Adsorption effects of the doping relevant peptides Insulin Lispro, Synachten, TB-500 and GHRP 5.

The tendency of peptides to adsorb to surfaces can raise a concern in variety of analytical fields where the qualitative/quantitative measurement of low concentration analytes (ng/mL-pg/mL) is required. To demonstrate the importance of using the optimal glassware/plasticware, four doping relevant model peptides (GHRP 5, TB-500, Insulin Lispro, Synachten) were chosen and their recovery from various surfaces were evaluated. Our experiments showed that choosing expensive consumables with low-bind characteristics is not beneficial in all cases. A careful selection of the consumables based on the evaluation of the physico/chemical features of the peptide is recommended.

An antibody-free, ultrafiltration-based assay for the detection of growth hormone-releasing hormones in urine at low pg/mL concentrations using nanoLC-HRMS/MS.

This work presents an ultrafiltration-based, validated method for the screening and confirmation of prohibited growth hormone-releasing hormone (GHRH) analogues (sermorelin/CJC-1293, sermorelin metabolite, CJC-1295 and tesamorelin) in urine by nanoLC-HRMS/MS. Sample preparation avoids the use of laborious antibody-based extraction approaches and consists solely of preconcentration by ultrafiltration. Even in the absence of immuno-affinity purification steps, high sensitivity was still ensured as limits of detection between 5 and 25 pg/mL and limits of identification between 25 and 50 pg/mL were established. The robustness of the miniaturized chromatographic setup was evaluated through the injection of 200 + preconcentrated urinary extracts. In a comparison with immuno-affinity purification, enhanced recoveries (59 - 115%) and similar sensitivity were achieved, yet at lower operational costs. Stability experiments showed the importance of the proper handling of urine samples to avoid degradation of these peptide hormones, especially for sermorelin and its metabolite which were found to rapidly degrade at temperatures > 4 °C and pH values < 7 in accordance with earlier studies. Without the need for specific antibodies, this method may be expanded to cover emerging peptide drugs (≥ ~3 kDa), as well as their metabolites in the future to facilitate coverage for this class of prohibited substances.

Doping control analysis of small peptides: A decade of progress.

Small peptides are handled in the field of sports drug testing analysis as a separate group doping substances. It is a diverse group, which includes but is not limited to growth hormone releasing-factors and gonadotropin-releasing hormone analogues. Significant progress has been achieved during the past decade in the doping control analysis of these peptides. In this article, achievements in the application of liquid chromatography-mass spectrometry-based methodologies are reviewed. To meet the augmenting demands for analyzing an increasing number of samples for the presence of an increasing number of prohibited small peptides, testing methods have been drastically simplified, whilst their performance level remained constant. High-resolution mass spectrometers have been installed in routine laboratories and became the preferred detection technique. The discovery and implementation of metabolites/catabolites in testing methods led to extended detection windows of some peptides, thus, contributed to more efficient testing in the anti-doping community.

Combining direct urinary injection with automated filtration and nanoflow LC-MS for the confirmatory analysis of doping-relevant small peptide hormones.

Nano-liquid chromatography (nanoLC) has proven itself as a powerful tool and its scope entails various applications in (bio)analytical fields. Operation at low (nL/min) flow rates in combination with reduced inner dimensions (ID < 100 µm), leads to significantly enhanced sensitivity when coupled with electrospray ionization-mass spectrometry (ESI-MS). Challenges that remain for the routine implementation of such miniaturized setups are related to clogging of the system and robustness in general, and thus the application of tedious sample preparation steps. To improve ruggedness, a filter placed upstream in the LC prevents particles from entering and clogging the system. This so-called online automatic filtration and filter back-flush (AFFL) system was combined with nanoLC and the direct injection principle for the sensitive confirmatory analysis of fifty different doping-relevant peptides in urine. The presented assay was fully validated for routine purposes according to selectivity and matrix interference, limit of identification (LOI), carryover, matrix effect, sample extract stability, analysis of educational external quality assessment (EQAS) samples, robustness of the online AFFL-setup and retention time stability. It was also fully compliant with the most recent minimum required performance levels (MRPL) and chromatographic/mass spectrometric identification criteria (IDCR), as imposed by the World Anti-Doping Agency (WADA). In the absence of labor-intensive sample preparation, the application of AFFL allowed for the injection of diluted urine samples without any noticeable pressure buildup in the nanoLC system. Contrary to earlier observations by our group and others, the addition of dimethylsulfoxide (DMSO) to the mobile phase did not enhance sensitivity in the presented nanoflow setup, yet was beneficial to reduce carry over. Although the robustness of the presented setup was evaluated only for the analysis of diluted urine samples, it is entirely conceivable that routine applications employing other matrices and currently running on analytical scale LC instruments could be transferred to micro/nanoLC scale systems to reach lower detection limits.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Urinary detection of rapid-acting insulin analogs in healthy humans.

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.

A high-throughput assay for the quantification of intact Insulin-like Growth Factor I in human serum using online SPE-LC-HRMS.

Quantification of IGF-I is relevant in both doping control as a biomarker of growth hormone (GH) misuse in sports, and in the clinical field for longitudinal follow-up of patients with disorders related to the GH axis. Currently, better standardization of IGF-I measurements using mass spectrometry is in our best interest as it would enable long-term monitoring of an athletes' IGF-I levels by its addition to the Athlete Biological Passport (ABP). Here, a simplified and rapid top-down LC-HRMS method for quantification of IGF-I in human serum is presented. A ten-minute precipitation-based offline sample preparation is combined with online sample clean-up and separation on a conventional LC, resulting in a total runtime of nine minutes in between injections. The method was validated in the relevant range of 50-1000 ng/mL for the following parameters: linearity, precision, bias, Limit Of Quantification (LOQ), carry-over, selectivity, recovery and ion suppression. As proof of concept, the presented LC-HRMS assay was compared with results from a previous inter-laboratory study on intact IGF-I quantification using four human GH administration samples. It was additionally compared with the IDS-iSYS immunoassay using 47 athlete serum samples, showing good overall agreement with a slight positive bias of 24.2 ng/mL for the LC-HRMS assay at a mean sample concentration of 234 ng/mL. Also, a discrepancy between commercially available IGF-I reference material for the calibration of quantitative assays is discussed. This is of importance if LC-MS assays for IGF-I are to be harmonized.

Peptide enrichment by ion-pair solid-phase extraction.

The technique of Solid-Phase Extraction (SPE) is widely used in various fields to concentrate samples and the search for tools to improve recoveries remains of outmost importance. The use of polymer based cartridges has become prevailing in a broad range of fields to enrich peptides from biological matrices. However, the existing SPE protocols are characterized by disparity. Ion-pairing (IP) reagents are commonly used in chromatographic applications, but their combination with SPE is less known. The aim of this study was to evaluate various SPE loading conditions, including the use of IP reagents, to improve the recoveries of nine selected peptide molecules. Control of pH and the use of IP reagents were found to be crucial to improve the enrichment of the peptides, especially cationic peptides, for which an up to ten-fold increase was observed. The practical potential of the presented theoretical findings were verified by employing IP-SPE for the development of an efficient extraction method for the doping relevant peptide Synacthen. 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 (20 pg/ml) and recovery (37%).

Utilizing ELISA-plate based immunopurification and liquid chromatography-tandem mass spectrometry for the urinary detection of short- and long acting human insulin analogues.

The measurement of human insulin and its synthetic analogues in biological matrices has become increasingly important not only in clinical fields but also in doping control. The use of insulin and its analogues have been included in the list of prohibited substances published by the World Anti-Doping Agency (WADA). This study describes a qualitative method for detection of insulin analogues (lispro, aspart, glulisine, glargine, degludec, detemir) in human urine. The sample preparation consists of a preconcentration step using ultrafiltration followed by an immunoaffinity extraction with an antibody precoated ELISA plate. The obtained extracts are analyzed by conventional high-performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS). The limits of detection range between 10 pg/ml and 150 pg/ml. The applicability of the method was proven by the analysis of real urine samples obtained from diabetic patients treated with synthetic insulin analogues.

DMSO Assisted Electrospray Ionization for the Detection of Small Peptide Hormones in Urine by Dilute-and-Shoot-Liquid-Chromatography-High Resolution Mass Spectrometry.

The mobile phase additive (DMSO) has been described as a useful tool to enhance electrospray ionization (ESI) of peptides and proteins. So far, this technique has mainly been used in proteomic/peptide research, and its applicability in a routine clinical laboratory setting (i.e., doping control analysis) has not been described yet. This work provides a simple, easy to implement screening method for the detection of doping relevant small peptides (GHRPs, GnRHs, GHS, and vasopressin-analogues) with molecular weight less than 2 kDa applying DMSO in the mobile phase. The gain in sensitivity was sufficient to inject the urine samples after a 2-fold dilution step omitting a time consuming sample preparation. The employed analytical procedure was validated for the qualitative determination of 36 compounds, including 13 metabolites. The detection limits (LODs) ranged between 50 and 1000 pg/mL and were compliant with the 2 ng/mL minimum detection level required by the World Anti-Doping Agency (WADA) for all the target peptides. To demonstrate the feasibility of the work, urine samples obtained from patients who have been treated with desmopressin or leuprolide and urine samples that have been declared as adverse analytical findings were analyzed. Graphical Abstract ᅟ.