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Development and application of a high-throughput sample cleanup process based on 96-well plate for simultaneous determination of 16 steroids in biological matrices using liquid chromatography–triple quadrupole mass spectrometry

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Abstract

A novel high-throughput sample pretreatment system was developed by the integration of protein precipitation (PP), phospholipid removal (PPR), and hollow fiber liquid-phase microextraction (HF-LPME) into two simple 96-well plates and a matching 96-grid lid. With this system, 16 steroids were separated from biological matrices of plasma, milk, and urine and analyzed by liquid chromatography–triple quadrupole mass spectrometry. In the tandem sample cleanup process, the prepositive PP and PPR step preliminarily removed some of the interferences from the biological matrices. The following HF-LPME step kept the residual interference out of the hollow fiber and enriched the steroids in the hollow fiber to achieve high sensitivity. By a series of method optimizations, acetonitrile was chosen as the crash solvent for PP and PPR. A mixture of octanol and toluene (1:1 v/v) was used as the acceptor phase for HF-LPME. The extraction was conducted at 80 rpm for 50 min in a donor phase containing 1 mL 20 % sodium chloride at 25 °C. Under these conditions, the limits of detection for the 16 steroids were 3.6–300.0 pg.mL-1 in plasma, 3.0–270.0 pg.mL-1 in milk, and 2.2–210.0 pg.mL-1 in urine. The recoveries of the 16 steroids were 81.9–97.9 % in plasma (relative standard deviation 1.0–8.0 %), 80.6–97.7 % in milk (relative standard deviation 0.8–5.4 %), and 87.3–98.7 % in urine (relative standard deviation 1.0–4.9 %). Further, the integrated 96-well platform of PP, PPR, and HF-LPME enabled us to run this assay in an automatic and high-throughput fashion. The reliability of the method was further corroborated by evaluation of its applicability in plasma and urine samples from volunteers and fresh bovine milk from local dairy enterprises.

The combined 96-throughput clean-up system for biological samples detection

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References

  1. Schänzer W (1996) Metabolism of anabolic androgenic steroids. Clin Chem 42:1001–1020

    Google Scholar 

  2. Shahidi NT (2001) A review of the chemistry, biological action, and clinical applications of anabolic-androgenic steroids. Clin Ther 23:1355–1390

    Article  CAS  Google Scholar 

  3. Sjöqvist F, Garle M, Rane A (2008) Use of doping agents, particularly anabolic steroids, in sports and society. Lancet 371:1872–1882

    Article  CAS  Google Scholar 

  4. Maravelias C, Dona A, Stefanidou M, Spiliopoulou C (2005) Adverse effects of anabolic steroids in athletes: a constant threat. Toxicol Lett 158:167–175

    Article  CAS  Google Scholar 

  5. Dohle GR, Smit M, Weber RFA (2003) Bilateral testicular microlithiasis predicts the presence of the precursor of testicular germ cell tumors in subfertile men. World J Urol 21:341–345

    Article  CAS  Google Scholar 

  6. Ferenchick GS (1991) Anabolic/androgenic steroid abuse and thrombosis: is there a connection? Med Hypotheses 35:27–31

    Article  CAS  Google Scholar 

  7. Clark AS, Henderson LP (2003) Behavioral and physiological responses to anabolic-androgenic steroids. Neurosci Biobehav Rev 27:413–436

    Article  CAS  Google Scholar 

  8. Wagner JC (1991) Enhancement of athletic performance with drugs. An overview. Sports Med 12:250–265

    Article  CAS  Google Scholar 

  9. Cowan DA, Kicman AT (1997) Doping in sport and society: misuse, analytical tests and legal aspects. Clin Chem 43:1110–1113

    CAS  Google Scholar 

  10. Hartgens F, Kuipers H (2004) Effects of androgenic-anabolic steroids in athletes. Sports Med 34:513–554

    Article  Google Scholar 

  11. Botrè F, Pavan A (2008) Enhancement drugs and the athlete. Neurol Clin 26:149–167

    Article  Google Scholar 

  12. International Olympic Committee (2012) IOC withdraws gold medal from shot put athlete Nadzeya Ostapchuk. http://www.olympic.org/news/ioc-withdraws-gold-medal-from-shot-put-athlete-nadzeya-ostapchuk/172684

  13. Fuh MR, Huang SY, Lin TY (2004) Determination of residual anabolic steroid in meat by gas chromatography-ion trap-mass spectrometer. Talanta 64:408–414

    Article  CAS  Google Scholar 

  14. Commission of the European Communities (1999) Council directive 96/22/EC. Off J Eur Communities L 125:9

    Google Scholar 

  15. The Ministry of Agriculture, People’s Republic of China, Regulation No. 193, 2002

  16. Aufartová J, Mahugo-Santana C, Sosa-Ferrera Z, Santana-Rodríguez JJ, Nováková L, Solich P (2011) Determination of steroid hormones in biological and environmental samples using green microextraction techniques: an overview. Anal Chim Acta 704:33–46

    Article  CAS  Google Scholar 

  17. Abdel-Khalik J, Björklund E, Hansen M (2013) Simultaneous determination of endogenous steroid hormones in human and animal plasma and serum by liquid or gas chromatography coupled to tandem mass spectrometry. J Chromatogr B 928:58–77

    Article  CAS  Google Scholar 

  18. Penning TM, Lee SH, Jin Y, Gutierrez A, Blair IA (2010) Liquid chromatography-mass spectrometry (LC-MS) of steroid hormone metabolites and its applications. J Steroid Biochem Mol Biol 121:546–555

    Article  CAS  Google Scholar 

  19. Huang XJ, Lin JB, Yuan DX, Hu RZ (2009) Determination of steroid sex hormones in wastewater by stir bar sorptive extraction based on poly(vinylpyridine-ethylene dimethacrylate) monolithic material and liquid chromatographic analysis. J Chromatogr A 1216:3508–3511

    Article  CAS  Google Scholar 

  20. Gañán J, Morante-Zarcero S, Gallego-Picó A, Garcinuño RM, Fernández-Hernando P, Sierra I (2014) Evaluation of a molecularly imprinted polymer for determination of steroids in goat milk by matrix solid phase dispersion. Talanta 126:157–169

    Article  CAS  Google Scholar 

  21. Xu X, Liang FH, Shi JY, Zhao X, Liu Z, Wu LJ, Song Y, Zhang HQ, Wang ZM (2013) Determination of hormones in milk by hollow fiber-based stirring extraction bar liquid-liquid microextraction gas chromatography mass spectrometry. Anal Chim Acta 790:39–46

    Article  CAS  Google Scholar 

  22. Liu W, Zhang L, Fan LB, Lin Z, Cai YM, Wei ZY, Chen GN (2012) An improved hollow fiber solvent-stir bar microextraction for the preconcentration of anabolic steroids in biological matrix with determination by gas chromatography-mass spectrometry. J Chromatogr A 1233:1–7

    Article  CAS  Google Scholar 

  23. Tomšíková H, Aufartová J, Solich P, Sosa-Ferrera Z, Santana-Rodríguez JJ, Novákova L (2012) High-sensitivity analysis of female-steroid hormones in environmental samples. Trends Anal Chem 34:35–58

    Article  CAS  Google Scholar 

  24. Gosetti F, Mazzucco E, Gennaro MC, Marengo E (2013) Ultra high performance liquid chromatography tandem mass spectrometry determination and profiling of prohibited steroids in human biological matrices. J Chromatogr B 927:22–36

    Article  CAS  Google Scholar 

  25. Mi XX, Li SC, Li YH, Wang KQ, Zhu D, Chen G (2014) Quantitative determination of 26 steroids in eggs from various species using liquid chromatography-triple quadrupole-mass spectrometry. J Chromatogr A 1356:54–63

    Article  CAS  Google Scholar 

  26. Keevil BG (2013) Novel liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for measuring steroids. Best Pract Res Clin Endocrinol Metab 27:663–674

    Article  CAS  Google Scholar 

  27. Eeckhaut AV, Lanckmans K, Sarre S, Smolders I (2009) Validation of bioanalytical LC–MS/MS assays: evaluation of matrix effects. J Chromatogr B 877:2198–2207

    Article  CAS  Google Scholar 

  28. Liu H, Dasgupta PK (1996) Analytical chemistry in a drop, solvent extraction in a microdrop. Anal Chem 68:1817–1821

    Article  CAS  Google Scholar 

  29. Jeannot MA, Cantwell FF (1996) Solvent microextraction into a single drop. Anal Chem 68:2236–2240

    Article  CAS  Google Scholar 

  30. Jeannot MA, Cantwell FF (1997) Mass transfer characteristics of solvent extraction into a single drop at the tip of a syringe needle. Anal Chem 69:235–239

    Article  CAS  Google Scholar 

  31. Pedersen-Bjergaard S, Rasmussen KE (1999) Liquid-liquid-liquid microextraction for sample preparation of biological fluids prior to capillary electrophoresis. Anal Chem 71:2650–2656

    Article  CAS  Google Scholar 

  32. Socas-Rodríguez B, Asensio-Ramos M, Hernández-Borges J, Rodríguez-Delgado MÁ (2014) Analysis of oestrogenic compounds in dairy products by hollow-fibre liquid-phase microextraction coupled to liquid chromatography. Food Chem 149:319–325

    Article  CAS  Google Scholar 

  33. Kawaguchi M, Takatsu A (2009) Miniaturized hollow fiber assisted liquid-phase microextraction and gas chromatography-mass spectrometry for the measurement of progesterone in human serum. J Chromatogr B 877:343–346

    Article  CAS  Google Scholar 

  34. Borijihan GR, Li YX, Gao JG, Bao JJ (2014) Development of a novel 96-well format for liquid-liquid microextraction and its application in the HPLC analysis of biological samples. J Sep Sci 37:1155–1161

    Article  CAS  Google Scholar 

  35. Bao JJ, Liu XJ, Zhang Y, Li YX (2014) The development of a novel high-throughput method for measuring octanol/water distribution coefficient based on hollow fiber membrane solvent microextraction technique. J Chromatogr B 967:183–189

    Article  CAS  Google Scholar 

  36. Bonfiglio R, King RC, Olah TV, Merkle K (1999) The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun Mass Spectrom 13:1175–1185

    Article  CAS  Google Scholar 

  37. Leinonen A, Vuorensola K, Lepola LM, Kuuranne T, Kotiaho T, Ketola RA, Kostiainen R (2006) Liquid-phase microextraction for sample preparation in analysis of unconjugated anabolic steroids in urine. Anal Chim Acta 559:166–172

    Article  CAS  Google Scholar 

  38. D.C. Jones, US Patent 7999084, 16 Aug 2011

  39. Audunsson GA (1986) Aqueous/aqueous extraction by means of a liquid membrane for sample cleanup and preconcentration of amines in a flow system. Anal Chem 58:2714–2723

    Article  CAS  Google Scholar 

  40. Schlüsener MP, Bester K (2005) Determination of steroid hormones, hormone conjugates and macrolide antibiotics in influents and effluents of sewage treatment plants utilising high-performance liquid chromatography/tandem mass spectrometry with electrospray and atmospheric pressure chemical ionization. Rapid Commun Mass Spectrom 19:3269–3278

    Article  CAS  Google Scholar 

  41. Farré M, Pérez S, Goncalves C, Alpendurada MF, Barceló D (2010) Green analytical chemistry in the determination of organic pollutants in the aquatic environment. Trends Anal Chem 29:1347–1362

    Article  CAS  Google Scholar 

  42. Arthur CL, Pawliszyn J (1990) Solid phase microextraction with thermal desorption using fused silica optical fibers. Anal Chem 62:2145–2148

    Article  CAS  Google Scholar 

  43. Marcos J, Renau N, Casals G, Segura J, Ventura R, Pozo OJ (2014) Investigation of endogenous corticosteroids profiles in human urine based on liquid chromatography tandem mass spectrometry. Anal Chim Acta 812:92–104

    Article  CAS  Google Scholar 

  44. Tölgyesi Á, Tölgyesi L, Sharma VK, Sohn M, Fekete J (2010) Quantitative determination of corticosteroids in bovine milk using mixed-mode polymeric strong cation exchange solid-phase extraction and liquid chromatography-tandem mass spectrometry. J Pharm Biomed 53:919–928

    Article  CAS  Google Scholar 

  45. Ray JA, Kushnir MM, Yost RA, Rockwood AL, Meikle AW (2015) Performance enhancement in the measurement of 5 endogenous steroids by LC-MS/MS combined with differential ion mobility spectrometry. Clin Chim Acta 438:330–336

    Article  CAS  Google Scholar 

  46. Almeida C, Nogueira JMF (2015) Determination of steroid sex hormones in real matrices by bar adsorptive microextraction (BAμE). Talanta 136:145–154

    Article  CAS  Google Scholar 

  47. European Medicines Agency (2009) Guideline on validation of bioanalytical methods. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf

  48. Liu HL, He CY, Wen DW, Liu HW, Liu F, Li K (2006) Extraction of testosterone and epitestosterone in human urine using 2-propanol-salt-H2O system. Anal Chim Acta 557:329–336

    Article  CAS  Google Scholar 

  49. Chiesa LM, Noblile M, Biolatti B, Pavlovic R, Panseri S, Cannizzo FT, Arioli F (2016) Detection of selected corticosteroids and anabolic steroids in calf milk replacers by liquid chromatography-electrospray ionization-tandem mass spectrometry. Food Control 61:196–203

    Article  CAS  Google Scholar 

  50. Brito DM, de Oca Porto RM, Correa Vidal MT, Ojeda RS, Pérez AR (2010) Excretion study of clomiphene in human urine. Evaluation of endogenous steroids profile after multiple oral doses. J Braz Chem Soc 21:12

    Article  Google Scholar 

  51. Guo F, Shao J, Liu Q, Shi JB, Jing GB (2014) Automated and sensitive determination off our anabolic androgenic steroids in urine by on line turbulent flow solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry: a novel approach for clinical monitoring and doping control. Talanta 125:432–438

    Article  CAS  Google Scholar 

  52. Regal P, Cepeda A, Fente C (2012) Development of an LC-MS/MS method to quantify sex hormones in bovine milk and influence of pregnancy in their levels. Food Addit Contam 29:770–779

    Article  CAS  Google Scholar 

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Acknowledgments

The authors appreciate the financial support from the National Natural Science Foundation of China (no. 21375093) and the Specialized Research Fund for the Doctoral Program of Higher Education (no. 20130032120081), Youth Fund of the Science and Technology Committee of Tianjin Municipal Government (no. 15JCQNJC43200) and the Independent Innovation Foundation of Tianjin University (no. 1102213).

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Authors and Affiliations

  1. Tianjin Key Laboratory for Modern Drug Delivery and High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin, 300072, China

    Guanzhong Luo & Youxin Li

  2. Tianjin Product Quality Inspection Technology Research Institute, 26, Kaihua Road, Huayuan Industrial Area, Tianjin, China

    Guanzhong Luo

  3. Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, 92 Weijin Road, Nankai District, Tianjin, 300072, China

    James J. Bao

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  1. Guanzhong Luo
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Correspondence to Youxin Li or James J. Bao.

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Luo, G., Li, Y. & Bao, J.J. Development and application of a high-throughput sample cleanup process based on 96-well plate for simultaneous determination of 16 steroids in biological matrices using liquid chromatography–triple quadrupole mass spectrometry. Anal Bioanal Chem 408, 1137–1149 (2016). https://doi.org/10.1007/s00216-015-9213-1

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