Quantifying Co in Doping Control Urine Samples – A Pilot Study
Transcription
Quantifying Co in Doping Control Urine Samples – A Pilot Study
Quantifying Co in Doping Control Urine Samples – A Pilot Study Daniel Kutscher,1 Oliver Krug,2 Mario Thevis,2 Shona McSheehy Ducos1 1 Thermo Fisher Scientific, Bremen, Germany; 2 German Sports University, Cologne, Germany Overview Results Purpose: To demonstrate the power of ICP-MS for the analysis of different elements in biological matrices and demonstrate the use of trace elemental analysis in clinical applications. The underlying mechanism in w improved performance of espec displayed in figure 2. In 2014, C World Anti-Doping Agency´s list so that dishonest sportsmen co option to increase their oxygen contrast, testing for elevated lev e.g. horse racing3. Methods: Total elemental determinations of Co and other trace elements in urine were performed using the Thermo Scientific™ iCAP™ Qc ICP-MS in a single, optimized operation mode. The excretion of Co in urine was measured in an elimination study after intake of Co2+ and vitamin B12 respectively. Results: Good agreement between the results determined using a certified reference material was achieved. The elimination study showed that the determination of Co in urine can be a potential tool to screen for its misuse as a performance enhancing substance. Introduction The role of Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in clinical research is relatively unknown in the mass spectrometry community. However, it is a powerful tool for trace elemental analysis thanks to high sensitivity and robustness when faced with high matrix samples. ICP-MS provides a fully quantitative technique for almost all elements in the periodic table, offers isotopic information and isotope ratio determinations and can also provide species specific information when coupled with a separation device such as Ion Chromatography (IC) or Liquid Chromatography (LC). The analysis of trace metals in urine is a way to address potentially harmful exposure to contaminants. One of the advantages for urine as a sample matrix is that the specimen is easily derived. Apart from screening for potential exposure to toxic elements like As, Cd, Hg and Pb, the determination of Co in urine has gained interest, as Co2+ could potentially be used as a performing enhancing substance in endurance sports1. Methods Sample Preparation Urine samples were diluted 10-fold in 2% HNO3, and 50 µl of an internal standard solution (5 ng.mL-1 Sc, Ge, 2.5 ng.mL-1 Rh and Ir) was added to each sample. Mass Spectrometry The iCAP Qc ICP-MS (Figure 1) was used for acquisition of all data. An SC-4Q autosampler (Elemental Scientific, Omaha, NE, USA) was used for all measurements. The iCAP Qc ICP-MS was operated in single He KED mode for all analyte ions. All instrumental parameters can be found in table 1. 2 Quantifying Co in Doping Control Urine Samples – A Pilot Study Performance Enhancemen FIGURE 2. Proposed mechan performance enhancement in • The effect of 5 µmol Co is eq Erythropoiesis as 1 IU rhEPO • Typically, 6-25 IU·L-1 of EPO • 150 mg CoCl2 leads to an inc 1.19 Mio cells·mm-2 in 7-22 d Analysis of Trace Elements in In a pilot study, approx. 200 sam and various other trace elemen levels of Co in urine. Verify - Co concentrations in urine were found range [0.1 to 2 ng· mL-1] All calibration standards were p and a six point calibration curve 0.025 to 25 ng·L-1 (verification o to 100 ng·mL-1 (elimination stud instrumental limit of detection fo ng L-1. UTAK® Urine Control Sa Range, UTAK Laboratories, Val regularly interspersed in the sam to verify the accuracy of the me for the repeated analysis of both NE, USA) was used for all measurements. The iCAP Qc ICP-MS was operated in single He KED mode for all analyte ions. All instrumental parameters can be found in table 1. and a six point calibration curve w 0.025 to 25 ng·L-1 (verification of re to 100 ng·mL-1 (elimination study). instrumental limit of detection for 5 ng L-1. UTAK® Urine Control Samp Range, UTAK Laboratories, Valenc regularly interspersed in the samp to verify the accuracy of the metho for the repeated analysis of both c batch (containing 70 samples) are TABLE 2. Results for the repeate controls as a quality control with all values given in µg·L-1. As Cd Ca Cr Co Cu 16.5 0.4 96 1.3 1.9 32 Maximum 14 19 0.34 0.46 82 110 1.1 1.5 1.6 2.2 27 37 Recovery Reference Minimum FIGURE 1. Thermo Scientific iCAP Qc ICP-MS. TABLE 1. Instrumental Conditions. 95% 98% 95% 95% 103% 1.8 6.6 1.6 4.6 3.3 2.9 Reference 118 4.6 490 6.2 7.6 81 Maximum 100 136 3.9 5.3 417 564 5.3 7.1 6.5 8.7 69 93 Recovery 94% 96% 91% 105% 98% 95% 3.6 2.9 1.7 1.9 1.6 3.2 Minimum Parameter Nebulizer Nebulizer Gas Flow Value PFA-ST 1.06 L·min-1 RF Power Interface Set-Up 1550 W Ni Cones, High Matrix Skimmer Insert QCell conditions Cell Gas Flow KED Voltage 94% RSD RSD IDL 4.8 mL·min-1 100% He 3V Data Analysis Thermo Scientific™ Qtegra™ Intelligent Scientific Data SolutionTM software was used for quantitative assessment of the data. 0.007 0.0006 0.3 0.001 0.0006 0.0008 Utak 12111 Normal Range Good agreement between the cert values was achieved. The low rela between the different replicates hig the method. The obtained results for both group ng·mL-1 for non elite athletes and 0 endurance athletes) were found to range, but showing a modest but s increase for professional enduranc sum test, p<0.01). However, strong concentrations were not observed. Thermo Scientific Poster Note • eWPC • PN43242-EN 0315S 3 Results he power of ICP-MS for the ts in biological matrices and e elemental analysis in clinical eterminations of Co and other e performed using the Thermo MS in a single, optimized ion of Co in urine was study after intake of Co2+ and The underlying mechanism in which Co2+ may lead to improved performance of especially endurance athletes is displayed in figure 2. In 2014, Co2+ was not listed on the World Anti-Doping Agency´s list of prohibited substances2, so that dishonest sportsmen could potentially use this option to increase their oxygen transport capacity. In contrast, testing for elevated levels of Co is already done in e.g. horse racing3. Feasibility of Co Urinary Co Anti Doping Testing In a second step, an eliminat verify that intake of Co2+ lead Co concentration. The results displayed in figure 3. Investigate excretio - 3 participants. s - 3 participants. mu Co2+ between the results reference material was udy showed that the can be a potential tool to erformance enhancing pled Plasma Mass linical research is relatively ometry community. However, elemental analysis thanks to ess when faced with high ovides a fully quantitative ments in the periodic table, nd isotope ratio determinations s specific information when evice such as Ion uid Chromatography (LC). s in urine is a way to address e to contaminants. One of the ample matrix is that the Apart from screening for elements like As, Cd, Hg and in urine has gained interest, used as a performing durance sports1. FIGURE 3. Proposed mech performance enhancement Performance Enhancement in Endurance Sports FIGURE 2. Proposed mechanism for Co2+ induced performance enhancement in enduring sports. • The effect of 5 µmol Co is equivalent to stimulation of Erythropoiesis as 1 IU rhEPO. • Typically, 6-25 IU·L-1 of EPO are circulating in humans. • 150 mg CoCl2 leads to an increase of RBC from 0.5 to 1.19 Mio cells·mm-2 in 7-22 days. Analysis of Trace Elements in Urine 10-fold in 2% HNO3, and 50 µl ion (5 ng.mL-1 Sc, Ge, 2.5 ed to each sample. re 1) was used for acquisition In a pilot study, approx. 200 samples were analyzed for Co and various other trace elements in order to verify baseline levels of Co in urine. Verify baseline Co levels in urine -100 healthy non-elite athletes - 96 endurance sports athletes - Co concentrations in urine were found to be in the typically observed range [0.1 to 2 ng· mL-1] The results show that the Co increased in comparison to th between 0.1-2.0 ng·mL-1. A s normalization of the determin to compensate for the specif are two conclusions to be dra • A strong increase of the ur observed within 6 h (betwe intake of Co2+. • Multiple doses of Co2+ lea observed even several da stopped. Furthermore, the excretion o upon regular (daily) intake of accounting for 22 µg Co). Ho seem to affect the urinary Co Conclusion • The iCAP Q ICP-MS is a elements in a challenging combination of a unique r single analysis mode for a method development and when it comes to routine samples. • The detection of Co in uri potential tool to investigat • Further investigation is re additionally influencing fa emental Scientific, Omaha, measurements. The iCAP Qc gle He KED mode for all All calibration standards were prepared in diluted nitric acid and a six point calibration curve was generated between 0.025 to 25 ng·L-1 (verification of reference levels) and 0.25 to 100 ng·mL-1 (elimination study). The attainable instrumental limit of detection for 59Co was found to be 0.6 can be found in table 1. ng L-1. UTAK® Urine Control Samples (Low and High Range, UTAK Laboratories, Valencia, CA, USA) were regularly interspersed in the sample list (every 10 samples) to verify the accuracy of the method. The obtained results – A Pilot Study 4 Quantifying Co in Doping Control Urine Samples for the repeated analysis of both control samples within a ntal Scientific, Omaha, urements. The iCAP Qc He KED mode for all be found in table 1. All calibration standards were prepared in diluted nitric acid and a six point calibration curve was generated between 0.025 to 25 ng·L-1 (verification of reference levels) and 0.25 to 100 ng·mL-1 (elimination study). The attainable instrumental limit of detection for 59Co was found to be 0.6 ng L-1. UTAK® Urine Control Samples (Low and High Range, UTAK Laboratories, Valencia, CA, USA) were regularly interspersed in the sample list (every 10 samples) to verify the accuracy of the method. The obtained results for the repeated analysis of both control samples within a batch (containing 70 samples) are displayed in table 2. As Reference telligent Scientific Data r quantitative assessment Cr Co Cu Fe Pb Mn Mo Ni Se Zn 0.4 96 1.3 1.9 32 30 0.6 3.5 52 2.6 40 269 Maximum 0.34 0.46 82 110 1.1 1.5 1.6 2.2 27 37 26 35 0.5 0.7 3.0 4.0 44 60 2.2 3.0 34 46 229 309 Recovery 94% 95% 98% 95% 95% 1.8 6.6 1.6 4.6 3.3 2.9 4.9 4.7 2.9 2.2 3.0 2.7 2.5 Minimum V Ca 14 19 ions. 8 mL·min-1 100% He Cd 16.5 Minimum RSD FA-ST 06 L·min-1 550 W Cones, High Matrix kimmer Insert • The iCAP Q ICP-MS is a perfe elements in a challenging mat combination of a unique robus single analysis mode for all an method development and help when it comes to routine anal samples. • The detection of Co in urine h potential tool to investigate its • Further investigation is require additionally influencing factors complementary sample matric TABLE 2. Results for the repeated analysis of urine controls as a quality control within one sample batch, all values given in µg·L-1. CAP Qc ICP-MS. alue Conclusion - Co concentrations in urine were found to be in the typically observed range [0.1 to 2 ng· mL-1] was used for acquisition Reference 103% 97% 122% 95% 94% 117% 90% 117% 118 4.6 490 6.2 7.6 81 565 137 4.2 71 27 55 859 Maximum 100 136 3.9 5.3 417 564 5.3 7.1 6.5 8.7 69 93 480 650 116 158 3.6 4.8 60 82 23 31 47 63 730 988 Recovery 94% 96% 91% 105% 98% 95% 87% 89% 3.6 2.9 1.7 1.9 1.6 RSD IDL 0.007 0.0006 0.3 3.2 3.1 1.0 References 1. O. Krug et al., Quantifying cobalt in a pilot study, Drug Testing Analysis 2. The Prohibited List, published by th https://www.wada-ama.org/en/resou medicine/prohibited-list 3. P. Bartley: in The Sidney Morning H 100% 95% 107% 91% 100% 4.1 3.8 1.7 5.2 2.8 0.001 0.0006 0.0008 0.002 0.0003 0.002 0.0007 0.002 0.02 0.006 Utak 12111 Normal Range Utak 12110 High Range Good agreement between the certified and the obtained values was achieved. The low relative standard deviation between the different replicates highlights the robustness of the method. Utak is the property of Utak Laboratories, Inc. All other trademarks subsidiaries. This information is not intended to encourage use of these product property rights of others. Presented at the European Winter Plasma Conference on Plasma The obtained results for both groups (mean values 0.28 ng·mL-1 for non elite athletes and 0.36 ng·mL-1 for listed endurance athletes) were found to be within the expected range, but showing a modest but statistically significant increase for professional endurance athletes (Wilcoxon rank sum test, p<0.01). However, strongly increased urinary Co concentrations were not observed. Thermo Scientific Poster Note • eWPC • PN43242-EN 0315S 5 which Co2+ may lead to ecially endurance athletes is Co2+ was not listed on the st of prohibited substances2, could potentially use this en transport capacity. In evels of Co is already done in Feasibility of Co Urinary Concentration as a Tool for Anti Doping Testing In a second step, an elimination study was performed to verify that intake of Co2+ leads to an increase of the urinary Co concentration. The results obtained for both groups are displayed in figure 3. Investigate excretion after intake of Co2+ - 3 participants. single dose; 500 µg - 3 participants. multiple doses of 500 µg Co2+ FIGURE 3. Proposed mechanism for Co2+ induced performance enhancement in enduring sports. ent in Endurance Sports nism for Co2+ induced in enduring sports. equivalent to stimulation of PO. O are circulating in humans. ncrease of RBC from 0.5 to days. in Urine amples were analyzed for Co nts in order to verify baseline y baseline Co levels in urine -100 healthy non-elite athletes - 96 endurance sports athletes d to be in the typically observed The results show that the Co concentration in urine is increased in comparison to the reference values found between 0.1-2.0 ng·mL-1. A similar picture was found after normalization of the determined Co concentration in order to compensate for the specific gravity of the samples. There are two conclusions to be drawn from the results: • A strong increase of the urinary Co concentration is observed within 6 h (between 40 and 318 ng·mL-1 ) after intake of Co2+. • Multiple doses of Co2+ lead to elevated levels of Co2+ observed even several days after the intake was stopped. Furthermore, the excretion of Co in urine was monitored upon regular (daily) intake of Vitamin B12 (500 µg·day-1, accounting for 22 µg Co). However, Vitamin B12 does not seem to affect the urinary Co level. Conclusion • The iCAP Q ICP-MS is a perfect tool to determine trace elements in a challenging matrix such as urine. The combination of a unique robust interface design and a single analysis mode for all analytes greatly simplifies method development and helps to improve throughput when it comes to routine analysis of a large number of samples. prepared in diluted nitric acid ve was generated between of reference levels) and 0.25 udy). The attainable for 59Co was found to be 0.6 Samples (Low and Co inHigh Doping Control Urine Samples A Pilotdetection Study 6 Quantifying • –The of Co in urine has been shown to be a alencia, CA, USA) were potential tool to investigate its abuse in sports doping. seem to affect the urinary Co level. eline Co levels in urine 100 healthy non-elite athletes 96 endurance sports athletes Conclusion n the typically observed red in diluted nitric acid generated between erence levels) and 0.25 he attainable Co was found to be 0.6 s (Low and High a, CA, USA) were list (every 10 samples) . The obtained results ntrol samples within a isplayed in table 2. • The iCAP Q ICP-MS is a perfect tool to determine trace elements in a challenging matrix such as urine. The combination of a unique robust interface design and a single analysis mode for all analytes greatly simplifies method development and helps to improve throughput when it comes to routine analysis of a large number of samples. • The detection of Co in urine has been shown to be a potential tool to investigate its abuse in sports doping. • Further investigation is required to elucidate the role of additionally influencing factors and to establish tests for complementary sample matrices like erythrocytes. d analysis of urine n one sample batch, e Pb Mn Mo Ni Se Zn 0 0.6 3.5 52 2.6 40 269 6 5 0.5 0.7 3.0 4.0 44 60 2.2 3.0 34 46 229 309 7% 122% 95% 94% 117% 90% 117% .9 4.7 2.9 2.2 3.0 2.7 2.5 65 137 4.2 71 27 55 859 80 50 116 158 3.6 4.8 60 82 23 31 47 63 730 988 7% 89% .1 1.0 References 1. O. Krug et al., Quantifying cobalt in doping control urine samples – a pilot study, Drug Testing Analysis 6 (2014), 1186-1190 2. The Prohibited List, published by the World Anti Doping Agency, https://www.wada-ama.org/en/resources/sciencemedicine/prohibited-list 3. P. Bartley: in The Sidney Morning Herald, February 4th 2014. 100% 95% 107% 91% 100% 4.1 3.8 1.7 5.2 2.8 002 0.0003 0.002 0.0007 0.002 0.02 0.006 Utak 12110 High Range ed and the obtained ve standard deviation lights the robustness of Utak is the property of Utak Laboratories, Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Presented at the European Winter Plasma Conference on Plasma Spectrochemistry, Münster, Germany 02/2015. (mean values 0.28 36 ng·mL-1 for listed e within the expected atistically significant athletes (Wilcoxon rank y increased urinary Co www.thermofisher.com ©2016 Thermo Fisher Scientific Inc. All rights reserved. Utak is the property of Utak Laboratories, Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Africa +43 1 333 50 34 0 Australia +61 3 9757 4300 Austria +43 810 282 206 Belgium +32 53 73 42 41 Canada +1 800 530 8447 China 800 810 5118 (free call domestic) 400 650 5118 PN43242-EN 09/16S Denmark +45 70 23 62 60 Europe-Other +43 1 333 50 34 0 Finland +358 9 3291 0200 France +33 1 60 92 48 00 Germany +49 6103 408 1014 India +91 22 6742 9494 Italy +39 02 950 591 Japan +81 45 453 9100 Korea +82 2 3420 8600 Latin America +1 561 688 8700 Middle East +43 1 333 50 34 0 Netherlands +31 76 579 55 55 New Zealand +64 9 980 6700 Norway +46 8 556 468 00 Russia/CIS +43 1 333 50 34 0 Singapore +65 6289 1190 Spain +34 914 845 965 Sweden +46 8 556 468 00 Switzerland +41 61 716 77 00 UK +44 1442 233555 USA +1 800 532 4752