Analysis of insulin and insulin analogs from dried blood spots by means of liquid chromatography–high resolution mass spectrometry

Abstract

While dried blood spot (DBS) analysis concerning low molecular mass molecules has become more and more established in various fields of analytical chemistry, the utility of DBS in determining peptides and proteins from DBS is yet comparably limited. In consideration of the fact that the apparent benefits of DBS sampling are similar for analytes of lower and higher molecular mass, dedicated (non-generic) sample preparation procedures are required that meet the needs for detecting peptidic drugs and hormones in DBS. The analysis of insulin and its synthetic analogs by mass spectrometry has received increased attention in several fields such as doping controls, forensics, and drug metabolism and pharmacokinetics studies. Hence, a strategy facilitating the analysis of insulin and its synthetic or animal analogs (human, Lispro, Aspart, Glulisine, Glargine, Detemir, Tresiba, and porcine and bovine insulin) from DBS was developed. The successful analysis of these substances at physiologically relevant concentrations was realized after ultrasonication-assisted extraction, immunoaffinity purification, and liquid chromatographic separation followed by high resolution mass spectrometric detection (with or without ion mobility). Assay validation demonstrated adequate sensitivity (LOD 0.5 ng/mL for most insulins), as well as precise (< 25%) andreproducible results for all included target insulins. Additionally, proof-of-principle data were obtained by the analysis of DBS samples obtained from healthy volunteers in non-fasting state as well as a sample from a diabetic volunteer treated with the fast acting analog insulin Aspart. KEYWORDS doping, dried blood spot (DBS), mass spectrometry, sports drug testing

Annual banned‐substance review – Analytical approaches in human sports drug testing

“…Another example for the expanding utility of DBS for routine doping controls was presented by Thomas et al, who demonstrated the applicability of the minimally invasive sampling technique to the analysis of insulin and insulin analogs . Employing citric acid‐pretreated DBS collection cards, spots of 20 μL volume were collected and subjected to an extraction protocol consisting of excising the entire spot, which was then extracted twice into 200 μL of a mixture of acetonitrile/3% acetic acid (60/40, v/v) and both volumes were combined and diluted with water prior to SPE.…”

Dried blood spots for anti‐doping: Why just going volumetric may not be sufficient

“…Dried blood spots (DBSs) are a promising sample collection strategy, which could complement the current strategy of urine and blood analysis for anti-doping. Applications have been demonstrated for many substances on the WADA Prohibited List, for in-and out-of-competition use (S0-S5 and M1-M3) and in-competition use (S6-S9, P1) 1 : S1 anabolic agents, 2,3 S2 peptides, hormones, growth factors, related substances and mimetics, [4][5][6][7][8][9][10] S3 beta agonists, 11 S4 hormone and metabolic modulators, 12,13 S5 diuretics and masking agents, 11 S6 simulants, 14 S7 nacotics, 15,16 S8 cannabinoids, 17 S9 glucocorticoids, 18 P1 beta-blockers, 11 and M1 manipulation of blood and blood components. [19][20][21] In October 2019, the World Anti-Doping Agency (WADA) together with seven anti-doping organizations (ADOs) announced the development and implementation of DBS testing for routine analysis in time for the 2022 Winter Olympic and Paralympic Games in Beijing.…”

Single‐dose administration of clenbuterol is detectable in dried blood spots

“…Dried blood spots (DBS) appear attractive as an alternative sample matrix for antidoping purposes due to the minimal invasiveness, potentially reduced costs for collection and transportation, 11 and the possibility to detect a wide range of doping substances. [11][12][13][14][15][16][17][18][19][20] While clenbuterol is detectable in DBS when it is added directly to the DBS card (i.e. a spiked DBS sample), 12 it is unexplored if and for how long the drug can be detected in DBS samples obtained from humans after clenbuterol ingestion.…”