Recommended criteria for the mass spectrometric identification of target peptides and proteins (&lt;8 kDa) in sports drug testing

Thevis, Mario; Loo, Joseph A.; Loo, Rachel R. Ogorzalek; Schänzer, Wilhelm

doi:10.1002/rcm.2848

Cited by 39 publications

(21 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These criteria are consistent with those proposed by other authors for the analysis of rhEPO analogues in the horse [27], but with the addition in the current study of a relative retention time tolerance due to the inclusion of an internal standard. Furthermore, in conjunction with the other factors described in the above ''Method Validation'', these criteria also fulfil the proposals of Thevis et al [31]. According to the identification points (IP) system proposed by the aforementioned authors (where a minimum score of 5 IP was suggested), the current confirmatory approach would lead to the allocation of 6.5 IP for each peptide (1 for the MS/ MS precursor ion, 1.5 for each of three product ions and 1 for immunoaffinity purification during sample preparation).…”

Section: Auto-injection Carryoversupporting

confidence: 52%

“…Thevis et al [31] have proposed criteria for the confirmatory detection of biological macromolecules (up to 8,000 kDa) in samples from human athletes by WADA laboratories. However, specific guidance notes for animal sports testing laboratories are currently lacking, so the following in house confirmatory criteria for the detection of rhEPO analogues in equine plasma and urine were applied:…”

Section: Auto-injection Carryovermentioning

confidence: 99%

“…Acceptance criteria for the measured m/z of the pseudomolecular ion was established as the presence of the most abundant isotope peak in the peptide charge envelope within ±m/z 0.1 of that of the authentic reference standard (equating to a deconvoluted molecular mass within ±0.2 or 0.3 Da for doubly and triply charged species, respectively, which fulfills the 0.5 Da acceptance criterion for molecular mass proposed for WADA laboratories by Thevis et al [31]). Acceptance criteria for the product ion spectra were the presence in the protein derived peptide mass spectrum of any ion present at greater than 10% of base peak in the authentic reference standard product ion spectrum.…”

Section: Comparison Of Peptide Data Generated From Intact Proteins Agmentioning

confidence: 99%

See 2 more Smart Citations

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

Scarth¹,

Seibert²,

Brown³

et al. 2011

Chromatographia

View full text Add to dashboard Cite

Recombinant human erythropoietin (rhEPO) analogues are known to have been used in horse sports for their assumed performance enhancing properties. Recently, several authors have published liquid chromatographictandem mass spectrometric (LC-MS/MS) methods for confirming the presence of rhEPO analogues in horse plasma. In the current study, an improved LC-MS/MS confirmatory procedure for rhEPO, darbepoetin (DPO) and continuous erythropoietin receptor activator (CERA) in horse plasma was developed and validated. The method was also adapted for and applied to urine samples for the first time. Similar to previously published plasma assays, the methods utilise size exclusion and immunoaffinity extraction prior to tryptic cleavage, enzymatic deglycosylation and LC-MS/MS analysis of the resulting signature tryptic peptides (rhEPO/CERA T5, rhEPO/CERA/DPO T6 and DPO T9). However, the novel application of UPLC chromatography significantly improves the run time of the method compared to nano-or micro-LC and its robustness compared to nano-LC. Furthermore, recombinant canine EPO was found to serve as an effective internal standard, thus allowing confidence in interpretation of the success/ failure of every step in the procedure. Limits of detection for confirming the presence of rhEPO, CERA and DPO in plasma were 0.1, 0.25 and 0.05 ng mL -1 , respectively, which were equal to or lower than limits achieved using previously published LC-MS/MS based methods. Limits of detection for confirming the presence of rhEPO, CERA and DPO in urine were 0.05, 0.15 and 0.025 ng mL -1 and the analysis of urine samples collected from horses administered rhEPO (Eprex TM ) or DPO (Aranesp TM ) demonstrated the use of this matrix as a suitable alternative in situations where plasma is not available.

show abstract

Section: Auto-injection Carryoversupporting

confidence: 52%

Section: Auto-injection Carryovermentioning

confidence: 99%

Section: Comparison Of Peptide Data Generated From Intact Proteins Agmentioning

confidence: 99%

See 1 more Smart Citation

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

Scarth¹,

Seibert²,

Brown³

et al. 2011

Chromatographia

View full text Add to dashboard Cite

show abstract

“…3 and 4). In order to fulfil suggested criteria for the identification of peptides and proteins in doping control analysis,37 the three most abundant multiply protonated molecules were additionally subjected to low‐energy CID without inducing dissociation of IGF‐1 and its analogues (Table 1). Owing to the considerable stability of the IGF‐1 molecule described earlier, these pseudo‐MS/MS experiments (e.g.…”

Section: Resultsmentioning

confidence: 99%

Quantification of human insulin‐like growth factor‐1 and qualitative detection of its analogues in plasma using liquid chromatography/electrospray ionisation tandem mass spectrometry

Bredehöft

Schänzer

Thevis

2008

Rapid Comm Mass Spectrometry

Self Cite

102

View full text Add to dashboard Cite

Human insulin-like growth factor-1 (IGF-1) is a peptide hormone that acts as a mediator of most of the somatotropic effects of growth hormone (GH). Therefore, it is supposed to be a biomarker indicating GH abuse in sports as well as diseases associated with a change in IGF-1 plasma concentration. It can be applied locally by injection to increase total protein and DNA content in tissues such as skeletal muscle -a highly desirable effect in various sports disciplines. In order to improve its growthpromoting properties, the primary structure of IGF-1 has been modified, yielding analogues such as des(1-3)IGF-1 and LONG TM R 3 IGF-1, which show a considerably reduced affinity to the respective binding proteins in plasma and, thus, an increased bioavailability at target tissues. Due to their capability to enhance performance, IGF-1 and its analogues belong to the prohibited list of the World Anti-Doping Agency. Hence, it was necessary to develop a reliable assay for the quantification of human IGF-1 as well as the detection of its derivatives. Immunoaffinity isolation and purification from 60 mL of plasma followed by liquid chromatography/electrospray ionisation tandem mass spectrometry enabled the unequivocal determination of all target analytes. Diagnostic product ions were characterised utilising an Orbitrap mass spectrometer with high resolution/high accuracy properties and employed for triple quadrupole MS/MS analysis. The described assay provided lower limits of detection (LLODs) between 20 and 50 ng/mL, recovery rates between 34-43% and a precision <15% at the LLOD as well as higher concentration levels. In order to prove the applicability of the developed assay, human plasma samples were analysed and the results were compared with the values obtained from a commercially available immunoradiometric assay (IRMA). Four of six samples resulted in concentration ratios with good correlation between both assays, whereas the absolute concentrations were lower for the presented procedure.

show abstract

“…This technique requires a highly effective sample preparation procedure to prevent the nano‐UPLC system from blocking and/or ion suppression effects 17. Immunoaffinity purification prior to LC provides a well‐established and powerful tool in peptide analysis and represents an additional dimension in the separation process 10, 18, 19…”

Section: Resultsmentioning

confidence: 99%

Determination of Synacthen in urine for sports drug testing by means of nano‐ultra‐performance liquid chromatography/tandem mass spectrometry

Thomas

Kohler

Schänzer

et al. 2009

Rapid Comm Mass Spectrometry

Self Cite

View full text Add to dashboard Cite

Doping control analysis of performance-enhancing peptides in urine represents a challenging requirement in modern sports drug testing. Low dosing, effective metabolism and short half-life lead to target concentrations in the low fmol/mL range in urine. Synthetic adrenocorticotropic hormone (1-24, Syn-ACTH-en) shares all these characteristics and improved analytical performance is required for its sufficient determination by means of liquid chromatography/tandem mass spectrometry (LC/MS/MS). The desired effects for cheating sportsmen are mainly due to enhanced release of corticosteroids as well as androgenic steroids into the circulation after systemic administration of the drug. Immunoaffinity purification with coated magnetic beads and subsequent liquid chromatography with nano-ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (high resolution/high mass accuracy) of Synacthen from urinary specimens is described in the present study. The general proof of principle was obtained by analysis of excretion study urine samples and validation was performed with focus on the limit of detection (3 pg/mL), linearity, precision (<20%), recovery ( approximately 30%), robustness, specificity and stability. For all experiments, the ACTH fragment 1-17 was used as the internal standard.

show abstract

Recommended criteria for the mass spectrometric identification of target peptides and proteins (<8 kDa) in sports drug testing

Cited by 39 publications

References 29 publications

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

Quantification of human insulin‐like growth factor‐1 and qualitative detection of its analogues in plasma using liquid chromatography/electrospray ionisation tandem mass spectrometry

Determination of Synacthen in urine for sports drug testing by means of nano‐ultra‐performance liquid chromatography/tandem mass spectrometry

Contact Info

Product

Resources

About