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An antibody-free, ultrafiltration-based assay for the detection of growth hormone-releasing hormones in urine at low pg/mL concentrations using nanoLC-HRMS/MS

Gilles Coppieters, Koen Deventer (UGent) , Michaël Polet (UGent) , Peter Van Eenoo (UGent) and Péter Judák
(2022) JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS. 214.
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Abstract
This work presents an ultrafiltration-based, validated method for the screening and confirmation of prohibited growth hormone-releasing hormone (GHRH) analogues (sermorelin/CJC-1293, sermorelin metabolite, CJC-1295 and tesamorelin) in urine by nanoLC-HRMS/MS. Sample preparation avoids the use of laborious antibody-based extraction approaches and consists solely of preconcentration by ultrafiltration. Even in the absence of immunoaffinity purification steps, high sensitivity was still ensured as limits of detection between 5 and 25 pg/mL and limits of identification between 25 and 50 pg/mL were established. The robustness of the miniaturized chromatographic setup was evaluated through the injection of 200 + preconcentrated urinary extracts. In a comparison with immuno-affinity purification, enhanced recoveries (59 - 115%) and similar sensitivity were achieved, yet at lower operational costs. Stability experiments showed the importance of the proper handling of urine samples to avoid degradation of these peptide hormones, especially for sermorelin and its metabolite which were found to rapidly degrade at temperatures > 4 C and pH values < 7 in accordance with earlier studies. Without the need for specific antibodies, this method may be expanded to cover emerging peptide drugs (>= similar to 3 kDa), as well as their metabolites in the future to facilitate coverage for this class of prohibited substances.
Keywords
Clinical Biochemistry, Spectroscopy, Drug Discovery, Pharmaceutical Science, Analytical Chemistry, GHRH, Peptides, Ultrafiltration, nanoLC-HRMS, Doping, Urine, CHROMATOGRAPHY-MASS SPECTROMETRY, IMMUNOAFFINITY PURIFICATION, DOPING CONTROL, IDENTIFICATION, LC, PEPTIDES, KDA

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MLA
Coppieters, Gilles, et al. “An Antibody-Free, Ultrafiltration-Based Assay for the Detection of Growth Hormone-Releasing Hormones in Urine at Low Pg/ML Concentrations Using NanoLC-HRMS/MS.” JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, vol. 214, 2022, doi:10.1016/j.jpba.2022.114726.
APA
Coppieters, G., Deventer, K., Polet, M., Van Eenoo, P., & Judák, P. (2022). An antibody-free, ultrafiltration-based assay for the detection of growth hormone-releasing hormones in urine at low pg/mL concentrations using nanoLC-HRMS/MS. JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 214. https://doi.org/10.1016/j.jpba.2022.114726
Chicago author-date
Coppieters, Gilles, Koen Deventer, Michaël Polet, Peter Van Eenoo, and Péter Judák. 2022. “An Antibody-Free, Ultrafiltration-Based Assay for the Detection of Growth Hormone-Releasing Hormones in Urine at Low Pg/ML Concentrations Using NanoLC-HRMS/MS.” JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 214. https://doi.org/10.1016/j.jpba.2022.114726.
Chicago author-date (all authors)
Coppieters, Gilles, Koen Deventer, Michaël Polet, Peter Van Eenoo, and Péter Judák. 2022. “An Antibody-Free, Ultrafiltration-Based Assay for the Detection of Growth Hormone-Releasing Hormones in Urine at Low Pg/ML Concentrations Using NanoLC-HRMS/MS.” JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 214. doi:10.1016/j.jpba.2022.114726.
Vancouver
1.
Coppieters G, Deventer K, Polet M, Van Eenoo P, Judák P. An antibody-free, ultrafiltration-based assay for the detection of growth hormone-releasing hormones in urine at low pg/mL concentrations using nanoLC-HRMS/MS. JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS. 2022;214.
IEEE
[1]
G. Coppieters, K. Deventer, M. Polet, P. Van Eenoo, and P. Judák, “An antibody-free, ultrafiltration-based assay for the detection of growth hormone-releasing hormones in urine at low pg/mL concentrations using nanoLC-HRMS/MS,” JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, vol. 214, 2022.
@article{8746139,
  abstract     = {{This work presents an ultrafiltration-based, validated method for the screening and confirmation of prohibited growth hormone-releasing hormone (GHRH) analogues (sermorelin/CJC-1293, sermorelin metabolite, CJC-1295 and tesamorelin) in urine by nanoLC-HRMS/MS. Sample preparation avoids the use of laborious antibody-based extraction approaches and consists solely of preconcentration by ultrafiltration. Even in the absence of immunoaffinity purification steps, high sensitivity was still ensured as limits of detection between 5 and 25 pg/mL and limits of identification between 25 and 50 pg/mL were established. The robustness of the miniaturized chromatographic setup was evaluated through the injection of 200 + preconcentrated urinary extracts. In a comparison with immuno-affinity purification, enhanced recoveries (59 - 115%) and similar sensitivity were achieved, yet at lower operational costs. Stability experiments showed the importance of the proper handling of urine samples to avoid degradation of these peptide hormones, especially for sermorelin and its metabolite which were found to rapidly degrade at temperatures > 4 C and pH values < 7 in accordance with earlier studies. Without the need for specific antibodies, this method may be expanded to cover emerging peptide drugs (>= similar to 3 kDa), as well as their metabolites in the future to facilitate coverage for this class of prohibited substances.}},
  articleno    = {{114726}},
  author       = {{Coppieters, Gilles and Deventer, Koen and Polet, Michaël and Van Eenoo, Peter and Judák, Péter}},
  issn         = {{0731-7085}},
  journal      = {{JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS}},
  keywords     = {{Clinical Biochemistry,Spectroscopy,Drug Discovery,Pharmaceutical Science,Analytical Chemistry,GHRH,Peptides,Ultrafiltration,nanoLC-HRMS,Doping,Urine,CHROMATOGRAPHY-MASS SPECTROMETRY,IMMUNOAFFINITY PURIFICATION,DOPING CONTROL,IDENTIFICATION,LC,PEPTIDES,KDA}},
  language     = {{eng}},
  pages        = {{6}},
  title        = {{An antibody-free, ultrafiltration-based assay for the detection of growth hormone-releasing hormones in urine at low pg/mL concentrations using nanoLC-HRMS/MS}},
  url          = {{http://doi.org/10.1016/j.jpba.2022.114726}},
  volume       = {{214}},
  year         = {{2022}},
}
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