TITLE

Identification of the long-acting erythropoiesis-stimulating agent darbepoetin alfa in human urine by liquid chromatography-tandem mass spectrometry

AUTHOR(S)
Okano, Masato; Sato, Mitsuhiko; Kageyama, Shinji
PUB. DATE
February 2014
SOURCE
Analytical & Bioanalytical Chemistry;Feb2014, Vol. 406 Issue 5, p1317
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
The misuse of recombinant human erythropoietin (rhEPO) increases the proliferation/production of erythrocytes, which enhance oxygen transport capacities, and has grave consequences with respect to human health and fairness in sports. For sports drug testing, the current analytical methods for rhEPOs are mainly gel electrophoretic methods, such as isoelectric focusing-polyacrylamide gel electrophoresis. Mass spectrometry is fundamentally necessary for the reliable identification of rhEPOs in doping control. In this study, a high-sensitivity and high-throughput mass spectrometric qualitative detection method for darbepoetin alfa in human urine was established by a bottom-up approach. The novel method involves the immunopurification of human urine (10 mL), protease digestion with endoproteinase Glu-C (V8-protease) in an ammonium bicarbonate buffer (pH 7.8) and ultra-performance liquid chromatography using a charged surface hybrid C column coupled with electrospray-ionisation high-sensitivity tandem mass spectrometry for improved selectivity of the target molecules. The specific fragment digested from darbepoetin alfa was TLQLHVDKAVSGLRSLTTLLRALGAQKE (V). The lower limit of detection of urinary darbepoetin alfa was 1.2 pg/mL. The limit of detection for the confirmation analysis was estimated to be 5 pg/mL. The developed method allows high-throughput confirmation analysis, namely 6 h for sample preparation and an analytical run time of only 10 min per sample; this high-throughput method dramatically decreases the workload in the laboratory. Darbepoetin alfa could be identified in human urine collected after the intravenous administration of 15 μg darbepoetin alfa ( n = 3). This mass spectrometric method is an innovative and powerful tool for detecting darbepoetin alfa in human urine for doping control testing. [Figure not available: see fulltext.]
ACCESSION #
94345494

 

Related Articles

  • Foreword. Carrera, Fernando // Nephrology Dialysis Transplantation;2007, Vol. 22 Issue suppl_4, piv1 

    An introduction to the journal is presented in which the editor discusses an article on haemoglobin (Hb) level stability because of erythropoiesis-stimulating agents (ESA), half-life of darbepoetin, and extended dosage of ESAs.

  • Erratum. Kim, Jin Young // Journal of Analytical Toxicology;Jul2014, Vol. 38 Issue 6, p385 

    A correction to the article "In Estimation of Measurement Uncertainty for the Quantification of 11-Nor-Delta 9-Tetrahydrocannabinol-9-Carboxylic Acid and Its Glucuronide in Urine Using Liquid Chromatography Tandem Mass Spectrometry" by J.Y. Kim et al. published in the 2014 issue is presented.

  • Erratum.  // European Surgical Research;Apr2016, Vol. 56 Issue 3/4, p180 

    A correction to the article "Darbepoetin-alfa promotes cell proliferation in established extrahepatic colorectal tumors after major hepatectomy" that was published in the previous issue of the journal is presented.

  • High Hematocrit Resulting from Administration of Erythropoiesis-stimulating Agents Is Not Fully Predictive of Mortality or Toxicities in Preclinical Species. Andrews, Dina A.; Pyrah, Ian T. G.; Boren, Babette M.; Tannehill-Gregg, Sarah H.; Lightfoot-Dunn, Ruth M. // Toxicologic Pathology;Apr2014, Vol. 42 Issue 3, p510 

    We conducted a retrospective analysis of publicly available preclinical toxicology studies with erythropoiesis-stimulating agents (ESAs) to examine common adverse events in rats, Beagle dogs, and cynomolgus monkeys. Mortality and/or thrombotic events were reported sporadically in a subset of...

  • Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP. De Bolós, C.; Giménez, E.; De Bolóz, C.; Belalcazar, V.; Andreau, D.; Borrás, E.; De La Torre, B. G.; Barbosa, J.; Segura, J.; Pascual, J. A. // Analytical & Bioanalytical Chemistry;Aug2007, Vol. 388 Issue 7, p1531 

    Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by...

  • Erythropoiesis stimulating agents: approaches to modulate activity. Sinclair, Angus M. // Biologics: Targets & Therapy;2013, Vol. 7, p161 

    Recombinant human erythropoietin (rHuEPO), such as the approved agents epoetin alfa and epoetin beta, has been used successfully for over 20 years to treat anemia in millions of patients. However, due to the relatively short half-life of the molecule (approximately 8 hours), frequent dosing may...

  • Determination of Urinary Biomarkers for Assessment of Short-Term Human Exposure to Aflatoxins in São Paulo, Brazil. Jager, Alessandra V.; Tonin, Fernando G.; Souto, Pollyana C. M. C.; Privatti, Rafaela T.; Oliveira, Carlos A. F. // Toxins;Jul2014, Vol. 6 Issue 7, p1996 

    In the present study, a longitudinal assessment was carried out to evaluate the short-term human exposure to aflatoxins in Pirassununga region, São Paulo, Brazil, by determination of urinary aflatoxins by a liquid chromatography coupled to mass sprectrometry (UPLC-MS/MS) method. Sixteen...

  • Reply to "A Novel 'Dilute-and-Shoot' Liquid Chromatography-Tandem Mass Spectrometry Method for the Screening of Antihypertensive Drugs in Urine". Kinnell, A.; Cole, R.; Thakrar, R.; Smith, P. R.; Patel, P.; Gupta, P. // Journal of Analytical Toxicology;Jan/Feb2017, Vol. 41 Issue 1, p80 

    A response from the author of the article "A Novel ‘Dilute-and-Shoot' Liquid Chromatography–Tandem Mass Spectrometry Method for the Screening of Antihypertensive Drugs in Urine" in the 2016 issue is presented.

  • Response to Kinnell letter. Lawson, Alexander James Frank; Shipman, Kate Elizabeth; George, Stephen Andrew; Dasgupta, Indranil // Journal of Analytical Toxicology;Jan/Feb2017, Vol. 41 Issue 1, p81 

    A response from the author of the article "A Novel ‘Dilute-and-Shoot' Liquid Chromatography–Tandem Mass Spectrometry Method for the Screening of Antihypertensive Drugs in Urine" in the 2016 issue is presented.

Share

Read the Article

Courtesy of THE LIBRARY OF VIRGINIA

Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics