was a need to develop a rapid qualitative analysis. For this reason, a liquid chromatography coupled with mass spectrometry (LC-MS/MS) method was optimized and validated for this group of substances: growth hormone releasing peptide (GHRP), hexarelin, melanotan-2, BPC 157, bremelanotide, thymosin Beta-4, thymosin alpha-1 and related substances. Our workplaces have seen a significant increase in the analysis of substances with a hormonal effect in the last two years. These are also small and medium peptides, which are part of the list of prohibited substances issued by the World Anti-Doping Agency from 1 January 2021. At the same time, the distribution of these substances and their storage in large quantities in the Czech Republic is prohibited. For this reason, a new LC-MS/MS method was developed to identify these substances. These are, for example, peptides GHRP (growth hormone - releasing peptides), Hexarelin, Melanotan, BPC 157, PT 141 (Bremelanotide), Thymosin Beta-4 (TB-500), Thymosin Alpha-1. Methods: For the identification of peptides in commercial preparations, the separation method of liquid chromatography (LC) on the reverse phase was used. The measurement was carried out under the following conditions: column - XBridge Premier BEH C18, 2.1 × 50 mm, 2.5 μm, mobile phase A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile, gradient elution, flow rate 0.2 ml/min, injection volume: 5 µl, column temperature 25 °C. Mass spectrometry was used for detection. The measurement was carried out using a mass spectrometer with low and high resolution, both by the method of direct injection into the mass spectrometer (Direct Injection, DI) or in conjunction with liquid chromatography (LC-MS). The measurement takes place in Full Scan mode using electrospray ionization in positive mode, the spectra are measured in the range of 100-3200 m/z. After measuring the MS spectra, the fragmentation spectra of the analytes were measured at different collision energies depending on the size of the peptide (10, 20, 35, 40, 60 eV). Results & Discussion: A new LC-MS/MS method was developed for the unambiguous identification of peptides GHRP 1, GHRP 2, GHRP 3, GHRP 4, GHRP 5, GHRP 6, Hexarelin, Melanotan 2, BPC 157, PT 141 (Bremelanotide), Thymosin Beta-4 (TB- 500) and Thymosin Alpha-1. The analysis of a significant amount of preparations using the developed method thus contributed to the successful detection of an international organized group engaged in the illegal production and distribution of substances with a hormonal effect. The perpetrators' profits from the sale of home-made products were several times higher than in the drug business.

Název v anglickém jazyce

Identification of selected substances with a hormonal effect (peptides) using LC-MS and MS/MS methods

Popis výsledku anglicky

The poster, which focused on the qualitative analysis of peptide hormones, was presented at the international conference called TIAFT (September 2024). Due to the increasing number of abuses of these substances in the Czech Republic, there was a need to develop a rapid qualitative analysis. For this reason, a liquid chromatography coupled with mass spectrometry (LC-MS/MS) method was optimized and validated for this group of substances: growth hormone releasing peptide (GHRP), hexarelin, melanotan-2, BPC 157, bremelanotide, thymosin Beta-4, thymosin alpha-1 and related substances. Our workplaces have seen a significant increase in the analysis of substances with a hormonal effect in the last two years. These are also small and medium peptides, which are part of the list of prohibited substances issued by the World Anti-Doping Agency from 1 January 2021. At the same time, the distribution of these substances and their storage in large quantities in the Czech Republic is prohibited. For this reason, a new LC-MS/MS method was developed to identify these substances. These are, for example, peptides GHRP (growth hormone - releasing peptides), Hexarelin, Melanotan, BPC 157, PT 141 (Bremelanotide), Thymosin Beta-4 (TB-500), Thymosin Alpha-1. Methods: For the identification of peptides in commercial preparations, the separation method of liquid chromatography (LC) on the reverse phase was used. The measurement was carried out under the following conditions: column - XBridge Premier BEH C18, 2.1 × 50 mm, 2.5 μm, mobile phase A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile, gradient elution, flow rate 0.2 ml/min, injection volume: 5 µl, column temperature 25 °C. Mass spectrometry was used for detection. The measurement was carried out using a mass spectrometer with low and high resolution, both by the method of direct injection into the mass spectrometer (Direct Injection, DI) or in conjunction with liquid chromatography (LC-MS). The measurement takes place in Full Scan mode using electrospray ionization in positive mode, the spectra are measured in the range of 100-3200 m/z. After measuring the MS spectra, the fragmentation spectra of the analytes were measured at different collision energies depending on the size of the peptide (10, 20, 35, 40, 60 eV). Results & Discussion: A new LC-MS/MS method was developed for the unambiguous identification of peptides GHRP 1, GHRP 2, GHRP 3, GHRP 4, GHRP 5, GHRP 6, Hexarelin, Melanotan 2, BPC 157, PT 141 (Bremelanotide), Thymosin Beta-4 (TB- 500) and Thymosin Alpha-1. The analysis of a significant amount of preparations using the developed method thus contributed to the successful detection of an international organized group engaged in the illegal production and distribution of substances with a hormonal effect. The perpetrators' profits from the sale of home-made products were several times higher than in the drug business.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10401 - Organic chemistry

Projekt

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Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2024

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů