chemicallytechFine Chemical TechnologiesТонкие химические технологии2410-65932686-7575MIREA – Russian Technological University (RTU MIREA).10.32362/2410-6593-2017-12-2-81-99chemicallytech-85Research ArticleCHEMISTRY AND TECHNOLOGY OF MEDICINAL COMPOUNDS AND BIOLOGICALLY ACTIVE SUBSTANCESХИМИЯ И ТЕХНОЛОГИЯ ЛЕКАРСТВЕННЫХ ПРЕПАРАТОВ И БИОЛОГИЧЕСКИ АКТИВНЫХ СОЕДИНЕНИЙDEVELOPMENT OF A TECHNIQUE FOR THE MASS-SPECTROMETRIC DETERMINATION OF GONADOTROPIN-RELEASING HORMONE AND ITS ANALOGS FOR DOPING CONTROLРАЗРАБОТКА МЕТОДИКИ МАСС-СПЕКТРОМЕТРИЧЕСКОГО ОПРЕДЕЛЕНИЯ РИЛИЗИНГ-ГОРМОНА ГОНАДОТРОПИНА И ЕГО АНАЛОГОВ В ЦЕЛЯХ ДОПИНГ-КОНТРОЛЯШестаковаК. М.ShestakovaK. M.

инженер отдела пептидного допинга и анализа крови

105005, Россия, Москва, Елизаветинский пер., д. 10, стр. 1

Moscow, 105005 Russia

noemail@neicon.ruДудкоГ. А.DudkoG. A.

начальник отдела хромато-масс-спектрометрических методов анализа

105005, Россия, Москва, Елизаветинский пер., д. 10, стр. 1

Moscow, 105005 Russia

noemail@neicon.ruДикунецМ. А.DikunetsM. A.

кандидат химических наук, директор

105005, Россия, Москва, Елизаветинский пер., д. 10, стр. 1

Moscow, 105005 Russia

dikunets@yandex.ruФедеральное государственное бюджетное учреждение «Антидопинговый Центр»РоссияFederal State Budgetary Institution “Antidoping Centre”Russian Federation2017280420171228199Copyright © Shestakova K.M., Dudko G.A., Dikunets M.A., 20172017Шестакова К.М., Дудко Г.А., Дикунец М.А.Shestakova K.M., Dudko G.A., Dikunets M.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.finechem-mirea.ru/jour/article/view/85

In the present study, a rapid, sensitive, and selective method for determination of several synthetic analogues of gonadotropin-releasing hormone in human urine by solid-phase extraction and ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was developed. Various parameters affecting sample preparation, LC separation, and MS/MS detection were investigated, and optimized conditions were identified. The UPLC-MS/MS system was equipped with an electrospray ion source operating in positive ion mode with selected reaction monitoring. Leuprolide-13С6 was used as internal standard for analytes quantitative assessment. The proposed method was validated considering the parameters specificity, linearity (0.1-10 ng/ml), recovery (52-98%), limit of detection (0.1 ng/ml), matrix effects and stability.

В настоящей работе представлена методика быстрого, чувствительного и селективного определения ряда синтетических аналогов рилизинг-гормона гонадотропина в моче человека с использованием твердофазной экстракции и сверхпроизводительной жидкостной хроматографии в сочетании с тандемной масс-спектрометрией (СВЭЖХ-МС/МС). Определены и оптимизированы параметры, влияющие на пробоподготовку, хроматографическое разделение и МС/МС-детектирование. Последнее проводили в условиях электрораспылительной ионизации с регистрацией положительно заряженных ионов в режиме мониторинга выбранных реакций. Для количественной оценки концентраций соединений в качестве внутреннего стандарта использовали леупролид-13С6. Предложенная методика была валидирована с учетом параметров специфичности, линейности (0.1-10 нг/мл), степени извлечения (52-98%), предела обнаружения (0.1 нг/мл), матричного эффекта и стабильности.

рилизинг-гормон гонадотропинаагонисты рилизинг-гормона гонадотропинатвердофазная экстракциясверхпроизводительная жидкостная хроматография в сочетании с тандемной масс-спектрометриейСВЭЖХ-МС/МСсверхпроизводительная жидкостная хроматография в сочетании с масс-спектрометрией высокого разрешенияСВЭЖХ-МСВРдопинг-контрольgonadotropin-releasing hormonegonadotropin-releasing hormone agonistssolidphase egonadotropin-releasing hormongonadotropin-releasing hormon agonistssolid-phase extractionultra-performance liquid chromatography coupled to tandem mass spectrometryUPLCMS/MSultra-performance liquid chromatography coupled to high resolution mass spectrometryUPLC-HRMSdoping controlReferencesMareck U., Geyer H., Opfermann G., Thevis M., Schänzer W. Factors influencing the steroid profile in doping control analysis // J. Mass Spectrom. 2008. V. 43. № 7. P. 877-891.Mareck U., Geyer H., Opfermann G., Thevis M., Schänzer W. Factors influencing the steroid profile in doping control analysis // J. Mass Spectrom. 2008. V. 43. № 7. P. 877–891.Handelsman D.J. The rationale for banning human chorionic gonadotropin and estrogen blockers in sport // J. Clin. Endocrinol. Metab. 2006. V. 91. № 5. P. 1646-1653.Handelsman D.J. The rationale for banning human chorionic gonadotropin and estrogen blockers in sport // J. Clin. Endocrinol. Metab. 2006. V. 91. № 5. P. 1646–1653.Stenman U.H., Hotakainen K., Alfthan H. Gonadotropins in doping: pharmacological basis and detection of illicit use // Br. J. Pharmacol. 2008. V. 154. № 3. P. 569-583.Stenman U.H., Hotakainen K., Alfthan H. Gonadotropins in doping: pharmacological basis and detection of illicit use // Br. J. Pharmacol. 2008. V. 154. № 3. P. 569–583.Gore A.C. Gonadotropin-releasing hormone (GnRH) neurons: gene expression and neuroanatomical studies // Prog. Brain Res. 2002. V. 141. P. 193-208.Gore A.C. Gonadotropin-releasing hormone (GnRH) neurons: gene expression and neuroanatomical studies // Prog. Brain Res. 2002. V. 141. P. 193–208.Esposito S., Deventer K., Geldof L., Van Eenoo P. In vitro models for metabolic studies of small peptide hormones in sport drug testing // J. Pept. Sci. 2015. V. 21. № 1. P. 1-9.Esposito S., Deventer K., Geldof L., Van Eenoo P. In vitro models for metabolic studies of small peptide hormones in sport drug testing // J. Pept. Sci. 2015. V. 21. № 1. P. 1–9.Richards S.L., Cawley A.T., Cavicchioli R., Suann C.J., Pickford R., Raftery M.J. Aptamer based peptide enrichment for quantitative analysis of gonadotropin-releasing hormone by LC-MS/MS // Talanta. 2016. V. 150. P. 671-680.Richards S.L., Cawley A.T., Cavicchioli R., Suann C.J., Pickford R., Raftery M.J. Aptamer based peptide enrichment for quantitative analysis of gonadotropin-releasing hormone by LC–MS/MS // Talanta. 2016. V. 150. P. 671–680.Thomas A., Walpurgis K., Krug O., Schänzer W., Thevis M. Determination of prohibited, small peptides in urine for sports drug testing by means of nano-liquid chromatography/benchtop quadrupole orbitrap tandem-mass spectrometry // J. Chromatogr. A 2012. V. 1259. P. 251-257.Thomas A., Walpurgis K., Krug O., Schänzer W., Thevis M. Determination of prohibited, small peptides in urine for sports drug testing by means of nano-liquid chromatography/benchtop quadrupole orbitrap tandem-mass spectrometry // J. Chromatogr. A 2012. V. 1259. P. 251–257.Thomas A., Geyer H., Kamber M., Schänzer W., Thevis M. Mass spectrometric determination of gonadotrophin-releasing hormone (GnRH) in human urine for doping control purposes by means of LC-ESI-MS/MS // J. Mass Spectrom. 2008. V. 43. № 7. P. 908-915.Thomas A., Geyer H., Kamber M., Schänzer W., Thevis M. Mass spectrometric determination of gonadotrophin-releasing hormone (GnRH) in human urine for doping control purposes by means of LC-ESI-MS/MS // J. Mass Spectrom. 2008. V. 43. № 7. P. 908–915.Handelsman D.J., Swerdloff R.S. Pharmacokinetics of gonadotropin-releasing hormone and its analogs // Endocr. Rev. 1986. V. 7. № 1. P. 95-105.Handelsman D.J., Swerdloff R.S. Pharmacokinetics of gonadotropin-releasing hormone and its analogs // Endocr. Rev. 1986. V. 7. № 1. P. 95–105.Yoshida T. Peptide separation by Hydrophilic-Interaction Chromatography: a review // J. Biochem. Biophys. Methods. 2004. V. 60. № 3. P. 265-280.Yoshida T. Peptide separation by HydrophilicInteraction Chromatography: a review // J. Biochem. Biophys. Methods. 2004. V. 60. № 3. P. 265–280.Sample preparation and chromatografic columns of company Waters // Analiticheskiy kontrol'. Farmatcevticheskaya otrasl' (Analytical Control. Pharmaceutical Industry). 2010. V. 23. № 6. P. 88-89 (in Russ.).Sample preparation and chromatografic columns of company Waters // Analiticheskiy kontrol'. Farmatcevticheskaya otrasl' (Analytical Control. Pharmaceutical Industry). 2010. V. 23. № 6. P. 88-89 (in Russ.).Garcia M.C. The effect of the mobile phase additives on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatographyelectrospray mass spectrometry // J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 2005. V. 825. № 2. P. 111-123.Garcia M.C. The effect of the mobile phase additives on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatographyelectrospray mass spectrometry // J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 2005. V. 825. № 2. P. 111–123.Roepstorff P., Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides // Biomed. Mass Spectrom. 1984. V. 11. № 11. P. 601.Roepstorff P., Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides // Biomed. Mass Spectrom. 1984. V. 11. № 11. P. 601.Johnson R.S., Martin S.A., Biemann K., Stults J.T., Throck Watson J. Novel fragmentation process of peptides by collision-induced decomposition in a tandem mass spectrometer: differentiation of leucine and isoleucine // Anal. Chem. 1987. V. 59. № 21. P. 2621-2625.Johnson R.S., Martin S.A., Biemann K., Stults J.T., Throck Watson J. Novel fragmentation process of peptides by collision-induced decomposition in a tandem mass spectrometer: differentiation of leucine and isoleucine // Anal. Chem. 1987. V. 59. № 21. P. 2621–2625.Bakhtiar R., Guan Z. Electron capture dissociation mass spectrometry in characterization of peptides and proteins // Biotechnol. Lett. 2006. V. 28. № 14. P. 1047-1059.Bakhtiar R., Guan Z. Electron capture dissociation mass spectrometry in characterization of peptides and proteins // Biotechnol. Lett. 2006. V. 28. № 14. P. 1047–1059.Sofianos Z.D., Katsila T., Kostomitsopoulos N., Balafas V., Matsoukas J., Tselios T., Tamvakopoulos C. In vivo evaluation and in vitro metabolism of leuprolide in mice - mass spectrometry-based biomarker measurement for efficacy and toxicity // J. Mass Spectrom. 2008. V. 43. № 10. P. 1381-1392.Sofianos Z.D., Katsila T., Kostomitsopoulos N., Balafas V., Matsoukas J., Tselios T., Tamvakopoulos C. In vivo evaluation and in vitro metabolism of leuprolide in mice – mass spectrometry-based biomarker measurement for efficacy and toxicity // J. Mass Spectrom. 2008. V. 43. № 10. P. 1381–1392.Michalet S., Favreau P., Stocklin R. Profiling and in vivo quantification of proteins by high resolution mass spectrometry: the example of goserelin, an analogue of luteinizing hormone-releasing hormone // Clin. Chem. Lab. Med. 2003. V. 41. № 12. P. 1589-1598.Michalet S., Favreau P., Stocklin R. Profiling and in vivo quantification of proteins by high resolution mass spectrometry: the example of goserelin, an analogue of luteinizing hormone-releasing hormone // Clin. Chem. Lab. Med. 2003. V. 41. № 12. P. 1589–1598.Kim H.K. Lee T.H., Suh J.H., Eom H.Y., Min J.W., Yeom H., Kim U., Jung H.J., Cha K.H., Choi Y.S., Youm J.R., Han S.B. Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of goserelin in rabbit plasma // J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 2010. V. 878. № 24. P. 2235-2242.Kim H.K. Lee T.H., Suh J.H., Eom H.Y., Min J.W., Yeom H., Kim U., Jung H.J., Cha K.H., Choi Y.S., Youm J.R., Han S.B. Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of goserelin in rabbit plasma // J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 2010. V. 878. № 24. P. 2235–2242.Bioanalysis of biotherapeutics summary [electronic resource] // Waters : [site] : - URL: http://www.waters.com/webassets/cms/library/docs/local_seminar_presentations/DA_Waters_Biotherapeutic_Bioanalysis_summary.pdf.Date of access: 15.08.2016.Bioanalysis of biotherapeutics summary [electronic resource] // Waters : [site] : ― URL: http://www.waters.com/webassets/cms/library/docs/local_seminar_presentations/DA_Waters_Biotherapeutic_Bioanalysis_summary.pdf. Date of access: 15.08.2016.

The authors declare that there are no conflicts of interest present.