Identification and quantification of the plasma volume expander dextran in human urine by liquid chromatography-tandem mass spectrometry of enzymatically derived isomaltose.
Plasma volume expanders are used in sports in order to control haematological parameters and/or to mask erythropoietin (EPO) misuse. A reliable method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for doping control purposes, enabling the identification and quantification of the plasma volume expander dextran in human urine. The dextran polymer was enzymatically hydrolysed by alpha-1,6-glucosidase (dextranase) followed by acetylation of the generated isomaltose subunits, allowing the chromatographic separation of different disaccharides, such as lactose, saccharose and isomaltose, as well as the identification and quantification of the analyte in human urine. The method was used to determine the basal concentration of isomaltose resulting from the enzymatic hydrolysis of polymeric 1,6-linked glucose in 238 routine doping control samples. In addition the concentration of dextran measured as isomaltose was estimated in seven urine specimens obtained from patients treated with dextran. Calibration curves for dextran were linear and reproducible. The inter- and intra-assay coefficients of variation for dextran ranged from 4.9 to 7.3% at three concentration levels between 53 and 1186 microg/mL. Recovery ranged from 97 to 112% (mean 106.9%). The assay limit of detection was 3.8 microg/mL and the lower limit of quantification was 12.5 microg/mL. In 96% of the investigated doping control samples, the concentrations of isomaltose were below the LLOQ of 12.5 microg/mL. Even the highest concentrations were approximately 100-300-fold lower than concentrations found in urine samples of patients after intravenous application of dextran. The presented results demonstrate the capability and reliability of the developed LC-MS/MS method for the identification and quantification of dextran in human urine and can be regarded as a method revealing the misuse of dextran in sports.
Guddat
Sven
S
Institute of Biochemistry, German Sport University, Cologne, Germany. s.guddat@biochem.dshs-koeln.de
Thevis
Mario
M
Schänzer
Wilhelm
W
D000368:Aged; D000369:Aged, 80 and over; D002853:Chromatography, Liquid / Q000379:methods*; D003910:Dextranase / Q000378:metabolism; D003911:Dextrans / Q000627:therapeutic use / Q000652:urine*; D004300:Doping in Sports / Q000517:prevention & control; D004355:Drug Stability; D005260:Female; D006801:Humans; D007534:Isomaltose / Q000032:analysis*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D010952:Plasma Substitutes / Q000032:analysis*; D012680:Sensitivity and Specificity
D003911:Dextrans; D010952:Plasma Substitutes; D007534:Isomaltose; D003910:Dextranase