Development of a multi‐functional concurrent assay using weak cation‐exchange solid‐phase extraction (WCX‐SPE) and reconstitution with a diluted sample aliquot for anti‐doping analysis

Kim, Yong-Seok; Jeon, Mijin; Min, Hophil; Son, Junghyun; Lee, Jaeick; Kwon, Oh‐Seung; Moon, Myeong Hee; Kim, Ki Hun

doi:10.1002/rcm.8119

Cited by 14 publications

(19 citation statements)

References 41 publications

(93 reference statements)

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“…Moreover, it was demonstrated that the implementation of lower molecular mass peptides (and peptide-related compounds) into comprehensive multi-target initial testing procedures is possible, either by simple dilute-and-inject approaches or combined analysis of SPE extracts with diluted urine specimens. [6][7][8] Although the provided limits of detection (LODs) meet World Anti-Doping Agency (WADA) requirements (minimum required performance level; MRPL = 2 ng/mL), the sensitivity of these assays is clearly inferior to peptide-specific stand-alone approaches, according to limitations of type and pH value of mobile phase as well as applied mass spectrometric acquisition modes.…”

Section: Introductionmentioning

confidence: 99%

Recent improvements in sports drug testing concerning the initial testing for peptidic drugs (< 2 kDa) – sample preparation, mass spectrometric detection, and data review

Görgens

Guddat

Thomas

et al. 2018

Drug Testing and Analysis

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In this work, a novel initial testing assay based on liquid chromatography-mass spectrometry is presented, enabling the detection of peptidic drugs and drug candidates (< 2 kDa) prohibited in sports. The assay covers representatives and metabolites of gonadotropin releasing hormone and its analogs (GnRHs), growth hormone secretagogues (GHS), growth hormone releasing peptides (GHRPs), and the Vasopressin-analog Desmopressin. The general objective of this work was to reduce sample preparation efforts to a minimum while preserving highest possible sensitivity and specificity of the assay, demonstrating limits of detection between 50 and 200 pg/mL.Here, a "dilute-and-inject" strategy provides the simplest conceivable sample preparation procedure. Furthermore, the combination of well-established strategies for the determination of peptides, such as two-dimensional liquid chromatography, dimethyl sulfoxide (DMSO)-assisted electrospray ionization, high resolution mass spectrometric detection and a tailored reporter template, which facilitates data review enormously, provides a high-throughput initial testing assay for lower molecular mass peptidic and peptide-related analytes.

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Section: Introductionmentioning

confidence: 99%

Recent improvements in sports drug testing concerning the initial testing for peptidic drugs (< 2 kDa) – sample preparation, mass spectrometric detection, and data review

Görgens

Guddat

Thomas

et al. 2018

Drug Testing and Analysis

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“…13 Among the several extraction methods, SPE involves less sample manipulation, which simplifies the pretreatment procedure and requires less proficiency from the analyst. 20 Mixed-mode weak cation exchange SPE (WCX-SPE) involves reverse phase and WCX that has proven to be an effective approach to extract peptides from urine. 5 Thomas et al reported on a WCX-SPE method to extract GHRPs from urine that inspired the extraction method utilized here.…”

Section: Sample Preparationmentioning

confidence: 99%

A high throughput approach for determination of dermorphin in human urine using liquid chromatography–mass spectrometry for doping control purposes

et al. 2020

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Dermorphin is a peptide with analgesic actions similar to morphine, but with greater effect and less potential to cause tolerance. The use of dermorphin has been documented in race horses, and its use in humans has already been reported. Considering the potential advantages from the use of dermorphin over morphine, a method to monitor it, and its main metabolite dermorphin (1-4) in humans becomes necessary for doping control. Here, we present two orthogonal methods for this purpose: a high-throughput liquid chromatography coupled to high-resolution mass spectrometry (HRMS) as an initial testing procedure and liquid chromatography-tandem mass spectrometry (MS/MS) in the selected reaction monitoring (SRM) acquisition mode for a confirmation procedure. For urine samples, pretreatment through a mixed-mode weak cation-exchange solid-phase extraction emerged as an effective approach to extract peptides from the biological sample. For the HRMS analysis, a full-MS scan acquisition mode was selected to detect the exact masses of dermorphin and dermorphin (1-4) at m/z 803.37226 and 457.20816, respectively. The SRM method used in the MS/MS confirmation protocol presented high specificity and sensitivity. The selected product ions for dermorphin were 602.2, 202.1 and 574.3 and for dermorphin (1-4) were 207.1, 223.1, and 235.1. Both methods were evaluated for specificity, repeatability, carryover, matrix effects, and recovery. No carryover and matrix effects were detected. The limit of detection for initial testing procedure and the limit of identification for confirmation procedure was 2.5 ng/ml. Also, specificity and robustness were acceptable for the application. Together, the developed methods proved to be efficient for the analysis of dermorphin and metabolite for human doping control purpose. K E Y W O R D S dermorphin, doping control, human urine, liquid chromatography, mass spectrometry 1 | INTRODUCTION Dermorphin is a heptapeptide (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) with analgesic effects similar to morphine that can be isolated from the skin of Phyllomedusa sauvagei, a South American species of frog. Whereas morphine is a partial agonist of μ-opioid receptors, dermorphin is a full agonist leading to a fourfold greater analgesic action than morphine when administered subcutaneously that

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“…These fall into several categories, each with distinct pharmacological profiles, chemical properties, and year‐by‐year increasingly specific requirements for their detection 2 . With recent technological advances in both liquid chromatography and mass spectrometry (LC‐MS), a great deal of effort has been directed toward development of multiclass initial testing procedures (ITP) to streamline analytical workflows in antidoping laboratories 3–32 …”

Section: Introductionmentioning

confidence: 99%

“…On the other hand, the second group does require preconcentration via liquid–liquid extraction (LLE) or solid phase extraction (SPE), which may or may not be preceded by enzymatic deconjugation. For this reason, multiclass ITPs had initially been developed utilizing triple quadrupole mass spectrometry (LC‐MS/MS) and two complimentary preparations such as dilute‐and‐shoot 3–12 and extraction‐based total fraction assays 13–32 . A comprehensive review has been provided by Thevis et al 33 wherein the former methods would typically cover diuretics, stimulants, narcotics, β‐blockers, and some of the β 2 ‐agonists, whereas the latter would focus on anabolic agents, hormone modulators, fentanyl analogs, synthetic cannabinoids, and glucocorticosteroids.…”

Section: Introductionmentioning

confidence: 99%

High‐throughput liquid chromatography tandem mass spectrometry assay as initial testing procedure for analysis of total urinary fraction

Sobolevsky¹,

Ahrens²

2020

Drug Testing and Analysis

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In the recent years, a lot of effort was put into the development of multiclass initial testing procedures (ITP) to streamline analytical workflow in antidoping laboratories. Here, a high‐throughput assay based on liquid chromatography–triple quadrupole mass spectrometry suitable for use as initial testing procedure covering multiple classes of compounds prohibited in sports is described. Employing a 96‐well plate packed with 10 mg of weak cation exchange polymeric sorbent, up to 94 urine samples and their associated positive and negative controls can be processed in less than 3 h with minimal labor. The assay requires a 0.5‐ml urine aliquot, which is subjected to enzymatic hydrolysis followed by solid phase extraction, evaporation, and reconstitution in a 96‐well collection plate. With a 10‐min run time, more than 100 analytes can be detected using electrospray ionization with polarity switching. The assay can be run nearly 24/7 with minimal downtime for instrument maintenance while detecting picogram amounts for the majority of analytes. Having analyzed approximately 28,000 samples, nearly 400 adverse analytical findings were found of which only one tenth were at or above 50% of the minimum required performance level established by the World Anti‐Doping Agency. Compounds most often identified were stanozolol, GW1516, ostarine, LGD4033, and clomiphene, with median estimated concentrations in the range of 0.02–0.09 ng/ml (either as parent drug or a metabolite). Our data demonstrate the importance of using a highly sensitive ITP to ensure efficient antidoping testing.

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Development of a multi‐functional concurrent assay using weak cation‐exchange solid‐phase extraction (WCX‐SPE) and reconstitution with a diluted sample aliquot for anti‐doping analysis

Cited by 14 publications

References 41 publications

Recent improvements in sports drug testing concerning the initial testing for peptidic drugs (< 2 kDa) – sample preparation, mass spectrometric detection, and data review

Recent improvements in sports drug testing concerning the initial testing for peptidic drugs (< 2 kDa) – sample preparation, mass spectrometric detection, and data review

A high throughput approach for determination of dermorphin in human urine using liquid chromatography–mass spectrometry for doping control purposes

High‐throughput liquid chromatography tandem mass spectrometry assay as initial testing procedure for analysis of total urinary fraction

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