madman
Super Moderator
Abstract
Kisspeptin-10 is a peptide hormone capable of increasing circulating follicle stimulating hormone, luteinizing hormone and testosterone levels in humans. Clinically, these effects suggest its use as a treatment for infertility. However, its testosterone-increasing effect indicates potential misuse in sports. As such, it is included in the 2024 World Anti-Doping Agency Prohibited List. This work describes the successful validation of an initial testing procedure (screening) and a confirmation procedure for kisspeptin-10 in urine using liquid chromatography–mass spectrometry. Additionally, kisspeptin-10 was incubated in human serum to mimic endogenous metabolism to improve method sensitivity, as previous research had demonstrated a rapid elimination time of only 30 min after injection (in rats). Four metabolites, corresponding to peptide fragments y9, y8, y7 and y5, were found and added to the ITP in full scan mode. A degradation product discovered during early experimentation was found to probably be caused by oxidation of the tryptophan residue into a kynurenine residue. Further research should elucidate the kinetic parameters of the reaction to improve product stability. Using the validated confirmation procedure, a blackmarket vial of kisspeptin-10 was analysed. The product contained no unexpected impurities, although it appeared to have undergone more degradation than the purchased reference standard.
1 | INTRODUCTION
Kisspeptins are a group of peptide products encoded by the KISS-1 gene, originally discovered as metastasis suppressors (Kotani et al., 2001). Further research demonstrated their ability to stimulate the release of the gonadotropins, follicle-stimulating hormone and luteinizing hormone, as well as increasing e circulating testosterone levels in human males (Dhillo et al., 2005; George et al., 2011). The gonadotropin-stimulating effects of kisspeptin-54 and kisspeptin-10,two major kisspeptin isoforms, indicate their potential use in treating infertility (Jayasena et al., 2015). However, as with gonadotropin releasing hormone itself and its agonist analogues, the testosterone stimulating effects of kisspeptin provoke potential misuse in sports where increased testosterone levels provide substantial benefit to athletes. As of 2024, the World Anti-Doping Agency (WADA, 2024) has included kisspeptin and its agonist analogues on their Prohibited List under section S2.2. Peptide hormones and releasing factors. Of the kisspeptin isoforms, kisspeptin-10 is the smallest peptide capable of activating its target receptor with full affinity and efficacy (Kotani et al., 2001). With a molecular weight below 2 kDa (1.302 kDa), it is considered a small peptide. Detection methods for small peptides, both initial testing procedures (ITPs, screening) and confirmation procedures (CPs), have been described by several research groups (Cuervo et al., 2017; Mazzarino et al., 2015; Okano et al., 2010;Thomas et al., 2016; Timms et al., 2014). Our group described ITP and CP methods using a dilute-and-shoot protocol including direct injection of the sample into a liquid chromatography–mass spectrometry (LC–MS) system (Coppieters et al., 2021; Judák et al., 2017). For the CP, nano-scale LC is used. The goal of this project was to include kisspeptin-10 in these two methods and for the very first time, describe the analysis of kisspeptin-10 in the frame of anti-doping. To improve the kisspeptin-10 detection window, metabolites were sought via serum incubation, as previous research in rats demonstrated rapid elimination after injection (Liu et al., 2013). After intravenous bolus administration of 1 mg/kg bodyweight, kisspeptin-10 became undetectable after just 30 min, indicating the need for targeting degradation products and metabolites.
Although anabolic androgenic steroids make up the bulk of (illegally) sold doping compounds, peptide hormones and proteins also account for a considerable portion, as demonstrated by the Norwegian doping control laboratory in 2015 (Hullstein et al., 2015). Analysis of black-market products confiscated by Norwegian police and custom authorities between 2011 and 2014 revealed that 28% contained peptide hormones or proteins. Further demonstrating the availability of illicit peptide hormones is a simple web search, where the wide array of illicit peptide hormones available for purchase through various online vendors becomes apparent. Among many, vials supposedly containing kisspeptin-10 are displayed. The safety concerns raised by the lack of quality assurance or relevant toxicological data regarding these products are well understood (Kimergård et al., 2014). During this project, a vial of kisspeptin-10 was purchased from such an online vendor and analysed using inhouse developed methods. Of note is the large online availability of specifically kisspeptin-10, whereas other isoforms are mostly available as reference standards at much higher prices.
3.5 | Black-market product
A vial supposedly containing kisspeptin-10, purchased from an online vendor, was confirmed to contain kisspeptin-10 using the validated CP. The product ion chromatograms generated by the CP are shown in Figure 9. When analysing the product in full-scan mode, except for the degradation product with m/z = 653.8209 also seen in the reference standard, no impurities or degradation products were seen in the black-market product. The purity of the product was further confirmed by performing proton nuclear magnetic resonance using a Bruker Avance Neo 400 MHz spectrometer (data not shown). Interestingly, the degradation product peak area relative to the kisspeptin-10 peak area was 3 times higher in the black-market product than in the reference standard. As described earlier, this could indicate improper storage (e.g. not frozen) or conversion during the production process. Even though it was transported as a lyophilized powder, perhaps degradation could still take place.Nonetheless, the black-market product contains kisspeptin-10 with no unexpected impurities, at 1/38th the cost of the reference standard.
4 | CONCLUSION
This paper describes the successful validation of kisspeptin-10 using in house developed ITP and CP methods. Noticed during the initial analysis of the reference standard, a degradation product with m/z = 653.8209 was investigated. The 4 Da mass shift was located on the third amino acid residue, a tryptophan, probably caused by an oxidation of this residue into a KYN residue. Further research into the kinetic parameters of this reaction is required to improve stability. After serum incubation, four kisspeptin-10 metabolites were found, corresponding to following peptide fragments: y9, y8, y7 both single and double charged and y5. These will be monitored in full-scan mode to improve method sensitivity. Finally, a black-market kisspeptin-10 vial was confirmed to contain only kisspeptin-10 and the expected degradation product, although more degradation had taken place compared with the reference standard.
Kisspeptin-10 is a peptide hormone capable of increasing circulating follicle stimulating hormone, luteinizing hormone and testosterone levels in humans. Clinically, these effects suggest its use as a treatment for infertility. However, its testosterone-increasing effect indicates potential misuse in sports. As such, it is included in the 2024 World Anti-Doping Agency Prohibited List. This work describes the successful validation of an initial testing procedure (screening) and a confirmation procedure for kisspeptin-10 in urine using liquid chromatography–mass spectrometry. Additionally, kisspeptin-10 was incubated in human serum to mimic endogenous metabolism to improve method sensitivity, as previous research had demonstrated a rapid elimination time of only 30 min after injection (in rats). Four metabolites, corresponding to peptide fragments y9, y8, y7 and y5, were found and added to the ITP in full scan mode. A degradation product discovered during early experimentation was found to probably be caused by oxidation of the tryptophan residue into a kynurenine residue. Further research should elucidate the kinetic parameters of the reaction to improve product stability. Using the validated confirmation procedure, a blackmarket vial of kisspeptin-10 was analysed. The product contained no unexpected impurities, although it appeared to have undergone more degradation than the purchased reference standard.
1 | INTRODUCTION
Kisspeptins are a group of peptide products encoded by the KISS-1 gene, originally discovered as metastasis suppressors (Kotani et al., 2001). Further research demonstrated their ability to stimulate the release of the gonadotropins, follicle-stimulating hormone and luteinizing hormone, as well as increasing e circulating testosterone levels in human males (Dhillo et al., 2005; George et al., 2011). The gonadotropin-stimulating effects of kisspeptin-54 and kisspeptin-10,two major kisspeptin isoforms, indicate their potential use in treating infertility (Jayasena et al., 2015). However, as with gonadotropin releasing hormone itself and its agonist analogues, the testosterone stimulating effects of kisspeptin provoke potential misuse in sports where increased testosterone levels provide substantial benefit to athletes. As of 2024, the World Anti-Doping Agency (WADA, 2024) has included kisspeptin and its agonist analogues on their Prohibited List under section S2.2. Peptide hormones and releasing factors. Of the kisspeptin isoforms, kisspeptin-10 is the smallest peptide capable of activating its target receptor with full affinity and efficacy (Kotani et al., 2001). With a molecular weight below 2 kDa (1.302 kDa), it is considered a small peptide. Detection methods for small peptides, both initial testing procedures (ITPs, screening) and confirmation procedures (CPs), have been described by several research groups (Cuervo et al., 2017; Mazzarino et al., 2015; Okano et al., 2010;Thomas et al., 2016; Timms et al., 2014). Our group described ITP and CP methods using a dilute-and-shoot protocol including direct injection of the sample into a liquid chromatography–mass spectrometry (LC–MS) system (Coppieters et al., 2021; Judák et al., 2017). For the CP, nano-scale LC is used. The goal of this project was to include kisspeptin-10 in these two methods and for the very first time, describe the analysis of kisspeptin-10 in the frame of anti-doping. To improve the kisspeptin-10 detection window, metabolites were sought via serum incubation, as previous research in rats demonstrated rapid elimination after injection (Liu et al., 2013). After intravenous bolus administration of 1 mg/kg bodyweight, kisspeptin-10 became undetectable after just 30 min, indicating the need for targeting degradation products and metabolites.
Although anabolic androgenic steroids make up the bulk of (illegally) sold doping compounds, peptide hormones and proteins also account for a considerable portion, as demonstrated by the Norwegian doping control laboratory in 2015 (Hullstein et al., 2015). Analysis of black-market products confiscated by Norwegian police and custom authorities between 2011 and 2014 revealed that 28% contained peptide hormones or proteins. Further demonstrating the availability of illicit peptide hormones is a simple web search, where the wide array of illicit peptide hormones available for purchase through various online vendors becomes apparent. Among many, vials supposedly containing kisspeptin-10 are displayed. The safety concerns raised by the lack of quality assurance or relevant toxicological data regarding these products are well understood (Kimergård et al., 2014). During this project, a vial of kisspeptin-10 was purchased from such an online vendor and analysed using inhouse developed methods. Of note is the large online availability of specifically kisspeptin-10, whereas other isoforms are mostly available as reference standards at much higher prices.
3.5 | Black-market product
A vial supposedly containing kisspeptin-10, purchased from an online vendor, was confirmed to contain kisspeptin-10 using the validated CP. The product ion chromatograms generated by the CP are shown in Figure 9. When analysing the product in full-scan mode, except for the degradation product with m/z = 653.8209 also seen in the reference standard, no impurities or degradation products were seen in the black-market product. The purity of the product was further confirmed by performing proton nuclear magnetic resonance using a Bruker Avance Neo 400 MHz spectrometer (data not shown). Interestingly, the degradation product peak area relative to the kisspeptin-10 peak area was 3 times higher in the black-market product than in the reference standard. As described earlier, this could indicate improper storage (e.g. not frozen) or conversion during the production process. Even though it was transported as a lyophilized powder, perhaps degradation could still take place.Nonetheless, the black-market product contains kisspeptin-10 with no unexpected impurities, at 1/38th the cost of the reference standard.
4 | CONCLUSION
This paper describes the successful validation of kisspeptin-10 using in house developed ITP and CP methods. Noticed during the initial analysis of the reference standard, a degradation product with m/z = 653.8209 was investigated. The 4 Da mass shift was located on the third amino acid residue, a tryptophan, probably caused by an oxidation of this residue into a KYN residue. Further research into the kinetic parameters of this reaction is required to improve stability. After serum incubation, four kisspeptin-10 metabolites were found, corresponding to following peptide fragments: y9, y8, y7 both single and double charged and y5. These will be monitored in full-scan mode to improve method sensitivity. Finally, a black-market kisspeptin-10 vial was confirmed to contain only kisspeptin-10 and the expected degradation product, although more degradation had taken place compared with the reference standard.
