Probing for peptidic drugs (2-10 kDa) in doping control blood samples.

Analytical method development and validation for the detection of prohibited bioactive peptides in blood samples using liquid chromatography coupled to high-resolution mass spectrometry.

“In the present study the applicability of a mixed‐mode anion‐exchange solid‐phase extraction (SPE) was shown to allow for an effective sample preparation adequate for subsequent LC‐MS analysis of doping control blood samples for several different prohibited peptides.”

Healthy male and female volunteers, a commercially available plasma pool, and insulin-dependent diabetic patients providing post-administration serum samples.

“The validation was carried out with EDTA plasma and serum samples from 10 healthy male and female volunteers without any medication within the last 24 h. Additionally, a commercially available plasma pool (Octaplas, Octapharma GmbH, Langenfeld, Germany) was used for selected validation parameters (e.g. recovery) due to the absence of endogenous insulin. Post‐administration serum samples were obtained from insulin‐dependent diabetics who regularly administer different synthetic insulin analogues”

10 healthy volunteers, 10 blank plasma samples, 10 fortified plasma samples, 6 technical replicates for recovery, 6 serum samples for robustness, and post-administration samples from diabetic patients.

“The validation was carried out with EDTA plasma and serum samples from 10 healthy male and female volunteers without any medication within the last 24 h.”

The study involved a 24-hour stability test in the autosampler and sample collection 2 to 3 hours after insulin administration in diabetic patients.

“Reinjection of the samples fortified at the MRPL after storage in the autosampler (set to 10°C) for 24 h showed good stability with a detection rate of 10/10.”

The developed mixed-mode solid-phase extraction method allowed for the co-extraction and analysis of multiple prohibited peptide classes (insulins, GH-RHs, IGFs, and MGFs) in blood samples, meeting WADA minimum required performance levels.

“Co‐extraction and analysis of several different peptides such as insulins (human, lispro, aspart, glulisine, tresiba, detemir, glargine, bovine insulin and porcine insulin), growth hormone releasing hormones (sermorelin, CJC‐1295 and tesamorelin), insulin‐like growth factors (long‐R3‐IGF‐I, R3‐IGF‐I and Des1‐3‐IGF‐I) and mechano growth factors (human MGF and MGF‐Goldspink) with criteria that fulfil the requirements of the WADA documents (TD2022 MRPL) for doping controls.”

The method demonstrated 100% detection rates at the minimum required performance levels for all target peptides in fortified samples, with limits of detection ranging from 25% to 50% of the MRPL for most peptides.

“It was found that for 12 out of the 18 target peptides the LOD range at 25% of the MRPL and for 16 out of the 18 target peptides at 50% of the respective MRPL.”

The proof of principle was confirmed by detecting synthetic insulin analogues (aspart and detemir) in post-administration serum samples from diabetic patients.

“The proof of principle was shown by the analysis of post administration samples after treatment with synthetic insulin analogues.”

The method is designed as an initial testing procedure and is not suitable for confirmatory analysis without immunoaffinity purification; additionally, not all target peptides were recovered satisfactorily, and some prohibited peptides like synacthen were excluded due to inadequate results.

“Noteworthy, not all target peptides were recovered satisfactorily with the chosen SPE‐based strategy and, thus, for example synacthen was not considered in this study.”