Detection of insulin analogues and large peptides >2 kDa in urine

Abstract

Insulin analogues and large bioactive peptides may be used by athletes to enhance performance and are banned by the World Anti‐Doping Agency (WADA). In addition to insulin analogues, the large peptides include a structurally diverse set of peptides including analogues of growth hormone releasing hormone (GHRH), insulin‐like growth factor‐1 (IGF‐1), and mechano‐growth factor (MGF). Detection of this class of peptides is difficult due to their absorptive losses and presence at very low concentrations in urine. In this report, a high throughput method is described that allows sensitive detection of four classes of large peptides in one assay. Sample extraction is performed by ultrafiltration to concentrate the urine followed by solid phase extraction in a 96‐well micro‐elution plate. Peptides in the urine samples are detected on a triple quadrupole mass spectrometer coupled to standard flow liquid chromatography. The method was validated and evaluated for limit of detection, limit of identification, specificity, precision, carryover, recovery, matrix interference, and post‐extraction stability. The limit of detection for insulin analogues is between 5 and 25 pg/ml and between 5 and 50 pg/ml for the other peptide classes. Specificity was good with no detection of interfering peaks in blank urine samples. Carryover from a high concentration sample was not observed and the post‐extraction stability was between 77% and 107%. The method was able to detect insulin analogues in three diabetic urine samples. Increased screening for this class of peptides will improve detection and deterrence.

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

“…Along with the development of increasingly powerful mass spectrometers with very high resolution (> 100,000 FWHM), it is now possible to simplify sample preparation to the extent that less pure extracts can be analysed without jeopardizing the required specificity 25 . Samples prepared in this way are generally not suitable for analysis with nano‐LC systems, but modern normal‐flow systems have demonstrated sufficient sensitivity and robustness to achieve the mandatory MRPLs in doping controls 7,8,14,16,19 . The present method is designed as an initial testing procedure, which ideally covers different (if not all) prohibited peptides in one analytical approach.…”

“…These methods are very specific and selective due to the complementary combination of immune extraction, liquid chromatographic separation and detection by (high‐resolution/tandem) MS. But due to the laborious sample preparation steps, recently also more simplified approaches were developed, which also meet the criteria outlined in mandatory WADA documents 16–19 . Most of these assays focus on urine analysis or are limited to one class of peptides (e.g.…”

Chromatographic–mass spectrometric analysis of peptidic analytes (2–10 kDa) in doping control urine samples

“…Performance‐enhancing peptidic drugs and drug candidates, which are prohibited in elite sports, are potentially detectable in blood samples as well as in urine samples. While blood samples own benefits regarding the expected higher concentrations as well as the detection of the non‐metabolised intact drug, urine samples still represent the most frequently collected specimens in doping controls due to good retrospectivity of low molecular doping agents 1–4 . Thus, effective and simplified detection methods for urine are still desirable for most doping control laboratories.…”

“…[6][7][8][9][10] Noteworthily, these hybrid methods are very specific by combining the selectivity of antibodies, the separation power of LC and high-resolution/high mass accuracy MS. Additionally, multiplex approaches are realised for several different classes of peptides, which enable already comprehensive initial testing procedures (ITPs). 2,11,12 These methods already reported on the option of replacing the immune extraction protocol by several other processing steps such as ultrafiltration, solid-phase extraction (SPE), etc. Nevertheless, most of these methods are characterised by laborious and costly sample preparation and impede the analysis of high sample numbers with short turnaround times (high throughput).…”

Annual banned‐substance review—Analytical approaches in human sports drug testing 2021/2022–

“…The target analytes including sermorelin, sermorelin metabolite (3-29-NH 2 ), CJC-1295, and tesamorelin were analyzed in PRM mode, and LODs between 5 and 25 pg/ml were reported, presumably facilitated by enhanced recoveries and superior chromatographic as well as ionization conditions, warranting further investigations also concerning additional metabolic products and TCs. Similarly, Cox et al reported on a multi-analyte ITP employing ultrafiltration (3 kDa cutoff)combined with mixed-anion exchange (MAX) SPE and LC-QqQ-MS analysis 70. A volume of 4 ml of urine was centrifuged, ultrafiltrated to a retentate volume of 100-250 μl, and solid-phase extracted using a microelution device, and the diluted eluate was injected onto a C-18 analytical column (2.1 Â 50 mm, 1.7 μm particle size).…”