Improved Sample Preparation Methods for Athlete Doping Analysis of Common Compounds in Urine by LCMS

Dr. Catrin Goebel2 , Lisa Wanders1, Sam Ellis1
1Thomson Instrument Company
2Australian Sports Drug Testing Laboratory in the National Measurement Institute Department of Industry


Anti-doping testing by urine analysis requires fast and robust screening methods with repeatable sample preparation. Since, every sample has to be screened, methods are designed to be sufficiently sensitive and specific to identify all suspect samples. One must be careful to minimize false suspects. Ensuring samples are spiked with internal standards accordingly will help verify that samples are being extracted and tested correctly and with accurate uniformity.

The Australian Sports Drug Testing Laboratory, our collaborators, have invested time in determining a limited number of comprehensive screening methods. These methods, using Thomson’s eXtreme Filter Vials (patented), comply with the World Anti-Doping Agency’s (WADA) Prohibited List.

In exploring new methods labs have looked at both detection and sample prep as routes to quicker and more accurate analysis. Liquid chromatography coupled with mass spectrometry detection is prevalent, superseding many of the gas chromatographic coupled with mass spectrometry methods because of the simpler sample preparation. Specifically, the anti-doping testing shown below consisted of sample preparation without the initial use of cumbersome traditional SPE methods, and instead consisted of the comparison of filtration techniques. Filter plates versus Thomson eXtreme Filter Vials (patented) were tested to determine which product allowed for a method of simple and quick urine analysis while complying with the WADA’s guidelines.


The experiments were performed at the National Measurement Institute (Australia) in the Sports Drug Testing Laboratory.

The 11.8 minute run time for the instrumental analysis meets the requirements of the WADA Technical Document- Minimum Required Performance Level (TD2013MRPL). This document details the analysis of a large number of analytes from the classes on the WADA Prohibited List, while meeting sensitivity requirements. The analytes included compounds in the following classes anabolic agents, B2-agonists, hormone antagonists and modulators, diuretics, stimulants, narcotics, glucocorticoids, B-blockers, etc.

Full Method

A comparison between sample preparation using filter plates sourced from several different manufactures, and Thomson eXtreme Filter Vials (patented) PVDF 0.2µm (85531-500) was conducted. The preparation with the Thomson eXtreme Filter Vials were automated using a Tecan robotics platform for liquid dispensing in the Thomson 48 position rack (#35010-RACK), and 48 position press (#35015).

Direct Urine Preparation

  1. Label each eXtreme Filter Vial with sample/quality control sample information.
  2. Pipette 200 µL of each sample into labeled eXtreme Filter Vial.
  3. Add 200 µL of the Mefruside Internal Standard (300 ng/mL in 0.5% formic acid) to each filter vial cup.
  4. Place the eXtreme Filter Vial tops onto each vial and press shut.


UPLC coupled to High Resolution Mass Spectrometry with an electrospray source in full scan mode. Data acquisition in both positive and negative polarity modes within a single 11.8 min chromatographic run.

  • Column: C18, 2.1mm × 50mm, 1.7µm
  • Column Temperature: 30 °C
  • Flow rate: 300µL/min
  • Mobile Phase:
    • A: 0.3% aqueous Formic Acid in Water
    • B: 0.3% Formic Acid in Acetonitrile
  • Gradient:
    • TimeA%B%
  • Injection volume: 10µL
  • Sample tray temperature: 18°C
  • Column Temperature: 30°C
  • Method run time: 11.8 minutes
  • Gas: UHP Nitrogen


The Thomson eXtreme Filter Vials (patented) PVDF 0.2 µm (85531-500) performed the best in compound extraction and identification while allowing the end user to follow the WADA validated method. The elimination of SPE steps from laboratory methods is a large time saver, and enables urine-direct-injection solely using Thomson eXtreme Filter Vials for filtration. Together the Thomson 48 position Filter Vial Press and automation enabled 48 position rack equaled timing of filter plate methodology but provided the best extraction and identification of all filter types. A total of 180 compounds can be identified through the screening analysis with the Thomson eXtreme Filter Vials (patented) PVDF 0.2µm (85531-500).

The method presented is being used for the analysis of athlete’s urine samples for banned substances at the Australian Sports Drug Testing Laboratory.

Comparison of Filter Types

athlete dopeing elements

Linearity of The Analysis Method Was Assessed Over a Range From 25% To 200% Of MRPL With R2 Generally Being Greater Than 0.98


124 Confirmed Compounds

  1. 5-Hydroxyindapamide
  2. Bisdesmethylsibutramine
  3. Desmethylsibutramine
  4. Exemestane
  5. Mefruside (+)
  6. Mefruside (-)
  7. D3-epitestosterone glucuronide
  8. D3-epitestosteronea
  9. AICAR
  10. GW1516
  11. Atenolol
  12. Bisoprolol
  13. Esmolol
  14. Metipranolol
  15. Nadolol
  16. Nadoxolol
  17. Oxprenolol
  18. Clenbuterol
  19. Gestrinone
  20. Methyldienolone
  21. Methyltrienolone
  22. Metribolone
  23. Tetrahydrogestrinone
  24. Tibolone
  25. Zilpaterol
  26. 3’-Hydroxystanozolol
  27. 4’-Hydroxystanozolol
  28. Bambuterol
  29. Formoterol
  30. Salbutamol
  31. Salmeterol
  32. Terbutaline
  33. Andarine
  34. Exemestane metabolite
  35. Aminoglutethimide
  36. Raloxifene
  37. Fulvestrant
  38. GW1516 (501516)
  39. Methazolamide
  40. Piretanide
  41. Quinethazone
  42. Spironolactone
  43. Trichlormethiazide
  44. Acetazolamide
  45. Althiazide
  46. Amiloride
  47. Bendroflumethiazide
  48. Benzthiazide
  49. Bumetanide
  50. Canrenone
  51. Chlorexolone
  52. Chlorothiazide
  53. Chlorthalidone
  54. Clopamide
  55. Probenecid
  56. Cyclopenthiazide
  57. Cyclothiazide
  58. Dichlorphenamide
  59. Epitizide
  60. Eplenerone
  61. Etacrynic acid (frag?)
  62. Furosemide
  63. Hydrochlorothiazide
  64. Mefruside metabolite 2
  65. Indapamide
  66. Metolazone
  67. Polythiazide
  68. Torasemide
  69. Triamterene
  70. Xipamide
  71. Caffeine
  72. Cis-4-Methylaminorex
  73. Cotinine (Nicotine metab)
  74. MBDB
  75. Methoxyamphetamine
  76. Methylenedioxyethylamphetamine
  77. Adrafinil
  78. Amiphenazole
  79. Amphetamine
  80. Benzoylecgonine
  81. Benzylpiperazine
  82. Carphedon
  83. Cathine
  84. Crotethamide
  85. Cyclazodone
  86. Ephedrine
  87. Phenylpropanolamine
  88. Pseudoepherine
  89. Etamivan
  90. Etilefrine
  91. Fenetylline
  92. Hydroxy mesocarb
  93. Isometheptene
  94. MDA
  95. MDMA
  96. Methylphenidate
  97. Modafinil
  98. Modafinil Acid (metabolite)
  99. Nikethamide
  100. Oxilofrine
  101. Pemoline
  102. Pentetrazol
  103. Phenmetrazine
  104. Pholedrine
  105. p-Hydroxy amphetamine
  106. Ritalinic Acid
  107. nor-Selegiline
  108. Methylecgonine
  109. Codeine
  110. Hydromorphone
  111. Morphine
  112. JWH018 N-(5-hydroxypentyl) metabolite
  113. JWH073 N-butanoic acid metabolite
  114. Budesonide
  115. Cortisol
  116. Cortisone
  117. Flumethasone
  118. Fluticasone propionate metabolite
  119. Methylprednisolone
  120. 16a-OH-Prednisolone
  121. Prednisolone
  122. Sildenafil
  123. Tadalafil
  124. Vardenafil


We would like to thank Dr. Catrin Goebel, Director, of Australian Sports Drug Testing Laboratory in the National Measurement Institute, Department of Industry (a WADA accredited laboratory in Australia) for her extensive testing. Dr. Goebel is also an Executive member of World Association of Anti-Doping Scientist.

Thomson Instrument Company is not affiliated with Australian Sports Drug Testing Laboratory or World Anti-Doping Agency.
World Anti-Doping Agency and Australian Sports Drug Testing Laboratory is not affiliated, nor endorses Thomson’s products.


Improved Sample Preparation Methods for Athlete Doping Analysis of Common Compounds in Urine by LCMS


The Australian Sports Drug Testing Laboratory, ... invested time in determining a limited number of comprehensive screening methods. These methods, using Thomson’s eXtreme|FV® ... , comply with the World Anti-Doping Agency’s (WADA) Prohibited List.

PDF Application

Product Used in Application

eXtreme|FV® - PVDF 0.2µm

eXtreme|FV® - PVDF 0.2µm

Pre-Split Septum, Red Cap


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