Urinary detection of rapid‐acting insulin analogs in healthy humans

Abstract

Human insulin and its synthetic analogs are considered as life‐saving drugs for people suffering from diabetes mellitus. Next to the therapeutic use, scientific and non‐scientific literature (e.g. bodybuilding forums; antidoping intelligence and investigation reports) indicate that these prohibited substances are used as performance enhancing agents. In the present report, the development and validation of a sensitive analytical strategy is described for the urinary detection of three rapid‐acting insulin analogs (Lispro, Aspart, Glulisine). The method is based on sample purification by the combination of ultrafiltration and immunoaffinity purification and subsequent analysis by nano‐flow liquid chromatography coupled to high resolution mass spectrometry. Next to the results on different validation parameters (LOD: 10 pg/mL; recovery: 25–48%; matrix effect: −3‐(−8) %), data on urinary elimination times, which were obtained in the frame of an administration study with the participation of healthy volunteers, are presented. The determined detection windows (~9 hours) are expected to help to evaluate current routine analytical methods and aim to aid doping authorities to set appropriate target windows for efficient testing.

On‐line reduction of insulin disulfide bonds with photoinduced radical reactions, upstream to nano liquid chromatography‐mass spectrometry

“…Only fragments of two consecutive amino acids (m/z 626.3 + = FVNQH and m/z 739.4 + = FVN-QHL) were frequently present with unstable intensities, insufficient for unsupervised and unambiguous identification. Neither of these fragments are utilized in earlier methods [16][17][18]. From LC-MS determination of insolution inter-chain cleaved insulin, the B chain of insulin was without exception successfully identified (Sequest-HT score = 471, n = 15) in the obtained mass fragmentation spectra (Figure 1C).…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…These LODs were shown to be adequate to determine the therapeutic use of 50 IU of insulin degludec 12 hr post‐administration. Probing for the detection windows of rapid‐acting insulins such as insulin lispro, insulin glulisine, and insulin aspart was the objective of a study by Judak et al 150 Healthy volunteers received a single subcutaneous dose of the respective synthetic insulin at 0.05 IU/kg bodyweight, and urine samples were collected prior to and up to 72 hr post‐injection. Similar to Mazzarino et al, urine samples were ultrafiltered (3‐kDa cut‐off), and retentates were immunoaffinity‐purified, here employing an anti‐insulin antibody‐coated ELISA well‐plate.…”

Facilitated Qualitative Determination of Insulin, Its Synthetic Analogs, and C-Peptide in Human Urine by Means of LC–HRMS

“…The present method is mainly developed in order to facilitate the analysis of urinary doping control samples for synthetic insulins. All published methods for the determination of insulin and C-peptide in urine by means of liquid chromatography/mass spectrometry mainly use immunoaffinity extraction of the target peptides from the matrix [ 3 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ]. These immunoaffinity-enriched sample aliquots are of highly purified quality and allow for nanoscale liquid chromatography.…”